SENECA VALLEY VIRUS BASED COMPOSITIONS AND METHODS FOR TREATING DISEASE

Abstract

The present invention relates to a novel RNA picornavirus that is called Seneca Valley virus ("SVV"). The invention provides isolated SVV nucleic acids and proteins encoded by these nucleic acids. Further, the invention provides antibodies that are raised against the SVV proteins. Because SVV has the ability to selectively kill some types of tumors, the invention provides methods of using SVV and SVV polypeptides to treat cancer. Because SVV specifically targets certain tumors, the invention provides methods of using SVV nucleic acids and proteins to detect cancer. Additionally, due to the information provided by the tumor-specific mechanisms of SVV, the invention provides methods of making new oncolytic virus derivatives and of altering viruses to have tumor-specific tropisms.

Description

[0001] This application is a continuation of U.S. Ser. No. 13/209,124, which was filed on Aug. 12, 2011, which is a continuation of U.S. Ser. No. 12/576,296, which was filed on Oct. 9, 2009, now U.S. Pat. No. 8,039,606 issued on Oct. 18, 2011, which is a continuation of U.S. Ser. No. 11/335,891, which was filed on Jan. 19, 2006, now U.S. Pat. No. 7,638,318 issued on Dec. 29, 2009, which is a continuation-in-part of International Application No. PCT/US2004/031504 (International Publication No. WO 2005/030139), which was filed on Sep. 23, 2004, which claims priority to U.S. Ser. No. 60/506,182, which was filed on Sep. 26, 2003. This application also claims priority to U.S. Ser. No. 60/664,442, which was filed on Mar. 23, 2005 and U.S. Ser. No. 60/726,313, which was filed on Oct. 13, 2005. These applications are hereby incorporated by reference in their entirety.

[0002] This disclosure contains material that is subject to copyright protection. The copyright owner has no objection to the facsimile reproduction by anyone of the patent document or the patent disclosure, as it appears in the U.S. Patent and Trademark Office patent file or records, but otherwise reserves any and all copyright rights.

[0003] All patent applications, published patent applications, issued and granted patents, texts, and literature references cited in this specification are hereby incorporated herein by reference in their entirety to more fully describe the state of the art to which the present invention pertains.

BACKGROUND OF THE INVENTION

[0004] Virotherapy holds great promise for treating cancer. Oncolytic viruses, which aim to specifically infect and kill cancer cells, whether native and/or engineered, may be more efficacious and less toxic than alternative treatments, such as chemotherapy and radiation. In addition, oncolytic virus therapy that uses replication competent viruses is the only therapy known that can amplify the therapeutic at the pharmacologically desired site.

[0005] A key aspect of cancer therapy is to achieve a high rate of killing of cancer cells versus normal cells. Accomplishing this goal has been difficult for many reasons, including the wide array of cell types involved, the systemic dissemination of cancer cells due to metastases, and the narrow biological differences between normal and cancer cells. While progress has been made, much still needs to be done to improve upon current cancer therapies.

[0006] In the past, surgeons have tried to remove tumors surgically without substantially harming the patient. Even complete removal of a primary tumor does not ensure survival since earlier metastases to unknown sites in the body are left undetected. There is also some research suggesting that surgical intervention may enhance the growth of distant metastases due to removal of tumor cells producing angiogenesis inhibitors. Finally, in many cases, the tumor grows back at the original site after surgical removal. Radiation aims to selectively destroy the most rapidly proliferating cells at the expense of the others. However, tumor cells can escape radiation therapy either by becoming resistant or by being in a non-dividing state during treatment. In addition, radiation is not always selective in that many normal cells are actively dividing and killed by the treatment (cells in bone marrow, gastrointestinal cells, hair follicles, etc.).

[0007] Like radiation, chemotherapy is not completely selective and thus destroys many normal cells, and does not kill all tumor cells due to drug resistance and/or division state of the cell. Thus, chemotherapy and radiation therapies exploit a small differential sensitivity that exists between normal and cancer cells, giving them a narrow therapeutic index. A small therapeutic index is clearly an undesirable property of any modality to treat cancer. Therefore, novel cancer therapeutic approaches overcoming these limitations are desired.

[0008] One such novel approach is oncolytic virus therapy. Initially, replication-defective viruses carrying cytotoxic transgenes were utilized in attempts to treat cancer. However, they were found to be inefficient in transduction of tumors, inadequate spread within the tumor mass and not adequately selective toward cancers. To overcome this limitation, viruses were either modified to replicate selectively in tumor cells or viruses were discovered to have natural tumor-selective properties. These oncolytic viruses thus had the properties to replicate, spread, and kill tumor cells selectively through a tumor mass by locally injecting the virus or by systemically delivering the virus (FIG. 1).

[0009] Despite the early promise of this newly defined class of anti-cancer therapeutics, several limitations remain that may limit their use as a cancer therapeutic. Therefore, there is an ongoing need for novel oncolytic viruses that can be utilized for cancer therapy.

SUMMARY OF THE INVENTION

[0010] A novel RNA picornavirus has been discovered (hereafter referred to as Seneca Valley virus ("SVV")) whose native properties include the ability to selectively kill some types of tumors. As demonstrated below in the examples, SVV selectively kills tumor lines with neurotropic properties, in most cases with a greater than 10,000 fold difference in the amount of virus necessary to kill 50% of tumor cells versus normal cells (i.e., the EC₅₀ value). This result also translates in vivo, where tumor explants in mice are selectively eliminated. Further, in vivo results indicate that SVV is not toxic to normal cells, in that up to 1×10¹⁴ vp/kg (vector or virus particles per kilogram) systemically administered causes no mortality and no visible clinical symptoms in immune deficient or immune competent mice.

[0011] SVV elicits efficacy at doses as low as 1×10⁸ vp/kg; therefore, a very high therapeutic index of >100,000 is achieved. Efficacy is very robust in that 100% of large pre-established tumors in mice can be completely eradicated (see Example 11). This efficacy may be mediated with a single systemic injection of SVV without any adjunct therapy. Furthermore, SVV injected mice show neither clinical symptoms nor recurrence of tumors for at least 200 days following injection. SVV can also be purified to high titer and can be produced at >200,000 virus particles per cell in permissive cell lines. SVV-based viral therapy therefore shows considerable promise as a safe, effective and new line of treatment for selected types of cancers. Further, SVV has a small and easily manipulatable genome, simple and fast lifecycle, and a well-understood biology of replication, and thus is amenable to modification. These properties, at least in part, allow for methods that generate modified SVVs that have new cell or tissue specific tropisms, such that SVV-based therapy can be directed to new tumor types resistant to infection by the original SVV isolate.

[0012] Accordingly, the present invention provides an isolated nucleic acid comprising a nucleic acid sequence having at least 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99% sequence identity to SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 168, or a contiguous portion of any one of these sequences that is at least 50 nucleotides in length, or 95% identical to a contiguous portion of any one of these sequences that is at least 10, 15 or 20 nucleotides in length. The isolated nucleic acids of the invention can be RNA or DNA.

[0013] For all aspects of the invention, an isolated nucleic acid can comprise a nucleic acid sequence having at least about 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to a contiguous portion of any one of the SVV nucleic acid SEQ ID NO sequences herein, wherein the contiguous portion is at least about 20, 25, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500, 750, 1000, 1250, 1500, 2000 or 2500 nucleotides in length, for example. The SVV nucleic acid SEQ ID NO sequences include, for example, SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21 and 168.

[0014] For all aspects of the invention, an isolated protein or peptide can comprise an amino acid sequence having at least about 50%, 55%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to a contiguous portion of any one of the SVV amino acid SEQ ID NO sequences herein, wherein the contiguous portion is at least at least about 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 125, 150, 175, 200, 225, 250, 275, 300, or 350 amino acids in length, for example. The SVV amino acid SEQ ID NO sequences include, for example, SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22 and 169.

[0015] In another aspect, the invention provides an isolated nucleic acid comprising a nucleic acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:168, or to a contiguous portion of SEQ ID NO:168 that is at least 20, 50, 100, or 200 nucleotides in length. The isolated nucleic acids can comprise specific portions of SEQ ID NO:168, including but not limited to: the 5' untranslated region (UTR) of SVV spanning nucleotides 1-666 of SEQ ID NO:168; the coding sequence for the SVV polyprotein spanning nucleotides 667-7209 of SEQ ID NO:168; the coding sequence for the leader peptide of SVV spanning nucleotides 667-903 of SEQ ID NO:168; the coding sequence for the SVV VP4 protein spanning nucleotides 904-1116 of SEQ ID NO:168; the coding sequence for the SVV VP2 protein spanning nucleotides 1117-1968 of SEQ ID NO:168; the coding sequence for the SVV VP3 protein spanning nucleotides 1969-2685 of SEQ ID NO:168; the coding sequence for the SVV VP1 protein spanning nucleotides 2686-3477 of SEQ ID NO:168; the coding sequence for the SVV 2A protein spanning nucleotides 3478-3504 of SEQ ID NO:168; the coding sequence for the SVV 2B protein spanning nucleotides 3505-3888 of SEQ ID NO:168; the coding sequence for the SVV 2C protein spanning nucleotides 3889-4854 of SEQ ID NO:168; the coding sequence for the SVV 3A protein spanning nucleotides 4855-5124 of SEQ ID NO:168; the coding sequence for the SVV 3B protein spanning nucleotides 5125-5190 of SEQ ID NO:168; the coding sequence for the SVV 3C protein spanning nucleotides 5191-5823 of SEQ ID NO:168; the coding sequence for the SVV 3D protein spanning nucleotides 5824-7209 of SEQ ID NO:168; and the 3'UTR of SVV spanning nucleotides 7210-7280 of SEQ ID NO:168.

[0016] In one aspect, the invention provides methods for using the SVV 2A, SVV leader peptide, or other SVV proteins or peptide portions thereof, to shut off host cell protein translation. In one aspect, such SVV proteins can be used to shut off host cell protein translation by interfering or inhibiting with the cap binding protein complex in the host cell.

[0017] In another aspect, the invention provides methods for using SVV 2A or other SVV proteins or peptide portions thereof in order to cleave a peptide or protein.

[0018] In other aspects, the invention provides an isolated nucleic acid that hybridizes under conditions of high, moderate stringency or low stringency to SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 168, or to a contiguous portion of any one of these sequences that is at least 50 nucleotides in length.

[0019] In another aspect, the invention provides a vector comprising a nucleic acid sequence having at least 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99% sequence identity to SEQ ID NOS 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 168, or to a contiguous portion of any one of these sequences that is at least 50 nucleotides in length. Vector compositions can also comprise the nucleic acid regions of SEQ ID NO:168 that code for SVV proteins.

[0020] The present invention also provides an isolated polypeptide encoded by a nucleic acid having at least 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99% sequence identity to a nucleic acid sequence comprising SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, or to a contiguous portion of any one of these sequences that is at least 50 nucleotides in length. The invention also provides an isolated polypeptide encoded by a nucleic acid having at least 95%, 96%, 97%, 98%, or 99% sequence identity to a nucleic acid region of SEQ ID NO:168 that encodes a SVV protein.

[0021] In one aspect, the invention provides an isolated polypeptide comprising an amino acid sequence having at least 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99% sequence identity to SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 169, or to a contiguous portion of any one of these sequences that is at least 10 amino acids in length.

[0022] In another aspect, the invention provides an isolated polypeptide comprising an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity to a contiguous portion of SEQ ID NO:169 that is at least 9, 10, 15, 20 or 50 amino acids in length. Exemplary contiguous portions of SEQ ID NO:169, include but are not limited to, regions that comprise a SVV protein, such as: the leader peptide spanning residues 1-79; VP4 spanning residues 80-150; VP2 spanning residues 151-434; VP3 spanning residues 435-673; VP1 spanning residues 674-937; 2A spanning residues 938-946; 2B spanning residues 947-1074; 2C spanning residues 1075-1396; 3A spanning residues 1397-1486; 3B spanning residues 1487-1508; 3C spanning residues 1509-1719; and 3D spanning residues 1720-2181.

[0023] In another aspect, the invention provides an isolated antibody which specifically binds a polypeptide comprising an amino acid sequence having at least 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99% sequence identity to SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 169, or to a contiguous portion of any one of these sequences that is at least 9, 10, 15, or amino acids in length. The isolated antibody can be generated such that it binds to any protein epitope or antigen of SEQ ID NOS:2 or 169. Further, the antibody can be a polyclonal antibody, a monoclonal antibody or a chimeric antibody.

[0024] In one aspect, the invention provides an isolated SVV or derivative or relative thereof, having a genomic sequence comprising a sequence that is at least 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99% identical to SEQ ID NO:1 or SEQ ID NO:168.

[0025] In another aspect, the invention provides an isolated virus having all the identifying characteristics and nucleic acid sequence of American Type Culture Collection (ATCC) Patent Deposit number PTA-5343. Some of the viruses of the present invention are directed to the PTA-5343 isolate, variants, homologues, relatives, derivatives and mutants of the PTA-5343 isolate, and variants, homologues, derivatives and mutants of other viruses that are modified in respect to sequences of SVV (both wild-type and mutant) that are determined to be responsible for its oncolytic properties.

[0026] The present invention further provides an isolated SVV comprising the following characteristics: a single stranded RNA genome (positive (+) sense strand) of ˜7.5 or of ˜7.3 kilobases (kb); a diameter of ˜27 nanometers (nm); a capsid comprising at least 3 proteins that have approximate molecular weights of about 31 kDa, 36 kDa and 27 kDa; a buoyant density of approximately 1.34 g/mL on cesium chloride (CsCl) gradients; and replication competence in tumor cells. In this aspect, the 31 kDa capsid protein (VP1) can comprise an amino acid sequence that is at least 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99% identical to SEQ ID NO:8 or residues 674-937 of SEQ ID NO:169; the 36 kDa capsid protein (VP2) can comprise an amino acid sequence that is at least 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99% identical to SEQ ID NO:4 or residues 151-434 of SEQ ID NO:169; and the 27 kDa capsid protein (VP3) can comprise an amino acid sequence that is at least 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99% identical to SEQ ID NO:6 or residues 435-673 of SEQ ID NO:169.

[0027] In another aspect, the invention provides an isolated SVV derivative or relative comprising the following characteristics: replication competence in tumor cells, tumor-cell tropism, and lack of cytolysis in normal cells. An SVV relative includes SVV-like picornaviruses, including viruses from the following USDA isolates: MN 88-36695, NC 88-23626, IA 89-47552, NJ 90-10324, IL 92-48963, CA 131395; LA 1278; IL 66289; IL 94-9356; MN/GA 99-29256; MN 99197; and SC 363649. If an SVV-like picornavirus does not naturally have the characteristics of replication competence in tumor cells, tumor-cell tropism, and lack of cytolysis in non-tumor cells, then the SVV-like picornavirus can be mutated such that these characteristics are obtained. Such mutations can be designed by comparing the sequence of the SVV-like picornavirus to SVV, and making mutations into the SVV-like picornavirus such that its amino acid sequence is identical or substantially identical (in a particular region) to SVV. In another aspect, the virus is replication competent in tumor cell types having neuroendocrine properties.

[0028] In other aspects, the present invention provides: a pharmaceutical composition comprising an effective amount of a virus of the invention and a pharmaceutically acceptable carrier; a cell comprising a virus of the invention; a viral lysate containing antigens of a virus of the invention; and an isolated and purified viral antigen obtained from a virus of the invention.

[0029] In yet another aspect, the invention provides a method of purifying a virus of the invention, comprising: infecting a cell with the virus; harvesting cell lysate; subjecting cell lysate to at least one round of gradient centrifugation; and isolating the virus from the gradient.

[0030] In another aspect, the invention provides a method for treating cancer comprising administering an effective amount of a virus or derivative thereof, so as to treat the cancer, wherein the virus has a genomic sequence that comprises a sequence that is at least 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99% identical to SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 168, or to a portion of SEQ ID NO:1 or SEQ ID NO:168. In one aspect, the invention provides a method for treating cancer a method for treating cancer comprising administering an effective amount of a virus or derivative thereof, so as to treat the cancer, wherein the virus has a genomic sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:1. The virus that has a genomic sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:1 can be, for example, a SVV mutant, a SVV-like picornavirus, or a cardiovirus. The SVV-like picornavirus can be, for example, a virus from one of the following isolates MN 88-36695, NC 88-23626, IA 89-47752, NJ 90-10324, IL 92-48963, CA 131395; LA 1278; IL 66289; IL 94-9356; MN/GA 99-29256; MN 99197; and SC 363649. The SVV-like picornaviruses can be wild-type or mutant.

[0031] In another aspect, the invention provides a method for treating cancer comprising administering an effective amount of a virus comprising a capsid encoding region that comprises a sequence that is at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NOS:3, 5, 7, nucleotides 904-3477 of SEQ ID NO:169, or to a contiguous portion thereof that is at least 75, 100, 200, or 500 nucleotides in length. The invention also provides a method for treating cancer comprising administering an effective amount of a virus comprising a capsid that comprises an amino acid sequence that is at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:4, 6, 8, residues 80-937 of SEQ ID NO:169, or a contiguous portion thereof that is at least 25, 50, or 100 amino acids in length.

[0032] In one aspect, the present invention provides a method for inhibiting cancer progression comprising contacting a cancer cell with a virus or derivative thereof, wherein the virus or derivative thereof specifically binds to the cancerous cell, wherein the virus has a genomic sequence that comprises a sequence that is at least 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99% identical to SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, or 168.

[0033] In another aspect, the invention provides a method for inhibiting cancer progression comprising contacting a cancer cell with a virus or derivative thereof, wherein the virus or derivative thereof specifically infects the cancerous cell, wherein the virus has a genomic sequence that comprises a sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 168, or to a contiguous portion of SEQ ID NO:168 that is at least 50, 100, 200, or 500 nucleotides in length.

[0034] In another aspect, the present invention provides a method for killing cancer cells comprising contacting a cancer cell with an effective amount of a virus or derivative thereof, wherein the virus has a genomic sequence that comprises a sequence that is at least 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99% identical to SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, or 168.

[0035] In another aspect, the present invention provides a method for killing cancer cells comprising contacting a cancer cell with an effective amount of a virus or derivative thereof,

[0036] wherein the virus has a genomic sequence that comprises a sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:168, or to a contiguous portion of SEQ ID NO:168 that is at least 50, 100, 200 or 500 nucleotides in length.

[0037] In these methods directed to cancer, the virus can be a picornavirus. The picornavirus can be a cardiovirus, erbovirus, aphthovirus, kobuvirus, hepatovirus, parechovirus, teschovirus, enterovirus, rhinovirus, SVV, or an SVV-like picornavirus. The cardiovirus can be selected from the group consisting of: vilyuisk human encephalomyelitis virus, Theiler's murine encephalomyelitis virus, and encephalomyocarditis virus. The SVV can be a virus having the ATCC deposit number PTA-5343 or a virus comprising a nucleic acid sequence that is at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:1 or SEQ ID NO:168, or to a contiguous portion thereof that is at least 50, 100, 200, or 500 nucleotides in length. The SVV-like picornavirus can be a virus comprising a nucleic acid sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:168, or to a contiguous portion thereof that is at least 50, 100, 200, or 500 nucleotides in length. The SVV-like picornavirus can be, for example, a virus from one of the following isolates MN 88-36695, NC 88-23626, IA 89-47752, NJ 90-10324, IL 92-48963, CA 131395; LA 1278; IL 66289; IL 94-9356; MN/GA 99-29256; MN 99197; and SC 363649. The SVV-like picornaviruses can be wild-type or mutant.

[0038] The present invention also provides a method of killing an abnormally proliferative cell comprising contacting the cell with a virus of the invention. In one aspect, the abnormally proliferative cell is a tumor cell. In various aspects of this method, the tumor cell is selected from the group consisting of: human small cell lung cancer, human retinoblastoma, human neuroblastoma, human medulloblastoma, mouse neuroblastoma, Wilms' tumor, and human non-small cell lung cancer.

[0039] The present invention also provides a method of treating a neoplastic condition in a subject comprising administering to the subject an effective amount of a virus of the invention to the mammal. In one aspect, the neoplastic condition is a neuroendocrine cancer. In another aspect, the subject is a mammal. In another aspect, the mammal is a human.

[0040] The present invention also provides a method of producing a virus of the invention, comprising: culturing cells infected with the virus under conditions that allow for replication of the virus and recovering the virus from the cells or the supernatant. In one aspect of this method, the cells are PER.C6 cells. In another aspect of this method, the cells are H446 cells. In the various aspects of this method, the cells may produce over 200,000 virus particles per cell.

[0041] In another aspect, the present invention provides a method for detecting a virus of the invention, comprising: isolating RNA from test material suspected to contain the virus of the invention; labeling RNA corresponding to at least 15 contiguous nucleotides of SEQ ID NO:1 or SEQ ID NO:168; probing the test material with the labeled RNA; and detecting the binding of the labeled RNA with the RNA isolated from the test material, wherein binding indicates the presence of the virus. In another aspect, the present invention provides a nucleic acid probe comprising a nucleotide sequence corresponding to at least 15 contiguous nucleotides of SEQ ID NO:1 or SEQ ID NO:168, or its complement.

[0042] The present invention also provides a method for making an oncolytic virus, the method comprising: (a) comparing a SVV genomic sequence with a test virus genomic sequence; (b) identifying at least a first amino acid difference between a polypeptide encoded by the SVV genomic sequence and a polypeptide encoded by the test virus genomic sequence; (c) mutating the test virus genomic sequence such that the polypeptide encoded by the test virus genomic sequence has at least one less amino acid difference to the polypeptide encoded by the SVV genomic sequence; (d) transfecting the mutated test virus genomic sequence into a tumor cell; and (e) determining whether the tumor cell is lytically infected by the mutated test virus genomic sequence. In one aspect, the amino acid(s) mutated in the test virus are amino acids in a structural region, such as in the capsid encoding region. In another aspect, the amino acids mutated in the test virus are amino acids in a non-structural region.

[0043] In one aspect of the method for making an oncolytic virus, the SVV genomic sequence is obtained from the isolated SVV having the ATCC deposit number PTA-5343 or from a virus comprising a sequence that is at least 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99% identical to SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 168, or a contiguous portion thereof. In one aspect, the SVV genomic sequence is obtained from the isolated SVV having the ATCC deposit number PTA-5343 or from a virus comprising a sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 168, or a contiguous portion thereof that is at least 50, 100, 200, or 500 nucleotides in length. In another aspect of this method, the step of mutating the test virus genomic sequence comprises mutating a cDNA having the test virus genomic sequence. In another aspect of this method, the step of transfecting the mutated test virus genomic sequence comprises transfecting RNA, wherein the RNA is generated from the cDNA having the mutated test virus genomic sequence.

[0044] In another aspect of the method for making an oncolytic virus, the test virus is a picornavirus. The test picornavirus can be a teschovirus, enterovirus, rhinovirus, cardiovirus, erbovirus, apthovirus, kobuvirus, hepatovirus, parechovirus or teschovirus. In another aspect, the test virus is a cardiovirus. In another aspect, the test virus is a SVV-like picornavirus. The SVV-like picornavirus can be, for example, a virus from one of the following isolates: MN 88-36695, NC 88-23626, IA 89-47552, NJ 90-10324, IL 92-48963, CA 131395; LA 1278; IL 66289; IL 94-9356; MN/GA 99-29256; MN 99197; and SC 363649. In another aspect, the amino acid differences identified in the methods for making an oncolytic virus are between a SVV capsid protein and a test virus capsid protein sequence. In another aspect for making an oncolytic virus, the test virus genomic sequence is selected from the group consisting of: Vilyuisk human encephalomyelitis virus, Theiler's murine encephalomyelitis virus, and encephalomyocarditis virus. In another aspect, the test virus genomic sequence is selected from an encephalomyocarditis virus. In yet another aspect, the encephalomyocarditis virus, the SVV-like picornavirus, or any other test virus can be selected from an isolate having a nucleic acid sequence comprising at least 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99% sequence identity to SVV of ATCC deposit number PTA-5343 or SEQ ID NO:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 168, or a contiguous portion thereof that is at least 50, 100, 200, or 500 nucleotides in length.

[0045] In another aspect of the method for making an oncolytic cardiovirus, the amino acid difference between the test virus and SVV is in a capsid protein region of SVV, wherein the amino acid difference is aligned within SVV SEQ ID NO:4, 6, 8, residues 80-937 of SEQ ID NO:169, residues 80-150 of SEQ ID NO:169, residues 151-434 of SEQ ID NO:169, residues 435-673 of SEQ ID NO:169, or residues 674-937 of SEQ ID NO:169.

[0046] The present invention also provides a method for making a mutant virus having an altered cell-type tropism, the method comprising: (a) creating a library of viral mutants comprising a plurality of nucleic acid sequences; (b) transfecting the library of viral mutants into a permissive cell, such that a plurality of mutant viruses is produced; (c) isolating the plurality of mutant viruses; (d) incubating a non-permissive cell with the isolated plurality of mutant viruses; and (e) recovering a mutant virus that was produced in the non-permissive cell, thereby making a mutant virus having an altered tropism. In one aspect, this method further comprises the steps of: (f) incubating the recovered mutant virus in the non-permissive cell; and (g) recovering a mutant virus that that was produced in the non-permissive cell. In another aspect, the method further comprises iteratively repeating steps (f) and (g). In another aspect, the library of viral mutants is created from a parental sequence comprising SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 168, or a contiguous portion thereof.

[0047] In one aspect of the method for making a mutant virus having an altered cell-type tropism, the incubating is conducted in a multi-well high-throughput platform wherein the platform comprises a different non-permissive cell-type in each well. In this aspect, the method can further comprise screening the platform to identify which wells contain a mutant virus that kills the cells. In another aspect, the screening is conducted by analyzing light absorbance in each well.

[0048] In another aspect of the method for making a mutant virus having an altered cell-type tropism, the non-permissive cell is a tumor cell.

[0049] In another aspect of the method for making a mutant virus having an altered cell-type tropism, the step of creating the library of viral mutants comprises: (i) providing a polynucleotide having a sequence identical to a portion of a genomic sequence of a virus; (ii) mutating the polynucleotide in order to generate a plurality of different mutant polynucleotide sequences; and (iii) ligating the plurality of mutated polynucleotides into a vector having the genomic sequence of the virus except for the portion of the genomic sequence of the virus that the polynucleotide in step (i) contains, thereby creating the library of viral mutants. In one aspect, the genomic sequence of a virus is from a picornavirus. In another aspect, the genomic sequence of a virus comprises a sequence that is at least 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99% identical to SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 168, or a contiguous portion thereof. In another aspect, the genomic sequence of a virus comprises a sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 168, or a contiguous portion thereof that is at least 50, 100, 200, or 500 nucleotides in length. In one aspect, the virus that comprises a sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to a contiguous portion of SEQ ID NO:168 that is at least 50, 100, 200, or 500 nucleotides in length is a SVV-like picornavirus. In another aspect, in the step of creating the library of viral mutants, the mutating of step (ii) is conducted by random insertion of nucleotides into the polynucleotide. In one aspect, the random insertion of nucleotides is conducted by trinucleotide-mutagenesis (TRIM). In another aspect, at least a portion of the nucleotides inserted into the polynucleotide encodes an epitope tag. In another aspect, in the step of creating the library of viral mutants, the mutating of step (ii) is conducted in a capsid encoding region of the polynucleotide.

[0050] The present invention also provides a method for making a mutant virus having an altered cell-type tropism, the method comprising: (a) creating a library of mutant polynucleotide sequences of a virus, wherein the creating comprises: providing a polynucleotide encoding a capsid region of the virus; mutating the polynucleotide in order to generate a plurality of different mutant capsid-encoding polynucleotide sequences; and ligating the plurality of mutated capsid-encoding polynucleotides into a vector having the genomic sequence of the virus except for the capsid-encoding region, thereby creating the library of mutant polynucleotide sequences of the virus; (b) transfecting the library of mutant polynucleotide sequences into a permissive cell, such that a plurality of mutant viruses is produced; (c) isolating the plurality of mutant viruses; (d) incubating a non-permissive cell with the isolated plurality of mutant viruses; and (e) recovering a mutant virus that that was produced in the non-permissive cell, thereby making a mutant virus having an altered tropism. In one aspect, the method further comprises the steps of: (f) incubating the recovered mutant virus in the non-permissive cell; and (g) recovering a mutant virus that that was produced in the non-permissive cell. In another aspect, the method further comprises iteratively repeating steps (f) and (g). In another aspect, the mutating is conducted by random insertion of nucleotides into the capsid-encoding polynucleotide. In another aspect, at least a portion of the nucleotides randomly inserted into the capsid-encoding polynucleotide encodes an epitope tag. In another aspect, the random insertion of nucleotides is conducted by TRIM. In another aspect, the plurality of different mutant capsid-encoding polynucleotide sequences comprises greater than 10⁸ or 10⁹ different capsid-encoding polynucleotide sequences. The library of mutant polynucleotide sequences can be from, for example, a cardiovirus or an SVV-like picornavirus.

[0051] In one aspect, a method for making a mutant SVV having an altered cell-type tropism comprises: (a) creating a cDNA library of SVV mutants; (b) generating SVV RNA from the cDNA library of SVV mutants; (c) transfecting the SVV RNA into a permissive cell, such that a plurality of mutant SVV is produced; (d) isolating the plurality of mutant SVV; (e) incubating a non-permissive tumor cell with the isolated plurality of mutant SVV; and (f) recovering a mutant SVV that lytically infects the non-permissive tumor cell, thereby making a mutant SVV having an altered tropism. In another aspect, the method further comprises the steps of: (g) incubating the recovered mutant SVV in the non-permissive cell; and (h) recovering a mutant SVV that lytically infects the non-permissive tumor cell. In another aspect, the method further comprises iteratively repeating steps (g) and (h). In one aspect, the incubating is conducted in a multi-well high-throughput platform wherein the platform comprises a different non-permissive tumor cell-type in each well. In another aspect, the method further comprises screening the platform to identify which wells contain a mutant SVV that lytically infects the cells. In another aspect, the screening is conducted by analyzing light absorbance in each well. In one aspect, the cDNA library of SVV mutants comprises a plurality of mutant SVV capsid polynucleotide sequences. In another aspect, the plurality of mutant SVV capsid polynucleotide sequences is generated by random insertion of nucleotides. In another aspect, at least a portion of the sequence of the nucleotides randomly inserted encodes an epitope tag. In another aspect, the random insertion of nucleotides is conducted by TRIM. In another aspect, the cDNA library of SVV mutants is generated from a SVV of ATCC deposit number PTA-5343. In another aspect, the cDNA library of SVV mutants is generated from a SVV comprising a sequence having at least 99%, 95%, 90%, 85%, 80%, 75%, 70%, or 65% sequence identity to SEQ ID NO:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 168, or to a contiguous portion thereof that is at least 50, 100, 200, or 500 nucleotides in length. In one aspect, the cDNA library of SVV mutants is generated from an SVV comprising a sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:168, or to a contiguous portion thereof that is at least 50, 100, 200, or 500 nucleotides in length. In another aspect, the non-permissive tumor cell is a tumor cell-line or a tumor cell-type isolated from a patient.

[0052] The present invention also provides a method for making a mutant virus having a tumor cell-type tropism in vivo, the method comprising: (a) creating a library of viral mutants comprising a plurality of nucleic acid sequences; (b) transfecting the library of viral mutants into a permissive cell, such that a plurality of mutant viruses is produced; (c) isolating the plurality of mutant viruses; (d) administering the isolated plurality of mutant viruses to a mammal with a tumor, wherein the mammal is not a natural host of the unmutated form of the mutant virus; and (e) recovering a virus that replicated in the tumor, thereby making a mutant virus having a tumor cell-type tropism in vivo. In one aspect, the step of creating a library of viral mutants comprises: providing a polynucleotide encoding a capsid region of a virus; mutating the polynucleotide in order to generate a plurality of different mutant capsid-encoding polynucleotide sequences; and ligating the plurality of mutated capsid-encoding polynucleotides into a vector having the genomic sequence of the virus except for the capsid-encoding region, thereby creating the library of viral mutants. In another aspect, the virus recovered in step (e) lytically infects cells of the tumor. In another aspect for a method for making a mutant virus having a tumor cell-type tropism in vivo, the tumor is a xenograft, a syngeneic tumor, an orthotopic tumor or a transgenic tumor. In another aspect, the mammal is a mouse.

[0053] For all the methods of the present invention, the virus can be a picornavirus. The picornavirus can be a cardiovirus, erbovirus, aphthovirus, kobuvirus, hepatovirus, parechovirus, teschovirus, entrovirus, rhinovirus, or a virus belonging to the genus to which SVV belongs. The virus can be a cardiovirus. The virus can be an SVV-like picornavirus. The virus can be SVV. The SVV can be a SVV having the ATCC Patent Deposit No. PTA-5343 or a SVV comprising a sequence that is at least 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99% identical to SEQ ID NO:1, 3, 6, 7, 9, 11, 13, 15, 17, 19, 21, 168, or a contiguous portion thereof. Further, the cardiovirus can be selected from the group consisting of: vilyuisk human encephalomyelitis virus, Theiler's murine encephalomyelitis virus, and encephalomyocarditis virus. In one aspect, the SVV-like picornavirus is selected from the group of isolates consisting of: MN 88-36695, NC 88-23626, IA 89-47552, NJ 90-10324, IL 92-48963, CA 131395; LA 1278; IL 66289; IL 94-9356; MN/GA 99-29256; MN 99197; and SC 363649. In another aspect, the present invention encompasses any virus that is selected from an isolate having at least 99%, 95%, 90%, 85%, 80%, 75%, 70%, or 65% sequence identity to SVV of ATCC deposit number PTA-5343 or SEQ ID NO:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 168, or a contiguous portion thereof or is otherwise considered related to SVV to by sequence homology.

[0054] In another aspect, the present invention encompasses any virus having at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% nucleic acid sequence identity to SVV of ATCC deposit number PTA-5343, to SEQ ID NO:168, or to a contiguous portion of SEQ ID NOS: 1 or 168 that is at least 100, 200, 300, 400, 500, 750, 1000, 1500, or 2000 nucleotides in length.

[0055] The present invention also provides an oncolytic virus made by any of the methods for making a mutant virus disclosed herein. In one aspect, the present invention provides a method for treating a patient with an oncolytic virus, the method comprising: (a) inactivating an oncolytic virus made by any of the methods for making a mutant virus disclosed herein, such that the oncolytic virus is non-infectious and the tropism of the oncolytic virus is unaffected; and (b) administering the irradiated oncolytic virus to a patient afflicted with a tumor. In another aspect, the method for treating a patient further comprises attaching a toxin to the inactivated oncolytic virus.

[0056] In another aspect, the present invention provides a method for treating a patient with a tumor with SVV, the method comprising: (a) inactivating a SVV such that the virus is non-infectious and the tropism is unaffected; and (b) administering the inactivated SVV in a patient afflicted with a tumor. In another aspect, the method for treating a patient with a tumor with SVV further comprises attaching a toxin to the inactivated SVV.

[0057] In another aspect, the present invention provides a SVV composition comprising an inactivated SVV or attenuated SVV. In another aspect, the present invention provides a SVV comprising an epitope tag incorporated in the capsid region.

[0058] The present invention also provides a method for treating a patient with a tumor with SVV, the method comprising: (a) creating a mutant SVV comprising an epitope tag encoded in the capsid; (b) attaching a toxin to the epitope tag; and (c) administering the mutant SVV with the attached toxin to a patient afflicted with a tumor. In one aspect, the creating comprises: inserting an oligonucleotide encoding an epitope tag into a capsid-encoding region polynucleotide of SVV. In one aspect, the mutant SVV does not have an altered cell-type tropism. In another aspect, the method further comprises inactivating the mutant SVV such that the mutant SVV is not infectious or cannot replicate.

[0059] The present invention also provides a method for detecting a tumor cell in a sample comprising: (a) isolating a tumor sample from a patient; (b) incubating the tumor sample with an epitope-tagged SVV; and (c) screening the tumor sample for bound SVV by detecting the epitope tag.

[0060] In one aspect, the invention provides a method for detecting a tumor cell in vivo comprising: (a) administering to a patient an inactivated epitope-tagged SVV, wherein a label is conjugated to the epitope-tag; and (b) detecting the label in the patient. In the methods for detecting a tumor cell of the present invention, the SVV can be a mutant SVV generated by the methods disclosed herein.

[0061] In one aspect, the invention provides a method for detecting a tumor cell in a sample comprising: (a) isolating a cell sample from a subject; (b) incubating the cell sample with SVV (or an SVV-like picornavirus); (c) incubating the cell sample from step (b) with an antibody specific to SVV (or an antibody specific to an SVV-like picornavirus); and (d) screening the cell sample for bound antibody, wherein bound antibody indicates that the sample contains a tumor cell.

[0062] In one aspect, the invention provides a method for determining whether a subject is candidate for SVV therapy, the method comprising: (a) isolating a cell from the subject; (b) incubating the cell with SVV; (c) incubating the sample from step (b) with an anti-SVV antibody; and (d) detecting for the presence of the anti-SVV antibody on or in the cell, wherein a positive detection indicates that the subject is a candidate for SVV therapy.

[0063] Screening a cell sample for bound antibody or detecting for the presence of an anti-SVV antibody can be conducted by adding a secondary antibody that can bind to the constant regions or non-epitope binding regions of the anti-SVV antibody, wherein the secondary antibody is conjugated or labeled with a detectable marker. The detectable marker can be, for example, a fluorophore such as fluorescein. When a secondary antibody is labeled with a detectable marker, the detectable marker can be detected, for example, by fluorescent microscopy. The cell from the subject can be from a tissue biopsy from the subject. The tissue biopsy can be from a tumor in the subject or from a region in the subject that is suspected to contain tumor cells. SVV directly labeled with fluorophore can also be used in identification of tumor cells.

[0064] Further, the methods for treating neoplastic conditions, for detecting neoplastic conditions and for producing SVV, apply to wild-type SVV, mutant (including modified or variant) SVV, relatives of SVV, SVV-like picornaviruses, and other tumor-specific viruses of the invention.

[0065] The viruses of the present invention, and the compositions thereof, can be used in the manufacture of a medicament for treating the diseases mentioned herein. Further, the viruses and composition thereof of the invention can be used for the treatment of the diseases mentioned herein. Thus, in one aspect of the present invention, the present invention provides the use of SVV (or mutants, derivatives, relatives, and compositions thereof) for the treatment of cancer or in the manufacture of a medicament for treating cancer.

[0066] SVV and SVV-like viruses for gene therapy: Replication defective SVV expressing gene(s) of interest can be used to deliver genes to correct genetic disorders. SVV and SVV-like viruses can also be used as delivery vehicle for siRNA to prevent any specific gene expression. Replication defective viruses can be grown in complementing cell lines and/or in the presence of a helper virus to provide for missing functions in the recombinant virus.

[0067] IRES of picornaviruses known to play a role in expression of genes in a tissue specific manner. IRES of SVV and SVV-like viruses can be used to replace IRES of other picornaviruses. This strategy can be used to generate viruses with altered tissue tropism. In one aspect, the invention provides an IRES of SVV or an IRES from an SVV-related virus for the purpose of expressing two genes from a single promoter in a tissue specific manner.

[0068] Self-cleavage properties of 2A protease of SVV can be used to express more than one gene in equal amounts using single promoter and transcription termination signal sequences. In one aspect, the invention provides a self-cleaving 2A peptide of SVV or of an SVV-related virus for the purpose of expressing of two or more proteins in equal amounts under the control of single promoter and a single poly(A) signal. In another aspect, the invention provides the use of an SVV or an SVV-related virus 3C protease to cleave polypeptides for production of proteins from a eukaryotic cell. In another aspect, the invention provides for the use of an SVV or an SVV-like virus leader peptide to cause shut off of cell protein synthesis in tumor cells or another cell type of interest.

[0069] Virus like particles of SVV can be generated and used as vaccines and identify a particular cell type in a mixed population of cells.

Deposit Information

[0070] The following material has been deposited with the American Type Culture Collection (ATCC), 10801 University Blvd., Manassas, Va., 20110-2209, U.S.A., under the terms of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure. All restrictions on the availability of the deposited material will be irrevocably removed upon the granting of a patent. Material: Seneca Valley Virus (SVV). ATCC Patent Deposit Number: PTA-5343. Date of Deposit: Jul. 25, 2003.

BRIEF DESCRIPTION OF THE DRAWINGS

[0071] FIG. 1 shows a schematic of virotherapy using oncolytic viruses. Oncolytic viruses have the properties to replicate, spread and kill tumor cells selectively through a tumor mass by locally injecting the virus or by systemically delivering the virus.

[0072] FIG. 2 shows purified SVV stained with uranyl acetate and examined by transmission electron microscopy. Spherical virus particles are about 27 nm in diameter.

[0073] FIG. 3 is an electron micrograph of an SVV-infected PER.C6 cell that has a large crystalline inclusion and large vesicular bodies.

[0074] FIG. 4A shows an analysis of SVV RNA. SVV genomic RNA is extracted using guanidium thiocyanate and a phenol extraction method using Trizol (Invitrogen Corp., Carlsbad, Calif.). RNA is resolved through a 1.25% denaturing agarose gel. The band is visualized by ethidium bromide (EtBr) staining and photographed. In lane 2, a predominant band of SVV genomic RNA is observed, indicating that the size of the full-length SVV genome is about 7.5 kilobases.

[0075] FIG. 4B is a schematic showing the genome structure and protein products generated from polyprotein processing for picornaviruses, including SVV.

[0076] FIGS. 5A-5E presents the nucleotide sequence of SVV (SEQ ID NO:1) and the encoded amino acid sequence (SEQ ID NO:2). The stop codon is depicted by a "*" at positions 5671-3. As a general note, in sequence disclosures that include positions where the exact nucleotide is being confirmed, these positions are represented by an "n". Therefore, in codons that possess an "n", the relevant amino acid is depicted by a "x".

[0077] FIGS. 6A-6D presents the nucleotide sequence (SEQ ID NO:1) of the majority of the full-length genome of SVV. The nucleotide sequence was derived from the SVV isolate having the ATCC Patent Deposit Number: PTA-5343. Date of Deposit: Jul. 25, 2003.

[0078] FIGS. 7A-7B presents the amino acid sequence (SEQ ID NO:2) encoded by SEQ ID NO:1.

[0079] FIG. 8 presents the nucleotide sequence (SEQ ID NO:3) of the partial 1B or VP2 encoding region of SVV. This sequence is identical to nucleotides 4-429 of SEQ ID NO:1.

[0080] FIG. 9 presents the amino acid sequence (SEQ ID NO:4) of the partial SVV VP2 protein that is encoded by SEQ ID NO:3. The sequence listed in SEQ ID NO:4 is identical to amino acids 2-143 of SEQ ID NO:2.

[0081] FIG. 10 presents the nucleotide sequence (SEQ ID NO:5) of the 1C or VP3 encoding region of SVV. This sequence is identical to nucleotides 430-1146 of SEQ ID NO:1.

[0082] FIG. 11 presents the amino acid sequence (SEQ ID NO:6) of the SVV VP3 protein that is encoded by SEQ ID NO:5. The sequence listed in SEQ ID NO:6 is identical to amino acids 144-382 of SEQ ID NO:2.

[0083] FIG. 12 presents the nucleotide sequence (SEQ ID NO:7) of the 1D or VP1 encoding region of SVV. This sequence is identical to nucleotides 1147-1923 of SEQ ID NO:1.

[0084] FIG. 13 presents the amino acid sequence (SEQ ID NO:8) of the SVV VP1 protein that is encoded by SEQ ID NO:7. The sequence listed in SEQ ID NO:8 is identical to amino acids 383-641 of SEQ ID NO:2.

[0085] FIG. 14 presents the nucleotide sequence (SEQ ID NO:9) of the 2A encoding region of SVV. This sequence is identical to nucleotides 1924-1965 of SEQ ID NO:1.

[0086] FIG. 15 presents the amino acid sequence (SEQ ID NO:10) of the SVV 2A protein that is encoded by SEQ ID NO:9. The sequence listed in SEQ ID NO:10 is identical to amino acids 642-655 of SEQ ID NO:2.

[0087] FIG. 16 presents the nucleotide sequence (SEQ ID NO:11) of the 2B encoding region of SVV. This sequence is identical to nucleotides 1966-2349 of SEQ ID NO:1.

[0088] FIG. 17 presents the amino acid sequence (SEQ ID NO:12) of the SVV 2B protein that is encoded by SEQ ID NO:11. The sequence listed in SEQ ID NO:12 is identical to amino acids 656-783 of SEQ ID NO:2.

[0089] FIG. 18 presents the nucleotide sequence (SEQ ID NO:13) of the 2C encoding region of SVV. This sequence is identical to nucleotides 2350-3315 of SEQ ID NO:1.

[0090] FIG. 19 presents the amino acid sequence (SEQ ID NO:14) of the SVV 2C protein that is encoded by SEQ ID NO:13. The sequence listed in SEQ ID NO:14 is identical to amino acids 784-1105 of SEQ ID NO:2.

[0091] FIG. 20 presents the nucleotide sequence (SEQ ID NO:15) of the 3A encoding region of SVV. This sequence is identical to nucleotides 3316-3585 of SEQ ID NO:1.

[0092] FIG. 21 presents the amino acid sequence (SEQ ID NO:16) of the SVV 3A protein that is encoded by SEQ ID NO:15. The sequence listed in SEQ ID NO:16 is identical to amino acids 1106-1195 of SEQ ID NO:2.

[0093] FIG. 22 presents the nucleotide sequence (SEQ ID NO:17) of the 3B encoding region of SVV. This sequence is identical to nucleotides 3586-3651 of SEQ ID NO:1.

[0094] FIG. 23 presents the amino acid sequence (SEQ ID NO:18) of the SVV 3B protein that is encoded by SEQ ID NO:17. The sequence listed in SEQ ID NO:18 is identical to amino acids 1196-1217 of SEQ ID NO:2.

[0095] FIG. 24 presents the nucleotide sequence (SEQ ID NO:19) of the 3C encoding region of SVV. This sequence is identical to nucleotides 3652-4284 of SEQ ID NO:1.

[0096] FIG. 25 presents the amino acid sequence (SEQ ID NO:20) of the SVV 3C protein that is encoded by SEQ ID NO:19. The sequence listed in SEQ ID NO:20 is identical to amino acids 1218-1428 of SEQ ID NO:2.

[0097] FIG. 26 presents the nucleotide sequence (SEQ ID NO:21) of the 3D encoding region of SVV. This sequence is identical to nucleotides 4285-5673 of SEQ ID NO:1.

[0098] FIG. 27 presents the amino acid sequence (SEQ ID NO:22) of the SVV 3D protein that is encoded by SEQ ID NO:21. The sequence listed in SEQ ID NO:22 is identical to amino acids 1429-1890 of SEQ ID NO:2.

[0099] FIGS. 28A-28H present an amino acid sequence alignment between SVV SEQ ID NO:2 and various members of the Cardiovirus genus, such as Encephalomyocarditis virus (EMCV; species Encephalomyocarditis virus), Theiler's murine encephalomyocarditis virus (TMEV; species Theilovirus), a rat TMEV-like agent (TLV; species Theilovirus), and Vilyuisk human encephalomyelitis virus (VHEV; species Theilovirus). The specific sequences of the various Cardioviruses are presented in: SEQ ID NOS: 23 (EMCV-R), 24 (EMCV-PV21), 25 (EMCV-B), 26 (EMCV-Da), 27 (EMCV-Db), 28 (EMCV-PV2), 29 (EMCV-Mengo), 30 (TMEV/DA), 31 (TMEV/GDVII), 32 (TMEV/BeAn8386), 33 (TLV-NGS910) and 34 (VHEV/Siberia-55).

[0100] Number positions in FIG. 28 do not correspond to the numbering of the sequence listings. The "I" symbol indicates cleavage sites where the polyprotein is cleaved into its final functional products. For example, the alignment between positions 1 and 157 is in the 1A (VP4) region. The alignment between positions: 159 and 428 is in the 1B (VP2) region; 430 and 668 is in the 1C (VP3) region; 670 and 967 is in the 1D (VP1) region; 969 and 1111 is in the 2A region; 1112 and 1276 is in the 2B region; 1278 and 1609 is in the 2C region; 1611 and 1700 is in the 3A region; 1702 and 1723 is in the 3B region; 1725 and 1946 is in the 3C region; 1948 and 2410 is in the 3D region. The alignment also shows regions of potential conservation or similarity between the viral sequences as can be determined by standard sequence analysis programs. The alignments were generated using BioEdit 5.0.9 and Clustal W 1.81.

[0101] FIG. 29 lists the final polypeptide products of SVV with respect to SEQ ID NO:2. Some conserved motifs are bolded and underlined: 2A/2B "cleavage" (NPGP (SEQ ID NO:111)); 2C ATP-binding (GxxGxGKS/T (SEQ ID NO:112) and hyhyhyxxD); 3B (VPg)/RNA attachment residue (Y); 3C (pro) active site residues (H, C, H); 3D (pol) motifs (KDEL/IR (SEQ ID NO:113), PSG, YGDD (SEQ ID NO:114), FLKR (SEQ ID NO:115)).

[0102] FIG. 30 lists the picornavirus species that were used in sequence analyses with SEQ ID NOS:1 and 2 to determine the phylogenetic relationship between SVV and these picornaviruses (see Example 4, Part I).

[0103] FIG. 31 shows the phylogenetic relationship between SVV (SEQ ID NO:4) and other picornaviruses in view of VP2 sequence analyses. The figure shows a bootstrapped neighbor-joining tree (see Example 4, Part I).

[0104] FIG. 32 shows a bootstrapped neighbor-joining tree for VP3 between SVV (SEQ ID NO:6) and other picornaviruses (see Example 4, Part I).

[0105] FIG. 33 shows a bootstrapped neighbor-joining tree for VP1 between SVV (SEQ ID NO:8) and other picornaviruses (see Example 4, Part I).

[0106] FIG. 34 shows a bootstrapped neighbor-joining tree for P1 (i.e., 1A, 1B, 1C and 1D) between SVV (i.e., partial P1--amino acids 2-641 of SEQ ID NO:2) and other picornaviruses (see Example 4, Part I).

[0107] FIG. 35 shows a bootstrapped neighbor-joining tree for 2C between SVV (SEQ ID NO:14) and other picornaviruses (see Example 4, Part I).

[0108] FIG. 36 shows a bootstrapped neighbor-joining tree for 3C (pro) between SVV (SEQ ID NO:20) and other picornaviruses (see Example 4, Part I).

[0109] FIG. 37 shows a bootstrapped neighbor-joining tree for 3D (pol) between SVV (SEQ ID NO:22) and other picornaviruses (see Example 4, Part I).

[0110] FIG. 38 presents the actual amino acid percent identities of VP2 between SVV (SEQ ID NO:4) and other picornaviruses (see Example 4, Part I).

[0111] FIG. 39 presents the actual amino acid percent identities of VP3 between SVV (SEQ ID NO:6) and other picornaviruses (see Example 4, Part I).

[0112] FIG. 40 presents the actual amino acid percent identities of VP1 between SVV (SEQ ID NO:8) and other picornaviruses (see Example 4, Part I).

[0113] FIG. 41 presents the actual amino acid percent identities of P1 between SVV (partial capsid or P1--amino acids 2-641 of SEQ ID NO:2) and other picornaviruses (see Example 4, Part I).

[0114] FIG. 42 presents the actual amino acid percent identities of 2C between SVV (SEQ ID NO:14) and other picornaviruses (see Example 4, Part I).

[0115] FIG. 43 presents the actual amino acid percent identities of 3C (pro) between SVV (SEQ ID NO:20) and other picornaviruses (see Example 4, Part I).

[0116] FIG. 44 presents the actual amino acid percent identities of 3D (pol) between SVV (SEQ ID NO:22) and other picornaviruses (see Example 4, Part I).

[0117] FIG. 45 shows the VP2 (˜36 kDa), VP1 (˜31 kDa) and VP3 (˜27 kDa) proteins of SVV as analyzed by SDS-PAGE. Purified SVV was subjected to SDS-PAGE and proteins were visualized by silver stain. Lane "MWt" is molecular weight markers; lane "SVV" contains structural proteins of SVV. The sizes of three molecular weight markers and the names of viral proteins are also given.

[0118] FIGS. 46A-46B show the amounts of SVV in blood and tumor following systemic administration (Example 7). H446 tumor bearing nude mice were treated with SVV at a dose of 1×10¹²/kg by tail vein injection. The mice were bled at 0, 1, 3, 6, 24, 48, 72 hours and at 7 days post-injection, and the plasma was separated from the blood immediately after collection, diluted in infection medium, and used to infect PER.C6 cells. The tumors were harvested at 6, 24, 48, 72 hours and at 7 days post-injection. The tumors were cut into small sections and suspended in one mL of medium and CVL was made.

[0119] FIGS. 46C-46D presents data relating to SVV clearance in vivo. The figures show that SVV exhibits a substantially longer resident time in the blood compared to similar doses of i.v. adenovirus (Example 7), and therefore SVV has a slower clearance rate than adenovirus in vivo. Following a single intravenous (i.v.) dose, SVV remains present in the blood for up to 6 hours (FIG. 46C; FIG. 46C is a duplication of FIG. 46A for comparison purposes to FIG. 46D), whereas adenovirus is cleared or depleted from the blood in about an hour (FIG. 46D).

[0120] FIG. 47 shows immunohistochemistry and hematoxylin and eosin (H&E) staining of H446 xenograft sections (Example 7). H446 tumor bearing nude mice were treated with Hank's balanced salt solution (HBSS) or SVV at a dose of 1×10¹² vp/kg by tail vein injection. The mice were sacrificed at 3 days post-injection and the tumors were collected. The virus proteins in the tumor cells are visualized by immunohistochemistry using SVV-specific mouse antibodies (upper panels). The general morphology of H446 tumor cells collected from HBSS or SVV treated mice were stained by H&E stain (lower panels).

[0121] FIG. 48 shows SVV mediated cytotoxicity in primary human hepatocytes (Example 9). Primary human hepatocytes plated in collagen coated 12-well plates were infected with SVV at 1, 10 and 100 and 1000 particles per cell (ppc). Three days after infection, the cell associated lactate dehydrogenase (LDH) and LDH in the culture supernatant were measured separately. Percent cytotoxicity was determined as a ration of LDH units in supernatant over maximal cellular LDH plus supernatant LDH.

[0122] FIG. 49 shows virus production by SVV in selected cell lines. To assess the replicative abilities of SVV, selected normal cells and tumor cells were infected with SVV at one virus particle per cell (ppc) (Example 9). After 72 hours, cells were harvested and CVL was assayed for titer on PER.C6 cells. For each cell line, the efficiency of SVV replication was expressed as plaque forming units per milliliter (pfu/ml).

[0123] FIG. 50 shows toxicity in nude and CD1 mice according to body weights (Example 10). The mean body weight of mice in each treatment group were measured different days post virus administration. Mice were injected with a single dose of SVV or PBS by tail vein on day 1.

[0124] FIG. 51 shows efficacy in a H446 xenograft model. H446 tumors are established in nude mice and the mice are sorted into groups (n=10) and treated via tail vein injection with either HBSS or six different doses of SVV (Example 11). On study day 20, five mice from the HBSS group that bear large tumors (mean tumor volume=2000 mm³) were injected with 1×10¹¹ vp/kg (indicated by an arrow). Data is expressed as mean tumor volume+standard deviation (SD).

[0125] FIG. 52 shows a picture of H446 xenograft nude mice that have been untreated or treated with SVV (Example 11). The efficacy of SVV is very robust in that 100% of large pre-established tumors were completely eradicated. SVV-treated mice show neither clinical symptoms nor recurrence of tumors for at least 200 days following injection.

[0126] FIG. 53 presents data relating to SVV tumor specificity and efficacy in vitro (Example 11). The graphs show cell survival following incubation of either H446 human small cell lung carcinoma (SCLC) tumor cells (top graph) or normal human H460 cells (bottom graph). SVV specifically killed the tumor cells with an EC₅₀ of approximately 10^-3 particles per cell. In contrast, normal human cells were not killed at any concentration of SVV.

[0127] FIG. 54 depicts a representative plasmid containing the complete genome of SVV (Example 15). The presence of the T7 promoter on the vector upstream of the SVV sequence allows for the in vitro transcription of the SVV sequence such that SVV RNA molecules can be generated.

[0128] FIG. 55 depicts a schematic for the construction of a full-length and functional genomic SVV plasmid and subsequent SVV virus production (Example 16). To obtain a functional genomic SVV clone, the complete genome of a SVV can be cloned into a vector with a T7 promoter. This can be accomplished by making cDNA clones of the virus, sequencing them and cloning contiguous pieces into one plasmid, resulting in the plasmid depicted "pSVV". The plasmid with the full genome of SVV can then be reverse-transcribed to generate SVV RNA. The SVV RNA is then transfected into permissive mammalian cells and SVV virus particles can then be recovered and purified.

[0129] FIG. 56 depicts a schematic for the construction of a vector ("pSVV capsid") containing the coding sequence (i.e., coding regions for 1A-1D) for the SVV capsid (Example 16). The pSVV capsid can then be used to generate a library of SVV capsid mutants.

[0130] FIG. 57 shows one method of mutating the SVV capsid for the generation of a library of SVV capsid mutants (Example 16). The figure illustrates the insertion of an oligonucleotide sequence at random sites of the plasmid. The oligonucleotides can be random oligonucleotides, oligonucleotides with known sequences, or an oligonucleotide encoding an epitope tag. In the figure, the restriction enzyme CviJI randomly cleaves the pSVV capsid DNA. Linearized pSVV capsid DNA that has been cut at a single site is isolated and purified from a gel, and ligated with oligonucleotides.

[0131] FIG. 58 presents a scheme to generate a library of full-length SVV mutants comprising sequence mutations in the capsid encoding region (Example 16). For example, the capsid encoding region from a pSVV capsid mutant library (generated according to the scheme depicted in FIG. 57, for example) is isolated by restriction digestion and gel purification. The vector containing the full-length SVV sequence is also digested such that the capsid encoding region is cut out. The capsid encoding region from the pSVV capsid mutant library is then ligated to the pSVV vector that is missing its wild-type capsid sequence, thereby generating a library of full-length SVV mutants (the "pSVVFL" vector) having a plurality of mutations in the capsid encoding region.

[0132] FIG. 59 presents a general method for producing the SVV virus particles comprising a library of capsid mutations (Example 16). The pSVVFL vector is reverse-transcribed to generate SVV RNA. The SVV RNA is transfected into permissive cells, wherein SVV mutant virus particles are produced. The virus particles lyse the cells and a population of SVV virus particles comprising a plurality of capsid variants, "SVV capsid library," are isolated.

[0133] FIG. 60 shows a general method for screening SVV capsid mutants that can specifically infect tumor cells while being unable to infect non-tumor cells. The SVV capsid library is incubated with a tumor cell line or tissue of interest. After an initial incubation period, the cells are washed such that SVV virus particles that were unable to gain entry into the cells are eliminated. The cells are then maintained in culture until viral lysis is observed. Culture supernatant is then collected to isolate SVV capsid mutants that were able to lytically infect the tumor cell. These viruses can then be grown-up by infecting a known permissive cell-line prior to a counter-screen. A counter-screen is performed by incubating the SVV capsid mutant viruses that were able to infect the tumor cell with normal cells. Only those viruses that remain unbound in the supernatant are collected, thereby isolating mutant SVV viruses that have tumor-specificity. This process can be repeated to further refine the isolation of SVV tumor-specific viruses.

[0134] FIG. 61 shows a traditional method for testing whether virus mutants can bind and/or infect cell lines. Traditional methods require what are often inefficient methods for growing cell-lines, i.e. flasks, such that a mass-screen of a library of virus mutants in relation to a number of different cell-lines becomes burdensome.

[0135] FIG. 62 shows a high-throughput method of the invention for screening virus mutants that have the ability to specifically infect different cell-lines (Example 16). In this figure, a number of different tumor cell-lines are grown in a 384 well-plate. To each well, a sample of a virus is added (for example, a sample of a SVV capsid library). From those wells which show cytopathic effects, the media is collected such that any viruses in the media can be amplified by infecting permissive cell lines (for example, for SVV, H446 or PER.C6) in flasks or large tissue culture plates. The viruses are grown such that the RNA can be isolated and the sequence analyzed to determine the encoded peptide sequence inserted by the oligonucleotide-insertion mutagenesis of the capsid. The peptide itself can then be tested to determine whether it can bind to a tumor cell-type specifically.

[0136] FIG. 63 shows another high-throughput screening schematic (Example 16). Tumor and normal cell lines are grown in multi-well plates. Viruses are added to each well to test whether the cells are killed by virus-mediated lysis. Cytopathic effects can be quickly assayed by reading the light-absorbance in each well. Viruses from the wells showing cytopathic effects are grown up and tested in further in vitro (re-testing of tumor and normal cell lines) and in vivo models (testing whether the virus can kill explanted tumors in mice).

[0137] FIG. 64 shows that SVV capsid mutants (SEQ ID NOS: 45-48, respectively, in order of appearance) having new tumor-specific tropisms can be analyzed to generate tumor-selective peptides. Those SVV capsid mutants that enable the specific infection of a tumor cell line are sequenced to determine the peptide encoded by the oligonucleotide insertion. An amino acid consensus sequence can thereby be determined from the successful capsid mutants. Peptides having the consensus sequence can then be tested to determine whether they can bind specifically to the tumor cell-type in question. Tumor-selective peptides can then be attached to toxins or drugs in order to serve as tumor-specific targeting vehicles.

[0138] FIG. 65 illustrates that an SVV capsid library can be first tested in vivo. Mice (including normal, athymic, nude, CD-1 transgenics, etc.) can be explanted with a specific tumor. These mice are then injected with a SVV derivative library, such as a SVV capsid library. At certain time points, tumor cells are recovered from the mice, such that in those mice that display the elimination of a tumor, viruses will be isolated from initial tumor samples and grown-up in permissive cell lines.

[0139] FIG. 66 shows a clinical testing program for the SVV derivatives of the present invention.

[0140] FIG. 67 illustrates that SVV derivatives (with new tumor tropisms) encoding epitope tags in their capsid can be used for a variety of purposes. They can be used as a screening reagent to detect whether a specific tumor cell is present in tissue samples by assaying for the presence of the epitope. Alternatively, toxins or other therapeutics can be attached to the epitope tag, and the virus then administered to patients. Further, wild-type or derivative SVV can be irradiated or inactivated such that the virus particle itself is used as a therapeutic device. Either the virus particle induces cellular apoptosis due to the presence of apoptosis-inducing peptides, or the particle can have a toxin or some other therapeutic attached such that the virus is used a specific-targeting delivery device.

[0141] FIG. 68 shows the basic life-cycle of the picornavirus.

[0142] FIG. 69 shows a comparison of the polypeptide lengths of SVV compared to other picornaviruses.

[0143] FIG. 70 lists an amino acid comparison of the picornavirus 2A-like NPG/P proteins (SEQ ID NOS: 49-110, respectively, in order of appearance). The sequence for SVV is listed at residues 635-656 of SEQ ID NO:2.

[0144] FIGS. 71A and 71B list the amino acid sequence (SEQ ID NO:23) for EMCV-R.

[0145] FIGS. 72A and 72B list the amino acid sequence (SEQ ID NO:24) for EMCV-PV21 (Accession CAA52361).

[0146] FIGS. 73A and 73B list the amino acid sequence (SEQ ID NO:25) for EMCV-B (Accession P17593).

[0147] FIGS. 74A and 74B list the amino acid sequence (SEQ ID NO:26) for EMCV-Da (Accession P17594).

[0148] FIGS. 75A and 75B list the amino acid sequence (SEQ ID NO:27) for EMCV-Db.

[0149] FIGS. 76A and 76B list the amino acid sequence (SEQ ID NO:28) for EMCV-PV2 (Accession CAA60776).

[0150] FIGS. 77A and 77B list the amino acid sequence (SEQ ID NO:29) for EMCV-mengo (Accession AAA46547).

[0151] FIGS. 78A and 78B list the amino acid sequence (SEQ ID NO:30) for TMEV/DA (Accession AAA47928).

[0152] FIGS. 79A and 79B list the amino acid sequence (SEQ ID NO:31) for TMEV/GDVII (Accession AAA47929).

[0153] FIGS. 80A and 80B list the amino acid sequence (SEQ ID NO:32) for TMEV/BeAn8386 (Accession AAA47930).

[0154] FIGS. 81A and 81B list the amino acid sequence (SEQ ID NO:33) for TLV-NGS910 (Accession BAC58035).

[0155] FIG. 82 lists the amino acid sequence (SEQ ID NO:34) for VHEV/Siberia-55 (Accession AAA47931).

[0156] FIGS. 83A-83H present the full-length genomic sequence of SVV (SEQ ID NO:168) and the encoded polyprotein amino acid sequence (SEQ ID NO:169), where this full-length genomic sequence was obtained from SVV viruses grown from the SVV isolate having ATCC Patent Deposit Number PTA-5343. Specific features of the SVV genomic sequence, such as the specific coding regions for proteins cleaved from the polyprotein sequence are described herein.

[0157] FIGS. 84A-84D present the full-length genomic sequence of SVV (SEQ ID NO:168). The sequence was obtained from SVV grown from the SVV isolate having ATCC Patent Deposit Number PTA-5343.

[0158] FIGS. 85A-85B present the amino acid sequence of the full-length polyprotein of SVV (SEQ ID NO:169) encoded by the nucleotides 667-7209 of SEQ ID NO:168.

[0159] FIG. 86 provides a phylogenetic analysis or epidemiology of SVV with respect to the full-length genome and polyprotein sequence of SVV from SEQ ID NOS:168 and 169. SVV is a unique virus, phylogenetically similar to cardioviruses, but in a separate tree. The SVV-like picornaviruses are most likely in the same tree or genus as SVV due to the high level of sequence identity between SVV and the SVV-like picornaviruses (see FIGS. 87-89) and due to the ability of antibodies raised against SVV-like picornaviruses to bind SVV (and vice versa) (see Example 4, Part III, Serum Studies).

[0160] FIGS. 87A-87D show a nucleic acid sequence comparison between SVV and some SVV-like picornaviruses in the areas of the P1 structural region and 2A. In particular, the comparison is in the VP2(partial)-VP3-VP1-2A (partial) regions. The listed SVV sequence is SEQ ID NO:170; the listed sequence for isolate IA 89-47752 is SEQ ID NO:171; the listed sequence for isolate CA 131395 is SEQ ID NO:172; the listed sequence for isolate NC 88-23626 is SEQ ID NO:173; the listed sequence for isolate MN 88-36695 is SEQ ID NO:174; the listed sequence for isolate NJ 90-10324 is SEQ ID NO:175; the listed sequence for isolate IL 92-48963 is SEQ ID NO:176; the listed sequence for isolate LA 1278 (97-1278) is SEQ ID NO:177; and the listed consensus sequence is SEQ ID NO:178.

[0161] FIG. 88 shows a nucleic acid sequence comparison between SVV and isolates IA 89-47752 and CA 131395 in the 2C coding region (partial). The listed SVV sequence is SEQ ID NO:179; the listed sequence for isolate IA 89-47752 is SEQ ID NO:180; the listed sequence for isolate CA 131395 is SEQ ID NO:181; and the listed consensus sequence is SEQ ID NO:182.

[0162] FIGS. 89A-89B show a nucleic acid sequence comparison between SVV and isolates NC 88-23626, MN 88-36695, IA 89-47752, NJ 90-10324, IL 92-48963, LA 97-1278, and CA 131395 in the 3D polymerase coding region (partial) and 3' UTR region. The listed sequences are SVV (SEQ ID NO:183), NC 88-23626 (SEQ ID NO:184), MN 88-36695 (SEQ ID NO:185), IA 89-47752 (SEQ ID NO:186), NJ 90-10324 (SEQ ID NO:187), IL 92-48963 (SEQ ID NO:188), LA 97-1278 (SEQ ID NO:189), CA 131395 (SEQ ID NO:190), and consensus sequence (SEQ ID NO:191).

[0163] FIGS. 90A-90E show that a single dose of SVV is efficacious in reducing the size and preventing the growth of explanted tumors in mice. FIG. 90A shows that SVV can reduce the size and prevent the growth of explanted H446 human SCLC tumors (ED₅₀=0.0007). FIG. 90B shows that SVV can reduce the size and prevent the growth of explanted Y79 human retinoblastoma tumors (ED₅₀=0.0007). FIG. 90C shows that SVV can reduce the size and prevent the growth of explanted H69AR human SCLC-MDR (multi drug resistant) tumors (ED₅₀=0.05). FIG. 90D shows that SVV can reduce the size and prevent the growth of explanted H1299 human HSCLC tumors (ED₅₀=4.8). FIG. 90E shows that SVV can reduce the size and prevent the growth of explanted N1E-115 murine neuroblastoma tumors in A/J mice (normal immunocompetent mice) (ED₅₀=0.001).

[0164] FIG. 91 show a molecular model of the EMCV and TMEV capsid structures in comparison with the sequence of SVV. A molecular model in conjunction with the use of algorithms for antigenic prediction allows for peptide sequences to be chosen for polyclonal antibody generation. β-sheets are shown in brown, α-helices are shown in green, and a 12-mer peptide sequence chosen for polyclonal generation is shown in yellow. The particular sequence (in the VP2 region) was chosen because it presents good surface exposure according to the model.

[0165] FIGS. 92A-92D show the specificity of polyclonal antibodies against SVV. FIG. 92A is a negative control, and presents an immunofluorescence image of cells infected with SVV that are stained with non-specific anti-mouse sera and secondary antibody. FIGS. 92B and 92C show immunofluorescence images of cells infected with SVV that are stained with mouse anti-SVV sera diluted 1:50 and secondary antibody (anti-mouse Ig conjugated to fluorescein). FIG. 92D shows that polyclonal anti-SVV antibodies can be used in viral binding assays; the image shows an immunofluorescence image of SVV concentrated in an outline around a cell because the cell was put on ice to prevent SVV internalization.

[0166] FIG. 93 shows the results of a neutralization assay of GP102 sera on SVV (see Example 18). The neutralization titer (calculated as the highest dilution that neutralizes the virus is 100%) is 1:100.

[0167] FIG. 94 shows the results of a neutralization assay of anti-SVV antisera on MN 88-36695 (see Example 18). The neutralization titer is 1:560.

[0168] FIG. 95A and FIG. 95B depict neighbor-joining trees. These trees were constructed using PHYLIP (Phylogeny Inference Package Computer Programs for Inferring Phylogenies) and show the relationship between SVV and seven SVV-like picornaviruses when comparing sequences from regions in P1 and partial 2A (FIG. 95A) and in the 3' end of the genome (FIG. 95B).

DETAILED DESCRIPTION OF THE INVENTION

[0169] The terms "virus," "viral particle," "virus particle," and "virion" are used interchangeably.

[0170] The terms "vector particle" and "viral vector particle" are interchangeable and are to be understood broadly--for example--as meaning infectious viral particles that are formed when, e.g., a viral vector of the invention is transduced or transfected into an appropriate cell or cell line for the generation of infectious particles.

[0171] The terms "derivative," "mutant," "variant" and "modified" are used interchangeably to generally indicate that a derivative, mutant, variant or modified virus can have a nucleic acid or amino acid sequence difference in respect to a template viral nucleic acid or amino acid sequence. For example, a SVV derivative, mutant, variant or modified SVV may refer to a SVV that has a nucleic acid or amino acid sequence difference with respect to the wild-type SVV nucleic acid or amino acid sequence of ATCC Patent Deposit Number PTA-5343.

[0172] An "SVV-like picornavirus" as used herein can have at least about 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to SVV at the nucleotide level (see SEQ ID NO:168, FIG. 84, and FIG. 83 for the SVV full-length genomic sequence), where the sequence comparison is not limited to a whole-genome analysis, but can be focused on a particular region of the genome, such as the 5'UTR, structural encoding regions, non-structural encoding regions, 3'UTR, and portions thereof. The particular length of the genome for sequence comparison that is adequate to determine relatedness/likeness to SVV is known to one skilled in the art, and the adequate length can vary with respect to the percentage of identity that is present. The length for sequence comparison can be, for example, at least 20, 50, 100, 200, 300, 400, 500, 750, 1000, 1500, 2000, or 2500 nucleotides. Where the length is shorter, one skilled in the art understands, for example, that the identity between sequences can be higher in order to consider the two sequences to be related. However, such guidance is qualified at least with respect to considerations of sequence conservation, in that certain regions of the genome are more conserved than others between related species. Additionally, if an antiserum generated from a virus can neutralize SVV infection of an SVV permissive cell line, then the virus is considered to be an SVV-like picornavirus. Additionally, if an antiserum generated from a virus can neutralize SVV infection of an SVV permissive cell line, and that antiserum can also bind to other viruses (for example, if the antiserum can be used in indirect immunofluorescence assays to detect virus), then the other viruses that can be bound by the antiserum are considered to be SVV-like picornaviruses. For purposes of the invention, SVV-like picornaviruses can include cardioviruses. Exemplary SVV permissive cells or cell lines include, but are not limited to, Y79, NCI-H446, N1E-115, NCI-H1770, NCI-H82, PER.C6®, NCI-H69AR, SK-NEP-1, IMR-32, NCI-H187, NCI-H209, HCC33, NCI-H1184, D283 Med, SK-N-AS, BEK PCB3E1, ST, NCI-H1299, DMS 153, NCI-H378, NCI-H295R, BEK, PPASMC, PCASMC, PAoSMC, NCI-H526, OVCAR-3, NCI-H207, ESK-4, SW-13, 293, Hs 578T, HS 1.Tes, and LOX IMVI.

[0173] As used herein, the terms "cancer," "cancer cells," "neoplastic cells," "neoplasia," "tumor," and "tumor cells," are used interchangeably, and refer to cells that exhibit relatively autonomous growth, so that they exhibit an aberrant growth phenotype characterized by a significant loss of control of cell proliferation. Neoplastic cells can be malignant or benign. According to the present invention, one type of preferred tumor cells are those with neurotropic properties.

[0174] The terms "identical" or percent "identity" in the context of two or more nucleic acid or protein sequences, refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same, when compared and aligned for maximum correspondence, as measured using a sequence comparison algorithm such as Protein-Protein BLAST (Protein-Protein BLAST of GenBank databases (Altschul, S.F., Gish, W., Miller, W., Myers, E. W. & Lipman, D. J. (1990) "Basic local alignment search tool." J. Mol. Biol. 215:403-410)) or by visual inspection. The BLAST algorithm is described in Altschul et al., J. Mol. Biol., 215:403-410 (1990), and publicly available BLAST software is provided through the National Center for Biotechnology Information (NCBI) (http://www.ncbi.nlm.nih.gov/).

[0175] For example, as used herein, the term "at least 90% identical to" refers to percent identities from 90 to 100 relative to the reference polypeptides (or polynucleotides). Identity at a level of 90% or more is indicative of the fact that, assuming for exemplification purposes a test and reference polypeptide length of 100 amino acids are compared, no more than 10% (i.e., 10 out of 100) amino acids in the test polypeptide differs from that of the reference polypeptide. Similar comparisons can be made between a test and reference polynucleotide. Such differences can be represented as point mutations randomly distributed over the entire length of an amino acid sequence or they can be clustered in one or more locations of varying length up to the maximum allowable, e.g., 10 out of 100 amino acid differences (90% identity). Differences are defined as nucleic acid or amino acid substitutions, insertions or deletions. At the level of identities above about 85-90%, the result should be independent of the program and gap parameters set; such high levels of identity can be assessed readily, often without relying on software.

[0176] The concepts of "high stringency," "intermediate stringency" or "low stringency" refer to nucleic acid hybridization conditions. High stringency conditions refers to conditions that require a greater identity between a target's nucleic acid sequence and a probe's nucleic acid sequence in order for annealing or hybridization to occur between the target and the probe. Low stringency conditions refer to conditions that require a lower identity between a target's nucleic acid sequence and a probe's nucleic acid sequence in order for annealing or hybridization to occur between the target and the probe. Stringency conditions can be controlled by the salt concentration of the buffer or by the temperature at which the hybridization is carried out, where higher salt concentrations result in less stringent conditions and where higher temperatures result in more stringent conditions. Although stringency conditions will vary based on the length and nucleic acid content of the sequences undergoing hybridization, representative conditions of high, intermediate and low stringency are described in the following exemplary conditions. A commonly used hybridization buffer is SSC (sodium chloride sodium citrate) with a 20× stock concentration corresponding to 0.3 M trisodium citrate and 3 M NaCl. For high stringency conditions, the working concentration of SSC can be 0.1×-0.5×(1.5-7.5 mM trisodium citrate, 15-75 mM NaCl) with the hybridization temperature set at 65° C. Intermediate conditions typically utilize a 0.5×-2×SSC concentration (7.5-30 mM trisodium citrate, 75-300 mM NaCl) at a temperature of 55-62° C. Hybridizations conducted under low stringency conditions can use a 2×-5×SSC concentration (30-75 mM trisodium citrate, 300-750 mM NaCl) at a temperature of 50-55° C. Note that these conditions are merely exemplary and are not to be considered limitations.

Seneca Valley Virus (SVV):

[0177] SVV is a novel, heretofore undiscovered RNA virus, and with respect to previously characterized picornaviruses, SVV is most closely related to members from the genus Cardiovirus in the family Picornaviridae (see International Application No. PCT/US2004/031594). The results of sequence analyses between SVV and other cardioviruses are discussed in PCT/US2004/031594, which is hereby incorporated by reference in its entirety. Since the time of the sequence analysis of SVV described in PCT/US2004/031594, the Picornavirus Study Group has initiated discussion as to whether SVV will be a member of a new genus. FIG. 86 presents a genetic relationship tree between members of the family Picornaviridae.

[0178] From initial sequence comparisons to known picornaviruses (see International Application No. PCT/US2004/031504), there were two phylogenetic classification options: (1) to include SVV as a new species in the genus Cardiovirus; or (2) assign SVV to a new genus. At that time and for the International application, SVV was designated to be a novel member of the genus Cardiovirus. After further analyses however, it has been found that several characteristics of SVV differ with that of cardioviruses. For example, some cardiovirus genomes contain an extended internal poly(C) tract in their 5' UTRs. SVV does not contain a poly(C) tract. From the additional 5' sequence information, the Internal Ribosome Entry Sequence (IRES) of SVV has been mapped and compared to other picornaviruses, and it has been determined that the SVV IRES is Type IV, whereas cardiovirus IRES's are Type II. The cardioviruses have a long (150 amino acid (aa)) 2A protease while SVV has a short (9 aa) 2A protease. The size of this protein as well as others (Leader peptide, 3A) differs significantly between SVV and cardioviruses. From the study of other picornaviruses, it is know that these proteins are likely involved in host cell interactions including tropism and virulence. Lastly, it is now thought that the overall sequences differ too much in a number of genome regions and SVV should therefore be considered to form a new genus. Additionally, multiple unique picornaviruses have been discovered at the USDA that are more similar to SVV than SVV is to other cardioviruses. Therefore, it has been decided by the Executive Committee of the International Committee for the Taxonomy of Viruses (ICVT) based on recommendations made by the Picornavirus Study Group that SVV will make up a new species of picornavirus, named Seneca Valley virus. However, currently, SVV and these unique USDA picornaviruses (herein referred to as being members of the group of SVV-like picornaviruses) are currently unassigned to any genus.

[0179] Several of the SVV-like picornaviruses discovered at the USDA are about 95-98% identical to SVV at the nucleotide level (for example, see FIGS. 87-89). Antisera against one virus (MN 88-36695) neutralizes SVV, and this virus is reactive to other antisera that can neutralize SVV. The SVV-like picornaviruses were isolated from pigs, and thus, pigs are likely a permissive host for SVV and other SVV-like viruses. Examples of SVV-like picornaviruses isolated from pigs include, but are not limited to, the following USDA isolates MN 88-36695, NC 88-23626, IA 89-47552, NJ 90-10324, IL 92-48963, CA 131395; LA 1278; IL 66289; IL 94-9356; MN/GA 99-29256; MN 99197; and SC 363649. SVV-like picornaviruses may also include cardioviruses closely related to SVV (as determined by sequence analysis or by cross-reactivity to antibodies raised against SVV antigens). Thus, for purposes of the present invention, SVV can be considered: (1) to be closely related to (or to be a member of) the genus Cardiovirus of the family Picornaviridae, and (2) to be a member of a new genus of the family Picornaviridae, where members of the new genus can include SVV and SVV-like picornaviruses not classified to be members of other genuses.

[0180] SVV, like cardioviruses, can be distinguished from other picornaviruses by special features of their genome organization, common pathological properties, and the dissociability of their virions at pHs between 5 and 7 in 0.1M NaCl (Scraba, D. et al., "Cardioviruses (Picornaviridae)," in Encyclopedia of Virology, 2nd Edition, R. G. Webster and A. Granoff, Editors, 1999). The genome of SVV consists of one single-stranded positive (+) sense strand RNA molecule having a size of 7,310 nucleotides including a poly(A) tail of 30 nucleotides in length (see FIGS. 83A-83H; FIGS. 84A-84D; SEQ ID NO:168). As SVV is a picornavirus, it has a number of features that are conserved in all picornaviruses: (i) genomic RNA is infectious, and thus can be transfected into cells to bypass the virus-receptor binding and entry steps in the viral life cycle; (ii) a long (about 600-1200 bp) untranslated region (UTR) at the 5' end of the genome (for SVV, nucleotides 1-666 of SEQ ID NO:168), and a shorter 3' untranslated region (about 50-100 bp; for SVV, nucleotides 7210-7280 of SEQ ID NO:168; (iii) the 5' UTR contains a clover-leaf secondary structure known as the internal ribosome entry site (IRES) (which can be, for example, from about nucleotide 300 to about nucleotide 366 of SEQ ID NO:168); cardioviruses have a Type II IRES and SVV has a Type IV IRES; (iv) the rest of the genome encodes a single polyprotein (for SVV, nucleotides 667-7209 of SEQ ID NO:168 encode the polyprotein (SEQ ID NO:169)) and (v) both ends of the genome are modified, the 5' end by a covalently attached small, basic protein, "Vpg," and the 3' end by polyadenylation (for SVV, nucleotides 7281-7310 of SEQ ID NO:168).

[0181] The invention provides the isolated SVV virus (ATCC Patent Deposit number PTA-5343) and the complete genomic content of SVV therefrom. At first, the largest SVV genomic fragment that was sequenced is an isolated SVV nucleic acid, derived from the PTA-5343 isolate, that comprises the majority of the SVV genomic sequence, and is listed in FIGS. 5A-5E and FIGS. 6A-6D, and has the designation of SEQ ID NO:1 herein. Translation of this nucleotide sequence shows that the majority of the single polyprotein of SVV is encoded by SEQ ID NO:1. The amino acid sequence encoded by nucleotides 1 to 5673 of SEQ ID NO:1 is listed in FIGS. 5A-E and FIGS. 7A-7B has the designation of SEQ ID NO:2 herein. The full-length genome or what appears to be the full-length genome has since been obtained, and is listed in FIGS. 83A-83H and SEQ ID NO:168. Nucleotides 667-7209 encode the full-length polyprotein of SVV, and the amino acid sequence of the polyprotein is listed in FIGS. 83A-83H and SEQ ID NO:169.

[0182] The invention provides isolated (or purified) portions of SEQ ID NO:1, including SEQ ID NOS:3, 5, 7, 9, 11, 13, 15, 17, 19 and 21, and isolated portions of SEQ ID NO:168, including the 5'UTR region (1-666), coding region for the leader peptide (667-903), coding region for the VP4 protein (904-1116), coding region for the VP2 protein (1117-1968), coding region for the VP3 protein (1969-2685), coding region for the VP1 protein (2686-3474), coding region for the coding region for the 2A protein (3478-3504), coding region for the 2B protein (3505-3888), coding region for the 2C protein (3889-4854), coding region for the 3A protein (4855-5124), coding region for the 3B protein (5125-5190), coding region for the 3C protein (5191-5823), coding region for the 3D protein (5824-7209), and the 3'UTR region including the poly(A) tail (7210-7310). The invention also provides isolated nucleic acids that are portions of the specified portions listed above. The invention also provides mutants or derivatives of such isolated portions. The isolated portions of SEQ ID NOS:1 and 168 can be subcloned into expression vectors such that polypeptides encoded by these portions can be isolated. Further encompassed by the invention are isolated nucleic acids that can hybridize to SEQ ID NO:1 or SEQ ID NO:168, or any portion thereof, under high, moderate or low stringency conditions. The following table lists the nucleotides of SEQ ID NO:168 that encode the SVV proteins. The invention provides isolated (or purified) SVV proteins or portions thereof. The table also lists the amino acid sequences of the SVV proteins with respect to the polyprotein sequence listed in SEQ ID NO:169.

TABLE-US-00001 TABLE A SVV Genome and Protein Features SVV Location in SEQ ID feature Location in SEQ ID NO: 168 NO: 169 5'UTR 1-666 N/A (not allowed) Leader 667-903 (coding sequence for Leader 1-79 peptide) VP4 904-1116 (coding sequence for VP4) 80-150 VP2 1117-1968 (coding sequence for VP2) 151-434 VP3 1969-2685 (coding sequence for VP3) 435-673 VP1 2686-3474 or 3477 (coding sequence for 674-936 or 937 VP1) 2A 3478-3504 (coding sequence for 2A) 938-946 2B 3505-3888 (coding sequence for 2B) 947-1074 2C 3889-4854 (coding sequence for 2C) 1075-1396 3A 4855-5124 (coding sequence for 3A) 1397-1486 3B 5125-5190 (coding sequence for 3B) 1487-1508 3C 5191-5823 (coding sequence for 3C) 1509-1719 3D 5824-7209 (coding sequence for 3D) 1720-2181 3'UTR 7210-7310 N/A

[0183] The invention provides an isolated SVV leader sequence peptide with the amino acid sequence of residues 1-79 of SEQ ID NO:169, which is encoded by nucleotides 667-903 of SEQ ID NO:168.

[0184] The invention provides an isolated SVV VP4 (1A) protein with the amino acid sequence of residues 80-150 of SEQ ID NO:169, which is encoded by nucleotides 904-1116 of SEQ ID NO:168.

[0185] The invention provides an isolated SVV VP2 (1B) protein with the amino acid sequence of residues 151-434 of SEQ ID NO:169, which is encoded by nucleotides 1117-1968 of SEQ ID NO:168. The invention also provides an isolated partial SVV VP2 (1B) protein with the amino acid sequence of SEQ ID NO:4, as listed in FIG. 9 (which corresponds to amino acids 2-143 of SEQ ID NO:2). The amino acid sequence of the partial SVV VP2 protein is encoded by the nucleic acid sequence of SEQ ID NO:3, as listed in FIG. 8 (which corresponds to nucleotides 4-429 of SEQ ID NO:1).

[0186] The invention provides an isolated SVV VP3 (1C) protein with the amino acid sequence of residues 435-673 of SEQ ID NO:169, which is encoded by nucleotides 1969-2685 of SEQ ID NO:168. The invention also provides an isolated SVV VP3 (1C) protein with the amino acid sequence of SEQ ID NO:6, as listed in FIG. 11 (which corresponds to amino acids 144-382 of SEQ ID NO:2). The amino acid sequence of the SVV VP3 protein is encoded by the nucleic acid sequence of SEQ ID NO:5, as listed in FIG. 10 (which corresponds to nucleotides 430-1146 of SEQ ID NO:1).

[0187] The invention provides an isolated SVV VP1 (1D) protein with the amino acid sequence of residues 674-937 of SEQ ID NO:169, which is encoded by nucleotides 2686-3477 of SEQ ID NO:168. The invention also provides an isolated SVV VP1 (1D) protein with the amino acid sequence of SEQ ID NO:8, as listed in FIG. 13 (which corresponds to amino acids 383-641 of SEQ ID NO:2). The amino acid sequence of the SVV VP1 protein is encoded by the nucleic acid sequence of SEQ ID NO:7, as listed in FIG. 12 (which corresponds to nucleotides 1147-1923 of SEQ ID NO:1).

[0188] The invention provides an isolated SVV 2A protein with the amino acid sequence of residues 938-946 of SEQ ID NO:169, which is encoded by nucleotides 3478-3504 of SEQ ID NO:168. The invention also provides an isolated SVV 2A protein with the amino acid sequence of SEQ ID NO:10, as listed in FIG. 15 (which corresponds to amino acids 642-655 of SEQ ID NO:2). The amino acid sequence of the SVV 2A protein is encoded by the nucleic acid sequence of SEQ ID NO:9, as listed in FIG. 14 (which corresponds to nucleotides 1924-1965 of SEQ ID NO:1).

[0189] The invention provides an isolated SVV 2B protein with the amino acid sequence of residues 947-1074 of SEQ ID NO:169, which is encoded by nucleotides 3505-3888 of SEQ ID NO:168. The present invention also provides an isolated SVV 2B protein with the amino acid sequence of SEQ ID NO:12, as listed in FIG. 17 (which corresponds to amino acids 656-783 of SEQ ID NO:2). The amino acid sequence of the SVV 2B protein is encoded by the nucleic acid sequence of SEQ ID NO:11, as listed in FIG. 16 (which corresponds to nucleotides 1966-2349 of SEQ ID NO:1).

[0190] The invention provides an isolated SVV 2C protein with the amino acid sequence of residues 1075-1396 of SEQ ID NO:169, which is encoded by nucleotides 3889-4854 of SEQ ID NO:168. The invention also provides an isolated SVV 2C protein with the amino acid sequence of SEQ ID NO:14, as listed in FIG. 19 (which corresponds to amino acids 784-1105 of SEQ ID NO:2). The amino acid sequence of the SVV 2B protein is encoded by the nucleic acid sequence of SEQ ID NO:13, as listed in FIG. 18 (which corresponds to nucleotides 2350-3315 of SEQ ID NO:1).

[0191] The invention provides an isolated SVV 3A protein with the amino acid sequence of residues 1397-1486 of SEQ ID NO:169, which is encoded by nucleotides 4855-5124 of SEQ ID NO:168. The invention also provides an isolated SVV 3A protein with the amino acid sequence of SEQ ID NO:16, as listed in FIG. 21 (which corresponds to amino acids 1106-1195 of SEQ ID NO:2). The amino acid sequence of the SVV 3A protein is encoded by the nucleic acid sequence of SEQ ID NO:15, as listed in FIG. 20 (which corresponds to nucleotides 3316-3585 of SEQ ID NO:1).

[0192] The invention provides an isolated SVV 3B (VPg) protein with the amino acid sequence of residues 1487-1508 of SEQ ID NO:169, which is encoded by nucleotides 5125-5190 of SEQ ID NO:168. The invention also provides an isolated SVV 3B protein with the amino acid sequence of SEQ ID NO:18, as listed in FIG. 23 (which corresponds to amino acids 1196-1217 of SEQ ID NO:2). The amino acid sequence of the SVV 3B protein is encoded by the nucleic acid sequence of SEQ ID NO:17, as listed in FIG. 22 (which corresponds to nucleotides 3586-3651 of SEQ ID NO:1).

[0193] The invention provides an isolated SVV 3C ("pro" or "protease") protein with the amino acid sequence of residues 1509-1719 of SEQ ID NO:169, which is encoded by nucleotides 5191-5823 of SEQ ID NO:168. The invention also provides an isolated SVV 3C protein with the amino acid sequence of SEQ ID NO:20, as listed in FIG. 25 (which corresponds to amino acids 1218-1428 of SEQ ID NO:2). The amino acid sequence of the SVV 3C protein is encoded by the nucleic acid sequence of SEQ ID NO:19, as listed in FIG. 24 (which corresponds to nucleotides 3652-4284 of SEQ ID NO:1).

[0194] The invention provides an isolated SVV 3D ("pol" or "polymerase") protein with the amino acid sequence of residues 1720-2181 of SEQ ID NO:169, which is encoded by nucleotides 5824-7209 of SEQ ID NO:168. The invention also provides an isolated SVV 3D protein with the amino acid sequence of SEQ ID NO:22, as listed in FIG. 27 (which corresponds to amino acids 1429-1890 of SEQ ID NO:2). The amino acid sequence of the SVV 3C protein is encoded by the nucleic acid sequence of SEQ ID NO:19, as listed in FIG. 24 (which corresponds to nucleotides 4285-5673 of SEQ ID NO:1; nucleotides 5671-5673, "tga," code for a stop-codon, which is depicted in the amino acid sequence listings as an asterisk "*").

[0195] The nucleic acids of the present invention include both RNA and DNA forms, and implicitly, the complementary sequences of the provided listings.

[0196] Thus, the isolated SVV nucleic acid depicted by SEQ ID NO:168 has a length of 7,310 nucleotides that encodes a polyprotein with the amino acid sequence depicted by SEQ ID NO:169. The isolated SVV nucleic acid depicted by SEQ ID NO:1 has a length of 5,752 nucleotides that encodes a polypeptide with the amino acid sequence depicted by SEQ ID NO:2. The SVV genomic sequence is translated as a single polyprotein that is cleaved into various downstream "translation products." The present invention encompasses all nucleic acid fragments of SEQ ID NO: 168 and SEQ ID NO:1, and all polypeptides encoded by such fragments.

[0197] The full-length SVV polyprotein amino acid sequence is depicted by SEQ ID NO:169 and is encoded by nucleotides 667-7209 of SEQ ID NO:168. The majority of the full-length SVV polyprotein amino acid sequence is encoded by nucleotides 1-5673 of SEQ ID NO:1. The polyprotein is cleaved into three precursor proteins, P1, P2 and P3 (see FIG. 4B). P1, P2 and P3 are further cleaved into smaller products. The cleavage products of the structural region P1 (1ABCD; or the capsid region) are 1ABC, VP0, VP4, VP2, VP3 and VP 1. The cleavage products of the non-structural protein P2 (2ABC) are 2A, 2BC, 2B and 2C. The cleavage products of the non-structural region P3 polyprotein (3ABCD) are 3AB, 3CD, 3A, 3C, 3D, 3C', and 3D'.

[0198] In certain embodiments, the invention provides isolated nucleic acids that comprise: (i) the coding sequence of 1ABCD or the capsid region (nucleotides 904-3477 of SEQ ID NO:168); (ii) the coding sequence of 1ABC (nucleotides 904-2685 of SEQ ID NO:168); (iii) the coding sequence of VP0 (nucleotides 904-1968 of SEQ ID NO:168); (iv) the coding sequence of 2ABC (nucleotides 3478-4854 of SEQ ID NO:168; nucleotides 1924-3315 of SEQ ID NO:1); (v) the coding sequence of 2BC (nucleotides 3505-4854 of SEQ ID NO:168; nucleotides 1966-3315 of SEQ ID NO:1); (iii) the coding sequence of 3ABCD (nucleotides 4855-7209 of SEQ ID NO:168; nucleotides 3316-5673 of SEQ ID NO:1); (iv) the coding sequence of 3AB (nucleotides 4855-5190 of SEQ ID NO:168; nucleotides 3316-3651 of SEQ ID NO:1); and (v) the coding sequence of 3CD (nucleotides 5191-7209 of SEQ ID NO:168; nucleotides 3652-5673 of SEQ ID NO:1). The invention also provides isolated proteins or peptides encoded by the coding sequences described above, including fragments thereof.

[0199] The basic capsid structure of picornaviruses consists of a densely packed icosahedral arrangement of 60 protomers, each consisting of 4 polypeptides, VP1, VP2, VP3 and VP4, all of which are derived from the cleavage of the original protomer, VP0. The SVV virus particle is about 27 nm in diameter (see FIG. 2), which is consistent with the size of other picornavirus particles, which are about 27-30 nm in diameter.

[0200] The kinetics of picornavirus replication is rapid, the cycle being completed in about 5-10 hours (typically by about 8 hours) (see FIG. 68 for a schematic of the picornavirus replication cycle). Upon receptor binding, the genomic RNA is released from the particle into the cytoplasm. Genomic RNA is then translated directly by polysomes, but in about 30 minutes after infection, cellular protein synthesis declines sharply, almost to zero. This phenomenon is called "shutoff," and is a primary cause of cytopathic effects (cpe). Shutoff appears to be due to cleavage of the host cell's 220 kDa cap-binding complex (CBC) that is involved in binding the m7G cap structure at the 5' end of all eukaryotic mRNA during initiation of translation. The cleavage of the CBC appears to be caused by the 2A protease.

[0201] The 5' UTR contains the IRES. Normally, eukaryotic translation is initiated when ribosomes bind to the 5' methylated cap and then scans along the mRNA to find the first AUG initiation codon. The IRES overcomes this process and allows Picornavirus RNA's to continue to be translated after degradation of CBC. In one embodiment, the invention provides for an isolated nucleic acid comprising the SVV IRES, wherein the IRES is contained within the 5'UTR. In one embodiment, the SVV IRES can be from nucleotides 300-366 of SEQ ID NO:168. The 5'UTR of SVV is present at nucleotides 1-666 of SEQ ID NO:168.

[0202] The virus polyprotein is initially cleaved by the 2A protease into polyproteins P1, P2 and P3 (see FIG. 4B). Further cleavage events are then carried out by 3C, the main picornavirus protease. One of the cleavage products made by 3C is the virus RNA-dependent RNA polymerase (3D), which copies the genomic RNA to produce a negative (-) sense strand. The (-) sense strand forms the template for the (+) strand (genomic) RNA synthesis. Some of the (+) strands are translated to produce additional viral proteins are some (+) strands are packaged into capsids to form new virus particles.

[0203] The (+) strand RNA genome is believed to be packaged into preformed capsids, although the molecular interactions between the genome and the capsid are not clear. Empty capsids are common in all picornavirus infections. The capsid is assembled by cleavage of the P1 polyprotein precursor into a protomer consisting of VP0, VP3, and VP1, which join together enclosing the genome. Maturation and infectivity of the virus particle relies on an internal autocatalytic cleavage of VP0 into VP2 and VP4. Release of newly formed virus particles occurs when the cell lyses.

[0204] The present invention also provides an isolated virus having all the identifying characteristics and nucleic acid sequence of ATCC Patent Deposit number PTA-5343. Viruses of the present invention can be directed to the PTA-5343 isolate, variants, homologues, derivatives and mutants of the PTA-5343 isolate, and variants, homologues, derivatives and mutants of other picornaviruses that are modified in respect to sequences of SVV (both wild-type as disclosed herein and mutant) that are determined to be responsible for its oncolytic properties.

[0205] The present invention further provides antibodies that are specific against: the isolated SVV having the ATTC Patent Deposit number PTA-5343, and epitopes from the isolated SVV proteins having the amino acid sequences SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, and 169 (including entire polyprotein, VP4, VP2, VP3, VP1, 2A, 2B, 2C, 3A, 3B, 3C, 3D, and portions thereof; see Table A supra for amino acids in SEQ ID NO:169 that make up these proteins). The invention also includes antibodies that are specific against epitopes from the proteins that are encoded by fragments or portions of SEQ ID NO:1 or SEQ ID NO:168.

[0206] Comparative analyses of the RNA sequences from a variety of cardiovirus isolates have shown >45% nucleotide identity between genomes. Cardioviruses can be subclassified into the EMC-like viruses ("EMCV"--such as, Mengo, B, R; and also MM, ME, Columbia-SK), the Theiler's-like viruses ("TMEV"--such as, BeAn, DA and GD VII strains), and the Vilyuisk viruses.

[0207] In analyzing the SVV sequence to other viruses, it appears that SVV is a cardiovirus (see Example 4 and Figures referenced therein). If EMCV and TMEV are taken as the standard cardioviruses, SVV is clearly not a typical cardiovirus. However, even these two viruses have their differences, notably in the 5' UTR (Pevear et al., 1987, J. Virol., 61: 1507-1516). Phylogenetically SVV clusters with EMCV and TMEV in much of its polyprotein (P1, 2C, 3C^pro and 3D^pol regions; see FIGS. 31-37), indicating that SVV is most likely a cardiovirus.

[0208] SVV is phylogenetically similar to cardioviruses, but it has now been determined to be in a separate tree (see FIG. 86). SVV can be in a separate genus because: (1) SVV IRES is Type IV (cardiovirus IRES are Type II); (2) multiple unique viruses ("SVV-like picornaviruses") are more similar to SVV than SVV is to other cardioviruses (see Example 18 and FIGS. 87-89); and antibodies that can neutralize SVV infection of permissive cell lines or were raised against SVV are able to bind to the SVV-like picornaviruses. Thus, an SVV-like picornavirus can be used in any of the present methods, including the methods to treat cancer, where it is determined that the SVV-like picornavirus is naturally oncolytic or is made to be oncolytic (for example, by designing mutations in the SVV-like picornavirus genome based on the SVV sequence). In one embodiment, MN 88-36696 is used in the present methods to treat cancer.

Methods for Treating Cancer:

[0209] The present invention provides methods for cancer therapy using viruses modified in view of the oncolytic properties of SVV, including picornaviruses (including SVV-like picornaviruses), derivatives, variants, mutants or homologues thereof. The present invention shows that wild-type SVV (i.e., ATTC Patent Deposit number PTA-5343) has the ability to selectively kill some types of tumors. For example, SVV can selectively kill tumor cells that have neurotropic or neuroendocrine properties, including small cell lung cancer (SCLC) and neuroblastomas. Other examples of neuroendocrine tumors that are contemplated to be treated by the viruses of the present invention include, but are not limited to: adrenal pheochromocytomas, gastrinomas (causing Zollinger-Ellison syndrome), glucagonomas, insulinomas, medullary carcinomas (including medullary thyroid carcinoma), multiple endocrine neoplasia syndromes, pancreatic endocrine tumors, paragangliomas, VIPomas (vasoactive intestinal polypeptide tumor), islet cell tumors, and pheochromocytoma.

[0210] In one embodiment, the invention provides methods for treating or reducing neuroendocrine tumors by administering to a subject SVV or an SVV-like picornavirus, where the neuroendocrine tumor expresses (or overexpresses) one or more neuroendocrine tumor markers, including but not limited to, NTR (Neurotensin receptor), ATOH (, GL11, Myc, GRP receptors, GRP, Neuronal enolase (neuron specific enolase (NSE)), carcinoembryonic antigen (CEA), chromoganin A, NCAM, IgF2, BCL-2, sonic hedgehog pathway, and a chemokine receptor.

[0211] Also encompassed in the present invention are the four types of neuroendocrine lung tumors. The most serious type, small cell lung cancer (SCLC), is among the most rapidly growing and spreading of all cancers. Large cell neuroendocrine carcinoma is a rare cancer that, with the exception of the size of the cells forming the cancer, is very similar to SCLC in its prognosis and in how patients are treated. Carcinoid tumors, also known as carcinoids, comprise the other 2 types of lung neuroendocrine cancer. These two types are typical carcinoid and atypical carcinoid.

[0212] Not being bound by theory, the ability of SVV to specifically kill tumor cells may include, but is not limited to: selective replication, cell protein synthesis shut-off, apoptosis, lysis via tumor-selective cell entry, tumor-selective translation, tumor-selective proteolysis, tumor-selective RNA replication, and combinations thereof.

[0213] SVV has many advantageous characteristics over other oncolytic viruses, including modified adenoviruses, for example: (i) SVV has a very high selectivity for cancers with neural properties, including SCLC, Wilms' tumor, retinoblastoma, and neuroblastoma--for example, SVV shows a greater than 10,000-fold selectivity toward neuroendocrine tumor cells; (ii) SVV has been shown to have a 1,000 fold better cell-killing specificity than chemotherapy treatments; (iii) SVV exhibits no overt toxicity in mice following systemic administration with as high as 10¹⁴ viral particles per kilogram; (iv) the efficacy of SVV is very robust in that 100% of large pre-established tumors can be eradicated in mice, with no recurrence of tumor growth; (v) SVV can be purified to high titer and can be produced at more than 200,000 particles per cell in permissive cell lines; (vi) SVV has a small size (the SVV virus particle is less than 30 nm in diameter) enabling better penetration and spread in tumors than other oncolytic viruses, (vii) SVV replicates quickly (less than 12 hours) and (viii) no modification of SVV is necessary for its use as a specific anti-tumor agent.

[0214] Further, initial studies (see Example 6) indicate some additional factors that make SVV an advantageous tool for oncolytic viral therapy: (i) human serum samples do not contain neutralizing antibodies directed against SVV; (ii) SVV is not inhibited by complement; and (iii) SVV does not produce hemagglutination of human erythrocytes. All of these factors contribute to the fact that SVV exhibits a longer circulation time in vivo than other oncolytic viruses (for example, see Example 7).

[0215] The present invention provides methods for selectively killing a neoplastic cell in a cell population that comprises contacting an effective amount of SVV with said cell population under conditions where the virus can transduce the neoplastic cells in the cell population, replicate and kill the neoplastic cells. Besides methods where SVV kills tumor cells in vivo, the present methods encompass embodiments where the tumors can be: (1) cultured in vitro when infected by SVV; (2) cultured in the presence of non-tumor cells; and (3) the cells are mammalian (both tumor and non-tumor cells), including where the cells are human cells. The in vitro culturing of cells and infection by SVV can have various applications. For example, in vitro infection be used as a method to produce large amounts of SVV, as method for determining or detecting whether neoplastic cells are present in a cell population, or as a method for screening whether a mutant SVV can specifically target and kill various tumor cell or tissue types.

[0216] The present invention further provides an ex vivo method of treating cancer wherein cells are isolated from a human cancer patient, cultured in vitro, infected with a SVV which selectively kills the cancer cells, and the non-tumor cells are introduced back to the patient. Alternatively, cells isolated form a patient can be infected with SVV and immediately introduced back to the patient as a method for administering SVV to a patient. In one embodiment, the cancer cells are of a hematopoietic origin. Optionally, the patient may receive treatment (e.g., chemotherapy or radiation) to destroy the patient's tumor cell in vivo before the cultured cells are introduced back to the patient. In one embodiment, the treatment may be used to destroy the patient's bone marrow cells.

[0217] Polymer coated SVV can be used to target the SVV to any specific cell type. This coating strategy can also be used to overcome antibodies to SVV.

[0218] SVV possesses potent antitumor activity against tumor cell-types with neural characteristics. SVV does not exhibit cytolytic activity against tested normal human. Further SVV is not cytotoxic to primary human hepatocytes. Table 1 below summarizes initial studies that have been conducted to determine the in vitro cytolytic potency of SVV against selected tumor cell types.

TABLE-US-00002 TABLE 1 SVV Cytolytic Potency Against Selected Tumor Cell-Types Cell Line Cell Type EC₅₀ (VP/cell) H446 Human SCLC 0.0012 PER.C6 Human Embryonic Retinoblast 0.02 H69AR SCLC-Multidrug Resistant 0.035 293 AD5 DNA Transformed Human 0.036 Kidney Y79 Human Retinoblastoma 0.00035 IMR32 Human Brain Neuroblastoma 0.035 D283 Med Human Brain Cerebellar 0.25 Medulloblastoma SK-N-AS Human Brain Neuroblastoma 0.474 N1E-115 Mouse Neuroblastoma 0.0028 BEKPCB3E1 Bovine embryonic Kidney cells 0.99 transformed with Ad5E1 H1299 Human non-SCLC 7.66 ST Porcine Testis 5.9 DMS153 Human SCLC 9.2 BEK Bovine Embryonic Kidney 17.55 M059K Human Brain Malignant 1,061 Glioblastoma PK15 Porcine Kidney 1,144 FBRC Fetal Bovine Retina 10,170 HCN-1A Human Brain 23,708 H460 Human LCLC >30,000 (inactive) Neuro 2A Mouse Neuroblastoma >30,000 (inactive) DMS79 Human SCLC >30,000 (inactive) H69 Human SCLC >30,000 (inactive) C8D30 Mouse Brain >30,000 (inactive) MRC-5 Human Fetal Lung Fibroblast >30,000 (inactive) HMVEC Neonatal vascular endothelial cells >30,000 (inactive) HMVEC Adult vascular endothelial cells >30,000 (inactive) A375-S2 Human Melanoma >30,000 (inactive) SK-MEL-28 Melanoma >30,000 (inactive) PC3 Human prostate cancer >30,000 (inactive) PC3M2AC6 Human prostate cancer >30,000 (inactive) LnCap Human Prostate cancer >30,000 (inactive) DU145 Human prostate cancer >30,000 (inactive)

[0219] Table 1-A below provides a list of cell lines that are permissive are non-permissive to SVV infection. The Table shows the cytolytic potency and selectivity of SVV.

TABLE-US-00003 TABLE 1 In Vitro Cytolytic Potency and Selectivity of SVV Cell Line Species Stage State Organ Type Metastatic Site EC50* PERMISSIVE Y79 Human Adult Cancer Eye, Retina Retinoblastoma 0.00035, 0.0007 NCI-H446 Human Adult Metastatic Lung Variant Small Cell Pleural effusion 0.0012, 0.002, Cancer Lung Carcinoma 0.0007 (SCLC) N1E-115 Murine Adult Cancer Brain Neuroblastoma 0.0028, 0.001 NCI-H1770 Human Adult Metastatic Lung Non-Small Cell Lymph Node 0.00724 Cancer Lung Carcinoma (NSCLC) NCI-H82 Human Adult Metastatic Lung Variant Small Cell Pleural effusion 0.015 Cancer Lung Carcinoma (SCLC) PER.C6 ® Human Fetal Cancer Eye, Retina Retinoblast 0.02, 0.0049 NCI-H69AR Human Adult Cancer Lung Small Cell Lung 0.035, 0.05 Carcinoma, multi-drug resistant (SCLC) SK-NEP-1 Human Adult Metastatic Kidney Wilms' Tumor Pleural effusion 0.03 Cancer IMR-32 Human Adult Cancer Brain Neuroblastoma 0.035, 0.0059, 0.05 NCI-H187 Human Adult Metastatic Lung Classic Small Cell Pleural effusion 0.00343 Cancer Lung Carcinoma (SCLC) NCI-H209 Human Adult Metastatic Lung Small Cell Lung Bone Marrow 0.04 Cancer Carcinoma (SCLC) NCI-H1184 Human Adult Metastatic Lung Small Cell Lung Lymph Node 0.155 Cancer Carcinoma (SCLC) D283 Med Human Adult Metastatic Brain, Medulloblastoma Peritoneum 0.25 Cancer Cerebellum SK-N-AS Human Adult Metastatic Brain Neuroblastoma Bone Marrow 0.474 Cancer BEK PCB3E1 Bovine Fetal Normal, Ad5 Kidney Ad5E1 transformed 0.99 transformed ST Porcine Fetal Normal, Testis 5.9 immortalized NCI-H1299 Human Adult Metastatic Lung Large Cell Lung Lymph Node 7.66, 4.8 Cancer Carcinoma DMS 153 Human Adult Metastatic Lung Small Cell Lung Liver 9.2 Cancer Carcinoma (SCLC) NCI-H295R Human Adult Cancer Adrenal Gland, Adrenocortical 16.5 Cortex Carcinoma BEK Bovine Fetal Normal, Kidney 17.55 immortalized PPASMC Porcine Adult Normal, Lung, Smooth Muscle Cells 18.4 Primary Pulmonary Artery PCASMC Porcine Adult Normal, Heart, Coronary Smooth Muscle Cells 11.9 Primary Artery PAoSMC Porcine Adult Normal, Heart, Aorta Smooth Muscle Cells 88 Primary NCI-H526 Human Adult Metastatic Lung Variant Small Cell Bone Marrow 46.4 Cancer Lung Carcinoma (SCLC) OVCAR-3 Human Adult Cancer Ovary Adenocarcinoma 39 ESK-4 Porcine Fetal Normal, Kidney Fibroblast 60 Immortalized SW-13 Human Adult Cancer Adrenal Gland, Small Cell <100 Cortex Adenocarcinoma 293 Human Fetal Normal, Ad5 Kidney Ad5 transformed 0.036, 184.8 transformed Hs 578T Human Adult Cancer Breast Carcinoma 273 Hs 1.Tes Human Fetal Normal, Testis 416 Immortalized LOX IMVI Human Adult Cancer Skin Melanoma 569 PK(15) Porcine Adult Normal, Kidney 1144, 129 Immortalized NON PERMISSIVE WI-38 Human Fetal Normal, Lung Fibroblast >10,000 Immortalized IMR-90 Human Fetal Normal, Lung Fibroblast >10,000 Immortalized MRC-5 Human Fetal Normal, Lung Fibroblast >10,000 Immortalized HCN-1A Human Adult Normal, Brain, Cortical >10,000 Immortalized Neuron HMVEC Human Adult Normal, Skin Microvascular >10,000 (neonatal) Primary Endothelial Cells HMVEC Human Adult Normal, Skin Microvascular >10,000 Primary Endothelial Cells HUVEC Human Adult Normal, Umbilical Vein Endothelial Cells >10,000 Primary HRE Human Adult Normal, Kidney Epithelial Cells >10,000 Primary HRCE Human Adult Normal, Kidney Cortical Epithelial Cells >10,000 Primary PHH Human Adult Normal, Liver Hepatocyte >10,000 Primary HCASMC-c Human Adult Normal, Heart, Coronary Smooth Muscle Cells >10,000 Primary Artery HCAEC Human Adult Normal, Heart, Coronary Endothelial Cells >10,000 Primary Artery HAEC Human Adult Normal, Heart, Aorta Endothelial Cells >10,000 Primary HAoSMC-c Human Adult Normal, Heart, Aorta Smooth Muscle Cells >10,000 Primary NHA Human Adult Normal, Brain Astrocytes 1713 Primary HPASMC Human Adult Normal, Lung Smooth Muscle Cells >10,000 Primary PBMC Human Adult Normal, Peripheral Blood Mononuclear Cells >10,000 Primary SF-295 Human Adult Cancer Brain Glioblastoma >10,000 U251 Human Adult Cancer Brain Glioblastoma >10,000 SF-539 Human Adult Cancer Brain Glioblastoma >10,000 SNB-19 Human Adult Cancer Brain Glioblastoma >10,000 SF-268 Human Adult Cancer Brain Glioblastoma 3103 U-118MG Human Adult Cancer Brain Glioblastoma, >10,000 Astrocytoma SNB-75 Human Adult Cancer Brain Astrocytoma >10,000 M059K Human Adult Cancer Brain, Glial Cell Malignant 1061 Glioblastoma KK Human Adult Cancer Brain, Glial Cell Glioblastoma >10,000 HCC-2998 Human Adult Cancer Colon Carcinoma >10,000 KM12 Human Adult Cancer Colon Carcinoma >10,000 HT-29 Human Adult Cancer Colon Adenocarcinoma >10,000 HCT 116 Human Adult Cancer Colon Carcinoma >10,000 HCT-15 Human Adult Cancer Colon Carcinoma >10,000 COLO 205 Human Adult Metastatic Colon Adenocarcinoma Ascites >10,000 Cancer SW620 Human Adult Metastatic Colon Colorectal Carcinoma Lymph Node 6503, >10,000 Cancer PC3M-2AC6 Human Adult Cancer Prostate >10,000 PC3M-2AC6 + Human Adult Cancer Prostate ND 2-AP PC-3 Human Adult Metastatic Prostate Adenocarcinoma Bone >10,000 Cancer LNCaP.FGC Human Adult Metastatic Prostate Adenocarcinoma Lymph Node >10,000 Cancer DU 145 Human Adult Metastatic Prostate Adenocarcinoma Brain >10,000 Cancer Hep3B Human Adult Cancer Liver Hepatocellular >10,000 Carcinoma Hep G2 Human Adult Cancer Liver Hepatocellular >10,000 Carcinoma 786-O Human Adult Cancer Kidney Clear Cell >10,000 Adenocarcinoma TK-10 Human Adult Cancer Kidney Carcinoma >10,000 RXF 393 Human Adult Cancer Kidney Carcinoma >10,000 UO-31 Human Adult Cancer Kidney Carcinoma >10,000 SN12C Human Adult Cancer Kidney Carcinoma >10,000 A-498 Human Adult Cancer Kidney Carcinoma >10,000 ACHN Human Adult Cancer Kidney Carcinoma >10,000 SW839 Human Adult Cancer Kidney Renal Clear Cell >10,000 Adenocarcinoma Caki-1 Human Adult Metastatic Kidney Clear Cell Skin >10,000 Cancer Adenocarcinoma 5637 Human Adult Cancer Bladder Carcinoma >10,000 NCI-H1339 Human Adult Cancer Lung >10,000 NCI-H1514 Human Adult Cancer Lung >10,000 A549 Human Adult Cancer Lung Carcinoma >10,000 S8 Human Adult Cancer Lung Carcinoma >10,000 NCI-H727 Human Adult Cancer Lung Carcinoid >10,000 NCI-H835 Human Adult Cancer Lung Carcinoid >10,000 UMC-11 Human Adult Cancer Lung Carcinoid >10,000 DMS 114 Human Adult Cancer Lung Small Cell Lung >10,000 Carcinoma (SCLC) DMS 53 Human Adult Cancer Lung Small Cell Lung >10,000 Carcinoma (SCLC) NCI-H69 Human Adult Cancer Lung Small Cell Lung >10,000 Carcinoma (SCLC) NCI-H2195 Human Adult Metastatic Lung Small Cell Lung Bone Marrow >10,000 Cancer Carcinoma (SCLC) DMS 79 Human Adult Metastatic Lung Small Cell Lung Pleural effusion >10,000 Cancer Carcinoma (SCLC) NCI-H146 Human Adult Metastatic Lung Classic Small Cell Bone Marrow >10,000 Cancer Lung Carcinoma (SCLC) NCI-H1618 Human Adult Metastatic Lung Classic Small Cell Bone Marrow >10,000 Cancer Lung Carcinoma (SCLC) NCI-H345 Human Adult Metastatic Lung Classic Small Cell Bone Marrow >10,000 Cancer Lung Carcinoma (SCLC) HOP-62 Human Adult Cancer Lung Non-Small Cell >10,000 Lung Carcinoma (NSCLC) EKVX Human Adult Cancer Lung Non-Small Cell >10,000 Lung Carcinoma (NSCLC) HOP-92 Human Adult Cancer Lung Non-Small Cell >10,000 Lung Carcinoma (NSCLC) NCI-H522 Human Adult Cancer Lung Non-Small Cell >10,000 Lung Carcinoma (NSCLC) NCI-H23 Human Adult Cancer Lung Non-Small Cell >10,000 Lung Carcinoma (NSCLC) NCI-H322M Human Adult Cancer Lung Non-Small Cell >10,000 Lung Carcinoma (NSCLC) NCI-H226 Human Adult Metastatic Lung Squamous Cell Pleural effusion >10,000 Cancer Carcinoma, Mesothelioma (NSCLC) NCI-H460 Human Adult Metastatic Lung Large Cell Lung Pleural effusion >10,000 Cancer Carcinoma HeLa, HeLa Human Adult Cancer Cervix Adenocarcinoma >10,000 S3 CCRF-CEM Human Adult Cancer Peripheral Acute Lymphoblastic >10,000 Blood, T Leukemia (ALL) lymphoblast MOLT-4 Human Adult Cancer Peripheral Acute Lymphoblastic >10,000 Blood, T Leukemia (ALL) lymphoblast RPMI 8226 Human Adult Cancer Peripheral Plasmacytoma, >10,000 Blood, B Myeloma lymphocyte SR Human Adult Metastatic Lymphoblast Large Cell Pleural effusion >10,000 Cancer Lymphoblastic Lymphoma HL-60(TB) Human Adult Cancer Peripheral Acute Promyelocytic >10,000 Blood, Leukemia (APL) Promyleoblast K-562 Human Adult Metastatic Bone Marrow Chronic Myelogenous Pleural effusion >10,000 Cancer Leukemia (CML) UACC-257 Human Adult Cancer Skin Melanoma >10,000 M14 Human Adult Cancer Skin Melanoma >10,000 UACC-62 Human Adult Cancer Skin Melanoma 6614 SK-MEL-2 Human Adult Cancer Skin Malignant Melanoma >10,000 SK-MEL-28 Human Adult Cancer Skin Malignant Melanoma >10,000 A375.S2 Human Adult Cancer Skin Malignant Melanoma >10,000 SK-MEL-28 Human Adult Cancer Skin Malignant Melanoma >10,000 SK-MEL-5 Human Adult Metastatic Skin Malignant Melanoma Lymph Node >10,000 Cancer MALME-3M Human Adult Metastatic Skin Malignant Melanoma Lung >10,000 Cancer

BT-549 Human Adult Cancer Breast Ductal Carcinoma >10,000 NCI/ Human Adult Cancer Breast Carcinoma >10,000 ADR-RES MCF7 Human Adult Metastatic Breast Adenocarcinoma Pleural effusion >10,000 Cancer MDA-MB-231 Human Adult Metastatic Breast Adenocarcinoma Pleural effusion >10,000 Cancer T-47D Human Adult Metastatic Breast Ductal Carcinoma Pleural effusion >10,000 Cancer MDA-MB-435 Human Adult Metastatic Breast Ductal Pleural effusion >10,000 Cancer Adenocarcinoma IGR-OV1 Human Adult Cancer Ovary Carcinoma >10,000 OVCAR-4 Human Adult Cancer Ovary Adenocarcinoma >10,000 OVCAR-5 Human Adult Cancer Ovary Adenocarcinoma >10,000 OVCAR-8 Human Adult Cancer Ovary Adenocarcinoma >10,000 SK-OV-3 Human Adult Metastatic Ovary Adenocarcinoma Ascites >10,000 Cancer BxPC-3 Human Adult Cancer Pancreas Adenocarcinoma >10,000 AsPC-1 Human Adult Metastatic Pancreas Adenocarcinoma Ascites >1000 Cancer NCI-H295 Human Adult Cancer Adrenal Gland, Adrenocortical >10,000 Cortex Carcinoma TT Human Adult Cancer Thyroid Medullary Carcinoma >10,000 C8-D30 Murine Adult Normal Brain, >10,000 Cerebellum LLC1 Murine Adult Cancer Lung Lewis Lung Carcinoma >10,000 RM-1 Murine Adult Cancer Prostate >10,000 MLTC-1 Murine Adult Cancer Testis Leydig Cell Tumor >10,000 KLN 205 Murine Adult Cancer Lung Squamous Cell >10,000 Carcinoma CMT-64 Murine Adult Cancer Lung Small Cell Lung >10,000 Carcinoma (SCLC) CMT-93 Murine Adult Cancer Rectum Polyploid Carcinoma >10,000 B16-F0 Murine Adult Cancer Skin Melanoma >10,000 RM-2 Murine Adult Cancer Prostate >10,000 RM-9 Murine Adult Cancer Prostate >10,000 Neuro-2A Murine Adult Cancer Brain Neuroblastoma >10,000 FBRC Bovine Fetal Eye, Retina >10,000 MDBK Bovine Adult Normal, Kidney >10,000 Immortalized CSL 503 Ovine Adult Normal, Lung Ad5E1 >10,000 Immortalized transformed OFRC Ovine Adult Normal, Eye, Retina Ad5E1 >10,000 Immortalized transformed PC-12 Rat Adult Cancer Adrenal Gland Pheochromocytoma >10,000 Vero Monkey Adult Normal, Kidney >10,000 Immortalized PAOEC Porcine Adult Normal, Heart, Aorta Endothelial Cells >10,000 Primary PCAEC Porcine Adult Normal, Heart, Coronary Endothelial Cells >10,000 Primary Artery PPAEC Porcine Adult Normal, Lung, Endothelial Cells >10,000 Primary Pulmonary Artery TBD NCI-H289 Human Adult Cancer Lung TBD NCI-H1963 Human Adult Cancer Lung Small Cell Lung TBD Carcinoma (SCLC) NCI-H2227 Human Adult Cancer Lung Small Cell Lung TBD Carcinoma (SCLC) NCI-H378 Human Adult Metastatic Lung Classic Small Cell Pleural effusion TBD Cancer Lung Carcinoma (SCLC) NCI-H2107 Human Adult Metastatic Lung Small Cell Lung Bone Marrow TBD Cancer Carcinoma (SCLC) HCC970 Human Adult Metastatic Lung Small Cell Lung Bone Marrow TBD Cancer Carcinoma (SCLC) HCC33 Human Adult Metastatic Lung Small Cell Lung Pleural effusion <1000/TBD Cancer Carcinoma (SCLC) BON Human Adult Cancer Pancreas Carcinoid TBD H1T-T15 Hamster Adult Normal, Pancreas Islets of Langerhans, TBD Immortalized b-cell *EC50 determined after 3 days except where noted

[0220] Table 1-A lists the results of SVV permissivity experiments on 165 primary cells and cell lines, representing 22 tissues from 8 different species. The results indicate that virtually all adult normal are nonpermissive for SVV. Thirteen primary adult human cell cultures tested were nonpermissive. Of the twelve bovine, ovine, porcine and primate normal cell cultures tested, only three cell cultures were permissive, which were porcine smooth muscle cells. This result is consistent with the hypothesis that the natural host for SVV may be pigs. Besides the porcine smooth muscle cells, only neuroendocrine cancer cell lines or most fetal lines were permissive.

[0221] Murine studies (see Examples) show that SVV can specifically kill tumors with great efficacy and specificity in vivo. These in vivo studies show that SVV has a number of advantages over other oncolytic viruses. For example, one important factor affecting the ability of an oncolytic tumor virus to eradicate established tumors is viral penetration. In studies with adenoviral vectors, Ad5 based vectors had no effect on SCLC tumor development in athymic mice. Based on immunohistochemical results, adenovirus did not appear to penetrate the established tumors. In contrast, SVV was able to eliminate H446 SCLC tumors in athymic nude mice following a single systemic administration. SVV has a small size (<30 nm in diameter) enabling better penetration and spread in tumor tissue than other viruses, and thus, the small size of SVV may contribute to its ability to successfully penetrate and eradicate established tumors.

[0222] Additional in vivo tests demonstrate the efficacy of a single intravenous dose of SVV in murine tumor models using athymic nude mice and immunocompetent mice. The tumor models tested were: (1) H446 (human SCLC); (2) Y79 (human retinoblastoma); (3) H69AR (human multi-drug resistant SCLC); (4) H1299 (human NSCLC); and (5) N1E-115 (murine neuroblastoma). The results of these tests are shown in FIGS. 90A-E and Example 11. The tests demonstrate efficacy of a single intravenous dose of SVV in all models and show an agreement between relative ranks of in vitro ED₅₀ and in vivo efficacy in human xenograft models. The results in the N1E-115 immunocompetent murine neuroblastoma model shows that SVV can be efficacious against tumors in subjects with normal immune systems.

[0223] Chemoresistance is a major issue facing any patient that receives chemotherapy as a facet of cancer therapy. Patients that become chemoresistant often, if not always, have a much poorer prognosis and may be left with no alternative therapy. It is well known that one of the major causes of chemoresistance is the expression, over expression, or increased activity of a family of proteins called Multiple Drug Resistant proteins (MRPs). Applicants have found that a sensitivity of certain tumor cells for SVV is also correlated with the chemoresistant state of cancer cells and MRP expression. H69 is a chemosensitive (adriamycin) cell line that is resistant to SVV in vitro, whereas H69AR is a chemoresistant cell line that overexpresses MRPs and is sensitive to SVV (see Table 1). Evidence indicates that overexpression of MRPs, including MDR, correlates with sensitivity of cells to SVV killing. Thus, in one embodiment, the present invention provides a method for treating cancer wherein SVV kills cells overexpressing an MRP.

[0224] The invention also provides methods for treating diseases that are a result of abnormal cells, such as abnormally proliferative cells. The method comprises contacting said abnormal cells with SVV in a manner that results in the destruction of a portion or all of the abnormal cells. SVV can be used to treat a variety of diseases that are a result of abnormal cells. Examples of these diseases include, but are not limited to, cancers wherein the tumor cells display neuroendocrine features and neurofibromatosis.

[0225] Neuroendocrine tumors can be identified by a variety of methods. For example, neuroendocrine tumors produce and secrete a multitude of peptide hormones and amines. Some of these substances cause a specific clinical syndrome: carcinoid, Zollinger-Ellison, hyperglycemic, glucagonoma and WDHA syndrome. Specific markers for these syndromes are basal and/or stimulated levels of urinary 5-HIAA, serum or plasma gastrin, insulin, glucagon and vasoactive intestinal polypeptide, respectively. Some carcinoid tumors and about one third of endocrine pancreatic tumors do not present any clinical symptoms and are called `nonfunctioning` tumors. Therefore, general tumor markers such as chromogranin A, pancreatic polypeptide, serum neuron-specific enolase and subunits of glycoprotein hormones have been used for screening purposes in patients without distinct clinical hormone-related symptoms. Among these general tumor markers chromogranin A, although its precise function is not yet established, has been shown to be a very sensitive and specific serum marker for various types of neuroendocrine tumors. This is because it may also be elevated in many cases of less well-differentiated tumors of neuroendocrine origin that do not secrete known hormones. At the moment, chromogranin A is considered the best general neuroendocrine serum or plasma marker available both for diagnosis and therapeutic evaluation and is increased in 50-100% of patients with various neuroendocrine tumors. Chromogranin A serum or plasma levels reflect tumor load, and it may be an independent marker of prognosis in patients with midgut carcinoids.

[0226] The invention also provides a pharmaceutical composition comprising SVV and a pharmaceutically acceptable carrier. Such compositions, which can comprise an effective amount of SVV in a pharmaceutically acceptable carrier, are suitable for local or systemic administration to individuals in unit dosage forms, sterile parenteral solutions or suspensions, sterile non-parenteral solutions or oral solutions or suspensions, oil in water or water in oil emulsions, and the like. Formulations for parenteral and non-parenteral drug delivery are known in the art. Compositions also include lyophilized and/or reconstituted forms of SVV. Acceptable pharmaceutical carriers are, for example, saline solution, protamine sulfate (Elkins-Sinn, Inc., Chemy Hill, N.J.), water, aqueous buffers, such as phosphate buffers and Tris buffers, or Polybrene (Sigma Chemical, St. Louis, Mo.) and phosphate-buffered saline and sucrose. The selection of a suitable pharmaceutical carrier is deemed to be apparent to those skilled in the art from the teachings contained herein. These solutions are sterile and generally free particulate matter other than SVV. The compositions may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions such as pH adjusting and buffering agents, toxicity adjusting agents and the like, for example, sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate, etc. Excipients that enhance infection of cells by SVV may be included.

[0227] SVV is administered to a host or subject in an amount that is effective to inhibit, prevent or destroy the growth of the tumor cells through replication of the virus in the tumor cells. Methods that utilize SVV for cancer therapy include systemic, regional or local delivery of the virus at safe, developable, and tolerable doses to elicit therapeutically useful destruction of tumor cells. Even following systemic administration, the therapeutic index for SVV is at least 10, preferably at least 100 or more preferably at least 1000. In general, SVV is administered in an amount of between 1×10⁸ and 1×10¹⁴ vp/kg. The exact dosage to be administered is dependent upon a variety of factors including the age, weight, and sex of the patient, and the size and severity of the tumor being treated. The viruses may be administered one or more times, which may be dependent upon the immune response potential of the host. Single or multiple administrations of the compositions can be carried out with dose levels and pattern being selected by the treating physician. If necessary, the immune response may be diminished by employing a variety of immunosuppressants, so as to permit repetitive administration and/or enhance replication by reducing the immune response to the viruses. Anti-neoplastic viral therapy of the present invention may be combined with other anti-neoplastic protocols. Delivery can be achieved in a variety of ways, employing liposomes, direct injection, catheters, topical application, inhalation, etc. Further, a DNA copy of the SVV genomic RNA, or portions thereof, can also be a method of delivery, where the DNA is subsequently transcribed by cells to produce SVV virus particles or particular SVV polypeptides.

[0228] A therapeutically effective dose refers to that amount of the virus that results in amelioration of symptoms or a prolongation of survival in a patient. Toxicity and therapeutic efficacy of viruses can be determined by standard procedures in cell cultures or experimental animals, e.g., for determining the LD₅₀ (the dose lethal to 50% of the population of animals or cells; for viruses, the dose is in units of vp/kg) and the ED₅₀ (the dose--vp/kg--therapeutically effective in 50% of the population of animals or cells) or the EC₅₀ (the effective concentration--vp/cell (see Table 1 for example)--in 50% of the population of animals or cells). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio between LD₅₀ and ED₅₀ or EC₅₀. Viruses which exhibit high therapeutic indices are preferred. The data obtained from these cell culture assays and animal studies can be used in formulating a range of dosage for use in human. The dosage of viruses lies preferably within a range of circulating concentrations that include the ED₅₀ or EC₅₀ with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized.

[0229] In yet another aspect, a method for treating a host organism having a neoplastic condition is provided, comprising administering a therapeutically effective amount of a viral composition of the invention to said host organism. In one embodiment, the neoplastic tissue is abnormally proliferating, and the neoplastic tissue can be malignant tumor tissue. Preferably, the virus is distributed throughout the tissue or tumor mass due to its capacity for selective replication in the tumor tissue. Neoplastic conditions potentially amenable to treatment with the methods of the invention include those with neurotropic properties.

Methods for Producing the Viruses of the Present Invention:

[0230] Methods for producing the present viruses to very high titers and yields are additional aspects of the invention. As stated, SVV can be purified to high titer and can be produced at more than 200,000 particles per cell in permissive cell lines. Cells that are capable of producing high quantities of viruses include, but are not limited to, PER.C6 (Fallaux et al., Human Gene Therapy, 9:1909-1917, 1998), H446 (ATCC#HTB-171) and the other cell lines listed in Table 1 where the EC₅₀ value is less than 10.

[0231] For example, the cultivation of picornaviruses can be conducted as follows. The virus of interest is plaque purified and a well-isolated plaque is picked and amplified in a permissive cell line, such as PER.C6. A crude virus lysate (CVL) from the infected cells can be made by multiple cycles of freeze and thaw, and used to infect large numbers of permissive cells. The permissive cells can be grown in various tissue culture flasks, for example, 50×150 cm² flasks using various media, such as Dulbecco's modified Eagle medium (DMEM, Invitrogen, Carlsbad, Calif.)) containing 10% fetal bovine serum (Biowhitaker, Walkersvile, Md.) and 10 mM magnesium chloride (Sigma, St Louis, Mo.). The infected cells can be harvested between 12 and 48 hours after infection or when complete cytopathic effects (CPE) are noticed, and are collected by centrifugation at 1500 rpm for 10 minutes at 4° C. The cell pellet is resuspended in the cell culture supernatant and is subjected to multiple cycles of freeze and thaw. The resulting CVL is clarified by centrifugation at 1500 rpm for 10 minutes at 4° C. Virus can be purified by gradient centrifugation. For example, two rounds of CsCl gradients can suffice for SVV purification: a one-step gradient (density of CsCl 1.24 g/ml and 1.4 g/ml) followed by one continuous gradient centrifugation (density of CsCl 1.33 g/ml). The purified virus concentration is determined spectrophotometrically, assuming 1A260=9.5×10¹² particles (Scraba D. G., and Palmenberg, A. C. 1999. Cardioviruses (Picornaviridae). In: Encyclopedia of Virology, Second edition, R. G. Webster and A Granoff, Eds). Infectivity titers of purified virus are also determined by a standard plaque and/or tissue culture infective dose 50 (TCID₅₀) assay using PER.C6 or any other suitable cell type. The yield of SVV from PER.C6 cells are greater than 200,000 particles per cell with particles to PFU ratio of about 100. The yields of SVV from other permissive cells (H446-ATCC#HTB-171) may be at least this high or higher. SVV can also be purified by column chromatography.

[0232] In addition, several steps in a commercially attractive large scale Good Manufacturing Processes (GMP) are applicable to the purification of SVV. The invention also contemplates methods for purifying SVV that are based on methods for purifying adenoviruses. These methods include isolating SVV based on its density, since SVV has a very similar density to adenovirus and can be co-purified with adenovirus.

Methods for Detecting and Studying Tumors:

[0233] The present invention provides methods for detecting tumor or neoplastic cells in a patient using the viruses of the present invention. Cellular samples can be obtained from a patient and screened by incubating the sample with an epitope-tagged SVV (or other tumor-specific viruses provided by the invention, i.e., tumor-specific mutant cardioviruses), and then screening the sample for bound SVV by detecting the epitope tag. Alternatively, the sample can be screened by detecting whether the SVV causes any cellular lysis. If SVV does cause cellular lysis, or if SVV can bind specifically to cells in the sample, this would indicate the possibility that the sample contains neoplastic or tumor cells known to be capable of being bound and/or infected by SVV.

[0234] Additionally, SVV can be used in a method for detecting a tumor cell in vivo. In such a method, epitope-tagged SVV can first be inactivated in a manner where SVV can still bind to tumor cells specifically but cannot replicate. Tumor cells that have bound SVV can be detected by assaying for the epitope tag. Detection of the epitope tag can be accomplished by antibodies that specifically bind the epitope, where the antibodies are either labeled (for example, fluorescently) or where the antibodies can then be detected by labeled secondary antibodies.

[0235] The present methods of detection encompass detecting any type of tumor or neoplastic cell that is specifically targeted by any virus of the present invention. Specific tumor types include, for example, neuroendocrine-type tumors, such as retinoblastomas, SCLC, neuroblastomas glioblastomas and medulloblastomas.

[0236] The present invention also provides the use of SVV as a tool to study tumor cells. SVV selectively destroys some tumor cell types, and has very little, if any, toxic effects on non-tumor cells. Because of these characteristics, SVV can be used to study tumors and possibly discover a new tumor specific gene and/or pathway. In other words, there is some characteristic of the tumor cells that allows replication of SVV, wherein normal cells do not exhibit said characteristic. Upon identification of a new tumor specific gene and/or pathway, therapeutic antibodies or small molecules can then be designed or screened to determine whether these reagents are anti-tumor agents.

[0237] The present invention also provides a method for identifying all types of cancers that respond to SVV. In one embodiment, the method for identifying SVV-responsive cells comprises obtaining cells, contacting said cells with SVV and detecting cell killing or detecting viral replication. Cell killing can be detected using various methods known to one skilled in the art (e.g., MTS assay, see High-Throughput section herein). Methods of detecting virus replication are also known to one skilled in the art (e.g., observance of CPE, plaque assay, DNA quantification methods, FACS to detect quantity of virus in the tumor cells, RT-PCR assays to detect viral RNA, etc.). In one embodiment, the cells are cancer cells. Examples of cancer cells include, but are not limited to, established tumor cell lines and tumor cells obtained from a mammal. In one embodiment, the mammal is a human. In a further embodiment, the cells are cancer cells obtained from a human cancer patient.

[0238] The method for identifying SVV-responsive cancer cells may be used to discover tumor cell lines or tumor tissues that are permissive for SVV replication. Also, by determining the characteristics of permissive tumor cells, one may be able to identify characteristics of tumor cells that cause the cells to be selectively killed by SVV. The discovery of these characteristics could lead to new targets for cancer drugs. Also, the methods for identifying SVV responsive cancer cells could be used as a screen for human cancer patients who would benefit from treatment with SVV.

[0239] For example, antibodies against SVV or an SVV-like picornavirus (polyclonal, monoclonal, etc.) can be used in a viral binding assay to pre-screen patients prior to SVV or SVV-like picornavirus therapy. The pre-screening can be conducted generally as follows: (1) cells from a patient are isolated, the cells can be from a tumor biopsy for example, (2) the cells are stained with anti-SVV or anti-SVV-like picornavirus antibodies, (3) a secondary antibody conjugated with a marker (such as fluoroscein or some other detectable dye or fluorophore) that is specific to the anti-SVV or anti-SVV-like picornavirus antibodies is added (for example, if the antibodies were raised in a rabbit, then the secondary antibody would be specific for rabbit immunoglobulins), and (4) detection for the marker is conducted--for example, fluorescence microscopy can be conducted where the marker is fluorescein. (Step 3 is optional if the anti-SVV or anti-SVV-like picornavirus antibodies are directly conjugated, i.e., where the antibodies are monoclonal. If the antibodies are polyclonal, indirect immunofluorescence--use of a secondary antibody--is suggested.) If the patient's tumor cells are permissive for SVV or SVV-like picornavirus infection, then the patient is a candidate for SVV or SVV-like picornavirus therapy. In a virus binding assay, the patient's tumor cells can be determined to be permissive for SVV if the cells are positive for antibody staining. For example, FIGS. 92B-92C shows immunofluorescent images of cells permissive for SVV and have been infected with SVV.

[0240] In pre-screening patients with a viral binding assay, the cell sample from the patient can also be a tissue section of a tissue suspected to contain tumor cells. The tissue section can then be prepared into sections and incubated with SVV prior to histochemistry with anti-SVV or anti-SVV-like picornavirus antibodies.

[0241] The invention also provides methods of detecting SVV. In one embodiment, the detection assay is based on antibodies specific to SVV polypeptide epitopes. In another embodiment, the detection assay is based on the hybridization of nucleic acids. In one embodiment, RNA is isolated from SVV, labeled (e.g., radioactive, chemiluminsecence, fluorescence, etc.) to make a probe. RNA is then isolated from test material, bound to nitrocellulose (or a similar or functionally equivalent substrate), probed with the labeled SVV RNA, and the amount of bound probe detected. Also, the RNA of the virus may be directly or indirectly sequenced and a PCR assay developed based on the sequences. In one embodiment, the PCR assay is a real time PCR assay.

Methods for Making Viruses with Altered Tropism:

[0242] The present invention provides methods for constructing SVV mutants (or variants or derivatives) where these mutants have an altered cell-type tropism. SVV-like picornaviruses may also be mutated in order to provide a particular cell-type tropism. Specifically, SVV and SVV-like picornavirus mutants are selected for their ability to specifically bind and/or kill tumor or neoplastic cells that are known to be resistant to wild-type SVV or wild-type SVV-like picornavirus binding.

[0243] The native or wild-type SVV has a simple genome and structure that allow the modification of the native virus to target other cancer indications. These new derivatives have an expanded tropism toward non-neural cancers and still maintain the high therapeutic index found in the native SVV. One possible means of targeting is the inclusion of tissue-specific peptides or ligands onto the virus.

[0244] To select cancer-targeting viral candidates, the present invention provides methods to construct and screen an oncolytic virus library with a genetic insertion that encodes a random peptide sequence in the capsid region of the native SVV. A random peptide library with a diversity of 10⁸ is believed to be sufficient and should yield peptides that specifically direct the virus to tumor tissue.

[0245] Various studies have shown that tumor cells exhibit different characteristics from normal cells such as: (1) tumor cells have more permeable cell membranes; (2) tumors have specific stromal cell types such as angiogenic endothelial cells which have previously been shown to express cell type specific receptors; and (3) tumor cells differentially express certain receptors, antigens and extracellular matrix proteins (Arap, W. et al., Nat. Med., 2002, 8(2): 121-127; Kolonin, M. et al., Curr. Opin. Chem. Biol., 2001, 5(3): 308-313; St. Croix, B. et al., Science, 2000, 289(5482): 1997-1202). These studies demonstrated that tumor and normal tissues are distinct at the molecular level and targeted drug delivery and treatment of cancer is feasible. Specifically, several peptides selected by homing to blood vessels in mouse models have been used for targeted delivery of cytotoxic drugs (Arap, W. et al., Science, 1998, 279(5349): 377-380), pro-apoptotic peptides (Ellerby, H. M. et al., Nat. Med., 1999, 17(8): 768-774), metalloprotease inhibitor (Koivunen, E. et al., Nat. Biotechnol, 1999, 17(8): 768-774), cytokine (Curnis, F. et al., Nat. Biotechnol., 2000, 18(11): 1185-1190), fluorophores (Hong. F. D. and Clayman, G. L., Cancer Res., 2000, 60(23): 6551-6556) and genes (Trepel, M. et al., Hum. Gene Ther., 2000, 11(14): 1971-1981). The tumor-targeting peptides have proven to increase the efficacy and lower the toxicity of the parental drugs.

[0246] A library of SVV derivatives can be generated by the insertion of a random peptide sequence into the capsid region of the virus. As shown in FIG. 57, a vector is first generated that contains the SVV capsid region, i.e., "pSVV capsid." This capsid vector can then be mutagenized, for example, by cutting the vector with a restriction enzyme that cuts DNA at random positions, i.e., CviJI (a blunt cutter). The vector is cut at numerous positions, and DNA that has been cut only once by CviJI can be isolated by gel-purification (see FIG. 57). This isolated population of DNA contains a plurality of species that have been cut in the capsid region at different locations. This population is then incubated with oligonucleotides and ligase, such that a percentage of the oligonucleotides will be ligated into the capsid region of the vector at a number of different positions. In this manner, a library of mutant SVV capsids can be generated.

[0247] The oligonucleotides that are inserted into the capsid encoding region can be either random oligonucleotides, non-random oligonucleotides (i.e., the sequence of the oligonucleotide is pre-determined), or semi-random (i.e., a portion of the oligonucleotide is pre-determined and a portion has a random sequence). The non-random aspect of the contemplated oligonucleotides can comprise an epitope-encoding region. Contemplated epitopes include, but are not limited to, c-myc--a 10 amino acid segment of the human protooncogene myc (EQKLISEEDL (SEQ ID NO: 35); HA--haemoglutinin protein from human influenza hemagglutinin protein (YPYDVPDYA (SEQ ID NO: 36)); and His₆ (SEQ ID NO:116)--a sequence encoding for six consecutive histidines.

[0248] The library of mutant capsid polynucleotides (for example, "pSVV capsid library" in FIG. 57) can then be digested with restriction enzymes such that only the mutant capsid encoding region is excised. This mutant capsid encoding region is then ligated into a vector containing the full-length genomic sequence minus the capsid encoding region (see FIG. 58, for example). This ligation generates a vector having a full-length genomic sequence, where the population (or library) of vectors comprise a plurality of mutant capsids. In FIG. 58, this library of SVV mutants comprising different capsids is denoted as "pSVVFL capsid." The pSVVFL capsid vector library is then linearized and in vitro transcribed in order to generate mutant SVV RNA (see FIG. 59). The mutant SVV RNA is then transfected into a permissive cell line such that those SVV sequences that do not possess a debilitating mutation in its capsid are translated by the host cells to produce a plurality of mutant SVV particles. In FIG. 59, the plurality of mutant SVV particles are denoted as a "SVV capsid library."

[0249] The peptide encoded by the oligonucleotide inserted into the capsid encoding region can serve as a targeting moiety for specific viral infection. The viruses that target a specific type of cancer would selectively infect only those cancer cells that have a receptor to the peptide, replicate in those cells, kill those cells, and spread to only those same types of cells. This methodology enables the identification of novel tumor-targeting peptides and ligands, tumor-selective receptors, therapeutic SVV derivatives and other virus derivatives, including picornavirus derivatives.

[0250] In vitro and in vivo screening of SVV mutant libraries have several advantages over other technologies such as peptide bead libraries and phage display. Unlike these other technologies, the desirable candidate here, i.e. an SVV derivative that selectively binds to a cancer cell, will replicate in situ. This replication-based library approach has numerous advantages over prior methods of discovering new cell binding moieties, such as phage display. First, the screening of a SVV library is based on replication. Only the desired viral derivatives can replicate in the target tissue, in this case certain cancer cells. The screening/selection process will yield very specific viral candidates that have both the targeting peptide moiety and may be a cancer therapeutic itself. On the contrary, phage display screens will only result in binding events and yields only the targeting peptide candidates. Thus, SVV library screening offers a much faster and selective approach. Second, during in vitro or in vivo phage display screening, phages recovered from the target cells have to be amplified in bacteria, while SVV derivatives can be directly recovered and purified from infected cells (or from the culture supernatant of lytically infected cells). Third, SVV has a smaller genome that renders easier manipulability; thus it is possible to randomly insert the genetic information into the capsid region to ensure an optimized insertion. Therefore, construction and screening of the SVV library has a high possibility to produce highly effective viral derivatives. These derivatives are designed and screened to specifically infect cancers with non-neural properties.

[0251] The insertion of oligonucleotides into the capsid encoding region will result in the generation of some defective mutants. Mutants may be defective in the sense that the insertion of an oligonucleotide sequence can result in a stop codon, such that the viral polyprotein will not be produced. Also, mutants may be defective in the sense that the insertion of an oligonucleotide sequence may result in the alteration of the capsid structure such that capsid can no longer be assembled. To decrease the probability that the insertion of oligonucleotide sequences may result in stop codon or untenable capsid structure, random oligonucleotides can be designed such that they do not encode for stop codons or for certain amino acids using methods such as TRIM.

[0252] To determine whether there is an optimal insertion point in the capsid region for oligonucleotides, one can generate an RGD-SVV library (see Example 16). The polynucleotide encoding the SVV capsid is randomly cut, for example, with CviJI. The randomly linearized capsid polynucleotides are then ligated to oligonucleotides encoding at least the RGD amino acid sequence (arginine-glycine-aspartic acid). These RGD-capsid sequences are then ligated into SVV full-length sequence vectors that are missing the capsid sequence. RGD-SVV derivatives viruses are produced and tested for their ability to infect and replicate in certain integrin-expressing cell lines (as the RGD peptide has been shown to target entities to integrin receptors). The RGD-SVV derivatives that are successful in infecting the integrin-expressing cell lines are then analyzed to determine whether there is a predominant insertion site for the RGD oligonucleotide. This site can then be used for site-directed insertion of random, non-random or semi-random oligonucleotides.

[0253] Further, in comparing portions of the capsid encoding region between SVV and other picornaviruses (see FIG. 28), there are various non-boxed regions between the viruses where the sequence similarity is at its lowest. These regions may be important in contributing to the different tropisms between the viruses. Thus, these regions may be candidate locations for oligonucleotide insertion mutagenesis of the SVV capsid (and for other viral capsids).

Inactivated SVV as a Tumor-Specific Therapeutic:

[0254] Since SVV and SVV-capsid derivatives can target specific tumor cell-types and/or tissues, the SVV particle itself can be used as a delivery vehicle for therapeutics. In such a method, the need for the oncolytic abilities of SVV becomes optional, as the delivered therapeutic can kill the targeted tumor cell.

[0255] For example, the wild-type SVV can be inactivated such that the virus no longer lyses infected cells, but where the virus can still specifically bind and enter targeted tumor cell-types. There are many standard methods known in the art to inactivate the replicative functions of viruses. For example, whole virus vaccines are inactivated by formalin or β-propiolactone such that the viruses cannot replicate. The wild-type SVV may itself contain peptides that cause the apoptosis of cells. Alternatively, SVV can be irradiated. However, irradiated viruses should first be tested to ensure that they are still capable of specifically targeting tumor cells, as certain irradiation conditions may cause protein, and thus capsid, alterations. Further, mutant SVVs can be generated where the packaging signal sequence is deleted. These SVV mutants are able to specifically bind and enter target cells, but replicated SVV genomic RNA will not be packaged and assembled into capsids. However, this method may prove to be useful as initial entry of these mutant SVVs will cause host-protein synthesis shut-off such that tumor-cell death is still achieved.

[0256] Derivative SVVs having mutant capsids can also be inactivated and used to kill cancer cells. Derivative SVVs with oligonucleotides encoding epitope tags inserted into the capsid region can be used as vehicles to deliver toxins to tumor cells. As described herein, derivative SVVs can be randomly mutagenized and screened for tumor-specific tropisms. Toxins can be attached to the epitope tags, such that the virus delivers the toxin to tumor cells. Alternatively, therapeutic antibodies that specifically bind to the epitope tag can be used, such that the virus delivers the therapeutic antibody to the tumor cell.

High-Throughput Screening:

[0257] The present invention encompasses high-throughput methods for screening viruses that have the ability to specifically infect different cell-lines. The specificity of infection can be detected by assaying for cytopathic effects. For example, a number of different tumor cell-lines can be grown in different wells of a multi-well plate that is amenable for high-throughput screening, for example a 384 well-plate. To each well, a sample of virus is added to test whether the cells are killed by virus-mediated lysis. From those wells that show cytopathic effects, the media is collected such that any viruses in the media can be amplified by infecting permissive cell lines in flasks or large tissue culture plates. The viruses are grown such that the RNA can be isolated and the sequence analyzed to determine sequence mutations that may be responsible for providing a tumor cell-type specific tropism for a virus.

[0258] Various colorimetric and fluorometric methods can quickly assay cytopathic effects, including fluorescent-dye based assays, ATP-based assays, MTS assays and LDH assays. Fluorescent-dye based assays can include nucleic acid stains to detect dead-cell populations, as cell-impermeant nucleic acid stains can specifically detect dead-cell populations. If it is desired to simultaneously detect both live-cell and dead-cell populations, nucleic acid stains can be used in combination with intracellular esterase substrates, membrane-permeant nucleic acid stains, membrane potential-sensitive probes, organelle probes or other cell-permeant indicators to detect the live-cell population. For example, Invitrogen (Carlsbad, Calif.) offers various SYTOX® nucleic acid stains that only penetrate cells with compromised plasma membranes. Ethidium bromide and propidium iodide can also be used to detect dead or dying cells. These stains are high-affinity nucleic acid stains that can be detected by any light-absorbance reader

[0259] For example, lysis can be based on the measurement of lactate dehydrogenase (LDH) activity released from the cytosol of damaged cells into the supernatant. To detect the presence of LDH in cell culture supernatants, a substrate mixture can be added such that LDH will reduce the tetrazolium salt INT to formazan by a coupled enzymatic reaction. The formazan dye can then be detected by a light-absorbance reader. Alternatively, an MTS assay [3-(4,5-dimethylthiazol-2-yl)-5(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)- -2H-tetrazolium, inner salt] using phenzine methosulfate (PMS) as the electron coupling reagent can also be used to detect cytotoxicity. Promega (Madison, Wis.) offers a CellTiter 96® AQ_ueous One Solution Cell Proliferation Assay kit where the solution reagent is added directly to culture wells, incubated for 1-4 hours and then absorbance is recorded at 490 nm. The quantity of formazan product as measured by the amount of 490 nm absorbance is directly proportional to the number of living cells in culture.

[0260] There are numerous high-throughput devices for reading light-absorbance. For example, SpectraMax Plus 384 Absorbance Platereader (Molecular Devices) can detect wavelengths from 190-1000 nm in 1 nm increments. The device can read 96-well microplates in 5 seconds and 384-well microplates in 16 seconds for ultra fast sample throughput.

[0261] Virus replication can also be assayed as an indication of successful infection, and such detection methods can be used in a high-throughput manner. For example, real-time RT-PCR methods can be used to detect the presence of virus transcripts in cell-culture supernatants. Upon reverse-transcription of viral RNA into cDNA, the cDNA can be amplified and detected by PCR with the use of double-stranded DNA-binding dyes (for example, SYBR® Green, Qiagen GmbH, Germany). The amount of PCR product can then be directly measured using a fluorimeter.

[0262] Viruses from the wells showing cytopathic effects are grown up and tested in further in vitro (re-testing of tumor and normal cell lines) and in vivo models (testing whether the virus can kill explanted tumors in mice).

Antibodies:

[0263] The present invention is also directed to antibodies that specifically bind to the viruses of the present invention, including the proteins of the viruses. Antibodies of the present invention include naturally occurring as well as non-naturally occurring antibodies, including, for example, single chain antibodies, chimeric antibodies, bifunctional antibodies and humanized antibodies, as well as antigen-binding fragments thereof. Such non-naturally occurring antibodies can be constructed using solid phase peptide synthesis, can be produced recombinantly or can be obtained, for example, by screening combinatorial libraries consisting of variable heavy chains and variable light chains (Huse et al., Science 246:1275-1281, 1989). These and other methods of making, for example, chimeric, humanized, CDR-grafted, single chain, and bifunctional antibodies are well known to those skilled in the art (Winter and Harris, Immunol. Today 14:243-246, 1993; Ward et al., Nature 341:544-546, 1989; Harlow and Lane, Antibodies: A laboratory manual, Cold Spring Harbor Laboratory Press, 1988); Hilyard et al., Protein Engineering: A practical approach, IRL Press 1992; Borrabeck, Antibody Engineering, 2d ed., Oxford University Press 1995). Antibodies of the invention include intact molecules as well as fragments thereof, such as Fab, F(ab')2, and Fv which are capable of binding to an epitopic determinant present in a polypeptide of the present invention.

[0264] Where a peptide portion of a SVV polypeptide of the invention (i.e., any peptide fragment from SEQ ID NO:2 or SEQ ID NO:169) or peptide portion of another viral polypeptide of the invention used as an immunogen for antibody generation is non-immunogenic, it can be made immunogenic by coupling the hapten to a carrier molecule such as bovine serum albumin (BSA) or keyhole limpet hemocyanin (KLH), or by expressing the peptide portion as a fusion protein. Various other carrier molecules and methods for coupling a hapten to a carrier molecule are well known in the art (for example, by Harlow and Lane, supra, 1988). Methods for raising polyclonal antibodies, for example, in a rabbit, goat, mouse or other mammal, are well known in the art (see, for example, Green et al., "Production of Polyclonal Antisera," in Immunochemical Protocols, Manson, ed., Humana Press 1992, pages 1-5; Coligan et al., "Production of Polyclonal Antisera in Rabbits, Rats, Mice and Hamsters," in Curr. Protocols Immunol. (1992), section 2.4.1).

[0265] Monoclonal antibodies also can be obtained using methods that are well known and routine in the art (Kohler and Milstein, Nature 256:495, 1975; Coligan et al., supra, 1992, sections 2.5.1-2.6.7; Harlow and Lane, supra, 1988). For example, spleen cells from a mouse immunized with a virus, viral polypeptide or fragment thereof, can be fused to an appropriate myeloma cell line to produce hybridoma cells. Cloned hybridoma cell lines can be screened using, for example, labeled SVV polypeptide to identify clones that secrete monoclonal antibodies having the appropriate specificity, and hybridomas expressing antibodies having a desirable specificity and affinity can be isolated and utilized as a continuous source of the antibodies. Polyclonal antibodies similarly can be isolated, for example, from serum of an immunized animal. Such antibodies, in addition to being useful for performing a method of the invention, also are useful, for example, for preparing standardized kits. A recombinant phage that expresses, for example, a single chain antibody also provides an antibody that can be used for preparing standardized kits. Monoclonal antibodies, for example, can be isolated and purified from hybridoma cultures by a variety of well established techniques, including, for example, affinity chromatography with Protein-A SEPHAROSE gel, size exclusion chromatography, and ion exchange chromatography (Barnes et al., in Meth. Mol. Biol. 10:79-104, Humana Press 1992); Coligan et al., supra, 1992, see sections 2.7.1-2.7.12 and sections 2.9.1-2.9.3).

[0266] An antigen-binding fragment of an antibody can be prepared by proteolytic hydrolysis of a particular antibody, or by expression of DNA encoding the fragment. Antibody fragments can be obtained by pepsin or papain digestion of whole antibodies by conventional methods. For example, antibody fragments can be produced by enzymatic cleavage of antibodies with pepsin to provide a 5S fragment denoted F(ab')2. This fragment can be further cleaved using a thiol-reducing agent, and optionally a blocking group for the sulfhydryl groups resulting from cleavage of disulfide linkages, to produce 3.5S Fab' monovalent fragments. Alternatively, an enzymatic cleavage using pepsin produces two monovalent Fab' fragments and an Fc fragment directly (see, for example, Goldenberg, U.S. Pat. No. 4,036,945 and U.S. Pat. No. 4,331,647; Nisonhoff et al., Arch. Biochem. Biophys. 89:230. 1960; Porter, Biochem. J. 73:119, 1959; Edelman et al., Meth. Enzymol., 1:422 (Academic Press 1967); Coligan et al., supra, 1992, see sections 2.8.1-2.8.10 and 2.10.1-2.10.4).

[0267] Another example of an antigen binding fragment of an antibody is a peptide coding for a single complementarity determining region (CDR). CDR peptides can be obtained by constructing polynucleotides encoding the CDR of an antibody of interest. Such polynucleotides can be prepared, for example, using the polymerase chain reaction to synthesize a variable region encoded by RNA obtained from antibody-producing cells (for example, Larrick et al., Methods: A Companion to Methods in Enzymology 2:106, 1991).

[0268] The antibodies of the invention are suited for use, for example, in immunoassays in which they can be utilized in liquid phase or bound to a solid phase carrier. In addition, the antibodies in these immunoassays can be detectably labeled in various ways. Examples of types of immunoassays which can utilize antibodies of the invention are competitive and non-competitive immunoassays in either a direct or indirect format. Examples of such immunoassays are the radioimmunoassay (RIA) and the sandwich (immunometric) assay. Detection of the antigens using the antibodies of the invention can be done utilizing immunoassays which are run in either the forward, reverse, or simultaneous modes, including immunohistochemical assays on physiological samples. Those of skill in the art will know, or can readily discern, other immunoassay formats without undue experimentation.

[0269] There are many different labels and methods of labeling antibodies known to those of ordinary skill in the art. Examples of the types of labels which can be used in the present invention include enzymes, radioisotopes, fluorescent compounds, colloidal metals, chemiluminescent compounds, phosphorescent compounds, and bioluminescent compounds. Those of ordinary skill in the art will know of other suitable labels for binding to the antibody, or alternatively to the antigen, or will be able to ascertain such, using routine experimentation.

[0270] As various changes can be made in the above methods and compositions without departing from the scope and spirit of the invention as described, it is intended that all subject matter contained in the above description, shown in the accompanying drawings, or defined in the appended claims be interpreted as illustrative, and not in a limiting sense.

EXAMPLES

[0271] The examples described below are provided to illustrate the present invention and are not included for the purpose of limiting the invention.

Example 1

Amplification and Purification of Virus

[0272] Cultivation of SVV in PER.C6 cells: SVV is plaque purified once and a well isolated plaque is picked and amplified in PER.C6 cells (Fallaux et al., 1998). A crude virus lysate (CVL) from SVV infected PER.C6 cells is made by three cycles of freeze and thaw and used to infect PER.C6 cells. PER.C6 cells are grown in 50×150 cm² T.C. flasks using Dulbecco's modified Eagle medium (DMEM, Invitrogen, Carlsbad, Calif., USA)) containing 10% fetal bovine serum (Biowhitaker, Walkersvile, Md., USA) and 10 mM magnesium chloride (Sigma, St Louis, Mo., USA). The infected cells harvested 30 hr after infection when complete CPE is noticed and are collected by centrifugation at 1500 rpm for 10 minutes at 4° C. The cell pellet is resuspended in the cell culture supernatant (30 ml) and is subjected to three cycles of freeze and thaw. The resulting CVL is clarified by centrifugation at 1500 rpm for 10 minutes at 4° C. Virus is purified by two rounds of CsCl gradients: a one-step gradient (density of CsCl 1.24 g/ml and 1.4 g/ml) followed by one continuous gradient centrifugation (density of CsCl 1.33 g/ml). The purified virus concentration is determined spectrophotometrically, assuming 1A₂₆₀=9.5×10¹² particles (Scraba D. G., and Palmenberg, A. C. 1999. Cardioviruses (Picornaviridae). In: Encyclopedia of Virology, Second edition, R. G. Webster and A Granoff, Eds). Titers of purified virus are also determined by a standard plaque assay using PER.C6 cells. The yield of SVV from PER.C6 cells are greater than 200, 000 particles per cell with particles to PFU ratio of about 100. The yields of SVV from other permissive cells (H446-ATCC# HTB-171) may be at least this high or higher.

Example 2

Electron Microscopy

[0273] SVV is mounted onto formvar carbon-coated grids using the direct application method, stained with uranyl acetate, and examined in a transmission electron microscope. Representative micrographs of the virus are taken at high magnification. For the transmission electron microscope, ultra-thin sections of SVV-infected PER.C6 cells are cut from the embedded blocks, and the resulting sections are examined in the transmission electron microscope.

[0274] The purified SVV particles are spherical and about 27 nm in diameter, appearing singly or in small aggregates on the grid. A representative picture of SVV is shown in FIG. 2. In some places, broken viral particles and empty capsids with stain penetration are also seen. Ultrastructural studies of infected PER.C6 cells revealed crystalline inclusions in the cytoplasm. A representative picture of PER.C6 cells infected with SVV is shown in FIG. 3. The virus infected cells revealed a few large vesicular bodies (empty vesicles).

Example 3

Nucleic Acid Isolation of SVV

[0275] RNA Isolation: SVV genomic RNA was extracted using guanidium thiocyanate and a phenol extraction method using Trizol (Invitrogen). Isolation was performed according to the supplier's recommendations. Briefly, 250 μl of the purified SVV was mixed with 3 volumes TRIZOL and 240 μl of chloroform. The aqueous phase containing RNA was precipitated with 600 μl isopropanol. The RNA pellet was washed twice with 70% ethanol, dried and dissolved in DEPC-treated water. The quantity of RNA extracted was estimated by optical density measurements at 260 nm. An aliquot of RNA was resolved through a 1.25% denaturing agarose gel (Cambrex Bio Sciences Rockland Inc., Rockland, Me. USA) and the band was visualized by ethidium bromide staining and photographed (FIG. 4).

[0276] cDNA synthesis: cDNA of the SVV genome was synthesized by RT-PCR. Synthesis of cDNA was performed under standard conditions using 1 μg of RNA, AMV reverse transcriptase, and random 14-mer oligonucleotide or oligo-dT. Fragments of the cDNA were amplified, cloned into plasmids and the clones are sequenced.

Example 4

SVV Sequence Analysis and Epidemiology

[0277] Part I: SVV SEQ ID NO:1

[0278] The nucleotide sequence of SVV SEQ ID NO:1 was analyzed to determine its evolutionary relationship to other viruses. The translated product (SEQ ID NO:2) for this ORF was picornavirus-like and reached from the middle of VP2 to the termination codon at the end of the 3D polymerase and was 1890 amino acids in length (FIG. 5A-5E and 7A-7B). The 3' untranslated region (UTR), nucleotides 5671-5734, which follows the ORF is 64 nucleotides (nt) in length, including the termination codon and excluding the poly(A) tail of which 18 residues are provided (FIG. 5E).

[0279] Preliminary comparisons (not shown) of three partial genome segments of SVV had revealed that SVV was most closely related members of the genus Cardiovirus (family Picornaviridae). Therefore an alignment of the polyprotein sequences of SVV, encephalomyocarditis virus (EMCV; species Encephalomyocarditis virus, Theiler's murine encephalomyelitis virus (TMEV; species Theilovirus), Vilyuisk human encephalomyelitis virus (VHEV; species Theilovirus) and a rat TMEV-like agent (TLV; species Theilovirus) was constructed (FIG. 28). From this alignment, the SVV polyprotein processing was compared to the polyprotein processing of the most closely related members of the Cardiovirus genus. Cleavage sites between the individual polypeptides is demarcated by the "I" character in FIG. 28.

[0280] In picornaviruses, most polyprotein cleavages are carried out by one or more virus-encoded proteases, although in cardio-, aphtho-, erbo- and teschoviruses the cleavage between P1-2A and 2B is carried out by a poorly understood cis-acting mechanism related to the 2A sequence itself and critically involving the sequence "NPG/P", where "I" represents the break between the 2A and 2B polypeptides (Donnelly et al., 1997, J. Gen. Virol. 78: 13-21). One of the parechoviruses, Ljungan virus, has this sequence (NPGP) present upstream of a typical parechovirus 2A and is either an additional 2A or is the C-terminal end of the P1 capsid region. In all nine currently recognised picornavirus genera, 3C^pro carries out all but the cis-acting self-cleaving reactions (i.e. 2A cleaves at its N-terminus in entero- and rhinoviruses and L cleaves at its C-terminus inaphthoviruses and erboviruses). The post-assembly cleavage of the capsid polypeptide VP0 to VP4 and VP2 is not carried out by 3C^pro, but by an unknown mechanism which may involve the virus RNA. The VP0 cleavage does not occur in parechoviruses and kobuviruses. The normal cardiovirus 3C^pro cleavage site has either a glutamine (Q) or glutamate (E) at the -1 position and glycine (G), serine (S), adenine (A) or asparagine (N) at the +1 position (Table 2). The cleavages of the SVV polyprotein conform to this pattern except for the VP3/VP1 site which is histidine (H)/serine (S) (Table 2); however, H/S is probably present as the cleavage site between 3A and 3B.sup.VPg in at least one strain of equine rhinitis A virus (ERAV; genus Aphthovirus) (Wutz et al., 1996, J. Gen. Virol. 77:1719-1730).

TABLE-US-00004 TABLE 2 Cleavage sites of SVV and cardioviruses Between SVV EMCV TMEV Rat TLV VHEV L VP4 Not LQ/GN PQ/GN PQ/GN PQ/GN known (SEQ (SEQ (SEQ (SEQ ID NO: ID NO: ID NO: ID NO: 125) 138) 152) 163) VP4 VP2 Not LA/DQ LL/DQ LL/DQ LL/DE known (SEQ (SEQ (SEQ (SEQ ID NO: ID NO: ID NO: ID NO: 126) 139) 153) 164) LM/DQ (SEQ ID NO: 140) VP2 VP3 EQ/GP RQ/SP AQ/SP PQ/SP PQ/SP (SEQ (SEQ (SEQ (SEQ (SEQ ID NO: ID NO: ID NO: ID NO: ID NO: 117) 127) 141) 154) 165) VP3 VP1 FH/ST PQ/GV PQ/GV PQ/GV PQ/GV (SEQ (SEQ (SEQ (SEQ (SEQ ID NO: ID NO: ID NO: ID NO: ID NO: 118) 128) 142) 155) 166) PQ/GI (SEQ ID NO: 143) PQ/GS (SEQ ID NO: 144) VP1 2A KQ/KM LE/SP LE/NP LQ/NP LE/NP (SEQ (SEQ (SEQ (SEQ (SEQ ID NO: ID NO: ID NO: ID NO: ID NO: 119) 129) 145) 156) 167) 2A 2B NPG/P* NPG/P* NPG/P* NPG/P* Nk (SEQ (SEQ (SEQ (SEQ ID NO: ID NO: ID NO: ID NO: 111) 130) 146) 157) 2B 2C MQ/GP QQ/SP PQ/GP AQ/SP Nk (SEQ (SEQ (SEQ (SEQ ID NO: ID NO: ID NO: ID NO: 120) 131) 147) 158) 2C 3A LQ/SP AQ/GP AQ/SP AQ/SP Nk (SEQ (SEQ (SEQ (SEQ ID NO: ID NO: ID NO: ID NO: 121) 132) 148) 159) AQ/AP (SEQ ID NO: 133) 3A 3B SE/NA EQ/GP EQ/AA EQ/AA Nk (SEQ (SEQ (SEQ (SEQ ID NO: ID NO: ID NO: ID NO: 122) 134) 149) 160) 3B 3C MQ/QP IQ/GP IQ/GG IQ/GG Nk (SEQ (SEQ (SEQ (SEQ ID NO: ID NO: ID NO: ID NO: 123) 135) 150) 161) VQ/GP (SEQ ID NO: 136) 3C 3D MQ/GL PQ/GA PQ/GA PQ/GA Nk (SEQ (SEQ (SEQ (SEQ ID NO: ID NO: ID NO: ID NO: 124) 137) 151) 162) *,the break between 2A and 2B is not a cleavage event

[0281] Primary cleavages (P1/P2 and P2/P3) of SVV: These primary cleavage events are predicted to occur in a similar fashion to cardio-, aphtho-, erbo- and teschoviruses, involving separation of P1-2A from 2B by a novel mechanism involving the sequence NPG/P (SEQ ID NO:111) and a traditional cleavage event by 3C^pro between 2BC and P3 (Table 2).

[0282] P1 cleavages: Cleavages within the SVV P1 capsid coding region were relatively easy to predict by alignment with sequence with EMCV and TMEV (Table 2).

[0283] P2 cleavages: The 2C protein is involved in RNA synthesis. The 2C polypeptide of SVV contains NTP-binding motifs GxxGxGKS/T (SEQ ID NO:112) (domain A) and hyhyhyxxD (in which by is any hydrophobic residue; domain B) present in putative helicases and all picornavirus 2Cs (FIG. 29).

[0284] P3 cleavages: Prediction of the P3 cleavage sites was also relatively straightforward. Little is known about the function of the 3A polypeptide. However, all picornavirus 3A proteins contain a putative transmembrane alpha-helix. Primary sequence identity is low in this protein between SVV and cardioviruses (See FIG. 28 between positions 1612 to 1701).

[0285] The genome-linked polypeptide, VPg, which is encoded by the 3B region, shares few amino acids in common with the other cardioviruses, however, the third residue is a tyrosine, consistent with its linkage to the 5' end of the virus genome (Rothberg et al., 1978). See FIG. 28 between positions 1703 and 1724.

[0286] The three-dimensional structure of four picornavirus 3C cysteine proteases have been solved and the active-site residues identified (HAV, Allaire et al., 1994, Nature, 369: 72-76; Bergmann et al., 1997, J. Virol., 71: 2436-2448; PV-1, Mosimann et al., 1997, J. Mol. Biol., 273: 1032-1047; HRV-14, Matthews et al., 1994, Cell, 77: 761-771; and HRV-2, Matthews et al., 1999, Proc. Natl. Acad. Sci. USA, 96: 11000-11007). The cysteine bolded in FIG. 29 is the nucleophile, while the first bolded histidine is the general base and the specificity for glutamine residues is defined mainly by the second bolded histidine; all three residues are conserved in the SVV sequence (FIG. 29) and all other known picornaviruses (FIG. 28; for 3C sequence comparison see between positions 1726 and 1946).

[0287] The 3D polypeptide is the major component of the RNA-dependent RNA polymerase and SVV contains motifs conserved in picorna-like virus RNA-dependent RNA polymerases, i.e. KDEL/IR (SEQ ID NO:113), PSG, YGDD (SEQ ID NO:114) and FLKR (SEQ ID NO:115) (FIG. 3; FIG. 28 between positions 1948 and 2410).

[0288] Myristoylation of the N-terminus of P1: In most picornaviruses the P1 precursor polypeptide is covalently bound by its N-terminal glycine residue (when present the N-terminal methionine is removed) to a molecule of myristic acid via an amide linkage (Chow et al., 1987, Nature, 327: 482-486). Consequently the cleavage products VP0 and VP4 which contain the P1 N-terminus are also myristoylated. This myristoylation is carried out by myristoyl transferase which recognises an eight amino acid signal beginning with glycine. In picornaviruses, a five residue consensus sequence motif, G-x-x-x-T/S, has been identified (Palmenberg, 1989, In Molecular Aspects of Picornavirus Infection and Detection, pp. 211-241, Ed. Semler & Ehrenfeld, Washington D.C., Amer. Soc. for Micro.). Parechoviruses (Human parechovirus and Ljungan virus) as well as not having a maturation cleavage of VP0 are apparently not myristoylated, however, there appears to be some type of molecule blocking the N-terminus of VP0 for these viruses.

Comparisons of the Individual SVV Polypeptides with the Public Sequence Databases

[0289] Each of the SVV polypeptides (SEQ ID NOS: 4, 6, 8, 10, 12, 14, 16, 18, 20 and 22) were compared to the public protein sequence databases using the FASTA online program at the European Bioinformatics Institute (EBI; http://www.ebi.ac.uk/). The results (best matches) of these comparisons are shown in Table 3. The capsid polypeptides (VP2, VP3 and VP1) taken as a whole, along with 2C, 3C^pro and 3D^pol are most closely related to members of the cardiovirus genus, however, the short predicted 2A sequence is closer to that of Ljungan virus (genus Parechovirus). A more detailed comparison of the SVV 2A nucleotide sequence with similar sequences is shown in FIG. 28 (see also FIG. 70 for 2A-like NPG/P protein comparison).

TABLE-US-00005 TABLE 3 Database matches of individual predicted polypeptides of Seneca Valley virus SVV Length % % identity Matched polypeptide (aa) identity ungapped aa overlap Organism protein L (Leader) No data -- -- -- -- -- VP4 (1A) No data -- -- -- -- -- VP2 (1B) >142 42.857 44.037 112 TMEV WW VP2 ~51 -- ~80 EMCV BEL-2887A/91 VP2 VP3 (1C) 239 44.068 46.637 236 EMCV ATCC VR-129B VP3 VP1 (1D) 259 31.086 36.404 267 EMCV M100/1/02 VP1 2A 14 71.429 71.429 14 Ljungan virus 174F 2A1 2B 128 39.286 41.509 56 Ureaplasma urealyticum Multiple banded antigen 2C 322 38.602 40.190 329 EMCV PV21 2C 3A 90 37.838 41.791 74 Chlorobium tepidum TLS* Enolase 2† 3B.sup.VPg 22 No -- -- -- -- matches 3C^pro 211 37.089 38.537 213 EMCV-R 3C protease 3D^pol 462 58.009 58.515 462 EMCV-PV21 3D polymerase *a photosynthetic, anaerobic, green-sulfur bacterium †2-phosphoglycerate dehydratase 2) (2-phospho-D-glycerate hydro-lyase 2

[0290] The significance of the matches of SVV 2B with Ureaplasma urealyticum multiple banded antigen or 3A with Chlorobium tepidum endolase 2 is not clear, however, these relationships maybe worthy of further investigation.

Phylogenetic Comparison of SVV Polypeptides with Other Picornaviruses

[0291] Those SVV polypeptides which could be aligned with the cardioviruses (VP2, VP3, VP1, 2C, 3C and 3D) were compared with the same proteins of representative members of each of the picornavirus species (Table 4). The programs BioEdit v5.0.9 (Hall, 1999, Nucl. Acids. Symp. Ser., 41: 95-98) and Clustal X v1.83 (Thompson et al., 1997, Nucl. Acids Res., 25:4876-4882) were used to make the alignments and to construct distance matrices and unrooted Neighbor-joining trees according to the algorithm of Saitou and Nei (Satiou and Nei, 1987, Mol. Biol. Evol., 4: 406-425). Confidence limits on branches were accessed by bootstrap resampling (1000 pseudo-replicates). The trees were drawn using TreeView 1.6.6 (Page, 1996) (FIGS. 31 to 37). The distance matrices used to construct the trees used values corrected for multiple substitutions, while FIGS. 38-44 show the actual percentage amino acid identities. Table 4 shows the current classification of the family Picornaviridae and the representative virus sequences used in these comparisons.

TABLE-US-00006 TABLE 4 The taxonomic classification of the picornaviruses used in the comparisons with SVV. Genus Species Representative virus Abbrev. Acc. No. Enterovirus Poliovirus Poliovirus 1 PV-1 V01149 Human enterovirus A Coxsackievirus A16 CV-A16 U05876 Human enterovirus B Coxsackievirus B5 CV-B5 X67706 Human enterovirus C Coxsackievirus A21 CV-A21 D00538 Human enterovirus D Enterovirus 70 EV-70 D00820 Simian enterovirus A Simian enterovirus A1 SEV-A AF201894 Bovine enterovirus Bovine enterovirus 1 BEV-1 D00214 Porcine enterovirus B Porcine enterovirus 9 PEV-9 AF363453 New genus? Not yet designated Simian virus 2* SV2 AY064708 Porcine enterovirus A Porcine enterovirus 8* PEV-8 AF406813 Rhinovirus Human rhinovirus A Human rhinovirus 2 HRV-2 X02316 Human rhinovirus B Human rhinovirus 14 HRV-14 K02121 Cardiovirus Encephalomyocarditis virus Encephalomyocarditis virus EMCV M81861 Theilovirus Theiler's murine encephalomyelitis TMEV M20562 virus Aphthovirus Foot-and-mouth disease virus Foot-and-mouth disease virus O FMDV-O X00871 Equine rhinitis A virus Equine rhinitis A virus ERAV X96870 Hepatovirus Hepatitis A virus Hepatitis A virus HAV M14707 Avian encephalomyelitis-like Avian encephalomyelitis virus AEV AJ225173 viruses Parechovirus Human parechovirus Human parechovirus 1 HPeV-1 L02971 Ljungan virus Ljungan virus LV AF327920 Kobuvirus Aichi virus Aichi virus AiV AB040749 Bovine kobuvirus Bovine kobuvirus BKV AB084788 Erbovirus Equine rhinitis B virus Equine rhinitis B virus 1 ERBV-1 X96871 Teschovirus Porcine teschovirus Porcine teschovirus 1 PTV-1 AJ011380 *the current taxonomic status of SV2 and PEV-8 places them in the enterovirus genus, however, it has been suggested that they may be reclassified in a new genus (Krumbholz et al., 2002; Oberste et al., 2003).

[0292] The trees of the individual capsid proteins (FIGS. 31 to 33) are not all representative of the tree produced when the data from all tree polypeptides is combined (FIG. 34). This is probably the result of difficulties in aligning the capsid polypeptides, particularly when they are not full length as is the case for VP2 (FIG. 31). However, the P1, 2C, 3C^pro and 3D^pol trees are all in agreement and show that SVV clusters with EMCV and TMEV.

Seneca Valley Virus as a Member of the Cardiovirus Genus

[0293] Clearly the 3D^pol of SVV is related to the cardioviruses, almost as closely as EMCV and TMEV are to each other (FIG. 37; FIG. 44). In the other polypeptides which are generally considered as being relatively conserved in picornaviruses, 2C and 3C, SVV is also most closely related to the cardioviruses although it is not as closely related to EMCV and TMEV as they are to each other (FIG. 42 and FIG. 43, respectively). In the outer capsid proteins (taken as a whole), SVV is also most closely related to the cardioviruses and has approximately the same relationship as the two aphthovirus species, Foot-and-mouth disease virus and Equine rhinitis A virus (˜33%). SVV diverges greatly from the cardioviruses in the 2B and 3A polypeptides and has no detectable relationship with any known picornavirus. However, this is not without precedent; avian encephalomyelitis virus differs considerably from hepatitis A virus (HAV) in 2A, 2B and 3A (Marvil et al., 1999, J. Gen. Virol., 80:653-662) but is tentatively classified within the genus Hepatovirus along with HAV.

[0294] Seneca Valley virus is clearly not a typical cardiovirus if EMCV and TMEV are taken as the standard. However, even these two viruses have their differences, notably in the 5' UTR (Pevear et al., 1987, J. Gen. Virol., 61: 1507-1516). However, phylogenetically SVV clusters with EMCV and TMEV in much of its polyprotein (P1, 2C, 3C^pro and 3D^pol regions). Ultimately, the taxonomic position of SVV within the Picornaviridae will be decided by the Executive Committee (EC) of the International Committee for the Taxonomy of Viruses (ICTV) following recommendations by the Picornaviridae Study Group and supporting published material. There are two options: i) include SVV as a new species in the cardiovirus genus; or ii) assign SVV to a new genus.

[0295] Part II: SVV SEQ ID NO:168

[0296] The full-length genome of SVV (FIGS. 83A-83H; SEQ ID NO:168; Example 15) allowed further epidemiological studies. The results of the further epidemiological studies are shown in FIG. 86, where SVV is shown to be genetically related to cardioviruses such as EMCV and TMEV, but in a separate tree.

[0297] The features of the SVV full-length genome with respect to its untranslated and coding regions are listed at Table A supra. The features of the full-length SVV in comparison to EMCV and TMEV-GDVII are listed in the table below.

TABLE-US-00007 EMCV EMCV TMEV-GDVII TMEV-GDVII SVV SVV [M81861] [M81861] [M20562] [M20562] Feature nt length aa length nt length aa length nt length aa length 5 `UTR 666 -- 833 -- 1068 -- Leader 237 79 201 67 228 76 VP4 213 71 210 70 213 71 VP2 852 284 768 256 801 267 VP3 717 239 693 231 696 232 VP1 792 264 831 277 828 276 2A 27 9 429 143 426 142 2B 384 128 450 150 381 127 2C 966 399 975 325 978 326 3A 270 90 264 88 264 88 3B 66 22 60 20 60 20 3D 1386 462 1380 460 1383 461 3` UTR 71 -- 126 -- 128 --

[0298] The cleavage sites of SVV (based on full-length sequence, see also bolded amino acids between at protein boundaries in FIGS. 83A-83H) are compared to the cleavage sites of other cardioviruses in the table below.

TABLE-US-00008 Between SVV EMCV TMEV Rat TLV VHEV L VP4 LQ/GN LQ/GN PQ/GN PQ/GN PQ/GN (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID NO: 192) NO: 192) NO: l93) NO: 193) NO: 193) VP4 VP2 LK/DH LA/DQ LL/DQ LL/DQ LL/DE (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID NO: 194) NO: 195) NO: 196) NO: 196) NO: 198) LM/DQ (SEQ ID NO: 197) VP2 VP3 EQ/GP RQ/SP AQ/SP PQ/SP PQ/SP (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID NO: 117) NO: 199) NO: 200) NO: 201) NO :201) VP3 VP1 FH/ST PQ/GV PQ/GV PQ/GV PQ/GV (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID NO: 118) NO: 202) NO: 202) NO: 202) NO: 202) PQ/GI (SEQ ID NO: 203) PQ/GS (SEQ ID NO: 204) VP1 2A MQ/SG LE/SP LE/NP LQ/NP LE/NP (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID NO: 205) NO: 206) NO: 207) NO: 208) NO: 207) 2A 2B NPG/P* NPG/P* NPG/P* NPG/P* unknown (SEQ ID (SEQ ID (SEQ ID (SEQ ID NO: 111) NO: 111) NO: 111) NO: 111) 2B 2C MQ/GP QQ/SP PQ/GP AQ/SP unknown (SEQ ID (SEQ ID (SEQ ID (SEQ ID NO: 120) NO: 209) NO: 210) NO: 200) 2C 3A LQ/SP AQ/GP AQ/SP AQ/SP unknown (SEQ ID (SEQ ID (SEQ ID (SEQ ID NO: 121) NO: 211) NO: 200) NO: 200) AQ/AP (SEQ ID NO: 212) 3A 3B SE/NA EQ/GP EQ/AA EQ/AA unknown (SEQ ID (SEQ ID (SEQ ID (SEQ ID NO: 122) NO: 213) NO: 214) NO: 214) 3B 3C MQ/QP IQ/GP IQ/GG IQ/GG unknown (SEQ ID (SEQ ID (SEQ ID (SEQ ID NO: 123) NO: 215) NO: 217) NO: 217) VQ/GP (SEQ ID NO: 216) 3C 3D MQ/GL PQ/GA PQ/GA PQ/GA unknown (SEQ ID (SEQ ID (SEQ ID (SEQ ID NO: 124) NO: 218) NO: 218) NO: 218) *ribosome skipping sequence

[0299] Multiple unique viruses were discovered at the USDA that are more similar to SVV than SVV is to other cardioviruses. These USDA virus isolates, herein considered to be members of the group called "SVV-like picornaviruses," are: MN 88-36695, NC 88-23626, IA 89-47552, NJ 90-10324, IL 92-48963, CA 131395; LA 1278; IL 66289; IL 94-9356; MN/GA 99-29256; MN 99197; and SC 363649. These SVV-like picornaviruses and SVV are considered to comprise a new picornavirus genus.

[0300] Each of these SVV-like picornaviruses are unique, and are about 95%-98% identical to SVV at the nucleotide level (see FIGS. 87-89 for nucleotide sequence comparisons between SVV and these USDA isolates).

[0301] Part III: Serum Studies

[0302] Pigs are a permissive host for the USDA virus isolates identified above. The isolate MN 88-36695 was inoculated into a gnobiotic pig and antisera generated (GP102). The antisera binds to all of the other USDA isolates listed above and to SVV. The antisera does not react with 24 common porcine virus pathogens indicating its specificity. Porcine sera was also tested for neutralizing antibodies to 1278 (Plum Island virus). Sera were collected in the US and 8/29 sera were positive with titers ranging from 1:57 to 1:36,500.

[0303] To test whether the pig is the natural source for SVV, serum samples from various animals were obtained and tested for their ability to act as neutralizing antibodies against SVV infection of permissive cells. The Serum Neutralization Assay is conducted as follows: (1) Dilute various serums 1:2 and 1:4; (2) Mix with 100 TCID₅₀ of virus (SVV; but any virus can be tested to determine whether a serum can neutralize its infection); (3) Incubate at 37° C. for 1 hour; (4) Add to 1×10⁴ PER.C6 cells (or other permissive cell type); (5) Incubate at 37° C. for 3 days; and (6) Measure CPE using MTS assay. The neutralization titer is defined as the highest dilution of sera that neutralizes SVV (or other virus in question) at 100%.

[0304] The serum neutralization results showed that there is a minimal or no presence of neutralizing antibodies in human and primate populations. In one experiment, 0/22 human sera contained neutralizing antibodies to SVV. In another experiment, only 1/28 human sera contained neutralizing antibodies. In a third experiment, 0/50 human sera from Amish farmers were neutralizing. In another experiment, 0/52 primate sera from four species were neutralizing.

[0305] The serum neutralization results showed that there is a prevalence of neutralizing antibodies in farm animal populations. In one experiment, 27/71 porcine sera from farms were neutralizing. In another experiment, 4/30 porcine sera from a disease-free farm were neutralizing. In another experiment, 10/50 bovine sera were neutralizing. In yet another experiment, 5/35 wild mouse sera were neutralizing. Because antibodies cross-reactive to SVV and/or SVV-like picornaviruses have been found in pigs, cows, and mice, these data indicate that SVV and/or SVV-like picornaviruses may be prevalent in a wide-variety of non-primate animals.

[0306] A crude viral lysate of MN 88-36695 was tested to assess its cytotoxicity ability on two cell lines permissive (NCI-H446; HEK293) for SVV and on two cell lines non-permissive (NCI-H460 and S8) for SVV. The cytotoxicity profile for MN 88-36695 was identical to SVV: the TCID50 for NCI-H446 was 1.6×10^-6; the TCID50 for HEK293 was 1.3×10^-2; and NCI-H460 and S8 were non-permissive for MN 88-36695. This data indicates that SVV-like picornaviruses have the potential to be used in the present methods directed to cancer therapy. In one embodiment, the invention provides for the use of the MN 88-36695 SVV-like picornavirus in any of the methods directed to cancer therapy, diagnosis, or screening.

[0307] Antisera to MN 88-36694 and SVV were tested in serum neutralization assays on each virus. Anti-SVV mouse serum was able to neutralize infection by both MN 88-36695 and SVV (neutralization titers on infection were 1:640 for MN 88-36695 and 1:1000 for SVV). Anti-MN 88-36695 gnobiotic pig serum was able to neutralize infection by both MN 88-36695 and SVV (neutralization titers on infection were 1:5120 for MN 88-36695 and 1:100 for SVV).

[0308] These data indicate that SVV is genetically and serologically linked to the porcine USDA virus isolates.

Example 4

SDS-PAGE and N-Terminal Sequence Analysis of SVV Capsid Proteins

[0309] Purified SVV is subjected to electrophoresis using NuPAGE pre-cast Bis-Tris polyacrylamide mini-gel electrophoresis system (Novex, San Diego, Calif., USA). One half of the gel is visualized by silver stain while the other half is used to prepare samples for amino acid sequencing of the N-termini of the capsid proteins. Prior to transfer of proteins to membrane, the gel is soaked in 10 mM CAPS buffer, pH 11, for 1 hour, and a PVDF membrane (Amersham) is wetted in methanol. Proteins are transferred to the PVDF membrane. After transfer, proteins are visualized by staining with Amido black for approximately 1 minute, and bands of interest are excised with a scalpel and air dried. The proteins can be subjected to automated N-terminal sequence determination by Edman degradation using a pulsed phase sequencer.

[0310] Three major structural proteins of the purified SVV are shown in FIG. 45 (approximately 36 kDa, 31 kDa, and 27 kDa).

Example 5

Assay for Neutralization Antibodies to SVV in Human Serum Samples

[0311] Preexisting antibodies to particular viral vectors may limit the use of such vectors for systemic delivery applications such as for treatment of metastatic cancer, because preexisting antibodies may bind to systemically delivered vectors and neutralize them before the vectors have a chance to transduce the targeted tissue or organ. Therefore, it is desirable to ensure that humans do not carry neutralization antibodies to viral vectors selected for systemic delivery. To determine whether human sera samples contain SVV-specific neutralizing antibodies, neutralization assays are carried out using randomly collected human sera samples.

[0312] Tissue culture infective dose 50: One day before the experiment, 180 μl of PER.C6 cell suspension containing 1×10⁴ cells are plated in 96-well tissue culture dish. The crude virus lysate (CVL) of SVV is diluted in log steps from 10^-0 to 10^-11 in DMEM medium (Dulbecco's Modified Eagle's Medium) and 20 μl of each dilution is transferred to three wells of a Falcon 96-well tissue culture plate containing PER.C6 cells. The plates are incubated at 37° C. in 5% CO₂ and read at 3 days for microscopic evidence of cytopathic effect (CPE), and the tissue culture infective dose 50 (TCID₅₀) is calculated.

[0313] Neutralization assay: First, 40 μl of medium is placed in all the wells and then 40 μl of heat-inactivated serum is added to the first well and mixed by pipeting, making a 1:4 dilution used for screening purposes. 40 μl is then transferred to the next well to perform a two-fold dilution of the serum samples. 40 μl of SVV virus, containing 100 TCID₅₀, is added to wells containing diluted serum samples. Plates are incubated at 37° C. for 1 hour. 40 μl of the mix is taken and transferred to a plate containing PER.C6 cells (1×10⁴ cells/160 μl/well). The plates are incubated at 37° C. for 3 days. After this time, the cultures are read microscopically for CPE.

[0314] In a representative neutralization assay performed as described above, twenty-two human sera samples randomly collected from USA, Europe and Japan were examined for SVV specific neutralizing antibodies. The serum samples were serially diluted and mixed with a fixed amount of SVV containing 100 TCID₅₀. Serum-virus mixtures were then used to infect PER.C6 cells and incubated for 24 hours. Neutralizing antibody titer was determined as the reciprocal of the highest dilution of serum able to block CPE formation. In this experiment, no dilution of serum blocked CPE formation indicating that the human serum samples did not contain SVV neutralizing antibodies.

[0315] Further SVV infection of PER.C6 was not inhibited by incubation with human blood (see Example 6), indicating that SVV infection was not inhibited by complement or by hemagglutination. As a result, SVV exhibits a longer circulation time in vivo than other oncolytic viruses, which is a significant problem with the use of oncolytic adenoviruses.

Example 6

Binding of SVV to Human Erythrocytes and Hemagglutination

[0316] Various viral serotypes have been shown to cause in vitro hemagglutination of erythrocytes isolated from blood of various animal species. Hemagglutination or binding to erythrocytes may cause toxicity in vivo and may also affect in vivo biodistribution and the efficacy of a viral vector. Therefore, it is desirable to analyze the erythrocyte agglutination properties of a viral vector selected for systemic administration to treat metastatic cancers.

[0317] Hemagglutination assay: To determine whether SVV causes agglutination of human erythrocytes, hemagglutination assays are carried out in U-bottom 96-well plates. Purified SVV is serially diluted in 25 μl PBS (Phosphate Buffered Saline) in duplicates, and an equal volume of 1% erythrocyte suspension is added to each well. Blood samples used for isolation of erythrocytes are obtained from healthy individuals with heparin as an anticoagulant. Erythrocytes are prepared by washing the blood three times in cold PBS to remove the plasma and the white blood cells. After the last wash, erythrocytes are suspended in PBS to make a 1% (V/V) cell suspension. The virus and erythrocytes are gently mixed and the plates are incubated at room temperature for 1 hour and monitored for a hemagglutination pattern.

[0318] Whole blood inactivation assay: To rule out direct inactivation of SVV by blood components, aliquots of virus are incubated with heparinized human blood belonging to A, B, AB and O blood groups or PBS for 30 minutes or 1 hour at room temperature prior to separation of plasma, after which PER.C6 cells are infected and titers are calculated.

[0319] In representative assays performed as described above, no hemagglutination of human erythrocytes of different blood groups (A, B, AB and O) was seen at any tested dilutions of SVV. A slight increase in the virus titer is noticed when SVV is mixed with blood human samples and incubated for 30 minutes and 1 hour, indicating that the virus is not inactivated by blood components but becomes more infectious under tested conditions.

Example 7

In Vivo Clearance

[0320] Blood circulation time: To determine the blood circulation time and the amount of the virus in the tumor, H446 tumor bearing nude mice were treated with SVV at a dose of 1×10¹² vp/kg by tail vein injection. The mice were bled at 0, 1, 3, 6, 24, 48, 72 hours and 7 days (189 hours) post-injection and the plasma was separated from the blood immediately after collection, diluted in infection medium, and used to infect PER.C6 cells. The injected mice were sacrificed at 6, 24, 48, 72 hours and 7 days post-injection and the tumors were collected. The tumors were cut into small sections and suspended in one ml of medium and subjected to three cycles of freeze and thaw to release the virus from the infected cells. Serial log dilutions of supernatants were made and assayed for titer on PER.C6 cells. SVV titers were expressed as pfu/ml. The tumor sections were also subjected to H&E staining and immunohistochemistry to detect the virus capsid proteins in the tumor.

[0321] The circulating levels of virus particles in the blood were determined based on the assumption that 7.3% of mouse body weight is blood. In representative assays performed as essentially as described above, within 6 hours of virus administration, the circulating levels of SVV reduced to zero particles and SVV was not detectable at later time points (FIG. 46A). In the tumor, SVV was detectable at 6 hours post-injection, after which the amount of the virus increased steadily by two logs (FIG. 46B). The virus was detectable in the tumor as late as 7 days postinjection (FIG. 46B). The tumor sections when subjected to immunohistochemistry, revealed SVV proteins in the tumor cells (FIG. 47, top panels). When stained by H&E, the tumor sections revealed several rounded tumor cells (FIG. 47, bottom panels).

[0322] SVV also exhibits a substantially longer resident time in the blood compared to similar doses of i.v. adenovirus. Following a single i.v. dose, SVV remains present in the blood for up to 6 hours (FIG. 46C; FIG. 46C is a duplication of FIG. 46A for comparison purposes to FIG. 46D), whereas adenovirus is cleared from the blood in about an hour (FIG. 46D).

Example 8

Tumor Cell Selectivity

[0323] In vitro cell killing activity of SVV: To determine the susceptibility of human, bovine, porcine, and mouse cells, normal and tumor cells were obtained from various sources and infected with SVV. All cell types were cultured in media and under the conditions recommended by the supplier. Primary human hepatocytes may be purchased from In Vitro Technologies (Baltimore, Md.) and cultured in Hepatocyte Culture Media (HCM®, BioWhittaker/Clonetics Inc., San Diego, Calif.).

[0324] In vitro cytopathic assay: To determine which types of cells are susceptible to SVV infection, monolayers of proliferating normal cells and tumor cells were infected with serial dilutions of purified SVV. The cells were monitored for CPE and compared with uninfected cells. Three days following infection, a MTS cytotoxic assay is performed and effective concentration 50 (Ec₅₀) values in particles per cell are calculated. See Tables 5 and 6 below and Table 1A supra.

TABLE-US-00009 TABLE 5 Cell lines with EC₅₀ values less than 100 EC50 number Cell lines with EC50 <1 H446 (human sclc) 0.001197 PERC6 0.01996 H69AR (sclc-multidrug resistant) 0.03477 293 (human kidney transformed with ad5E1) 0.03615 Y79 (human retinoblastoma) 0.0003505 IMR32 (human brain; neuroblastoma) 0.03509 D283med (human brain; cerebellum; 0.2503 medulloblastoma) SK-N-AS (human brain; neuroblastoma) 0.474 N1E-115 (mouse neuroblastoma) 0.002846 SK-NEP-1 (kidney, wilms' tumor, pleural 0.03434 effusion, human) BEKPCB3E1 (bovine embryonic kidney cells 0.99 transformed with ad5E1 Cell Lines with EC50 <10 (1-10) H1299 (human-non sclc) 7.656 ST (pig testes) 5.929 DMS 153 (human sclc) 9.233 Cell lines with EC50 <100 (10-100) BEK (bovine embryonic kidney) 17.55

TABLE-US-00010 TABLE 6 Cell lines with EC₅₀ values more than 1000 M059K (human brain; HUVEC (human vein endothelial CMT-64 (mouse-sclc) malignant glioblastoma) cells) KK (human glioblastoma) HAEC (human aortic endothelial LLC-1 (mouse-LCLC)) cells) U-118MG (human WI38 (human lung fibroblast) RM-1 (mouse-prostate) glioblastoma) DMS 79 (human sclc) MRC-5 (human lung fibroblast) RM-2 (mouse-prostate) H69 (human sclc) IMR90 (human lung fibroblast) RM-9 (mouse-prostate) DMS 114 (human sclc) HMVEC (human microvascular MLTC-1 (mouse-testes) endothelial cells-adult) DMS 53 (human sclc) HMVEC (human microvascular KLN-205 (mouse-sqcc) endothelial cells-neonatal) H460 (human-LCLC) HCN-1A (human brain) CMT-93 (mouse-rectal) A375-S2 (human HRCE (human renal cortical B16F0 (mouse melanoma) epithelial cells) melanoma) SK-MEL-28 (human Neuro-2A (mouse melanoma) neuroblastoma) PC3 (human prostate) C8D30 (mouse brain) PC3M2AC6 (human PK15 (pig-kidney) prostate) LNCaP (human prostate) FBRC (fetal bovine retina) DU145 (human prostate) MDBK (bovine kidney) Hep3B (human liver CSL 503 (sheep lung cells carcinoma) transformed with ad5E1) Hep2G (human liver OFRC (ovine fetal retina carcinoma) cells) SW620 (human-colon) SW839 (human kidney) 5637 (human bladder) HeLa S3 S8

[0325] The MTS assay was performed according to the manufacturer's instructions (CellTiter 96® AQ_ueous Assay by Promega, Madison, Wis.). The CellTiter 96® AQ_ueous Assay preferably uses the tetrazolium compound (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl- )-2H-tetrazolium, inner salt; MTS) and an electron coupling reagent, phenazine methosulfate (PMS). Contact-inhibited normal human cells evaluated in the study include: HUVEC (human umbilical vein endothelial cells), HAEC (human aortic endothelial cells, Clonetics/BioWhittaker #CC-2535), Wi38 (normal human embryo lung fibroblasts, ATCC #CCL-75), IMR90 (human normal lung fibroblasts, ATCC CCL-186), MRC-5 (human normal lung fibroblasts, ATCC, #CCL-171) and HRCE (human renal cortical epithelial cells, Clonetics/BioWhittaker #CC-2554).

[0326] SVV does not produce CPE in any of the above contact-inhibited normal cells. No virus-induced CPE was seen in the following human tumor cell lines: Hep3B (ATCC #HB-8064), HepG2 (human hepatocellular carcinoma, ATCC #HB-8065), LNCaP (human prostate carcinoma, ATCC #CRL-10995), PC3M-2AC6, SW620 (human colorectal adenocarcinoma, ATCC #CCL-227), SW 839 (human kidney adenocarcinoma, ATCC #HTB-49), 5637 (human urinary bladder carcinoma, ATCC #HTB-9), DMS-114 (small cell lung cancer, ATCC #CRL-2066), DMS 153 (human small cell lung cancer, ATCC #CRL-2064), A549 (human lung carcinoma, ATCC #CCL-185), HeLa S3 (human cervical adenocarcinoma, ATCC #CCL-2.2), NCI-H460 (human large cell lung cancer, ATCC #HTB-177), KK (glioblastoma), and U-118 MG (human glioblastoma, ATCC #HTB-15). Note--the cell lines in Table 6 with EC₅₀ values greater than 1000 are most likely not permissive for SVV replication and/or virion production; although the possibility remains that SVV can bind and enter into these cells but CPE is not observed because SVV replication cannot occur inside the cell or that replication does occur but CPE is not observed because there is some other post-entry block (i.e., no packaging of replicated SVV genomes into virions). However, considering the absence of CPE in these cell lines, these cell-lines, and potentially tumor-types thereof, are good candidates to test which cell and tumor-types are permissive or non-permissive for SVV replication. Although wild-type SVV is tumor-specific, and has been shown to target neuroendocrine tumors, including small cell lung cancer and neuroblastomas, there may be individual patients that have types of etiologies such that SVV is not permissive in their form of neuroendocrine tumor. Therefore, the invention does contemplate the generation of SVV derivatives that can kill tumor cell-types isolated from individual patients where the tumors are non-permissive to the wild-type SVV, and the tumor-types isolated from these individuals can include, for example, glioblastoma, lymphoma, small cell lung cancer, large cell lung cancer, melanoma, prostate cancer, liver carcinoma, colon cancer, kidney cancer, colon cancer, bladder cancer, rectal cancer and squamous cell lung cancer.

[0327] SVV-mediated cytotoxicity on primary human hepatocytes (In Vitro Technologies) was determined by LDH release assay (CytoTox® 96 Non-Radioactive Cytotoxicity Assay, Promega, #G1780). Primary human hepatocytes plated in collagen coated 12-well plates were infected with SVV at 1, 10 and 100 and 1000 particles per cell (ppc). After 3 hours of infection, the infection medium was replaced with 2 ml of growth medium and incubated for 3 days in a CO₂ incubator. The cell associated lactate dehydrogenase (LDH) and LDH in the culture supernatant was measured separately. Percent cytotoxicity is determined as a ratio of LDH units in supernatant over maximal cellular LDH plus supernatant LDH.

Percent cytotoxicity = LDH units in culture supernatant × 100 Sum of LDH units in supernatant and cell lysate ##EQU00001##

The data shown in FIG. 48 illustrates the absence of SVV mediated hepatoxicity at all tested multiplicity of infections.

Example 10

Virus Production Assay

[0328] To assess the replicative abilities of SVV, several selected contact-inhibited normal cells and actively dividing tumor cells were infected with SVV at one virus particle per cell (ppc). After 72 hours, cells and the medium were subjected to three freeze-thaw cycles and centrifuged to collect the supernatant. Serial log dilutions of supernatants were made and assayed for titer on PER.C6 cells. For each cell line, the efficiency of SVV replication was expressed as pfu/ml (FIG. 49).

Example 10

Toxicity

[0329] The maximum tolerated dose (MTD) is defined as the dosage immediately preceding the dose at which animals (e.g. mice) demonstrate a dose limiting toxicity (DLT) after the treatment with SVV. DLT is defined as the dose at which the animals exhibit a loss in body weight, symptoms, and mortality attributed to SVV administration during the entire duration of the study. Neutralizing antibodies to SVV were assessed at baseline, day 15, and day 21. Neutralization assays were carried as described earlier.

[0330] Escalating doses (1×10⁸-1×10¹⁴ vp/kg) of SVV were administered intravenously into both immune deficient nude and caesarean derived-1 (CD-1) out-bred immune competent mice purchased from Harlan Sprague Dawley (Indianapolis, Ind., USA) to determine the MTD with 10 mice per dose level. The virus was well-tolerated at all tested dose levels without exhibiting any clinical symptoms and without loss in body weight (FIG. 50). Mice were bled at day 15 and 21 and the sera was monitored for the presence of SVV-specific neutralizing antibodies in neutralization assays. SVV injected CD1 mice develop neutralizing antibodies and the titers range from 1/1024 to greater than 1/4096.

[0331] Another toxicity study was conducted on the immunocompetent mouse strain (A/J). It has been demonstrated that SVV exhibits cell killing activity and replication in N1E-115 cells (see Table 1). The murine cell line N1E-115 (a neuroblastoma cell line, i.e., neuroendocrine cancer) is derived from the A/J mouse strain. Thus, a syngeneic mouse model was established where N1E-115 cells were implanted subcutaneously in A/J mice to form tumors, and the mice were then treated with SVV to investigate its efficacy and toxicity.

[0332] In the A/J study, mice were i.v. injected with SVV to determine whether A/J mice can tolerate systemic administration of SVV. Blood hematology results were obtained to look for signs of toxicity, and serum chemistry results can also be obtained. The study design is shown in Table 7 below:

TABLE-US-00011 TABLE 7 A/J Study Design Dosage Dosage Group Animals Test Level Volume Dosing Necropsy # (Female) Article (particles/kg) (mL/kg) regimen Day 1 5 Vehicle 0 10 IV on Day 15 Day 1 2 5 SVV .sup. 10⁸ 10 IV on Day 15 Day 1 3 5 SVV .sup. 10¹¹ 10 IV on Day 15 Day 1 4 5 SVV .sup. 10¹⁴ 10 IV on Day 15 Day 1

[0333] The A/J mice were 8-10 week old females obtained from The Jackson Laboratory (Bar Harbor, Me.). SVV was prepared by storing isolated virions at -80° C. until use. SVV was prepared fresh by thawing on ice and diluting with HBSS (Hank's balanced salt solution). SVV was diluted to concentrations of 10⁷ particles/mL for group 2, 10¹⁰ particles/mL for group 3, and 10¹³ particle/mL for group 4. HBSS was used as the vehicle control for group 1. All dosing solutions were kept on wet ice until dosing.

[0334] SVV was administered to animals intravenous injection via the tail vein at a dose volume of 10 mL/kg body weight. Animals were weighed on the day of dosing and dose volumes were adjusted based on body weight (i.e., a 0.0200 kg mouse gets 0.200 mL of dosing solution). Mice were monitored twice daily for morbidity and mortality. Mice were weighed twice weekly. Information relating to moribund animals and animals exhibiting any unusual symptoms (physically or behaviorally) are recorded immediately.

[0335] Post-mortem observations and measurements entail the collection of blood from all surviving animals at terminal sacrifice for standard hematology and serum chemistry (AST, ALT, BUN, CK, LDH). The following organs are to be collected at sacrifice: brain, heart, lung, kidney, liver, and gonads. Half of each organ sample is snap frozen on dry ice and the other half will be placed in formalin.

[0336] Initial blood hematology results (CBC, differential) were obtained two weeks after SVV injection and the results are summarized below in Table 8 below. Five mice were tested from each test group (see Table 7):

TABLE-US-00012 TABLE 8 A/J Toxicity Results - Blood Hematology Test Group 1 Test Group 2 Test Group 3 Test Group 4 Body Weight Result ± SD (g): Day 0 21.48 ± 0.88 21.98 ± 1.93 22.58 ± 0.87 21.04 ± 1.67 Day 14 20.26 ± 0.93 20.92 ± 1.71 21.44 ± 0.84 21.26 ± 1.45 CBC Wet (Result ± SD (ref range)): White blood count 3.63 ± 1.57 4.5 ± 1.57 4.26 ± 0.94 4.72 ± 0.62 (THSN/UL) (2.60-10.69) (2.60-10.69) (2.60-10.69) (2.60-10.69) Red blood count 9.87 ± 0.03 9.49 ± 0.07 9.76 ± 0.37 9.71 ± 0.32 (MILL/UL) (6.4-9.4) (6.4-9.4) (6.4-9.4) (6.4-9.4) Hemoglobin 15.37 ± 0.06 14.78 ± 0.29 15.12 ± 0.66 15.02 ± 0.63 (GM/DL) (11.5-16.1) (11.5-16.1) (11.5-16.1) (11.5-16.1) Hematocrit (%) 46.03 ± 0.40 44.52 ± 0.49 45.7 ± 1.82 45.28 ± 1.69 (36.1-49.5) (36.1-49.5) (36.1-49.5) (36.1-49.5) MCV (FL) 46.67 ± 0.58 47.00 ± 0.0 47.0 ± 0.0 46.6 ± 0.55 (45.4-60.3) (45.4-60.3) (45.4-60.3) (45.4-60.3) MHC (PICO GM) 15.57 ± 0.06 15.70 ± 0.17 15.37 ± 0.06 15.43 ± 0.15 (14.1-19.3) (14.1-19.3) (14.1-19.3) (14.1-19.3) MCHC (%) 33.37 ± 0.12 33.14 ± 0.48 33.08 ± 0.22 33.14 ± 0.25 (25.4-34.1) (25.4-34.1) (25.4-34.1) (25.4-34.1) Platelet (THSN/UL) 885.33 ± 28.6 758.2 ± 146.2 874.8 ± 56.7 897.2 ± 105.4 (592-2972) (592-2972) (592-2972) (592-2972) Differential (Result ± SD (ref range)): Bands (THSN/UL) 0.0 0.0 0.0 0.0 (0.0-0.1) (0.0-0.1) (0.0-0.1) (0.0-0.1) Seg. Neutrophils 0.92 ± 0.27 1.16 ± 0.37 1.09 ± 0.38 0.96 ± 0.20 (THSN/UL) (0.13-2.57) (0.13-2.57) (0.13-2.57) (0.13-2.57) Lymphocytes 2.64 ± 1.26 2.98 ± 1.41 3.10 ± 0.56 3.70 ± 0.41 (THSN/UL) (1.43-9.94) (1.43-9.94) (1.43-9.94) (1.43-9.94) Monocytes 0.06 ± 0.04 0.15 ± 0.05 0.06 ± 0.03 0.05 ± 0.02 (THSN/UL) (0.0-0.39) (0.0-0.39) (0.0-0.39) (0.0-0.39) Eosinophils 0.01 ± 0.01 0.01 ± 0.01 0.01 ± 0.01 0.003 ± 0.01 (THSN/UL) (0.0-0.24) (0.0-0.24) (0.0-0.24) (0.0-0.24) Basophils 0.0 0.004 ± 0.005 0.0 0.0 (THSN/UL) (0.0-0.0) (0.0-0.0) (0.0-0.0) (0.0-0.0) Atypical Lympho. 0.0 0.0 0.0 0.0 (THSN/UL) (0.0-0.0) (0.0-0.0) (0.0-0.0) (0.0-0.0) Metamyelocytes 0.0 0.0 0.0 0.0 (THSN/UL) (0.0-0.0) (0.0-0.0) (0.0-0.0) (0.0-0.0) Myelocytes 0.0 0.0 0.0 0.0 (THSN/UL) (0.0-0.0) (0.0-0.0) (0.0-0.0) (0.0-0.0) NRBC (/100WBC) 0.0 0.0 0.0 0.0 (0.0-0.0) (0.0-0.0) (0.0-0.0) (0.0-0.0) Other (Result ± SD (ref range)): AST (SGOT) (U/L) 1762.8 ± 1129.8 899.0 ± 234.6 779.8 ± 312.2 843.2 ± 653.4 (72-288) (72-288) (72-288) (72-288) ALT (SGPT) (U/L) 2171.8 ± 2792.9 535.2 ± 272.8 555 ± 350.8 380.2 ± 385.7 (24-140) (24-140) (24-140) (24-140) BUN (MG/DL) 27.2 ± 0.8 24.8 ± 1.9 24.6 ± 5.5 28.2 ± 12.8 (9-28) (9-28) (9-28) (9-28) Creatine phospho- 28312.8 ± 20534.4 12194.4 ± 4049.2 10157 ± 5420.5 11829 ± 10363.9 kinase (U/L) (0-800) (0-800) (0-800) (0-800) LDH (U/L) 6650.2 ± 4788.6 3661.6 ± 933.6 3450.8 ± 972.6 2808.4 ± 1709.1 (260-680) (260-680) (260-680) (260-680) Hemolytic Index 706.6 ± 423.4 477.6 ± 195.7 589.6 ± 198.6 496.4 ± 321.1 (MG/DL HGB) (0-70) (0-70) (0-70) (0-70)

[0337] These results show that there are no abnormalities in blood hematology profiles obtained from mice treated with low, medium and high doses of SVV compared to blood hematology profiles obtained from untreated mice. From this study, it can be concluded that there are no measureable signs of toxicity following systemic administration of SVV, indicating that SVV is tolerated by A/J mice following i.v. injection.

Example 11

Efficacy

[0338] Athymic female nude mice (nu/nu) aged 6-7 weeks purchased from Harlan Sprague Dawley (Indianapolis, Ind.) were used in efficacy studies. Mice were injected subcutaneously with 5×10⁶ H446 cells into the right flank using manual restraint. Tumor sizes were measured regularly, and the volumes were calculated using the formula π/6×W×L², where L=length and W=width of the tumor. When the tumors reach approximately 100-150 mm³, mice (n=10) were randomly divided into groups. Mice were injected with escalating doses of SVV by tail vein injections at a dose volume of 10 ml/kg. A control group of mice was injected with an equivalent volume of HBSS. Dose escalation proceeds from 1×10⁷ to 1×10¹³ particles per kilogram body weight. Antitumoral efficacy was determined by measuring tumor volumes twice weekly following SVV administration. Complete response was defined as complete disappearance of xenograft; partial response as regression of the tumor volume by equal to or more than 50%; and no response as continuous growth of tumor as in the control group.

[0339] Tumors from mice treated with HBSS grew rapidly and the tumor volumes reached more than 2000 mm³ by study day 20 (FIG. 51; see line with open diamond). In contrast, mice given one systemic injection of SVV at all tested doses (with the exception of the lowest dose) became tumor free by study day 20. In the lowest dose group, 8 mice became tumor free, one mouse had a very large tumor and the other had a small palpable tumor (25 mm³) by study day 31. To evaluate the antitumor activity of SVV on large sized tumors, five mice from HBSS group bearing tumors >2000 mm³ were systemically injected with a single dose of 1×10¹¹ vp/kg on study day 20. For the duration of the follow-up period (11 days of after SVV injection), a dramatic regression of the tumor volumes were noted (FIG. 51).

[0340] Additional experiments to test the efficacy of a single intravenous dose of SVV was conducted in murine tumor models that express neuroendocrine markers. The tumor models tested included H446 (human SCLC) (see FIG. 90A), Y79 (human retinoblastoma) (see FIG. 90B), H69AR (human multi-drug resistant SCLC) (see FIG. 90C), H1299 (human NSCLC) (see FIG. 90D), and N1E-115 (murine neuroblastoma) (see FIG. 90E).

[0341] The results show that a single intravenous dose of SVV has efficacy in all of the murine neuroendocrine tumor models. The results also show that SVV is efficacious in the N1E-115 immunocompetent murine neuroblastoma model.

[0342] FIG. 52 shows a picture of mice that were "untreated" with SVV (i.e., treated with HBSS) or "treated" with SVV. As can be seen, the untreated mice had very large tumors and the treated mice showed no visible signs of tumor. Further, for unsacrificed mice treated with SVV, no tumor regrowth was observed for the duration of the study, 200 days.

[0343] In vitro efficacy data for SVV for specific tumor cell lines is shown in Tables 1, 1A, and 5. The data shows that SVV specifically infects particular tumor cell types and does not infect normal adult cells (except for porcine normal cells), a significant advantage over any other known oncolytic virus. SVV has been shown to have 1,000 times better cell killing specificity than chemotherapy treatments (cell killing specificity values for SVV have been shown to be greater than 10,000, whereas cell killing specificity values for chemotherapy are around 10).

[0344] Specific cytotoxic activity of SVV was demonstrated in H446 human SCLC cells. Following a two-day incubation with increasing concentrations of SVV, cell viability was determined. The results are shown in FIG. 53. FIG. 53 shows cell survival following incubation of SVV with either H446 SCLC tumor cells (top graph) or normal human H460 cells (bottom graph). SVV specifically killed the tumor cells with an EC₅₀ of approximately 10^-3 particles per cell. In contrast, normal human cells were not killed at any concentration of SVV. Further, as summarized in Tables 1, 1A-3, SVV was also cytotoxic toward a number of other tumor cell lines, including SCLC-multidrug resistant tumor cells, and some fetal cells and cell lines. The EC₅₀ values for SVV cytotoxicity for the other tumor cell lines ranged from 10^-3 to greater than 20,000 particles per cell. SVV was non-cytotoxic against a variety other non-neural tumors and normal human tissues. Additionally, SVV was not cytotoxic to primary human hepatocytes, as measured by LDH release at up to 1000 particles per cell (see FIG. 48).

Example 12

Biodistribution and Pharmacokinetic Study in Rodents

[0345] Pharmacokinetic and biodistribution study of SVV is performed in normal mice and immunocompromised athymic nude mice bearing H446 SCLC tumors. This study evaluates the biodistribution, elimination and persistence of SVV following a single intravenous administration to both normal and immunocompromised tumor-bearing mice. Groups of mice each receive a single i.v. dose of control buffer or one of three doses of SVV (10⁸, 10¹⁰, or 10¹² vp/kg) and are monitored for clinical signs. Blood samples are obtained from groups of 5 mice at 1, 6, 24 and 48 hours post dose, and at 1, 2, 4, and 12 weeks post dose. Dose levels include a known low efficacious dose and two higher dose levels to determine linearity of virus elimination. Groups of mice are sacrificed at 24 hours, and 2, 4 and 12 weeks post dose. Selected tissues, including liver, heart, lung, spleen, kidney, lymph nodes, bone marrow, brain and spinal cord tissues are aseptically collected and tested for the presence of SVV RNA using a validated RT-PCR assay.

[0346] Samples of urine and feces are obtained at sacrifice, at 24 hours, and at 2, 4 and 12 weeks post dose and are examined for the presence of infectious virus. The design of the experiments in this Example are shown in Table 9 below:

TABLE-US-00013 TABLE 9 Biodistribution of SVV in CD-1 Mice and Athymic Nude Mice Bearing SCLC Tumors # of # of Mice/ Dose Mice/Timepoint Timepoint Level for Blood for PCR Tissue Group Treatment (vp/kg) Route Sampling Distribution Normal CD-1 Mice 1 Saline 0 i.v. 5 5 2 SVV 10⁸ i.v. 5 5 3 SVV 10¹⁰ i.v. 5 5 4 SVV 10¹² i.v. 5 5 Athymic Tumor Bearing Mice 5 Saline 0 i.v. 5 5 6 SVV 10⁸ i.v. 5 5 7 SVV 10¹⁰ i.v. 5 5 8 SVV 10¹² i.v. 5 5

[0347] Acute i.v. toxicology studies were also performed in both normal and immunocompromised athymic nude mice bearing H446 SCLC tumors. Preliminary i.v. studies in normal and SCLC tumor bearing mice indicate safety of SVV at doses up to 10¹⁴ vp/kg. No adverse clinical signs were observed and there was no loss of body weight up to 2 weeks following a single i.v. dose of 10¹⁴ vp/kg.

Example 13

Viral Transmission Study in Normal Adult and Pregnant Mice

[0348] The purpose of this Example is to determine if SVV is transmissible following cohabitation of noninfected normal mice with mice injected with a high concentration of SVV. Because SVV does not replicate in normal, non-tumor bearing mice, tumor bearing mice can also be injected with high concentrations of SVV and subsequently exposed to normal, healthy animals to better simulate the clinical scenario. A secondary purpose is to assess the potential transmissibility of SVV from an infected female to an uninfected pregnant DAM, and subsequently to the developing fetus.

[0349] Three groups of five naive male and female CD-1 mice are exposed to a single mouse of the same sex infected with either 10⁸, 10¹⁰ or 10¹²/kg, and are monitored for the presence of SVV by blood sampling.

[0350] Similarly, an SVV exposed female is co-mingled with a number of timed pregnant females, and the ability of the virus to transmit from the infected female to an uninfected pregnant female, and subsequently to the developing fetus is determined

Example 14

Non-Human Primate Studies

[0351] The safety, toxicity and toxicokinetics of SVV are also determined in non-human primates. In a dose range-finding phase, individual monkeys receive a single i.v. dose of SVV at 10⁸ vp/kg and are closely monitored for clinical signs of infection or toxicity. If this dose is well tolerated, additional animals are treated with a higher i.v. dose until a dose of 10¹² vp/kg is achieved. Subsequently, the main study consists of groups of three male and female monkeys, and each monkey is dosed once weekly for six weeks with either vehicle alone or one of three doses of SVV and monitored for signs of toxicity. An additional two monkeys per sex are dosed with the vehicle alone and with the high dose level of SVV for six weeks, and are allowed an additional four weeks recovery prior to sacrifice.

[0352] Blood samples are obtained following dosing during week 1 and week 6. Clinical pathological and hematology blood samples are obtained prior to the initial dose and prior to sacrifice. Additional blood samples are obtained following each dose for assessing the presence of neutralizing antibodies to SVV.

[0353] Surviving monkeys are euthanized and subjected to a full gross necropsy and a full tissue list is collected from the main study and recovery monkeys. Tissues from the control and high dose groups are evaluated histopathologically. Urine and fecal samples are collected following dosing on weeks 1 and 6 and are evaluated for presence of infectious SVV. The overall design of this Example is shown in Table 10 below.

TABLE-US-00014 TABLE 10 Multiple Dose Toxicology Study of SVV in Primates Dose Range-finding Phase Group Treatment Dose (vp/kg) Route Males Females 1 SVV 10⁸ IV 1 1 2 SVV 10¹⁰* IV 1 1 3 SVV 10¹²* IV 1 1 Main Phase Dose Main Phase Recovery Group Treatment (vp/kg) Route Male Female Male Female 1 Control -- IV 3 3 2 2 2 SVV 10⁸*.sup. IV 3 3 -- -- 3 SVV 10¹⁰* IV 3 3 -- -- 4 SVV 10¹²* IV 3 3 2 2 *Doses can vary based on results of Dose Range-finding phase

Example 15

Construction of an Infectious Full-Length and Functional Genomic SVV Plasmid

[0354] With SEQ ID NO:1, only about 1.5-2 Kb of the 5' genomic sequence of SVV remains to be sequenced, representing the nucleotide region covering the 5' UTR, 1A (VP4) and part of 1B (VP2). To clone the 5' end missing in SEQ ID NO:1, polymerases that function at high temperatures and reagents that can enable a polymerase to read through secondary structures were used. Additional SVV cDNAs were prepared from isolated SVV of ATCC deposit number PTA-5343. SVV particles were infected into a permissive cell line, such as PER.C6, and viruses are isolated. Viral RNA was then recovered from the virus particles such that cDNA copies are made therefrom. Individual cDNA clones were sequenced, such that selected cDNA clones are combined into one full-length clone in a plasmid having a T7 promoter upstream of the 5' end of the SVV sequence. The full-length genomic sequence of SVV is listed in FIGS. 83A-83H and SEQ ID NO:168. The full-length SVV from this plasmid is reverse-transcribed, by utilizing T7 polymerase and an in vitro transcription system, in order to generate full-length RNA (see FIG. 55). The full-length RNA is then transfected into permissive cell lines to test the infectivity of the full-length clone (see FIG. 55).

[0355] The methodology was as follows.

[0356] RNA Isolation:

[0357] SVV genomic RNA was extracted using guanidium thiocyanate and a phenol extraction method using Trizol (Invitrogen). Briefly, 250 μl of the purified SVV (˜3×10¹² virus particles) was mixed with 3 volumes of Trizol and 240 μl of chloroform. The aqueous phase containing RNA was precipitated with 600 μl isopropanol. The RNA pellet was washed twice with 70% ethanol, dried and dissolved in sterile DEPC-treated water. The quantity of RNA extracted can be estimated by optical density measurements at 260 nm. An aliquot of RNA can be resolved through a 1.25% denaturing agarose gel (Cambrex Bio Sciences Rockland Inc., Rockland, Me. USA) and the band visualized by ethidium bromide staining and photographed.

[0358] cDNA Synthesis:

[0359] cDNA of the SVV genome was synthesized by RT-PCR. Synthesis of cDNA was performed under standard conditions using 1 μg of RNA, AMV reverse transcriptase, and oligo-dT primers. Random 14-mer oligonucleotide can also be used. Fragments of the cDNA were amplified and cloned into the plasmid pGEM-3Z (Promega) and the clones were sequenced. The sequence at the 5' end of the viral genome was cloned by RACE and the sequence determined Sequence data was compiled to generate the complete genome sequence of SVV.

[0360] Cloning of Full Length Genome:

[0361] Three cDNA fragments representing the full-length SVV genome were amplified by three PCR reactions employing six sets of SVV-specific primers. Turbo pfu polymerase (Stratagene) was used in PCR reactions. First, a fragment representing the 5' end of SVV genome was amplified with primers 5'SVV-A (SEQ ID NO:219) and SVV1029RT-R1 (SEQ ID NO:220) and the resulting fragment was cut with ApaI and EcoRI and gel purified. The gel purified fragment was ligated to Nde-ApaT7SVV (SEQ ID NO:221), an annealed oligo duplex containing engineered NdeI site at 5' end, T7 core promoter sequence in the middle and first 17 nucleotides of SVV with ready to use ApaI site at 3' end and cloned into Nde I and Eco RI sites of pGEM-3Z (Promega) by three-way ligation to generate pNTX-03. Second, a fragment representing 3' end of viral genome was amplified by PCR with primers SVV6056 (SEQ ID NO:222) and SVV7309NsiB (SEQ ID NO:223). The antisense primer, SVV7309NsiB was used to introduced poly(A) tail of 30 nucleotides in length and Nsi I recognition sequence at 3' end to clone into PstI site of pGEM-3Z plasmid. The resulting PCR product was digested with BamHI and gel purified. A fragment covering the internal part of the viral genome was amplified with primers SVV911L (SEQ ID NO:224) and SVV6157R (SEQ ID NO:225). The resulting PCR product was cut with EcoRI and BamHI and gel purified. The two gel purified fragments representing the middle and 3' end of SVV genome were cloned into EcoRI and SmaI sites of pGEM-4Z by three-way ligation to generate pNTX-02. To generate full-length SVV cDNA, pNTX-02 was digested with EcoRI and NsiI and the resulting 6.3 kb fragment was gel purified cloned into EcoRI and PstI sites of pNTX-03. The resulting full-length plasmid was called pNTX-04.

[0362] The full-length plasmid pNTX-04 was further modified at both 5' and 3' ends to facilitate in vitro transcription and rescuing of the virus following RNA transfection into PER.C6 cells. First, a SwaI restriction enzyme site was inserted immediately downstream of the poly(A) tail to liberate the 3' end of SVV-cDNA from the plasmid backbone prior to in vitro transcription. A PCR approach was used to insert the site utilizing a primer pair of SVV6056 (SEQ ID NO:222) and SVVSwaRev (SEQ ID NO:226) and pNTX-04 as template. The antisense primer SVV3SwaRev contained 58 nucleotides representing the 3' end of the SVV sequence and recognition sequences for SwaI and SphI restriction enzyme sites. The resulting PCR fragment was digested with BamHI and SphI and used to replace the corresponding fragment from pNTX-04 to generate pNTX-06. Second, an extra four nucleotides present between the T7 promoter transcription start site and 5' end of SVV cDNA in pNTX-06 were removed using annealed oligo duplex approach. The duplex nucleotides were engineered to contain KpnI recognition site, T7 core promoter sequence and the first 17 nucleotides of SVV with a ready to use ApaI site at the 3' end (SEQ ID NO:227). The annealed oligos were used to replace the corresponding portion of pNTX-06 using KpnI and ApaI sites to generate pNTX-07. Finally, a two base pair deletion noticed in the polymerase encoding region of pNTX-07 was restored by replacing BamHI and SphI fragment with a corresponding fragment amplified from SVV cDNA by PCR to generate pNTX-09.

[0363] In Vitro Transcription:

[0364] Infectivity of in vitro transcribed RNA was tested by first digesting pNTX-09 with SwaI to liberate 3' end of SVV sequence from plasmid backbone. The linearized plasmid was subjected to in vitro transcription using T7 polymerase (Promega).

[0365] Transfection of In Vitro Transcribed RNA into PER.C6 Cells:

[0366] One day prior to transfection, PER.C6 cells were plated in 6-well tissue-culture dishes. On the next day, Lipofetamine reagent (Invitrogen) was used to transfect in vitro transcribed RNA (1.5 ng) into the cells following the recommendations of the supplier. Cytopathic effect (CPE) due to virus production was noticed within 36 hour post-transfection. The transfected cells were subjected to three cycles of freeze-thaw and the viruses in lysate were further confirmed by infecting PER.C6 cells. Thus, the full-length SVV cDNA clone proved to be infectious.

[0367] As described above, the plasmid with the full-length genome of SVV can be reverse-transcribed following standard protocols. The viral RNA (100 ng) can be used to transfect any cell line known to be permissive for the native SVV, but the most efficient cell line for viral RNA transfection can be empirically determined among a variety of cell lines.

Example 16

Construction of an RGD-Displaying SVV Library

[0368] To find the optimal insertion position for the construction of SVV capsid mutants generated with random with oligonucleotides encoding random peptide sequences, a simple model system (RGD) is employed. RGD (arginine, glycine, aspartic acid) is a short peptide ligand that binds to integrins. A successful RGD-SVV derivative should contain the following characteristics: (1) the genetic insertion should not alter any of SVV's unique and desirable properties; and (2) a successful RGD derivative virus should have tropism toward α_Vβ₅ integrin containing cells.

[0369] A SVV plasmid containing just the contiguous capsid region will be singly cleaved at random positions and a short model peptide sequence, referred to as RGD, will be inserted at each position. The virus SVV-RGD library will be constructed from this plasmid library utilizing the general technology described in FIGS. 56 and 57.

[0370] Random insertion of the cRGD oligonucleotide into the capsid region is conducted. In brief, a plasmid is constructed that just encodes the contiguous 2.1 Kb capsid region of SVV (see FIG. 56, "pSVVcapsid"). A single random cleavage is made in pSVVcapsid by partially digesting the plasmid utilizing either CviJI or an endonuclease V method as described below (see FIG. 57). After isolating the single cleaved plasmid the RGD oligonucleotide will be inserted to create a pSVVcapsid-RGD library.

[0371] The restriction enzyme CviJI has several advantages over other random cleavage methods such as sonication or shearing. First, as CviJI is a blunt ended cutter no repair is necessary. Second, CviJI has been demonstrated to cleave at random locations such that no hot spots will occur. The procedure is also simple and rapid. Briefly, the concentration of CviJI and/or time of digestion are increasingly lowered until the majority of cleaved DNA is a linearized plasmid, i.e. a single cleavage. This can be observed by standard agarose gel electrophoresis as depicted in FIG. 57. The band is then isolated, purified and ligated with the RGD oligo.

[0372] Another method that may be utilized to randomly cleave DNA is the endonuclease V method (Kiyazaki, K., Nucleic Acids Res., 2002, 30(24): e139). Endonuclease V nicks uracil-containing DNA at the second or third phosphodiester bond 3' to uracil sites. This method is also expected to randomly cleave DNA, the frequency is simply determined by adjusting the concentration of dUTP in the polymerase chain reaction. Although the cleavage sites are always two or three bases downstream of a thymidine (substituted by uracil) site, this method is expected to produce much fewer hot and cold spots than other methodologies.

[0373] The randomly linearized plasmids are ligated with the cRGD oligonucleotides. The resultant pSVV capsid library is then amplified, such that a population of polynucleotides encoding the capsid region with randomly inserted cRGD regions can be purified (see FIGS. 57 and 58). The population of capsid polynucleotides is then subcloned into a vector containing the full-length SVV sequence minus the capsid region, such that a library of full-length SVV sequences are generated (where the library manifests sequence diversity in the capsid region as the cRGD sequence is randomly inserted). This library is then reverse transcribed into RNA, and the RNA is transfected into a permissive cell line to generate a population of SVV particles having different capsids (see FIG. 59). Once this RGD-SVV population of virus particles is recovered ("RGD-SVV library"), a number of viruses (i.e., 10 or more) will be randomly picked for sequencing to confirm the insertion of the RGD sequence and diversity of insertion site.

[0374] In Vitro Selection of the RGD-Displaying SVV Library.

[0375] The SVV-RGD library is screened to determine which insertion site enabled an expanded tropism of SVV. The RGD-SVV library is allowed to infect α_Vβ₅ integrin-expressing NSCLC lines (non-small cell lung cancer cell lines, i.e., A549 expressing α_Vβ₅). Only those SVV derivatives that contain a functional and properly displayed RGD motif can infect these cells and replicate.

[0376] In vitro screening is carried out by a high throughput automation system (TECAN) that is capable of liquid handling, concurrent incubation of 20 cell lines and measurement in 384-well plates (see FIG. 62 and FIG. 63). The cells are harvested 30 hr after infection when complete CPE is noticed and then cells are collected by centrifugation at 1500 rpm for 10 minutes at 4° C. The cell pellets are then resuspended in the cell culture supernatant and subjected to three cycles of freeze and thaw. The resulting suspension is clarified by centrifugation at 1500 rpm for 10 minutes at 4° C. Virus is purified by two rounds of CsCl gradients: a one-step gradient (density of CsCl 1.24 g/ml and 1.4 g/ml) followed by one continuous gradient centrifugation (density of CsCl 1.33 g/ml). The purified virus concentration is determined spectrophotometrically, assuming 1A₂₆₀=9.5×10¹² particles (Scraba, D. G. and Palmenberg, A. C., 1999). The process may be repeated multiple times until a sufficient amount of virus is recovered from α_Vβ₅ cells.

[0377] Analysis of Recovered RGD-SVV Derivatives.

[0378] A small pool of individual RGD-displaying SVV derivatives (about 10-50 different derivatives) are analyzed. The viral mixture is diluted and single viral particles are expanded for analysis. Each derivative is tested to determine whether they have gained the ability to infect α_Vβ₅-expressing cells efficiently and specifically. The capsid region of each derivative with this property is then be sequenced to determine the site of RGD insertion. The recovered cRGD-displaying SVV derivatives should possess the following properties: (1) the original properties of the virus are still intact; and (2) the derivatives have gained the ability to infect cells that express high levels of integrins that bind to RGD. This approach aims to identify one or more sites that enable an expanded tropism with RGD insertion, such that random oligonucleotides can be inserted into these sites to generate SVV derivatives with altered tropism.

[0379] The sequenced cRGD-SVV derivatives are numbered and ranked by their binding abilities to integrin. To test the binding activity, recombinant β₂ integrin is immobilized on a 96-well microtiter plate in PBS, washed twice with PBS, blocked with 3% BSA in PBS, and then incubated with a unique RGD-displaying virus. The native virus without peptide insertions is used as a negative control. After 1-5 hr of incubation, the wells are washed at least three times with PBS. Then, the viruses that are bound to the plate will be detected by anti-SVV antibodies. RGD peptide or antibodies against integrin should be able to compete with the binding of the RGD-SVV derivatives to the integrin-bound plate.

[0380] The cRGD-SVV derivatives (20) that have the strongest binding to integrin are analyzed to determine the `successful` location(s) of cRGD oligonucleotide insertion. The insertion sites provide insights into the tropism of SVV. Based on the analysis of the insertion sites and other known structures, an ideal location to place a random peptide library can be determined (this method is an alternative method for generating SVV derivatives, where oligonucleotides (known sequence or random sequence) are inserted into random locations in the capsid). SVV derivatives generated with random sequence oligonucleotides are constructed in essentially the same manner as described above for the RGD-SVV library, except for two additional and novel methodologies. To avoid unwanted stop codons and deleterious amino acid insertions (e.g. cysteines or prolines) within a desired coding region, TRIM (trinucleotide-mutagenesis) technology developed by Morphosys (Munich, Germany) can be used to generate random oligonucleotides for capsid insertion. TRIM utilizes tri-nucleotides which only code for amino acids at the desired position (Virnekas, B. et al., Nucleic Acids Res, 1994, 22(25): 5600-5607). The random-peptide displaying SVV with a diversity of 10⁸ is believed to be sufficient and expected to yield peptides that specifically direct the virus to targeted tumor tissues. Random-peptide displaying SVV is tested in vitro as described above, or in vivo using tumor-bearing mice.

Example 17

Serum Studies

[0381] Pigs are a permissive host for the USDA virus isolates identified above. The isolate MN 88-36695 was inoculated into a gnotobiotic pig and antisera generated (GP102). The antisera binds to all of the other USDA isolates listed above and to SVV. The antisera does not react with 24 common porcine virus pathogens indicating its specificity. Porcine sera was also tested for neutralizing antibodies to 1278 (Plum Island virus). Sera were collected in the US and 8/29 sera were positive with titers ranging from 1:57 to 1:36,500.

[0382] To test whether the pig is the natural source for SVV, serum samples from various animals were obtained and tested for their ability to act as neutralizing antibodies against SVV infection of permissive cells. The Serum Neutralization Assay is conducted as follows: (1) Dilute various serums 1:2 and 1:4 and serially in increasing dilutions if necessary; (2) Mix with 100 TCID₅₀ of virus (SVV; but any virus can be tested to determine whether a serum can neutralize its infection); (3) Incubate at 37° C. for 1 hour; (4) Add the mixture to 1×10⁴ PER.C6® cells (or other permissive cell type); (5) Incubate at 37° C. for 3 days; and (6) Measure CPE using a tetrazolium based dye cytotoxicity (such as MTS) assay. The neutralization titer is defined as the highest dilution of sera that neutralizes SVV (or other virus in question) at 100%.

[0383] The serum neutralization results showed that there is a minimal or no presence of neutralizing antibodies in human and primate populations. In one experiment, 0/22 human sera contained neutralizing antibodies to SVV. In another experiment, only 1/28 human sera contained neutralizing antibodies. In a third experiment, 0/50 human sera from Amish farmers were neutralizing. In another experiment, 0/52 primate sera from four species were neutralizing.

[0384] The serum neutralization results showed that there is a prevalence of SVV neutralizing antibodies in farm animal populations. In one experiment, 27/71 porcine sera from farms were neutralizing. In another experiment, 4/30 porcine sera from a disease-free farm were neutralizing. In another experiment, 10/50 bovine sera were neutralizing. In yet another experiment, 5/35 wild mouse sera were neutralizing.

[0385] Antisera to MN 88-36694 were tested in serum neutralization assays on SVV (see Example 2). Anti-MN 88-36695 gnotobiotic pig serum was able to neutralize infection by SVV (neutralization titer on infection was 1:100 for SVV). As stated above, the antisera binds to all of the other USDA isolates and to SVV, indicating that the herein disclosed USDA isolates are SVV-like picornaviruses due to their serological cross-reactivity with the gnotobiotic pig serum as measured in an indirect immunofluorescence assay.

[0386] These data indicate that SVV is genetically and serologically linked to the porcine USDA virus isolates.

Example 18

SVV and SVV-Like Picornaviruses

[0387] The grouping of the following isolates: MN 88-36695, NC 88-23626, IA 89-47552, NJ 90-10324, IL 92-48963, CA 131395; LA 1278; IL 66289; IL 94-9356; MN/GA 99-29256; MN 99197; and SC 363649, was deduced in part from indirect immunofluorescence experiments. Antisera GP102 was raised against isolate MN 88-36695 by inoculation of the virus into a gnotobiotic pig. The antisera binds to all twelve isolates demonstrating that they are serologically related to one another.

[0388] The GP102 antisera was tested in a neutralization assay with SVV. In this assay, serial dilutions of antisera are mixed with a known quantity of SVV (100 TCID₅₀). The mixtures are placed at 37° C. for 1 hour. An aliquot of the mixture is then added to 1×10⁴ PER.C6® cells, or another cell line that is also permissive for SVV, and the mixtures are placed at 37° C. for 3 days. The wells are then checked for a cytopathic effect of the virus (CPE). If the serum contains neutralizing antibodies, it would neutralize the virus and inhibit the infection of the PER.C6® cells by the virus. CPE is measured quantitatively by using a tetrazolium based dye reagent that changes absorbance based on the number of live cells present. The results are expressed as the percent of viable cells of an uninfected control vs. the log dilution of serum, and are shown in FIG. 93. This data indicates that SVV is serologically linked to the porcine USDA virus isolates.

[0389] Additionally, the viral lysate of MN 88-36695 was tested in cytotoxicity assays with four different cell lines and the results are shown in Table 4. The permissivity profile is identical to that of SVV in that NCI-H446 and HEK293 are permissive for SVV, and NCI-H460 and S8 are not. Additionally, MN 88-36695, like SVV, was cytotoxic to PER.C6® cells. Further, polyclonal antisera to SVV raised in mice was used in a neutralization assay along with MN 88-36695 virus. The results are shown in FIG. 94. The anti-SVV antisera neutralized MN 88-36695, further linking SVV to the USDA viruses serologically.

TABLE-US-00015 TABLE 11 MN 88-36695 Cytotoxicity Results Cell Line TCID50 (pfu/ml) Result NCI-H446 1.6 × 10-6 Permissive HEK293 1.3 × 10-2 Permissive NCI-H460 0 Nonpermissive S8 0 Nonpermissive

[0390] Partial genomic sequence analysis of several of the USDA isolates revealed that they are all very closely related to SVV (see FIGS. 87-89 for sequence alignments). Table 12 shows the percent sequence identity between SVV and six of the isolates. It was found that about 95-98% identity exists at the nucleotide (nt) level over 460 nt of the 3' end of the genome encoding 3D^pol and the 3'UTR (FIG. 89). Each of the USDA viruses is unique and is about 95-98% identical to SVV at the nucleotide level.

TABLE-US-00016 TABLE 12 Percent Sequence Identity Between SVV and Six USDA Isolates 1 2 3 4 5 6 7 Virus Name 96.5 99.1 97.2 97.0 97.4 97.0 1 NJ 90-10324 97.0 95.7 94.8 95.0 98.3* 2 CA 13195 97.6 97.2 97.6 97.2 3 IA 89-47752 95.4 96.1 96.3 4 IL 92-48963 98.9 95.2 5 MN 88-36695 95.4 6 NC 88-23626 7 SVV-001 (SVV)

[0391] Further sequencing of parts of the P1 (FIG. 87) and 2C (FIG. 88) genes of two of the isolates has confirmed this close relationship with SVV. The USDA isolates are more highly related to SVV than any other known viruses, including members of the genus Cardiovirus. Sequences from several regions of seven of the USDA viruses were compared with SVV and neighbor-joining trees were constructed (FIGS. 95A and 95B). These trees further confirm the high degree of relation between the viruses, and identifying CA 131395 as SVV's current closest relative.

Sequence CWU 1

1

22715752DNASeneca Valley Virusmodified_base(1)..(2)a, t, c or g 1nntctagccc accatggcaa caagaagagc ttacaggagc tgaatgaaga acagtgggtg 60gaaatgtctg acgattaccg gaccgggaaa aacatgcctt ttcagtctct tggcacatac 120tatcggcccc ctaactggac ttggggtccc aatttcatca acccctatca agtaacggtt 180ttcccacacc aaattctgaa cgcgagaacc tctacctcgg tagacataaa cgtcccatac 240atcggggaga cccccacgca atcctcagag acacagaact cctggaccct cctcgttatg 300gtgctcgttc ccctagacta taaggaagga gccacaactg acccagaaat tacattttct 360gtaaggccta caagtcccta cttcaatggg cttcgcaacc gctacacggc cgggacggac 420gaagaacagg ggcccattcc tacggcaccc agagaaaatt cgcttatgtt tctctcaacc 480ctccctgacg acactgtccc tgcttacggg aatgtgcgta cccctcctgt caattacctc 540cctggtgaaa taaccgacct tttgcaactg gcccgcatac ccactctcat ggcatttgag 600cgggtgcctg aacccgtgcc tgcctcagac acatatgtgc cctacgttgc cgttcccacc 660cagttcgatg acaggcctct catctccttc ccgatcaccc tttcagatcc cgtctatcag 720aacaccctgg ttggcgccat cagttcaaat ttcgccaatt accgtgggtg tatccaaatc 780actctgacat tttgtggacc catgatggcg agagggaaat tcctgctctc gtattctccc 840ccaaatggaa cgcaaccaca gactctttcc gaagctatgc agtgcacata ctctatttgg 900gacataggct tgaactctag ttggaccttc gtcgtcccct acatctcgcc cagtgactac 960cgtgaaactc gagccattac caactcggtt tactccgctg atggttggtt tagcctgcac 1020aagttgacca aaattactct accacctgac tgtccgcaaa gtccctgcat tctctttttc 1080gcttctgctg gtgaggatta cactctccgt ctccccgttg attgtaatcc ttcctatgtg 1140ttccactcca ccgacaacgc cgagaccggg gttattgagg cgggtaacac tgacaccgat 1200ttctctggtg aactggcggc tcctggccct aaccacacta atgtcaagtt cctgtttgat 1260cgatctcgat tattgaatgt aatcaaggta ctggagaagg acgccgtttt cccccgccct 1320ttccctacac aagaaggtgc gcagcaggat gatggttact tttgtcttct gaccccccgc 1380ccaacagtcg cttcccgacc cgccactcgt ttcggcctgt acgccaatcc gtccggcagt 1440ggtgttcttg ctaacacttc actggacttc aatttttata gcttggcctg tttcacttac 1500tttagatcgg accttgaggt tacggtggtc tcactagagc cggatctgga atttgctgta 1560gggtggtttc cttctggcag tgaataccag gcttccagct ttgtctacga ccagctgcat 1620gtgcccttcc actttactgg gcgcactccc cgcgctttcg ctagcaaggg tgggaaggta 1680tctttcgtgc tcccttggaa ctctgtctcg tctgtgctcc ccgtgcgctg ggggggggct 1740tccaagctct cttctgctac gcggggtcta ccggcgcatg ctgattgggg gactatttac 1800gcctttgtcc cccgtcctaa tgagaagaaa agcaccgctg taaaacacgt ggccgtgtac 1860attcggtaca agaacgcacg tgcctggtgc cccagcatgc ttccctttcg cagctacaag 1920cagaagatgc tgatgcaatc tggcgatatc gagaccaatc ctggtcctgc ttctgacaac 1980ccaattttgg agtttcttga agcagaaaat gatctagtca ctctggcctc tctctggaag 2040atggtgcact ctgttcaaca gacctggaga aagtatgtga agaacgatga tttttggccc 2100aatttactca gcgagctagt gggggaaggc tctgtcgcct tggccgccac gctatccaac 2160caagcttcag taaaggctct tttgggcctg cactttctct ctcgggggct caattacact 2220gacttttact ctttactgat agagaaatgc tctagtttct ttaccgtaga accacctcct 2280ccaccagctg aaaacctgat gaccaagccc tcagtgaagt cgaaattccg aaaactgttt 2340aagatgcaag gacccatgga caaagtcaaa gactggaacc aaatagctgc cggcttgaag 2400aattttcaat ttgttcgtga cctagtcaaa gaggtggtcg attggctgca ggcctggatc 2460aacaaagaga aagccagccc tgtcctccag taccagttgg agatgaagaa gctcgggcct 2520gtggccttgg ctcatgacgc tttcatggct ggttccgggc cccctcttag cgacgaccag 2580attgaatacc tccagaacct caaatctctt gccctaacac tggggaagac taatttggcc 2640caaagtctca ccactatgat caatgccaaa caaagttcag cccaacgagt tgaacccgtt 2700gtggtggtcc ttagaggcaa gccgggatgc ggcaagggct tggcctctac gttgattgcc 2760caggctgtgt ccaagcgcct ctatggctcc caaagtgtat attctcttcc cccagatcca 2820gatttcttcg atggatacaa aggacagttc gtgaccttga tggatgattt gggacaaaac 2880ccggatggac aagatttccc caccttttgt cagatggtgt cgaccgccca atttctcccc 2940aacatggcgg accttgcaga gaaagggcgt ccctttacct ccaatctcat cattgcaact 3000acaaatctcc cccacttcag tcctgtcacc attgctgatc cttctgcagt ctctcgccgt 3060atcaactacg atctgactct agaagtatct gaggcctaca agaaacacac acggctgaat 3120tttgacttgg ctttcaggcg cacagacgcc ccccccattt atccttttgc tgcccatgtg 3180ccctttgtgg acgtagctgt gcgcttcaaa aatggtcacc agaattttaa tctcctagag 3240ttggtcgatt ccatttgtac agacattcga gccaagcaac aaggtgcccg aaacatgcag 3300actctggttc tacagagccc caacgagaat gatgacaccc ccgtcgacga ggcgttgggt 3360agagttctct cccccgctgc ggtcgatgag gcgcttgtcg acctcactcc agaggccgac 3420ccggttggcc gtttggctat tcttgccaag ctaggtcttg ccctagctgc ggtcacccct 3480ggtctgataa tcttggcagt gggactctac aggtacttct ctggctctga tgcagaccaa 3540gaagaaacag aaagtgaggg atctgtcaag gcacccagga gcgaaaatgc ttatgacggc 3600ccgaagaaaa actctaagcc ccctggagca ctctctctca tggaaatgca acagcccaac 3660gtggacatgg gctttgaggc tgcggtcgct aagaaagtgg tcgtccccat taccttcatg 3720gttcccaaca gaccttctgg gcttacacag tccgctcttc tggtgaccgg ccggaccttc 3780ctaatcaatg aacatacatg gtccaatccc tcctggacca gcttcacaat ccgcggtgag 3840gtacacactc gtgatgagcc cttccaaacg gttcatttca ctcaccacgg tattcccaca 3900gatctgatga tggtacgtct cggaccgggc aattctttcc ctaacaatct agacaagttt 3960ggacttgacc agatgccggc acgcaactcc cgtgtggttg gcgtttcgtc cagttacgga 4020aacttcttct tctctggaaa tttcctcgga tttgttgatt ccgtcacctc tgaacaagga 4080acttacgcaa gactctttag gtacagggtg acgacctaca aaggatggtg cggctcggcc 4140ctggtctgtg aggccggtgg cgtccgacgc atcattggcc tgcattctgc tggcgccgcc 4200ggtatcggcg ccgggaccta tatctcaaaa ttaggactaa tcaaagccct gaaacacctc 4260ggtgaacctt tggccacaat gcaaggactg atgactgaat tagagcctgg aatcaccgta 4320catgtacccc ggaaatccaa attgagaaag acgaccgcac acgcggtgta caaaccggag 4380tttgagcctg ctgtgttgtc aaaatttgat cccagactga acaaggatgt tgacttggat 4440gaagtaattt ggtctaaaca cactgccaat gtcccttacc aacctccttt gttctacaca 4500tacatgtcag agtacgctca tcgagtcttc tccttcttgg ggaaagacaa tgacattctg 4560accgtcaaag aagcaattct gggcatcccc ggactagacc ccatggatcc ccacacagct 4620ccgggtctgc cttacgccat caacggcctt cgacgtactg atctcgtcga ttttgtgaac 4680ggtacagtag atgcggcgct ggctgtacaa atccagaaat tcttagacgg tgactactct 4740gaccatgtct tccaaacttt tctgaaagat gagatcagac cctcagagaa agtccgagcg 4800ggaaaaaccc gcattgttga tgtgccctcc ctggcgcatt gcattgtggg cagaatgttg 4860cttgggcgct ttgctgccaa gtttcaatcc catcctggct ttctcctcgg ctctgctatc 4920gggtctgacc ctgatgtttt ctggaccgtc ataggggctc aactcgaggg gagaaagaac 4980acgtatgacg tggactacag tgcctttgac tcttcacacg gcactggctc cttcgaggct 5040ctcatctctc actttttcac cgtggacaat ggttttagcc ctgcgctggg accgtatctc 5100agatccctgg ctgtctcggt gcacgcttac ggcgagcgtc gcatcaagat taccggtggc 5160ctcccctccg gttgtgccgc gaccagcctg ctgaacacag tgctcaacaa tgtgatcatc 5220aggactgctc tggcattgac ttacaaggaa tttgagtatg acacggttga tatcatcgcc 5280tacggtgacg accttctggt tggcacggat tacgatctgg acttcaatga ggtggcacga 5340cgcgctgcca agttggggta taagatgact cctgccaaca agggttctgt cttccctccg 5400acttcctctc tttccgatgc tgtttttcta aagcgcaaat tcgtccaaaa caacgacggc 5460ttatacaaac cagttatgga tttaaagaat ttggaagcca tgctctccta cttcaaacca 5520ggaacactac tcgagaagct gcaatctgtt tctatgttgg ctcaacattc tggaaaagaa 5580gaatatgata gattgatgca ccccttcgct gactacggtg ccgtaccgag tcacgagtac 5640ctgcaggcaa gatggagggc cttgttcgac tgacccagat agcccaaggc gcttcggtgc 5700tgccggcgat tctgggagaa ctcagtcgga acagaaaaaa aaaaaaaaaa aa 575221890PRTSeneca Valley VirusMOD_RES(1)variable amino acid 2Xaa Leu Ala His His Gly Asn Lys Lys Ser Leu Gln Glu Leu Asn Glu 1 5 10 15 Glu Gln Trp Val Glu Met Ser Asp Asp Tyr Arg Thr Gly Lys Asn Met 20 25 30 Pro Phe Gln Ser Leu Gly Thr Tyr Tyr Arg Pro Pro Asn Trp Thr Trp 35 40 45 Gly Pro Asn Phe Ile Asn Pro Tyr Gln Val Thr Val Phe Pro His Gln 50 55 60 Ile Leu Asn Ala Arg Thr Ser Thr Ser Val Asp Ile Asn Val Pro Tyr 65 70 75 80Ile Gly Glu Thr Pro Thr Gln Ser Ser Glu Thr Gln Asn Ser Trp Thr 85 90 95 Leu Leu Val Met Val Leu Val Pro Leu Asp Tyr Lys Glu Gly Ala Thr 100 105 110 Thr Asp Pro Glu Ile Thr Phe Ser Val Arg Pro Thr Ser Pro Tyr Phe 115 120 125 Asn Gly Leu Arg Asn Arg Tyr Thr Ala Gly Thr Asp Glu Glu Gln Gly 130 135 140 Pro Ile Pro Thr Ala Pro Arg Glu Asn Ser Leu Met Phe Leu Ser Thr 145 150 155 160Leu Pro Asp Asp Thr Val Pro Ala Tyr Gly Asn Val Arg Thr Pro Pro 165 170 175 Val Asn Tyr Leu Pro Gly Glu Ile Thr Asp Leu Leu Gln Leu Ala Arg 180 185 190 Ile Pro Thr Leu Met Ala Phe Glu Arg Val Pro Glu Pro Val Pro Ala 195 200 205 Ser Asp Thr Tyr Val Pro Tyr Val Ala Val Pro Thr Gln Phe Asp Asp 210 215 220 Arg Pro Leu Ile Ser Phe Pro Ile Thr Leu Ser Asp Pro Val Tyr Gln 225 230 235 240Asn Thr Leu Val Gly Ala Ile Ser Ser Asn Phe Ala Asn Tyr Arg Gly 245 250 255 Cys Ile Gln Ile Thr Leu Thr Phe Cys Gly Pro Met Met Ala Arg Gly 260 265 270 Lys Phe Leu Leu Ser Tyr Ser Pro Pro Asn Gly Thr Gln Pro Gln Thr 275 280 285 Leu Ser Glu Ala Met Gln Cys Thr Tyr Ser Ile Trp Asp Ile Gly Leu 290 295 300 Asn Ser Ser Trp Thr Phe Val Val Pro Tyr Ile Ser Pro Ser Asp Tyr 305 310 315 320Arg Glu Thr Arg Ala Ile Thr Asn Ser Val Tyr Ser Ala Asp Gly Trp 325 330 335 Phe Ser Leu His Lys Leu Thr Lys Ile Thr Leu Pro Pro Asp Cys Pro 340 345 350 Gln Ser Pro Cys Ile Leu Phe Phe Ala Ser Ala Gly Glu Asp Tyr Thr 355 360 365 Leu Arg Leu Pro Val Asp Cys Asn Pro Ser Tyr Val Phe His Ser Thr 370 375 380 Asp Asn Ala Glu Thr Gly Val Ile Glu Ala Gly Asn Thr Asp Thr Asp 385 390 395 400Phe Ser Gly Glu Leu Ala Ala Pro Gly Pro Asn His Thr Asn Val Lys 405 410 415 Phe Leu Phe Asp Arg Ser Arg Leu Leu Asn Val Ile Lys Val Leu Glu 420 425 430 Lys Asp Ala Val Phe Pro Arg Pro Phe Pro Thr Gln Glu Gly Ala Gln 435 440 445 Gln Asp Asp Gly Tyr Phe Cys Leu Leu Thr Pro Arg Pro Thr Val Ala 450 455 460 Ser Arg Pro Ala Thr Arg Phe Gly Leu Tyr Ala Asn Pro Ser Gly Ser 465 470 475 480Gly Val Leu Ala Asn Thr Ser Leu Asp Phe Asn Phe Tyr Ser Leu Ala 485 490 495 Cys Phe Thr Tyr Phe Arg Ser Asp Leu Glu Val Thr Val Val Ser Leu 500 505 510 Glu Pro Asp Leu Glu Phe Ala Val Gly Trp Phe Pro Ser Gly Ser Glu 515 520 525 Tyr Gln Ala Ser Ser Phe Val Tyr Asp Gln Leu His Val Pro Phe His 530 535 540 Phe Thr Gly Arg Thr Pro Arg Ala Phe Ala Ser Lys Gly Gly Lys Val 545 550 555 560Ser Phe Val Leu Pro Trp Asn Ser Val Ser Ser Val Leu Pro Val Arg 565 570 575 Trp Gly Gly Ala Ser Lys Leu Ser Ser Ala Thr Arg Gly Leu Pro Ala 580 585 590 His Ala Asp Trp Gly Thr Ile Tyr Ala Phe Val Pro Arg Pro Asn Glu 595 600 605 Lys Lys Ser Thr Ala Val Lys His Val Ala Val Tyr Ile Arg Tyr Lys 610 615 620 Asn Ala Arg Ala Trp Cys Pro Ser Met Leu Pro Phe Arg Ser Tyr Lys 625 630 635 640Gln Lys Met Leu Met Gln Ser Gly Asp Ile Glu Thr Asn Pro Gly Pro 645 650 655 Ala Ser Asp Asn Pro Ile Leu Glu Phe Leu Glu Ala Glu Asn Asp Leu 660 665 670 Val Thr Leu Ala Ser Leu Trp Lys Met Val His Ser Val Gln Gln Thr 675 680 685 Trp Arg Lys Tyr Val Lys Asn Asp Asp Phe Trp Pro Asn Leu Leu Ser 690 695 700 Glu Leu Val Gly Glu Gly Ser Val Ala Leu Ala Ala Thr Leu Ser Asn 705 710 715 720Gln Ala Ser Val Lys Ala Leu Leu Gly Leu His Phe Leu Ser Arg Gly 725 730 735 Leu Asn Tyr Thr Asp Phe Tyr Ser Leu Leu Ile Glu Lys Cys Ser Ser 740 745 750 Phe Phe Thr Val Glu Pro Pro Pro Pro Pro Ala Glu Asn Leu Met Thr 755 760 765 Lys Pro Ser Val Lys Ser Lys Phe Arg Lys Leu Phe Lys Met Gln Gly 770 775 780 Pro Met Asp Lys Val Lys Asp Trp Asn Gln Ile Ala Ala Gly Leu Lys 785 790 795 800Asn Phe Gln Phe Val Arg Asp Leu Val Lys Glu Val Val Asp Trp Leu 805 810 815 Gln Ala Trp Ile Asn Lys Glu Lys Ala Ser Pro Val Leu Gln Tyr Gln 820 825 830 Leu Glu Met Lys Lys Leu Gly Pro Val Ala Leu Ala His Asp Ala Phe 835 840 845 Met Ala Gly Ser Gly Pro Pro Leu Ser Asp Asp Gln Ile Glu Tyr Leu 850 855 860 Gln Asn Leu Lys Ser Leu Ala Leu Thr Leu Gly Lys Thr Asn Leu Ala 865 870 875 880Gln Ser Leu Thr Thr Met Ile Asn Ala Lys Gln Ser Ser Ala Gln Arg 885 890 895 Val Glu Pro Val Val Val Val Leu Arg Gly Lys Pro Gly Cys Gly Lys 900 905 910 Gly Leu Ala Ser Thr Leu Ile Ala Gln Ala Val Ser Lys Arg Leu Tyr 915 920 925 Gly Ser Gln Ser Val Tyr Ser Leu Pro Pro Asp Pro Asp Phe Phe Asp 930 935 940 Gly Tyr Lys Gly Gln Phe Val Thr Leu Met Asp Asp Leu Gly Gln Asn 945 950 955 960Pro Asp Gly Gln Asp Phe Ser Thr Phe Cys Gln Met Val Ser Thr Ala 965 970 975 Gln Phe Leu Pro Asn Met Ala Asp Leu Ala Glu Lys Gly Arg Pro Phe 980 985 990 Thr Ser Asn Leu Ile Ile Ala Thr Thr Asn Leu Pro His Phe Ser Pro 995 1000 1005 Val Thr Ile Ala Asp Pro Ser Ala Val Ser Arg Arg Ile Asn Tyr Asp 1010 1015 1020 Leu Thr Leu Glu Val Ser Glu Ala Tyr Lys Lys His Thr Arg Leu Asn 1025 1030 1035 1040Phe Asp Leu Ala Phe Arg Arg Thr Asp Ala Pro Pro Ile Tyr Pro Phe 1045 1050 1055 Ala Ala His Val Pro Phe Val Asp Val Ala Val Arg Phe Lys Asn Gly 1060 1065 1070 His Gln Asn Phe Asn Leu Leu Glu Leu Val Asp Ser Ile Cys Thr Asp 1075 1080 1085 Ile Arg Ala Lys Gln Gln Gly Ala Arg Asn Met Gln Thr Leu Val Leu 1090 1095 1100 Gln Ser Pro Asn Glu Asn Asp Asp Thr Pro Val Asp Glu Ala Leu Gly 1105 1110 1115 1120Arg Val Leu Ser Pro Ala Ala Val Asp Glu Ala Leu Val Asp Leu Thr 1125 1130 1135 Pro Glu Ala Asp Pro Val Gly Arg Leu Ala Ile Leu Ala Lys Leu Gly 1140 1145 1150 Leu Ala Leu Ala Ala Val Thr Pro Gly Leu Ile Ile Leu Ala Val Gly 1155 1160 1165 Leu Tyr Arg Tyr Phe Ser Gly Ser Asp Ala Asp Gln Glu Glu Thr Glu 1170 1175 1180 Ser Glu Gly Ser Val Lys Ala Pro Arg Ser Glu Asn Ala Tyr Asp Gly 1185 1190 1195 1200Pro Lys Lys Asn Ser Lys Pro Pro Gly Ala Leu Ser Leu Met Glu Met 1205 1210 1215 Gln Gln Pro Asn Val Asp Met Gly Phe Glu Ala Ala Val Ala Lys Lys 1220 1225 1230 Val Val Val Pro Ile Thr Phe Met Val Pro Asn Arg Pro Ser Gly Leu 1235 1240 1245 Thr Gln Ser Ala Leu Leu Val Thr Gly Arg Thr Phe Leu Ile Asn Glu 1250 1255 1260 His Thr Trp Ser Asn Pro Ser Trp Thr Ser Phe Thr Ile Arg Gly Glu 1265 1270 1275 1280Val His Thr Arg Asp Glu Pro Phe Gln Thr Val His Phe Thr His His 1285 1290 1295 Gly Ile Pro Thr Asp Leu Met Met Val Arg Leu Gly Pro Gly Asn Ser 1300 1305 1310 Phe Pro Asn Asn Leu Asp Lys Phe Gly Leu Asp Gln Met Pro Ala Arg 1315 1320 1325 Asn Ser Arg Val Val Gly Val Ser Ser Ser Tyr Gly Asn Phe Phe Phe 1330 1335 1340 Ser Gly Asn Phe Leu Gly Phe Val Asp Ser Val Thr Ser Glu Gln Gly 1345 1350 1355 1360Thr Tyr Ala Arg Leu Phe Arg Tyr Arg Val Thr Thr Tyr Lys Gly Trp 1365 1370 1375 Cys Gly Ser Ala Leu Val Cys Glu Ala Gly Gly Val Arg Arg Ile Ile 1380 1385 1390 Gly Leu His Ser Ala Gly Ala Ala Gly Ile Gly Ala Gly Thr Tyr

Ile 1395 1400 1405 Ser Lys Leu Gly Leu Ile Lys Ala Leu Lys His Leu Gly Glu Pro Leu 1410 1415 1420 Ala Thr Met Gln Gly Leu Met Thr Glu Leu Glu Pro Gly Ile Thr Val 1425 1430 1435 1440His Val Pro Arg Lys Ser Lys Leu Arg Lys Thr Thr Ala His Ala Val 1445 1450 1455 Tyr Lys Pro Glu Phe Glu Pro Ala Val Leu Ser Lys Phe Asp Pro Arg 1460 1465 1470 Leu Asn Lys Asp Val Asp Leu Asp Glu Val Ile Trp Ser Lys His Thr 1475 1480 1485 Ala Asn Val Pro Tyr Gln Pro Pro Leu Phe Tyr Thr Tyr Met Ser Glu 1490 1495 1500 Tyr Ala His Arg Val Phe Ser Phe Leu Gly Lys Asp Asn Asp Ile Leu 1505 1510 1515 1520Thr Val Lys Glu Ala Ile Leu Gly Ile Pro Gly Leu Asp Pro Met Asp 1525 1530 1535 Pro His Thr Ala Pro Gly Leu Pro Tyr Ala Ile Asn Gly Leu Arg Arg 1540 1545 1550 Thr Asp Leu Val Asp Phe Val Asn Gly Thr Val Asp Ala Ala Leu Ala 1555 1560 1565 Val Gln Ile Gln Lys Phe Leu Asp Gly Asp Tyr Ser Asp His Val Phe 1570 1575 1580 Gln Thr Phe Leu Lys Asp Glu Ile Arg Pro Ser Glu Lys Val Arg Ala 1585 1590 1595 1600Gly Lys Thr Arg Ile Val Asp Val Pro Ser Leu Ala His Cys Ile Val 1605 1610 1615 Gly Arg Met Leu Leu Gly Arg Phe Ala Ala Lys Phe Gln Ser His Pro 1620 1625 1630 Gly Phe Leu Leu Gly Ser Ala Ile Gly Ser Asp Pro Asp Val Phe Trp 1635 1640 1645 Thr Val Ile Gly Ala Gln Leu Glu Gly Arg Lys Asn Thr Tyr Asp Val 1650 1655 1660 Asp Tyr Ser Ala Phe Asp Ser Ser His Gly Thr Gly Ser Phe Glu Ala 1665 1670 1675 1680Leu Ile Ser His Phe Phe Thr Val Asp Asn Gly Phe Ser Pro Ala Leu 1685 1690 1695 Gly Pro Tyr Leu Arg Ser Leu Ala Val Ser Val His Ala Tyr Gly Glu 1700 1705 1710 Arg Arg Ile Lys Ile Thr Gly Gly Leu Pro Ser Gly Cys Ala Ala Thr 1715 1720 1725 Ser Leu Leu Asn Thr Val Leu Asn Asn Val Ile Ile Arg Thr Ala Leu 1730 1735 1740 Ala Leu Thr Tyr Lys Glu Phe Glu Tyr Asp Thr Val Asp Ile Ile Ala 1745 1750 1755 1760Tyr Gly Asp Asp Leu Leu Val Gly Thr Asp Tyr Asp Leu Asp Phe Asn 1765 1770 1775 Glu Val Ala Arg Arg Ala Ala Lys Leu Gly Tyr Lys Met Thr Pro Ala 1780 1785 1790 Asn Lys Gly Ser Val Phe Pro Pro Thr Ser Ser Leu Ser Asp Ala Val 1795 1800 1805 Phe Leu Lys Arg Lys Phe Val Gln Asn Asn Asp Gly Leu Tyr Lys Pro 1810 1815 1820 Val Met Asp Leu Lys Asn Leu Glu Ala Met Leu Ser Tyr Phe Lys Pro 1825 1830 1835 1840Gly Thr Leu Leu Glu Lys Leu Gln Ser Val Ser Met Leu Ala Gln His 1845 1850 1855 Ser Gly Lys Glu Glu Tyr Asp Arg Leu Met His Pro Phe Ala Asp Tyr 1860 1865 1870 Gly Ala Val Pro Ser His Glu Tyr Leu Gln Ala Arg Trp Arg Ala Leu 1875 1880 1885 Phe Asp 18903426DNASeneca Valley Virus 3ctagcccacc atggcaacaa gaagagctta caggagctga atgaagaaca gtgggtggaa 60atgtctgacg attaccggac cgggaaaaac atgccttttc agtctcttgg cacatactat 120cggcccccta actggacttg gggtcccaat ttcatcaacc cctatcaagt aacggttttc 180ccacaccaaa ttctgaacgc gagaacctct acctcggtag acataaacgt cccatacatc 240ggggagaccc ccacgcaatc ctcagagaca cagaactcct ggaccctcct cgttatggtg 300ctcgttcccc tagactataa ggaaggagcc acaactgacc cagaaattac attttctgta 360aggcctacaa gtccctactt caatgggctt cgcaaccgct acacggccgg gacggacgaa 420gaacag 4264142PRTSeneca Valley Virus 4Leu Ala His His Gly Asn Lys Lys Ser Leu Gln Glu Leu Asn Glu Glu 1 5 10 15 Gln Trp Val Glu Met Ser Asp Asp Tyr Arg Thr Gly Lys Asn Met Pro 20 25 30 Phe Gln Ser Leu Gly Thr Tyr Tyr Arg Pro Pro Asn Trp Thr Trp Gly 35 40 45 Pro Asn Phe Ile Asn Pro Tyr Gln Val Thr Val Phe Pro His Gln Ile 50 55 60 Leu Asn Ala Arg Thr Ser Thr Ser Val Asp Ile Asn Val Pro Tyr Ile 65 70 75 80Gly Glu Thr Pro Thr Gln Ser Ser Glu Thr Gln Asn Ser Trp Thr Leu 85 90 95 Leu Val Met Val Leu Val Pro Leu Asp Tyr Lys Glu Gly Ala Thr Thr 100 105 110 Asp Pro Glu Ile Thr Phe Ser Val Arg Pro Thr Ser Pro Tyr Phe Asn 115 120 125 Gly Leu Arg Asn Arg Tyr Thr Ala Gly Thr Asp Glu Glu Gln 130 135 140 5717DNASeneca Valley Virus 5gggcccattc ctacggcacc cagagaaaat tcgcttatgt ttctctcaac cctccctgac 60gacactgtcc ctgcttacgg gaatgtgcgt acccctcctg tcaattacct ccctggtgaa 120ataaccgacc ttttgcaact ggcccgcata cccactctca tggcatttga gcgggtgcct 180gaacccgtgc ctgcctcaga cacatatgtg ccctacgttg ccgttcccac ccagttcgat 240gacaggcctc tcatctcctt cccgatcacc ctttcagatc ccgtctatca gaacaccctg 300gttggcgcca tcagttcaaa tttcgccaat taccgtgggt gtatccaaat cactctgaca 360ttttgtggac ccatgatggc gagagggaaa ttcctgctct cgtattctcc cccaaatgga 420acgcaaccac agactctttc cgaagctatg cagtgcacat actctatttg ggacataggc 480ttgaactcta gttggacctt cgtcgtcccc tacatctcgc ccagtgacta ccgtgaaact 540cgagccatta ccaactcggt ttactccgct gatggttggt ttagcctgca caagttgacc 600aaaattactc taccacctga ctgtccgcaa agtccctgca ttctcttttt cgcttctgct 660ggtgaggatt acactctccg tctccccgtt gattgtaatc cttcctatgt gttccac 7176239PRTSeneca Valley Virus 6Gly Pro Ile Pro Thr Ala Pro Arg Glu Asn Ser Leu Met Phe Leu Ser 1 5 10 15 Thr Leu Pro Asp Asp Thr Val Pro Ala Tyr Gly Asn Val Arg Thr Pro 20 25 30 Pro Val Asn Tyr Leu Pro Gly Glu Ile Thr Asp Leu Leu Gln Leu Ala 35 40 45 Arg Ile Pro Thr Leu Met Ala Phe Glu Arg Val Pro Glu Pro Val Pro 50 55 60 Ala Ser Asp Thr Tyr Val Pro Tyr Val Ala Val Pro Thr Gln Phe Asp 65 70 75 80Asp Arg Pro Leu Ile Ser Phe Pro Ile Thr Leu Ser Asp Pro Val Tyr 85 90 95 Gln Asn Thr Leu Val Gly Ala Ile Ser Ser Asn Phe Ala Asn Tyr Arg 100 105 110 Gly Cys Ile Gln Ile Thr Leu Thr Phe Cys Gly Pro Met Met Ala Arg 115 120 125 Gly Lys Phe Leu Leu Ser Tyr Ser Pro Pro Asn Gly Thr Gln Pro Gln 130 135 140 Thr Leu Ser Glu Ala Met Gln Cys Thr Tyr Ser Ile Trp Asp Ile Gly 145 150 155 160Leu Asn Ser Ser Trp Thr Phe Val Val Pro Tyr Ile Ser Pro Ser Asp 165 170 175 Tyr Arg Glu Thr Arg Ala Ile Thr Asn Ser Val Tyr Ser Ala Asp Gly 180 185 190 Trp Phe Ser Leu His Lys Leu Thr Lys Ile Thr Leu Pro Pro Asp Cys 195 200 205 Pro Gln Ser Pro Cys Ile Leu Phe Phe Ala Ser Ala Gly Glu Asp Tyr 210 215 220 Thr Leu Arg Leu Pro Val Asp Cys Asn Pro Ser Tyr Val Phe His 225 230 235 7777DNASeneca Valley Virus 7tccaccgaca acgccgagac cggggttatt gaggcgggta acactgacac cgatttctct 60ggtgaactgg cggctcctgg ccctaaccac actaatgtca agttcctgtt tgatcgatct 120cgattattga atgtaatcaa ggtactggag aaggacgccg ttttcccccg ccctttccct 180acacaagaag gtgcgcagca ggatgatggt tacttttgtc ttctgacccc ccgcccaaca 240gtcgcttccc gacccgccac tcgtttcggc ctgtacgcca atccgtccgg cagtggtgtt 300cttgctaaca cttcactgga cttcaatttt tatagcttgg cctgtttcac ttactttaga 360tcggaccttg aggttacggt ggtctcacta gagccggatc tggaatttgc tgtagggtgg 420tttccttctg gcagtgaata ccaggcttcc agctttgtct acgaccagct gcatgtgccc 480ttccacttta ctgggcgcac tccccgcgct ttcgctagca agggtgggaa ggtatctttc 540gtgctccctt ggaactctgt ctcgtctgtg ctccccgtgc gctggggggg ggcttccaag 600ctctcttctg ctacgcgggg tctaccggcg catgctgatt gggggactat ttacgccttt 660gtcccccgtc ctaatgagaa gaaaagcacc gctgtaaaac acgtggccgt gtacattcgg 720tacaagaacg cacgtgcctg gtgccccagc atgcttccct ttcgcagcta caagcag 7778259PRTSeneca Valley Virus 8Ser Thr Asp Asn Ala Glu Thr Gly Val Ile Glu Ala Gly Asn Thr Asp 1 5 10 15 Thr Asp Phe Ser Gly Glu Leu Ala Ala Pro Gly Pro Asn His Thr Asn 20 25 30 Val Lys Phe Leu Phe Asp Arg Ser Arg Leu Leu Asn Val Ile Lys Val 35 40 45 Leu Glu Lys Asp Ala Val Phe Pro Arg Pro Phe Pro Thr Gln Glu Gly 50 55 60 Ala Gln Gln Asp Asp Gly Tyr Phe Cys Leu Leu Thr Pro Arg Pro Thr 65 70 75 80Val Ala Ser Arg Pro Ala Thr Arg Phe Gly Leu Tyr Ala Asn Pro Ser 85 90 95 Gly Ser Gly Val Leu Ala Asn Thr Ser Leu Asp Phe Asn Phe Tyr Ser 100 105 110 Leu Ala Cys Phe Thr Tyr Phe Arg Ser Asp Leu Glu Val Thr Val Val 115 120 125 Ser Leu Glu Pro Asp Leu Glu Phe Ala Val Gly Trp Phe Pro Ser Gly 130 135 140 Ser Glu Tyr Gln Ala Ser Ser Phe Val Tyr Asp Gln Leu His Val Pro 145 150 155 160Phe His Phe Thr Gly Arg Thr Pro Arg Ala Phe Ala Ser Lys Gly Gly 165 170 175 Lys Val Ser Phe Val Leu Pro Trp Asn Ser Val Ser Ser Val Leu Pro 180 185 190 Val Arg Trp Gly Gly Ala Ser Lys Leu Ser Ser Ala Thr Arg Gly Leu 195 200 205 Pro Ala His Ala Asp Trp Gly Thr Ile Tyr Ala Phe Val Pro Arg Pro 210 215 220 Asn Glu Lys Lys Ser Thr Ala Val Lys His Val Ala Val Tyr Ile Arg 225 230 235 240Tyr Lys Asn Ala Arg Ala Trp Cys Pro Ser Met Leu Pro Phe Arg Ser 245 250 255 Tyr Lys Gln 942DNASeneca Valley Virus 9aagatgctga tgcaatctgg cgatatcgag accaatcctg gt 421014PRTSeneca Valley Virus 10Lys Met Leu Met Gln Ser Gly Asp Ile Glu Thr Asn Pro Gly 1 5 10 11384DNASeneca Valley Virus 11cctgcttctg acaacccaat tttggagttt cttgaagcag aaaatgatct agtcactctg 60gcctctctct ggaagatggt gcactctgtt caacagacct ggagaaagta tgtgaagaac 120gatgattttt ggcccaattt actcagcgag ctagtggggg aaggctctgt cgccttggcc 180gccacgctat ccaaccaagc ttcagtaaag gctcttttgg gcctgcactt tctctctcgg 240gggctcaatt acactgactt ttactcttta ctgatagaga aatgctctag tttctttacc 300gtagaaccac ctcctccacc agctgaaaac ctgatgacca agccctcagt gaagtcgaaa 360ttccgaaaac tgtttaagat gcaa 38412128PRTSeneca Valley Virus 12Pro Ala Ser Asp Asn Pro Ile Leu Glu Phe Leu Glu Ala Glu Asn Asp 1 5 10 15 Leu Val Thr Leu Ala Ser Leu Trp Lys Met Val His Ser Val Gln Gln 20 25 30 Thr Trp Arg Lys Tyr Val Lys Asn Asp Asp Phe Trp Pro Asn Leu Leu 35 40 45 Ser Glu Leu Val Gly Glu Gly Ser Val Ala Leu Ala Ala Thr Leu Ser 50 55 60 Asn Gln Ala Ser Val Lys Ala Leu Leu Gly Leu His Phe Leu Ser Arg 65 70 75 80Gly Leu Asn Tyr Thr Asp Phe Tyr Ser Leu Leu Ile Glu Lys Cys Ser 85 90 95 Ser Phe Phe Thr Val Glu Pro Pro Pro Pro Pro Ala Glu Asn Leu Met 100 105 110 Thr Lys Pro Ser Val Lys Ser Lys Phe Arg Lys Leu Phe Lys Met Gln 115 120 125 13966DNASeneca Valley Virus 13ggacccatgg acaaagtcaa agactggaac caaatagctg ccggcttgaa gaattttcaa 60tttgttcgtg acctagtcaa agaggtggtc gattggctgc aggcctggat caacaaagag 120aaagccagcc ctgtcctcca gtaccagttg gagatgaaga agctcgggcc tgtggccttg 180gctcatgacg ctttcatggc tggttccggg ccccctctta gcgacgacca gattgaatac 240ctccagaacc tcaaatctct tgccctaaca ctggggaaga ctaatttggc ccaaagtctc 300accactatga tcaatgccaa acaaagttca gcccaacgag ttgaacccgt tgtggtggtc 360cttagaggca agccgggatg cggcaagggc ttggcctcta cgttgattgc ccaggctgtg 420tccaagcgcc tctatggctc ccaaagtgta tattctcttc ccccagatcc agatttcttc 480gatggataca aaggacagtt cgtgaccttg atggatgatt tgggacaaaa cccggatgga 540caagatttcc ccaccttttg tcagatggtg tcgaccgccc aatttctccc caacatggcg 600gaccttgcag agaaagggcg tccctttacc tccaatctca tcattgcaac tacaaatctc 660ccccacttca gtcctgtcac cattgctgat ccttctgcag tctctcgccg tatcaactac 720gatctgactc tagaagtatc tgaggcctac aagaaacaca cacggctgaa ttttgacttg 780gctttcaggc gcacagacgc cccccccatt tatccttttg ctgcccatgt gccctttgtg 840gacgtagctg tgcgcttcaa aaatggtcac cagaatttta atctcctaga gttggtcgat 900tccatttgta cagacattcg agccaagcaa caaggtgccc gaaacatgca gactctggtt 960ctacag 96614322PRTSeneca Valley Virus 14Gly Pro Met Asp Lys Val Lys Asp Trp Asn Gln Ile Ala Ala Gly Leu 1 5 10 15 Lys Asn Phe Gln Phe Val Arg Asp Leu Val Lys Glu Val Val Asp Trp 20 25 30 Leu Gln Ala Trp Ile Asn Lys Glu Lys Ala Ser Pro Val Leu Gln Tyr 35 40 45 Gln Leu Glu Met Lys Lys Leu Gly Pro Val Ala Leu Ala His Asp Ala 50 55 60 Phe Met Ala Gly Ser Gly Pro Pro Leu Ser Asp Asp Gln Ile Glu Tyr 65 70 75 80Leu Gln Asn Leu Lys Ser Leu Ala Leu Thr Leu Gly Lys Thr Asn Leu 85 90 95 Ala Gln Ser Leu Thr Thr Met Ile Asn Ala Lys Gln Ser Ser Ala Gln 100 105 110 Arg Val Glu Pro Val Val Val Val Leu Arg Gly Lys Pro Gly Cys Gly 115 120 125 Lys Gly Leu Ala Ser Thr Leu Ile Ala Gln Ala Val Ser Lys Arg Leu 130 135 140 Tyr Gly Ser Gln Ser Val Tyr Ser Leu Pro Pro Asp Pro Asp Phe Phe 145 150 155 160Asp Gly Tyr Lys Gly Gln Phe Val Thr Leu Met Asp Asp Leu Gly Gln 165 170 175 Asn Pro Asp Gly Gln Asp Phe Ser Thr Phe Cys Gln Met Val Ser Thr 180 185 190 Ala Gln Phe Leu Pro Asn Met Ala Asp Leu Ala Glu Lys Gly Arg Pro 195 200 205 Phe Thr Ser Asn Leu Ile Ile Ala Thr Thr Asn Leu Pro His Phe Ser 210 215 220 Pro Val Thr Ile Ala Asp Pro Ser Ala Val Ser Arg Arg Ile Asn Tyr 225 230 235 240Asp Leu Thr Leu Glu Val Ser Glu Ala Tyr Lys Lys His Thr Arg Leu 245 250 255 Asn Phe Asp Leu Ala Phe Arg Arg Thr Asp Ala Pro Pro Ile Tyr Pro 260 265 270 Phe Ala Ala His Val Pro Phe Val Asp Val Ala Val Arg Phe Lys Asn 275 280 285 Gly His Gln Asn Phe Asn Leu Leu Glu Leu Val Asp Ser Ile Cys Thr 290 295 300 Asp Ile Arg Ala Lys Gln Gln Gly Ala Arg Asn Met Gln Thr Leu Val 305 310 315 320Leu Gln 15270DNASeneca Valley Virus 15agccccaacg agaatgatga cacccccgtc gacgaggcgt tgggtagagt tctctccccc 60gctgcggtcg atgaggcgct tgtcgacctc actccagagg ccgacccggt tggccgtttg 120gctattcttg ccaagctagg tcttgcccta gctgcggtca cccctggtct gataatcttg 180gcagtgggac tctacaggta cttctctggc tctgatgcag accaagaaga aacagaaagt 240gagggatctg tcaaggcacc caggagcgaa 2701690PRTSeneca Valley Virus 16Ser Pro Asn Glu Asn Asp Asp Thr Pro Val Asp Glu Ala Leu Gly Arg 1 5 10 15 Val Leu Ser Pro Ala Ala Val Asp Glu Ala Leu Val Asp Leu Thr Pro 20 25 30 Glu Ala Asp Pro Val Gly Arg Leu Ala Ile Leu Ala Lys Leu Gly Leu 35 40 45 Ala Leu Ala Ala Val Thr

Pro Gly Leu Ile Ile Leu Ala Val Gly Leu 50 55 60 Tyr Arg Tyr Phe Ser Gly Ser Asp Ala Asp Gln Glu Glu Thr Glu Ser 65 70 75 80Glu Gly Ser Val Lys Ala Pro Arg Ser Glu 85 901766DNASeneca Valley Virus 17aatgcttatg acggcccgaa gaaaaactct aagccccctg gagcactctc tctcatggaa 60atgcaa 661822PRTSeneca Valley Virus 18Asn Ala Tyr Asp Gly Pro Lys Lys Asn Ser Lys Pro Pro Gly Ala Leu 1 5 10 15 Ser Leu Met Glu Met Gln 20 19633DNASeneca Valley Virus 19cagcccaacg tggacatggg ctttgaggct gcggtcgcta agaaagtggt cgtccccatt 60accttcatgg ttcccaacag accttctggg cttacacagt ccgctcttct ggtgaccggc 120cggaccttcc taatcaatga acatacatgg tccaatccct cctggaccag cttcacaatc 180cgcggtgagg tacacactcg tgatgagccc ttccaaacgg ttcatttcac tcaccacggt 240attcccacag atctgatgat ggtacgtctc ggaccgggca attctttccc taacaatcta 300gacaagtttg gacttgacca gatgccggca cgcaactccc gtgtggttgg cgtttcgtcc 360agttacggaa acttcttctt ctctggaaat ttcctcggat ttgttgattc cgtcacctct 420gaacaaggaa cttacgcaag actctttagg tacagggtga cgacctacaa aggatggtgc 480ggctcggccc tggtctgtga ggccggtggc gtccgacgca tcattggcct gcattctgct 540ggcgccgccg gtatcggcgc cgggacctat atctcaaaat taggactaat caaagccctg 600aaacacctcg gtgaaccttt ggccacaatg caa 63320211PRTSeneca Valley Virus 20Gln Pro Asn Val Asp Met Gly Phe Glu Ala Ala Val Ala Lys Lys Val 1 5 10 15 Val Val Pro Ile Thr Phe Met Val Pro Asn Arg Pro Ser Gly Leu Thr 20 25 30 Gln Ser Ala Leu Leu Val Thr Gly Arg Thr Phe Leu Ile Asn Glu His 35 40 45 Thr Trp Ser Asn Pro Ser Trp Thr Ser Phe Thr Ile Arg Gly Glu Val 50 55 60 His Thr Arg Asp Glu Pro Phe Gln Thr Val His Phe Thr His His Gly 65 70 75 80Ile Pro Thr Asp Leu Met Met Val Arg Leu Gly Pro Gly Asn Ser Phe 85 90 95 Pro Asn Asn Leu Asp Lys Phe Gly Leu Asp Gln Met Pro Ala Arg Asn 100 105 110 Ser Arg Val Val Gly Val Ser Ser Ser Tyr Gly Asn Phe Phe Phe Ser 115 120 125 Gly Asn Phe Leu Gly Phe Val Asp Ser Val Thr Ser Glu Gln Gly Thr 130 135 140 Tyr Ala Arg Leu Phe Arg Tyr Arg Val Thr Thr Tyr Lys Gly Trp Cys 145 150 155 160Gly Ser Ala Leu Val Cys Glu Ala Gly Gly Val Arg Arg Ile Ile Gly 165 170 175 Leu His Ser Ala Gly Ala Ala Gly Ile Gly Ala Gly Thr Tyr Ile Ser 180 185 190 Lys Leu Gly Leu Ile Lys Ala Leu Lys His Leu Gly Glu Pro Leu Ala 195 200 205 Thr Met Gln 210 211389DNASeneca Valley Virus 21ggactgatga ctgaattaga gcctggaatc accgtacatg taccccggaa atccaaattg 60agaaagacga ccgcacacgc ggtgtacaaa ccggagtttg agcctgctgt gttgtcaaaa 120tttgatccca gactgaacaa ggatgttgac ttggatgaag taatttggtc taaacacact 180gccaatgtcc cttaccaacc tcctttgttc tacacataca tgtcagagta cgctcatcga 240gtcttctcct tcttggggaa agacaatgac attctgaccg tcaaagaagc aattctgggc 300atccccggac tagaccccat ggatccccac acagctccgg gtctgcctta cgccatcaac 360ggccttcgac gtactgatct cgtcgatttt gtgaacggta cagtagatgc ggcgctggct 420gtacaaatcc agaaattctt agacggtgac tactctgacc atgtcttcca aacttttctg 480aaagatgaga tcagaccctc agagaaagtc cgagcgggaa aaacccgcat tgttgatgtg 540ccctccctgg cgcattgcat tgtgggcaga atgttgcttg ggcgctttgc tgccaagttt 600caatcccatc ctggctttct cctcggctct gctatcgggt ctgaccctga tgttttctgg 660accgtcatag gggctcaact cgaggggaga aagaacacgt atgacgtgga ctacagtgcc 720tttgactctt cacacggcac tggctccttc gaggctctca tctctcactt tttcaccgtg 780gacaatggtt ttagccctgc gctgggaccg tatctcagat ccctggctgt ctcggtgcac 840gcttacggcg agcgtcgcat caagattacc ggtggcctcc cctccggttg tgccgcgacc 900agcctgctga acacagtgct caacaatgtg atcatcagga ctgctctggc attgacttac 960aaggaatttg agtatgacac ggttgatatc atcgcctacg gtgacgacct tctggttggc 1020acggattacg atctggactt caatgaggtg gcacgacgcg ctgccaagtt ggggtataag 1080atgactcctg ccaacaaggg ttctgtcttc cctccgactt cctctctttc cgatgctgtt 1140tttctaaagc gcaaattcgt ccaaaacaac gacggcttat acaaaccagt tatggattta 1200aagaatttgg aagccatgct ctcctacttc aaaccaggaa cactactcga gaagctgcaa 1260tctgtttcta tgttggctca acattctgga aaagaagaat atgatagatt gatgcacccc 1320ttcgctgact acggtgccgt accgagtcac gagtacctgc aggcaagatg gagggccttg 1380ttcgactga 138922462PRTSeneca Valley Virus 22Gly Leu Met Thr Glu Leu Glu Pro Gly Ile Thr Val His Val Pro Arg 1 5 10 15 Lys Ser Lys Leu Arg Lys Thr Thr Ala His Ala Val Tyr Lys Pro Glu 20 25 30 Phe Glu Pro Ala Val Leu Ser Lys Phe Asp Pro Arg Leu Asn Lys Asp 35 40 45 Val Asp Leu Asp Glu Val Ile Trp Ser Lys His Thr Ala Asn Val Pro 50 55 60 Tyr Gln Pro Pro Leu Phe Tyr Thr Tyr Met Ser Glu Tyr Ala His Arg 65 70 75 80Val Phe Ser Phe Leu Gly Lys Asp Asn Asp Ile Leu Thr Val Lys Glu 85 90 95 Ala Ile Leu Gly Ile Pro Gly Leu Asp Pro Met Asp Pro His Thr Ala 100 105 110 Pro Gly Leu Pro Tyr Ala Ile Asn Gly Leu Arg Arg Thr Asp Leu Val 115 120 125 Asp Phe Val Asn Gly Thr Val Asp Ala Ala Leu Ala Val Gln Ile Gln 130 135 140 Lys Phe Leu Asp Gly Asp Tyr Ser Asp His Val Phe Gln Thr Phe Leu 145 150 155 160Lys Asp Glu Ile Arg Pro Ser Glu Lys Val Arg Ala Gly Lys Thr Arg 165 170 175 Ile Val Asp Val Pro Ser Leu Ala His Cys Ile Val Gly Arg Met Leu 180 185 190 Leu Gly Arg Phe Ala Ala Lys Phe Gln Ser His Pro Gly Phe Leu Leu 195 200 205 Gly Ser Ala Ile Gly Ser Asp Pro Asp Val Phe Trp Thr Val Ile Gly 210 215 220 Ala Gln Leu Glu Gly Arg Lys Asn Thr Tyr Asp Val Asp Tyr Ser Ala 225 230 235 240Phe Asp Ser Ser His Gly Thr Gly Ser Phe Glu Ala Leu Ile Ser His 245 250 255 Phe Phe Thr Val Asp Asn Gly Phe Ser Pro Ala Leu Gly Pro Tyr Leu 260 265 270 Arg Ser Leu Ala Val Ser Val His Ala Tyr Gly Glu Arg Arg Ile Lys 275 280 285 Ile Thr Gly Gly Leu Pro Ser Gly Cys Ala Ala Thr Ser Leu Leu Asn 290 295 300 Thr Val Leu Asn Asn Val Ile Ile Arg Thr Ala Leu Ala Leu Thr Tyr 305 310 315 320Lys Glu Phe Glu Tyr Asp Thr Val Asp Ile Ile Ala Tyr Gly Asp Asp 325 330 335 Leu Leu Val Gly Thr Asp Tyr Asp Leu Asp Phe Asn Glu Val Ala Arg 340 345 350 Arg Ala Ala Lys Leu Gly Tyr Lys Met Thr Pro Ala Asn Lys Gly Ser 355 360 365 Val Phe Pro Pro Thr Ser Ser Leu Ser Asp Ala Val Phe Leu Lys Arg 370 375 380 Lys Phe Val Gln Asn Asn Asp Gly Leu Tyr Lys Pro Val Met Asp Leu 385 390 395 400Lys Asn Leu Glu Ala Met Leu Ser Tyr Phe Lys Pro Gly Thr Leu Leu 405 410 415 Glu Lys Leu Gln Ser Val Ser Met Leu Ala Gln His Ser Gly Lys Glu 420 425 430 Glu Tyr Asp Arg Leu Met His Pro Phe Ala Asp Tyr Gly Ala Val Pro 435 440 445 Ser His Glu Tyr Leu Gln Ala Arg Trp Arg Ala Leu Phe Asp 450 455 460 232292PRTEncephalomyocarditis virus 23Met Ala Thr Thr Met Glu Gln Glu Thr Cys Ala His Ser Leu Thr Phe 1 5 10 15 Glu Glu Cys Pro Lys Cys Ser Ala Leu Gln Tyr Arg Asn Gly Phe Tyr 20 25 30 Leu Leu Lys Tyr Asp Glu Glu Trp Tyr Pro Glu Glu Leu Leu Thr Asp 35 40 45 Gly Glu Asp Asp Val Phe Asp Pro Glu Leu Asp Met Glu Val Val Phe 50 55 60 Glu Leu Gln Gly Asn Ser Thr Ser Ser Asp Lys Asn Asn Ser Ser Ser 65 70 75 80Glu Gly Asn Glu Gly Val Ile Ile Asn Asn Phe Tyr Ser Asn Gln Tyr 85 90 95 Gln Asn Ser Ile Asp Leu Ser Ala Asn Ala Ala Gly Ser Asp Pro Pro 100 105 110 Arg Thr Tyr Gly Gln Phe Ser Asn Leu Phe Ser Gly Ala Val Asn Ala 115 120 125 Phe Ser Asn Met Leu Pro Leu Leu Ala Asp Gln Asn Thr Glu Glu Met 130 135 140 Glu Asn Leu Ser Asp Arg Val Ser Gln Asp Thr Ala Gly Asn Thr Val 145 150 155 160Thr Asn Thr Gln Ser Thr Val Gly Arg Leu Val Gly Tyr Gly Thr Val 165 170 175 His Asp Gly Glu His Pro Ala Ser Cys Ala Asp Thr Ala Ser Glu Lys 180 185 190 Ile Leu Ala Val Glu Arg Tyr Tyr Thr Phe Lys Val Asn Asp Trp Thr 195 200 205 Ser Thr Gln Lys Pro Phe Glu Tyr Ile Arg Ile Pro Leu Pro His Val 210 215 220 Leu Ser Gly Glu Asp Gly Gly Val Phe Gly Ala Ala Leu Arg Arg His 225 230 235 240Tyr Leu Val Lys Thr Gly Trp Arg Val Gln Val Gln Cys Asn Ala Ser 245 250 255 Gln Phe His Ala Gly Ser Leu Leu Val Phe Met Ala Pro Glu Tyr Pro 260 265 270 Thr Leu Asp Ala Phe Ala Met Asp Asn Arg Trp Ser Lys Asp Asn Leu 275 280 285 Pro Asn Gly Thr Arg Thr Gln Thr Asn Lys Lys Gly Pro Phe Ala Met 290 295 300 Asp His Gln Asn Phe Trp Gln Trp Thr Leu Tyr Pro His Gln Phe Leu 305 310 315 320Asn Leu Arg Thr Asn Thr Thr Val Asp Leu Glu Val Pro Tyr Val Asn 325 330 335 Ile Ala Pro Thr Ser Ser Trp Thr Gln His Ala Ser Trp Thr Leu Val 340 345 350 Ile Ala Val Val Ala Pro Leu Thr Tyr Ser Thr Gly Ala Ser Thr Ser 355 360 365 Leu Asp Ile Thr Ala Ser Ile Gln Pro Val Arg Pro Val Phe Asn Gly 370 375 380 Leu Arg His Glu Thr Leu Ser Arg Gln Ser Pro Ile Pro Val Thr Ile 385 390 395 400Arg Glu His Ala Gly Thr Trp Tyr Ser Thr Leu Pro Asp Ser Thr Val 405 410 415 Pro Ile Tyr Gly Lys Thr Pro Val Ala Pro Ser Asn Tyr Met Val Gly 420 425 430 Glu Tyr Lys Asp Phe Leu Glu Ile Ala Gln Ile Pro Thr Phe Ile Gly 435 440 445 Asn Lys Ile Pro Asn Ala Val Pro Tyr Ile Glu Ala Ser Asn Thr Ala 450 455 460 Val Lys Thr Gln Pro Leu Ala Thr Tyr Gln Val Thr Leu Ser Cys Ser 465 470 475 480Cys Leu Ala Asn Thr Phe Leu Ala Ala Leu Ser Arg Asn Phe Ala Gln 485 490 495 Tyr Arg Gly Ser Leu Val Tyr Thr Phe Val Phe Thr Gly Thr Ala Met 500 505 510 Met Lys Gly Lys Phe Leu Ile Ala Tyr Thr Pro Pro Gly Ala Gly Lys 515 520 525 Pro Thr Ser Arg Asp Gln Ala Met Gln Ala Thr Tyr Ala Ile Trp Asp 530 535 540 Leu Gly Leu Asn Ser Ser Tyr Ser Phe Thr Val Pro Phe Ile Ser Pro 545 550 555 560Thr His Phe Arg Met Val Gly Thr Asp Gln Val Asn Ile Thr Asn Ala 565 570 575 Asp Gly Trp Val Thr Val Trp Gln Leu Thr Pro Leu Thr Tyr Pro Pro 580 585 590 Gly Cys Pro Thr Ser Ala Lys Ile Leu Thr Met Val Ser Ala Gly Lys 595 600 605 Asp Phe Ser Leu Lys Met Pro Ile Ser Pro Ala Pro Trp Ser Pro Gln 610 615 620 Gly Val Glu Asn Ala Glu Lys Gly Val Thr Glu Asn Thr Asn Ala Thr 625 630 635 640Ala Asp Phe Val Ala Gln Pro Val Tyr Leu Pro Glu Asn Gln Thr Lys 645 650 655 Val Ala Phe Phe Tyr Asn Arg Ser Ser Pro Ile Gly Ala Phe Thr Val 660 665 670 Lys Ser Gly Ser Leu Glu Ser Gly Phe Ala Pro Phe Ser Asn Gly Thr 675 680 685 Cys Pro Asn Ser Val Ile Leu Thr Pro Gly Pro Gln Phe Asp Pro Ala 690 695 700 Tyr Asp Gln Leu Arg Pro Gln Arg Leu Thr Glu Ile Trp Gly Asn Gly 705 710 715 720Asn Glu Glu Thr Ser Lys Val Phe Pro Leu Lys Ser Lys Gln Asp Tyr 725 730 735 Ser Phe Cys Leu Phe Ser Pro Phe Val Tyr Tyr Lys Cys Asp Leu Glu 740 745 750 Val Thr Leu Ser Pro His Thr Ser Gly Asn His Gly Leu Leu Val Arg 755 760 765 Trp Cys Pro Thr Gly Thr Pro Thr Lys Pro Thr Thr Gln Val Leu His 770 775 780 Glu Val Ser Ser Leu Ser Glu Gly Arg Thr Pro Gln Val Tyr Ser Ala 785 790 795 800Gly Pro Gly Ile Ser Asn Gln Ile Ser Phe Val Val Pro Tyr Asn Ser 805 810 815 Pro Leu Ser Val Leu Ser Ala Val Trp Tyr Asn Gly His Lys Arg Phe 820 825 830 Asp Asn Thr Gly Ser Leu Gly Ile Ala Pro Asn Ser Asp Phe Gly Thr 835 840 845 Leu Phe Phe Ala Gly Thr Lys Pro Asp Ile Lys Phe Thr Val Tyr Leu 850 855 860 Arg Tyr Lys Asn Lys Arg Val Phe Cys Pro Arg Pro Thr Val Phe Phe 865 870 875 880Pro Trp Pro Thr Ser Gly Asp Lys Ile Asp Met Thr Pro Arg Ala Gly 885 890 895 Val Leu Met Leu Glu Ser Pro Asn Ala Leu Asp Ile Ser Arg Thr Tyr 900 905 910 Pro Thr Leu His Val Leu Ile Gln Phe Asn His Arg Gly Leu Glu Val 915 920 925 Arg Leu Phe Arg His Gly His Phe Trp Ala Glu Thr Arg Ala Asp Val 930 935 940 Ile Leu Arg Ser Lys Thr Lys Gln Val Ser Phe Leu Ser Asn Gly Asn 945 950 955 960Tyr Pro Ser Met Asp Ser Arg Ala Pro Trp Asn Pro Trp Lys Asn Thr 965 970 975 Tyr Gln Ala Val Leu Arg Ala Glu Pro Cys Arg Val Thr Met Asp Ile 980 985 990 Tyr Tyr Lys Arg Val Arg Pro Phe Arg Leu Pro Leu Val Gln Lys Glu 995 1000 1005 Trp Pro Val Arg Glu Glu Asn Val Phe Gly Leu Tyr Arg Ile Phe Asn 1010 1015 1020 Ala His Tyr Ala Gly Tyr Phe Ala Asp Leu Leu Ile His Asp Ile Glu 1025 1030 1035 1040Thr Asn Pro Gly Pro Phe Met Phe Arg Pro Arg Lys Gln Val Phe Gln 1045 1050 1055 Thr Gln Gly Ala Ala Val Ser Ser Met Ala Gln Thr Leu Leu Pro Asn 1060 1065 1070 Asp Leu Ala Ser Lys Ala Met Gly Ser Ala Phe Thr Ala Leu Leu Asp 1075 1080 1085 Ala Asn Glu Asp Ala Gln Lys Ala Met Lys Ile Ile Lys Thr Leu Ser 1090 1095 1100 Ser Leu Ser Asp Ala Trp Glu Asn Val Lys Glu Thr Leu Asn Asn Pro 1105 1110 1115 1120Glu Phe Trp Lys Gln Leu Leu Ser Arg Cys Val Gln Leu Ile Ala Gly 1125 1130 1135 Met Thr Ile Ala Val Met His Pro Asp Pro Leu Thr Leu Leu Cys Leu 1140 1145 1150 Gly Thr Leu Thr Ala Ala Glu Ile Thr Ser Gln Thr Ser Leu Cys Glu 1155 1160 1165 Glu Ile Ala Ala Lys Phe Lys Thr Ile Phe Ile Thr Pro Pro Pro Arg 1170 1175 1180

Phe Pro Thr Ile Ser Leu Phe Gln Gln Gln Ser Pro Leu Lys Gln Val 1185 1190 1195 1200Asn Asp Ile Phe Ser Leu Ala Lys Asn Leu Asp Trp Ala Val Lys Thr 1205 1210 1215 Val Glu Lys Val Val Asp Trp Phe Gly Thr Trp Ile Val Gln Glu Glu 1220 1225 1230 Lys Glu Gln Thr Leu Asp Gln Leu Leu Gln Arg Phe Pro Glu His Ala 1235 1240 1245 Lys Arg Ile Ser Asp Leu Arg Asn Gly Met Ala Ala Tyr Val Glu Cys 1250 1255 1260 Lys Glu Ser Phe Asp Phe Phe Glu Lys Leu Tyr Asn Gln Ala Val Lys 1265 1270 1275 1280Glu Lys Arg Thr Gly Ile Ala Ala Val Cys Glu Lys Phe Arg Gln Lys 1285 1290 1295 His Asp His Ala Thr Ala Arg Cys Glu Pro Val Val Ile Val Leu Arg 1300 1305 1310 Gly Asp Ala Gly Gln Gly Lys Ser Leu Ser Ser Gln Val Ile Ala Gln 1315 1320 1325 Ala Val Ser Lys Thr Ile Phe Gly Arg Gln Ser Val Tyr Ser Leu Pro 1330 1335 1340 Pro Asp Ser Asp Phe Phe Asp Gly Tyr Glu Asn Gln Phe Ala Ala Ile 1345 1350 1355 1360Met Asp Asp Leu Gly Gln Asn Pro Asp Gly Ser Asp Phe Thr Thr Phe 1365 1370 1375 Cys Gln Met Val Ser Thr Thr Asn Phe Leu Pro Asn Met Ala Ser Leu 1380 1385 1390 Glu Arg Lys Gly Thr Pro Phe Thr Ser Gln Leu Val Val Ala Thr Thr 1395 1400 1405 Asn Leu Pro Glu Phe Arg Pro Val Thr Ile Ala His Tyr Pro Ala Val 1410 1415 1420 Glu Arg Arg Ile Thr Phe Asp Tyr Ser Val Ser Ala Gly Pro Val Cys 1425 1430 1435 1440Ser Lys Thr Glu Ala Gly Tyr Lys Val Leu Asp Val Glu Arg Ala Phe 1445 1450 1455 Arg Pro Thr Gly Glu Ala Pro Leu Pro Cys Phe Gln Asn Asn Cys Leu 1460 1465 1470 Phe Leu Glu Lys Ala Gly Leu Gln Phe Arg Asp Asn Arg Thr Lys Glu 1475 1480 1485 Ile Ile Ser Leu Val Asp Val Ile Glu Arg Ala Val Ala Arg Ile Glu 1490 1495 1500 Arg Lys Lys Lys Val Leu Thr Thr Val Gln Thr Leu Val Ala Gln Gly 1505 1510 1515 1520Pro Val Asp Glu Val Ser Phe His Ser Val Val Gln Gln Leu Lys Ala 1525 1530 1535 Arg Gln Gln Ala Thr Asp Glu Gln Leu Glu Glu Leu Gln Glu Ala Phe 1540 1545 1550 Ala Lys Val Gln Glu Arg Asn Ser Val Phe Ser Asp Trp Leu Lys Ile 1555 1560 1565 Ser Ala Met Leu Cys Ala Ala Thr Leu Ala Leu Ser Gln Val Val Lys 1570 1575 1580 Met Ala Lys Ala Val Lys Gln Met Val Lys Pro Asp Leu Val Arg Val 1585 1590 1595 1600Gln Leu Asp Glu Gln Glu Gln Gly Pro Tyr Asn Glu Thr Ala Arg Val 1605 1610 1615 Lys Pro Lys Thr Leu Gln Leu Leu Asp Ile Gln Gly Pro Asn Pro Val 1620 1625 1630 Met Asp Phe Glu Lys Tyr Val Ala Lys His Val Thr Ala Pro Ile Gly 1635 1640 1645 Phe Val Tyr Pro Thr Gly Val Ser Thr Gln Thr Cys Leu Leu Val Arg 1650 1655 1660 Gly Arg Thr Leu Val Val Asn Arg His Met Ala Glu Ser Asp Trp Thr 1665 1670 1675 1680Ser Ile Val Val Arg Gly Val Thr His Ala Arg Ser Thr Val Lys Ile 1685 1690 1695 Leu Ala Ile Ala Lys Ala Gly Lys Glu Thr Asp Val Ser Phe Ile Arg 1700 1705 1710 Leu Ser Ser Gly Pro Leu Phe Arg Asp Asn Thr Ser Lys Phe Val Lys 1715 1720 1725 Ala Gly Asp Val Leu Pro Thr Gly Ala Ala Pro Val Thr Gly Ile Met 1730 1735 1740 Asn Thr Asp Ile Pro Met Met Tyr Thr Gly Thr Phe Leu Lys Ala Gly 1745 1750 1755 1760Val Ser Val Pro Val Glu Thr Gly Gln Thr Phe Asn His Cys Ile His 1765 1770 1775 Tyr Lys Ala Asn Thr Arg Lys Gly Trp Cys Gly Ser Ala Leu Leu Ala 1780 1785 1790 Asp Leu Gly Gly Ser Lys Lys Ile Leu Gly Ile His Ser Ala Gly Ser 1795 1800 1805 Met Gly Ile Ala Ala Ala Ser Ile Val Ser Gln Glu Met Ile Arg Ala 1810 1815 1820 Val Val Asn Ala Phe Glu Pro Gln Gly Ala Leu Glu Arg Leu Pro Asp 1825 1830 1835 1840Gly Pro Arg Ile His Val Pro Arg Lys Thr Ala Leu Arg Pro Thr Val 1845 1850 1855 Ala Arg Gln Val Phe Gln Pro Ala Tyr Ala Pro Ala Val Leu Ser Lys 1860 1865 1870 Phe Asp Pro Arg Thr Glu Ala Asp Val Asp Glu Val Ala Phe Ser Lys 1875 1880 1885 His Thr Ser Asn Gln Glu Ser Leu Pro Pro Val Phe Arg Met Val Ala 1890 1895 1900 Lys Glu Tyr Ala Asn Arg Val Phe Thr Leu Leu Gly Lys Asp Asn Gly 1905 1910 1915 1920Arg Leu Thr Val Lys Gln Ala Leu Glu Gly Leu Glu Gly Met Asp Pro 1925 1930 1935 Met Asp Arg Asn Thr Ser Pro Gly Leu Pro Tyr Thr Ala Leu Gly Met 1940 1945 1950 Arg Arg Thr Asp Val Val Asp Trp Glu Ser Ala Thr Leu Ile Pro Phe 1955 1960 1965 Ala Ala Glu Arg Leu Arg Lys Met Asn Glu Gly Asp Phe Ser Glu Val 1970 1975 1980 Val Tyr Gln Thr Phe Leu Lys Asp Glu Leu Arg Pro Ile Glu Lys Val 1985 1990 1995 2000Gln Ala Ala Lys Thr Arg Ile Val Asp Val Pro Pro Phe Glu His Cys 2005 2010 2015 Ile Leu Gly Arg Gln Leu Leu Gly Lys Phe Ala Ser Lys Phe Gln Thr 2020 2025 2030 Gln Pro Gly Leu Glu Leu Gly Ser Ala Ile Gly Cys Asp Pro Asp Val 2035 2040 2045 His Trp Thr Ala Phe Gly Val Ala Met Gln Gly Phe Glu Arg Val Tyr 2050 2055 2060 Asp Val Asp Tyr Ser Asn Phe Asp Ser Thr His Ser Val Ala Met Phe 2065 2070 2075 2080Arg Leu Leu Ala Glu Glu Phe Phe Thr Pro Glu Asn Gly Phe Asp Pro 2085 2090 2095 Leu Thr Arg Glu Tyr Leu Glu Ser Leu Ala Ile Ser Thr His Ala Phe 2100 2105 2110 Glu Glu Lys Arg Phe Leu Ile Thr Gly Gly Leu Pro Ser Gly Cys Ala 2115 2120 2125 Ala Thr Ser Met Leu Asn Thr Ile Met Asn Asn Ile Ile Ile Arg Ala 2130 2135 2140 Gly Leu Tyr Leu Thr Tyr Lys Asn Phe Glu Phe Asp Asp Val Lys Val 2145 2150 2155 2160Leu Ser Tyr Gly Asp Asp Leu Leu Val Ala Thr Asn Tyr Gln Leu Asp 2165 2170 2175 Phe Asp Lys Val Arg Ala Ser Leu Ala Lys Thr Gly Tyr Lys Ile Thr 2180 2185 2190 Pro Ala Asn Lys Thr Ser Thr Phe Pro Leu Asn Ser Thr Leu Glu Asp 2195 2200 2205 Val Val Phe Leu Lys Arg Lys Phe Lys Lys Glu Gly Pro Leu Tyr Arg 2210 2215 2220 Pro Val Met Asn Arg Glu Ala Leu Glu Ala Met Leu Ser Tyr Tyr Arg 2225 2230 2235 2240Pro Gly Thr Leu Ser Glu Lys Leu Thr Ser Ile Thr Met Leu Ala Val 2245 2250 2255 His Ser Gly Lys Gln Glu Tyr Asp Arg Leu Phe Ala Pro Phe Arg Glu 2260 2265 2270 Val Gly Val Val Val Pro Ser Phe Glu Ser Val Glu Tyr Arg Trp Arg 2275 2280 2285 Ser Leu Phe Trp 2290 242292PRTEncephalomyocarditis virus 24Met Ala Thr Thr Met Glu Gln Glu Thr Cys Ala His Ser Leu Thr Phe 1 5 10 15 Glu Glu Cys Pro Lys Cys Ser Ala Leu Gln Tyr Arg Asn Gly Phe Tyr 20 25 30 Leu Leu Lys Tyr Asp Glu Glu Trp Tyr Pro Glu Glu Leu Leu Thr Asp 35 40 45 Gly Glu Asp Asp Val Phe Asp Pro Glu Leu Asp Met Glu Val Val Phe 50 55 60 Glu Leu Gln Gly Asn Ser Thr Ser Ser Asp Lys Asn Asn Ser Ser Ser 65 70 75 80Glu Gly Asn Glu Gly Val Ile Ile Asn Asn Phe Tyr Ser Asn Gln Tyr 85 90 95 Gln Asn Ser Ile Asp Leu Ser Ala Asn Ala Ala Gly Ser Asp Pro Pro 100 105 110 Arg Thr Tyr Gly Gln Phe Ser Asn Leu Phe Ser Gly Ala Val Asn Ala 115 120 125 Phe Ser Asn Met Leu Pro Leu Leu Ala Asp Gln Asn Thr Glu Glu Met 130 135 140 Glu Asn Leu Ser Asp Arg Val Ser Gln Asp Thr Ala Gly Asn Thr Val 145 150 155 160Thr Asn Thr Gln Ser Thr Val Gly Arg Leu Val Gly Tyr Gly Thr Val 165 170 175 His Asp Gly Glu His Pro Ala Ser Cys Ala Asp Thr Ala Ser Glu Lys 180 185 190 Ile Leu Ala Val Glu Arg Tyr Tyr Thr Phe Lys Val Asn Asp Trp Thr 195 200 205 Ser Thr Gln Lys Pro Phe Glu Tyr Ile Arg Ile Pro Leu Pro His Val 210 215 220 Leu Ser Gly Glu Asp Gly Gly Val Phe Gly Ala Ala Leu Arg Arg His 225 230 235 240Tyr Leu Val Lys Thr Gly Trp Arg Val Gln Val Gln Cys Asn Ala Ser 245 250 255 Gln Phe His Ala Gly Ser Leu Leu Val Phe Met Ala Pro Glu Tyr Pro 260 265 270 Thr Leu Asp Thr Phe Val Met Asp Asn Arg Trp Ser Lys Asp Asn Leu 275 280 285 Pro Asn Gly Ala Arg Thr Gln Thr Asn Lys Lys Gly Pro Phe Ala Met 290 295 300 Asp His Gln Asn Phe Trp Gln Trp Thr Leu Tyr Pro His Gln Phe Leu 305 310 315 320Asn Leu Arg Thr Asn Thr Thr Val Asp Leu Glu Val Pro Tyr Val Asn 325 330 335 Ile Ala Pro Thr Ser Ser Trp Thr Gln His Ala Ser Trp Thr Leu Val 340 345 350 Ile Ala Val Val Ala Pro Leu Thr Tyr Ser Thr Gly Ala Ser Thr Ser 355 360 365 Leu Asp Ile Thr Ala Ser Ile Gln Pro Val Arg Pro Val Phe Asn Gly 370 375 380 Leu Arg His Glu Thr Leu Ser Arg Gln Ser Pro Ile Pro Val Thr Ile 385 390 395 400Arg Glu His Ala Gly Thr Trp Tyr Ser Thr Leu Pro Asp Ser Thr Val 405 410 415 Pro Ile Tyr Gly Lys Thr Pro Val Ala Pro Ser Asn Tyr Met Val Gly 420 425 430 Glu Tyr Lys Asp Phe Leu Glu Ile Ala Gln Ile Pro Thr Phe Ile Gly 435 440 445 Asn Lys Ile Pro Asn Ala Val Pro Tyr Ile Glu Ala Ser Asn Thr Ala 450 455 460 Val Lys Thr Gln Pro Leu Ala Thr Tyr Gln Val Thr Leu Ser Cys Ser 465 470 475 480Cys Leu Ala Asn Thr Phe Leu Ala Ala Leu Ser Arg Asn Phe Ala Gln 485 490 495 Tyr Arg Gly Ser Leu Val Tyr Thr Phe Val Phe Thr Gly Thr Ala Met 500 505 510 Met Lys Gly Lys Phe Leu Ile Ala Tyr Thr Pro Pro Gly Ala Gly Lys 515 520 525 Pro Thr Ser Arg Asp Gln Ala Met Gln Ala Thr Tyr Ala Ile Trp Asp 530 535 540 Leu Gly Leu Asn Ser Ser Tyr Ser Phe Thr Val Pro Phe Ile Ser Pro 545 550 555 560Thr His Phe Arg Met Val Gly Thr Asp Gln Val Asn Ile Thr Asn Ala 565 570 575 Asp Gly Trp Val Thr Val Trp Gln Leu Thr Pro Leu Thr Tyr Pro Pro 580 585 590 Gly Cys Pro Thr Ser Ala Lys Ile Leu Thr Met Val Ser Ala Gly Lys 595 600 605 Asp Phe Ser Leu Lys Met Pro Ile Ser Pro Ala Pro Trp Ser Pro Gln 610 615 620 Gly Val Glu Asn Ala Glu Lys Gly Val Thr Glu Asn Ala Asp Ala Thr 625 630 635 640Ala Asp Phe Val Ala Gln Pro Val Tyr Leu Pro Glu Asn Gln Thr Lys 645 650 655 Val Ala Phe Phe Tyr Asp Arg Ser Ser Pro Ile Gly Ala Phe Thr Val 660 665 670 Gln Ser Gly Ser Leu Glu Ser Gly Phe Ala Pro Phe Ser Asn Lys Thr 675 680 685 Cys Pro Asn Ser Val Ile Leu Thr Pro Gly Pro Gln Phe Asp Pro Ala 690 695 700 Tyr Asp Gln Leu Arg Pro Gln Arg Leu Thr Glu Ile Trp Gly Asn Gly 705 710 715 720Asn Glu Glu Thr Ser Lys Val Phe Pro Leu Lys Ser Lys Gln Asp Tyr 725 730 735 Ser Phe Cys Leu Phe Ser Pro Phe Val Tyr Tyr Lys Cys Asp Leu Glu 740 745 750 Val Thr Leu Ser Pro His Thr Ser Gly Asn His Gly Leu Leu Val Arg 755 760 765 Trp Cys Pro Thr Gly Thr Pro Thr Lys Pro Thr Thr Gln Val Leu His 770 775 780 Glu Val Ser Ser Leu Ser Glu Gly Arg Thr Pro Gln Val Tyr Ser Ala 785 790 795 800Gly Pro Gly Ile Ser Asn Gln Ile Ser Phe Val Val Pro Tyr Asn Ser 805 810 815 Pro Leu Ser Val Leu Pro Ala Val Trp Tyr Asn Gly His Lys Arg Phe 820 825 830 Asp Asn Thr Gly Ser Leu Gly Ile Ala Pro Asn Ser Asp Phe Gly Thr 835 840 845 Leu Phe Phe Ala Gly Thr Lys Pro Asp Ile Lys Phe Thr Val Tyr Leu 850 855 860 Arg Tyr Lys Asn Met Arg Val Phe Cys Pro Arg Pro Thr Val Phe Phe 865 870 875 880Pro Trp Pro Thr Ser Gly Asp Lys Ile Asp Met Thr Pro Arg Ala Gly 885 890 895 Val Leu Met Leu Glu Ser Pro Asn Ala Leu Asp Ile Ser Arg Thr Tyr 900 905 910 Pro Thr Leu His Val Leu Ile Gln Phe Asn His Arg Gly Leu Glu Val 915 920 925 Arg Leu Phe Arg His Gly Gln Phe Trp Ala Glu Thr Arg Ala Asp Val 930 935 940 Ile Leu Arg Ser Lys Thr Lys Gln Val Ser Phe Leu Ser Asn Gly Asn 945 950 955 960Tyr Pro Ser Met Asp Ser Arg Ala Pro Trp Asn Pro Trp Lys Asn Thr 965 970 975 Tyr Gln Ala Val Leu Arg Ala Glu Pro Cys Arg Val Thr Met Asp Ile 980 985 990 Tyr Tyr Lys Arg Val Arg Pro Phe Arg Leu Pro Leu Val Gln Lys Glu 995 1000 1005 Trp Arg Val Arg Glu Glu Asn Val Phe Gly Leu Tyr Arg Ile Phe Asn 1010 1015 1020 Ala His Tyr Ala Gly Tyr Phe Ala Asp Leu Leu Ile His Asp Ile Glu 1025 1030 1035 1040Thr Asn Pro Gly Pro Phe Met Phe Arg Pro Arg Lys Gln Val Phe Gln 1045 1050 1055 Thr Gln Gly Ala Ala Val Ser Ser Met Ala Gln Thr Leu Leu Pro Asn 1060 1065 1070 Asp Leu Ala Ser Lys Ala Met Gly Ser Ala Phe Thr Ala Leu Leu Asp 1075 1080 1085 Ala Asn Glu Asp Ala Arg Lys Ala Met Lys Ile Ile Lys Thr Leu Ser 1090 1095 1100 Ser Leu Ser Asp Ala Trp Glu Asn Val Lys Glu Thr Leu Asn Asn Pro 1105 1110 1115 1120Glu Phe Trp Lys Gln Leu Leu Ser Arg Cys Val Gln Leu Ile Ala Gly 1125 1130 1135 Met Thr Ile Ala Val Met His Pro Asp Pro Leu Thr Leu Leu Cys Leu 1140 1145 1150 Gly Thr Leu Thr Ala Ala Glu Ile Thr Ser Gln Thr Ser Leu Cys Glu 1155 1160

1165 Glu Ile Ala Ala Lys Phe Lys Thr Ile Phe Ile Thr Pro Pro Pro Arg 1170 1175 1180 Phe Pro Thr Ile Ser Leu Phe Gln Gln Gln Ser Pro Leu Lys Gln Val 1185 1190 1195 1200Asn Asp Phe Phe Ser Leu Ala Lys Asn Leu Asp Trp Ala Val Lys Thr 1205 1210 1215 Val Glu Lys Val Val Asp Trp Phe Gly Thr Trp Ile Val Gln Glu Glu 1220 1225 1230 Lys Glu Gln Thr Leu Asp Gln Leu Leu Gln Arg Phe Pro Glu His Ala 1235 1240 1245 Lys Arg Ile Ser Asp Leu Arg Asn Gly Met Ala Ala Tyr Val Glu Cys 1250 1255 1260 Lys Glu Ser Phe Asp Phe Phe Glu Lys Leu Tyr Asn Gln Ala Val Lys 1265 1270 1275 1280Glu Lys Arg Thr Gly Ile Ala Ala Val Cys Glu Lys Phe Arg Gln Lys 1285 1290 1295 His Asp His Ala Thr Ala Arg Cys Glu Pro Val Val Ile Val Leu Arg 1300 1305 1310 Gly Asp Ala Gly Gln Gly Lys Ser Leu Ser Ser Gln Val Ile Ala Gln 1315 1320 1325 Ala Val Ser Lys Thr Ile Phe Gly Arg Gln Ser Val Tyr Ser Leu Pro 1330 1335 1340 Pro Asp Ser Asp Phe Phe Asp Gly Tyr Glu Asn Gln Phe Ala Ala Ile 1345 1350 1355 1360Met Asp Asp Leu Gly Gln Asn Pro Asp Gly Ser Asp Phe Thr Thr Phe 1365 1370 1375 Cys Gln Met Val Ser Thr Thr Asn Phe Leu Pro Asn Met Ala Ser Leu 1380 1385 1390 Glu Arg Lys Gly Thr Pro Phe Thr Ser Gln Leu Val Val Ala Thr Thr 1395 1400 1405 Asn Leu Pro Glu Phe Arg Pro Val Thr Ile Ala His Tyr Pro Ala Val 1410 1415 1420 Glu Arg Arg Ile Thr Phe Asp Tyr Ser Val Ser Ala Gly Pro Val Cys 1425 1430 1435 1440Ser Lys Thr Glu Ala Gly Tyr Lys Val Leu Asp Val Glu Arg Ala Phe 1445 1450 1455 Arg Pro Thr Gly Glu Ala Pro Leu Pro Cys Phe Gln Asn Asn Cys Leu 1460 1465 1470 Phe Leu Glu Lys Ala Gly Leu Gln Phe Arg Asp Asn Arg Thr Lys Glu 1475 1480 1485 Ile Ile Ser Leu Val Asp Val Ile Glu Arg Ala Val Ala Arg Ile Glu 1490 1495 1500 Arg Lys Lys Lys Val Leu Thr Thr Val Gln Thr Leu Val Ala Gln Ala 1505 1510 1515 1520Pro Val Asp Glu Val Ser Phe His Ser Val Val Gln Gln Leu Lys Ala 1525 1530 1535 Arg Gln Glu Ala Thr Asp Glu Gln Leu Glu Glu Leu Gln Glu Ala Phe 1540 1545 1550 Ala Lys Val Gln Glu Arg Asn Ser Val Phe Ser Asp Trp Leu Lys Ile 1555 1560 1565 Ser Ala Met Leu Cys Ala Ala Thr Leu Ala Leu Ser Gln Val Val Lys 1570 1575 1580 Met Ala Lys Ala Val Lys Gln Met Val Lys Pro Asp Leu Val Arg Val 1585 1590 1595 1600Gln Leu Asp Glu Gln Glu Gln Gly Pro Tyr Asn Glu Thr Ala Arg Ala 1605 1610 1615 Lys Pro Lys Thr Leu Gln Leu Leu Asp Ile Gln Gly Pro Asn Pro Val 1620 1625 1630 Met Asp Phe Glu Lys Tyr Val Ala Lys His Val Thr Ala Pro Ile Asp 1635 1640 1645 Phe Val Tyr Pro Thr Gly Val Ser Thr Gln Thr Cys Leu Leu Val Arg 1650 1655 1660 Gly Arg Thr Leu Ala Val Asn Arg His Met Ala Glu Ser Asp Trp Thr 1665 1670 1675 1680Ser Ile Val Val Arg Gly Val Thr His Ala Arg Ser Thr Val Lys Ile 1685 1690 1695 Leu Ala Ile Ala Lys Ala Gly Lys Glu Thr Asp Val Ser Phe Ile Arg 1700 1705 1710 Leu Ser Ser Gly Pro Leu Phe Arg Asp Asn Thr Ser Lys Phe Val Lys 1715 1720 1725 Ala Gly Asp Val Leu Pro Thr Gly Ala Ala Pro Val Thr Gly Ile Met 1730 1735 1740 Asn Thr Asp Ile Pro Met Met Tyr Thr Gly Thr Phe Leu Lys Ala Gly 1745 1750 1755 1760Val Ser Val Pro Val Glu Thr Gly Gln Thr Phe Asn His Cys Ile His 1765 1770 1775 Tyr Lys Ala Asn Thr Arg Lys Gly Trp Cys Gly Ser Ala Leu Leu Ala 1780 1785 1790 Asp Leu Gly Gly Ser Lys Lys Ile Leu Gly Ile His Ser Ala Gly Ser 1795 1800 1805 Met Gly Ile Ala Ala Ala Ser Ile Val Ser Gln Glu Met Ile Arg Ala 1810 1815 1820 Val Val Asn Ala Phe Glu Pro Gln Gly Ala Leu Glu Arg Leu Pro Asp 1825 1830 1835 1840Gly Pro Arg Ile His Val Pro Arg Lys Thr Ala Leu Arg Pro Thr Val 1845 1850 1855 Ala Arg Gln Val Phe Gln Pro Ala Tyr Ala Pro Ala Val Leu Ser Lys 1860 1865 1870 Phe Asp Pro Arg Thr Glu Ala Asp Val Asp Glu Val Ala Phe Ser Lys 1875 1880 1885 His Thr Ser Asn Gln Glu Ser Leu Pro Pro Val Phe Arg Met Val Ala 1890 1895 1900 Lys Glu Tyr Ala Asn Arg Val Phe Thr Leu Leu Gly Lys Asp Asn Gly 1905 1910 1915 1920Arg Leu Thr Val Lys Gln Ala Leu Glu Gly Leu Glu Gly Met Asp Pro 1925 1930 1935 Met Asp Arg Asn Thr Ser Pro Gly Leu Pro Tyr Thr Ala Leu Gly Met 1940 1945 1950 Arg Arg Thr Asp Val Val Asp Trp Glu Ser Ala Thr Leu Ile Pro Phe 1955 1960 1965 Ala Ala Glu Arg Leu Arg Lys Met Asn Glu Gly Asp Phe Ser Glu Val 1970 1975 1980 Val Tyr Gln Thr Phe Leu Lys Asp Glu Leu Arg Pro Ile Glu Lys Val 1985 1990 1995 2000Gln Ala Ala Lys Thr Arg Ile Val Asp Val Pro Pro Phe Glu His Cys 2005 2010 2015 Ile Leu Gly Arg Gln Leu Leu Gly Lys Phe Ala Ser Lys Phe Gln Thr 2020 2025 2030 Gln Pro Gly Leu Glu Leu Gly Ser Ala Ile Gly Cys Asp Pro Asp Val 2035 2040 2045 His Trp Thr Ala Phe Gly Val Ala Met Gln Gly Phe Glu Arg Val Tyr 2050 2055 2060 Asp Val Asp Tyr Ser Asn Phe Asp Ser Thr His Ser Val Ala Met Phe 2065 2070 2075 2080Arg Leu Leu Ala Glu Glu Phe Phe Thr Pro Glu Asn Gly Phe Asp Pro 2085 2090 2095 Leu Thr Arg Glu Tyr Leu Glu Ser Leu Ala Ile Ser Thr His Ala Phe 2100 2105 2110 Glu Glu Lys Arg Phe Leu Ile Thr Gly Gly Leu Pro Ser Gly Cys Ala 2115 2120 2125 Ala Thr Ser Met Leu Asn Thr Ile Met Asn Asn Ile Ile Ile Arg Ala 2130 2135 2140 Gly Leu Tyr Leu Thr Tyr Lys Asn Phe Glu Phe Asp Asp Val Lys Val 2145 2150 2155 2160Leu Ser Tyr Gly Asp Asp Leu Leu Val Ala Thr Asn Tyr Gln Leu Asp 2165 2170 2175 Phe Asp Lys Val Arg Ala Ser Leu Ala Lys Thr Gly Tyr Lys Ile Thr 2180 2185 2190 Pro Ala Asn Lys Thr Ser Thr Phe Pro Leu Asn Ser Thr Leu Glu Asp 2195 2200 2205 Val Val Phe Leu Lys Arg Lys Phe Lys Lys Glu Gly Pro Leu Tyr Arg 2210 2215 2220 Pro Val Met Asn Arg Glu Ala Leu Glu Ala Met Leu Ser Tyr Tyr Arg 2225 2230 2235 2240Pro Gly Thr Leu Ser Glu Lys Leu Thr Ser Ile Thr Met Leu Ala Val 2245 2250 2255 His Ser Gly Lys Gln Glu Tyr Asp Arg Leu Phe Ala Pro Phe Arg Glu 2260 2265 2270 Val Gly Val Val Val Pro Ser Phe Glu Ser Val Glu Tyr Arg Trp Arg 2275 2280 2285 Ser Leu Phe Trp 2290 252292PRTEncephalomyocarditis virus 25Met Ala Thr Thr Met Glu Gln Glu Ile Cys Ala His Ser Leu Thr Leu 1 5 10 15 Lys Gly Cys Pro Lys Cys Ser Ala Leu Gln Tyr Arg Asn Gly Phe Tyr 20 25 30 Leu Leu Lys Tyr Asp Glu Glu Trp Tyr Pro Glu Glu Leu Leu Thr Asp 35 40 45 Gly Glu Asp Asp Val Phe Asp Pro Glu Leu Asp Met Glu Val Val Phe 50 55 60 Glu Leu Gln Gly Asn Ser Thr Ser Ser Asp Lys Asn Asn Ser Ser Ser 65 70 75 80Asp Gly Asn Glu Gly Val Ile Ile Asn Asn Phe Tyr Ser Asn Gln Tyr 85 90 95 Gln Asn Ser Ile Asp Leu Ser Ala Asn Ala Thr Gly Ser Asp Pro Pro 100 105 110 Arg Thr Tyr Gly Gln Phe Ser Asn Leu Leu Ser Gly Ala Val Asn Ala 115 120 125 Phe Ser Asn Met Ile Pro Leu Leu Ala Asp Gln Asn Thr Glu Glu Met 130 135 140 Glu Asn Leu Ser Asp Arg Val Leu Gln Asp Thr Ala Gly Asn Thr Val 145 150 155 160Thr Asn Thr Gln Ser Thr Val Gly Arg Leu Val Gly Tyr Gly Ala Val 165 170 175 His Asp Gly Glu His Pro Ala Ser Cys Ala Asp Thr Ala Ser Glu Lys 180 185 190 Ile Leu Ala Val Glu Arg Tyr Tyr Thr Phe Lys Val Asn Asp Trp Thr 195 200 205 Ser Thr Gln Lys Pro Phe Glu Tyr Ile Arg Ile Pro Leu Pro His Val 210 215 220 Leu Ser Gly Glu Asp Gly Gly Val Phe Gly Ala Ala Leu Arg Arg His 225 230 235 240Tyr Leu Val Lys Thr Gly Trp Arg Val Gln Val Gln Cys Asn Ala Ser 245 250 255 Gln Phe His Ala Gly Ser Leu Leu Val Phe Met Ala Pro Glu Tyr Pro 260 265 270 Thr Leu Asp Ala Phe Ala Met Asp Asn Arg Trp Ser Lys Asp Asn Leu 275 280 285 Pro Asn Gly Thr Lys Thr Gln Thr Asn Arg Lys Gly Pro Phe Ala Met 290 295 300 Asp His Gln Asn Phe Trp Gln Trp Thr Leu Tyr Pro His Gln Phe Leu 305 310 315 320Asn Leu Arg Thr Asn Thr Thr Val Asp Leu Glu Val Pro Tyr Val Asn 325 330 335 Ile Ala Pro Thr Ser Ser Trp Thr Gln His Ala Ser Trp Thr Leu Val 340 345 350 Ile Ala Val Val Ala Pro Leu Thr Tyr Ser Thr Gly Ala Ser Thr Ser 355 360 365 Leu Asp Ile Thr Ala Ser Ile Gln Pro Val Arg Pro Val Phe Asn Gly 370 375 380 Leu Arg His Glu Thr Leu Ser Arg Gln Ser Pro Ile Pro Val Thr Ile 385 390 395 400Arg Glu His Ala Gly Thr Trp Tyr Ser Thr Leu Pro Asp Ser Thr Val 405 410 415 Pro Ile Tyr Gly Lys Thr Pro Val Ala Pro Ala Asn Tyr Met Val Gly 420 425 430 Glu Tyr Lys Asp Phe Leu Glu Ile Ala Gln Ile Pro Thr Phe Ile Gly 435 440 445 Asn Lys Ile Pro Asn Ala Val Pro Tyr Ile Glu Ala Ser Asn Thr Ala 450 455 460 Val Lys Thr Gln Pro Leu Ala Thr Tyr Gln Val Thr Leu Ser Cys Ser 465 470 475 480Cys Leu Ala Asn Thr Phe Leu Ala Ala Leu Ser Arg Asn Phe Ala Gln 485 490 495 Tyr Arg Gly Ser Leu Val Tyr Thr Phe Val Phe Thr Gly Thr Ala Met 500 505 510 Met Lys Gly Lys Phe Leu Ile Ala Tyr Thr Pro Pro Gly Ala Gly Lys 515 520 525 Pro Thr Ser Arg Asp Gln Ala Met Gln Ala Thr Tyr Ala Ile Trp Asp 530 535 540 Leu Gly Leu Asn Ser Ser Tyr Ser Phe Thr Val Pro Phe Ile Ser Pro 545 550 555 560Thr His Phe Arg Met Val Gly Thr Asp Gln Val Asn Ile Thr Asn Val 565 570 575 Asp Gly Trp Val Thr Val Trp Gln Leu Thr Pro Leu Thr Tyr Pro Pro 580 585 590 Gly Cys Pro Thr Ser Ala Lys Ile Leu Thr Met Val Ser Ala Gly Lys 595 600 605 Asp Phe Ser Leu Lys Met Pro Ile Ser Pro Ala Pro Trp Ser Pro Gln 610 615 620 Gly Val Glu Asn Ala Glu Arg Gly Val Thr Glu Asp Thr Asp Ala Thr 625 630 635 640Ala Asp Phe Val Ala Gln Pro Val Tyr Leu Pro Glu Asn Gln Thr Lys 645 650 655 Val Ala Phe Phe Tyr Asp Arg Ser Ser Pro Ile Gly Ala Phe Thr Val 660 665 670 Lys Ser Gly Ser Leu Glu Ser Gly Phe Thr Pro Phe Ser Asn Gln Thr 675 680 685 Cys Pro Asn Ser Val Ile Leu Thr Pro Gly Pro Gln Phe Asp Pro Ala 690 695 700 Tyr Asp Gln Leu Arg Pro Gln Arg Leu Thr Glu Ile Trp Gly Asn Gly 705 710 715 720Asn Glu Glu Thr Ser Lys Val Phe Pro Leu Lys Ser Lys Gln Asp Tyr 725 730 735 Ser Phe Cys Leu Phe Ser Pro Phe Val Tyr Tyr Lys Cys Asp Leu Glu 740 745 750 Val Thr Leu Ser Pro His Thr Ser Gly Asn His Gly Leu Leu Val Arg 755 760 765 Trp Cys Pro Thr Gly Thr Pro Thr Lys Pro Thr Thr Gln Val Leu His 770 775 780 Glu Val Ser Ser Leu Ser Glu Gly Arg Thr Pro Gln Val Tyr Ser Ala 785 790 795 800Gly Pro Gly Ile Thr Asn Gln Ile Ser Phe Val Val Pro Tyr Asn Ser 805 810 815 Pro Leu Ser Val Leu Pro Ala Val Trp Tyr Asn Gly His Lys Arg Phe 820 825 830 Asp Asn Thr Gly Ser Leu Gly Ile Ala Pro Asn Ser Asp Phe Gly Thr 835 840 845 Leu Phe Phe Ala Gly Thr Lys Pro Asp Ile Lys Phe Thr Val Tyr Leu 850 855 860 Arg Tyr Lys Asn Met Arg Val Phe Cys Pro Arg Pro Thr Val Phe Phe 865 870 875 880Pro Trp Pro Ser Ser Gly Asp Lys Ile Asp Met Thr Pro Arg Ala Gly 885 890 895 Val Leu Met Leu Glu Ser Pro Asn Ala Leu Asp Ile Ser Arg Thr Tyr 900 905 910 Pro Thr Leu His Ile Leu Ile Gln Phe Asn His Gly Gly Leu Glu Ile 915 920 925 Arg Leu Phe Arg His Gly Met Phe Trp Ala Glu Ala His Ala Asp Val 930 935 940 Ile Leu Arg Ser Arg Thr Lys Gln Ile Ser Phe Leu Asn Asn Gly Ser 945 950 955 960Phe Pro Ser Met Asp Ala Arg Ala Pro Trp Asn Pro Trp Lys Asn Thr 965 970 975 Tyr His Ala Val Leu Arg Ala Glu Pro Tyr Arg Val Thr Met Asp Val 980 985 990 Tyr His Lys Arg Ile Arg Pro Phe Arg Leu Pro Leu Val Gln Lys Glu 995 1000 1005 Trp Asn Val Arg Glu Glu Asn Val Phe Gly Leu Tyr Gly Ile Phe Asn 1010 1015 1020 Ala His Tyr Ala Gly Tyr Phe Ala Asp Leu Leu Ile His Asp Ile Glu 1025 1030 1035 1040Thr Asn Pro Gly Pro Phe Met Ala Lys Pro Lys Lys Gln Val Phe Gln 1045 1050 1055 Thr Gln Gly Ala Ala Val Ser Ser Met Ala Gln Thr Leu Leu Pro Asn 1060 1065 1070 Asp Leu Ala Ser Lys Val Met Gly Ser Ala Phe Thr Ala Leu Leu Asp 1075 1080 1085 Ala Asn Glu Asp Ala Gln Lys Ala Met Arg Ile Ile Lys Thr Leu Ser 1090 1095 1100 Ser Leu Ser Asp Ala Trp Glu Asn Val Lys Glu Thr Leu Asn Asn Pro 1105 1110 1115 1120Glu Phe Trp Lys Gln Leu Leu Ser Arg Cys Val Gln Leu Ile Ala Gly 1125 1130 1135 Met Thr Ile Ala Val Met His Pro Asp Pro Leu Thr Leu Leu Cys Leu 1140

1145 1150 Gly Thr Leu Thr Ala Ala Glu Ile Thr Ser Gln Thr Ser Leu Cys Glu 1155 1160 1165 Glu Ile Val Ala Lys Phe Lys Lys Ile Phe Thr Thr Pro Pro Pro Arg 1170 1175 1180 Phe Pro Thr Ile Ser Leu Phe Gln Gln Gln Ser Pro Leu Lys Gln Val 1185 1190 1195 1200Asn Asp Val Phe Ser Leu Ala Lys Asn Leu Asp Trp Ala Val Lys Thr 1205 1210 1215 Val Glu Lys Val Val Asp Trp Phe Gly Thr Trp Val Val Gln Glu Glu 1220 1225 1230 Lys Glu Gln Thr Leu Asp Gln Leu Leu Gln Arg Phe Pro Glu His Ala 1235 1240 1245 Lys Arg Ile Ser Asp Leu Arg Asn Gly Met Ser Ala Tyr Val Glu Cys 1250 1255 1260 Lys Glu Ser Phe Asp Phe Phe Glu Lys Leu Tyr Asn Gln Ala Val Lys 1265 1270 1275 1280Glu Lys Arg Thr Gly Ile Ala Ala Val Cys Glu Lys Phe Arg Gln Lys 1285 1290 1295 His Asp His Ala Thr Ala Arg Cys Glu Pro Val Val Ile Val Leu Arg 1300 1305 1310 Gly Asp Ala Gly Gln Gly Lys Ser Leu Ser Ser Gln Val Ile Ala Gln 1315 1320 1325 Ala Val Ser Lys Thr Ile Phe Gly Arg Gln Ser Val Tyr Ser Leu Pro 1330 1335 1340 Pro Asp Ser Asp Phe Phe Asp Gly Tyr Glu Asn Gln Phe Ala Ala Ile 1345 1350 1355 1360Met Asp Asp Leu Gly Gln Asn Pro Asp Gly Ser Asp Phe Thr Thr Phe 1365 1370 1375 Cys Gln Met Val Ser Thr Thr Asn Phe Leu Pro Asn Met Ala Ser Leu 1380 1385 1390 Glu Arg Asn Gly Thr Pro Phe Thr Ser Gln Ile Val Val Ala Thr Thr 1395 1400 1405 Asn Leu Pro Glu Phe Arg Pro Val Thr Ile Ala His Tyr Pro Ala Val 1410 1415 1420 Glu Arg Arg Ile Thr Phe Asp Tyr Ser Val Ser Ala Gly Pro Val Cys 1425 1430 1435 1440Ser Lys Thr Glu Ala Gly Tyr Lys Val Leu Asp Val Glu Arg Ala Phe 1445 1450 1455 Arg Pro Thr Gly Asp Ala Pro Leu Pro Cys Phe Gln Asn Asn Cys Leu 1460 1465 1470 Phe Leu Glu Lys Ala Gly Leu Gln Phe Arg Asp Asn Arg Thr Lys Glu 1475 1480 1485 Ile Leu Ser Leu Val Asp Val Ile Glu Arg Ala Val Ala Arg Ile Glu 1490 1495 1500 Arg Lys Lys Lys Val Leu Thr Thr Val Gln Thr Leu Val Ala Gln Ala 1505 1510 1515 1520Pro Val Asp Glu Val Ser Phe His Ser Val Val Gln Gln Leu Lys Ala 1525 1530 1535 Arg Gln Glu Ala Thr Asp Glu Gln Leu Glu Glu Leu Gln Glu Ala Phe 1540 1545 1550 Ala Lys Thr Gln Glu Arg Ser Ser Val Phe Ser Asp Trp Met Lys Ile 1555 1560 1565 Ser Ala Met Leu Cys Ala Ala Thr Leu Ala Leu Ser Gln Val Val Lys 1570 1575 1580 Met Ala Lys Thr Val Lys Gln Met Val Arg Pro Asp Leu Val Arg Val 1585 1590 1595 1600Gln Leu Asp Glu Gln Glu Gln Gly Pro Tyr Asn Glu Ala Val Arg Ala 1605 1610 1615 Lys Pro Lys Thr Leu Gln Leu Leu Asp Ile Gln Gly Pro Asn Pro Val 1620 1625 1630 Met Asp Phe Glu Lys Tyr Val Ala Lys Phe Val Thr Ala Pro Ile Asp 1635 1640 1645 Phe Val Tyr Pro Thr Gly Val Ser Thr Gln Thr Cys Leu Leu Val Lys 1650 1655 1660 Gly Arg Thr Leu Ala Val Asn Arg His Met Ala Glu Ser Asp Trp Ser 1665 1670 1675 1680Ser Ile Val Val Arg Gly Val Thr His Ala Arg Ser Thr Val Arg Ile 1685 1690 1695 Leu Ala Ile Ala Lys Ala Gly Lys Glu Thr Asp Val Ser Phe Ile Arg 1700 1705 1710 Leu Ser Ser Gly Pro Leu Phe Arg Asp Asn Thr Ser Lys Phe Val Lys 1715 1720 1725 Ala Asp Asp Val Leu Pro Ala Thr Ser Ala Pro Val Ile Gly Ile Met 1730 1735 1740 Asn Thr Asp Ile Pro Met Met Phe Thr Gly Thr Phe Leu Lys Ala Gly 1745 1750 1755 1760Val Ser Val Pro Val Glu Thr Gly Gln Thr Phe Asn His Cys Ile His 1765 1770 1775 Tyr Lys Ala Asn Thr Arg Lys Gly Trp Cys Gly Ser Ala Leu Leu Ala 1780 1785 1790 Asp Leu Gly Gly Lys Lys Lys Ile Leu Gly Met His Ser Ala Gly Ser 1795 1800 1805 Met Gly Arg Thr Ala Ala Ser Ile Val Ser Gln Glu Met Ile Cys Ala 1810 1815 1820 Val Val Ser Ala Phe Glu Pro Gln Gly Ala Leu Glu Arg Leu Pro Asp 1825 1830 1835 1840Gly Pro Arg Ile His Val Pro Arg Lys Thr Ala Leu Arg Pro Thr Val 1845 1850 1855 Ala Arg Arg Val Phe Gln Pro Ala Tyr Ala Pro Ala Val Leu Ser Lys 1860 1865 1870 Phe Asp Pro Arg Thr Glu Ala Asp Val Asp Glu Val Ala Phe Ser Lys 1875 1880 1885 His Thr Ser Asn Gln Glu Ser Leu Pro Pro Val Phe Arg Met Val Ala 1890 1895 1900 Lys Glu Tyr Ala Asn Arg Val Phe Thr Leu Leu Gly Arg Asp Asn Gly 1905 1910 1915 1920Arg Leu Thr Val Lys Gln Ala Leu Glu Gly Leu Glu Gly Met Asp Pro 1925 1930 1935 Met Asp Lys Asn Thr Ser Pro Gly Leu Pro Tyr Thr Ala Leu Gly Met 1940 1945 1950 Arg Arg Thr Asp Val Val Asp Trp Glu Ser Ala Thr Leu Ile Pro Tyr 1955 1960 1965 Ala Ala Asp Arg Leu Lys Lys Met Asn Glu Gly Asp Phe Ser Asp Ile 1970 1975 1980 Val Tyr Gln Thr Phe Leu Lys Asp Glu Leu Arg Pro Val Glu Lys Val 1985 1990 1995 2000Gln Ala Ala Lys Thr Arg Ile Val Asp Val Pro Pro Phe Glu His Cys 2005 2010 2015 Ile Leu Gly Arg Gln Leu Leu Gly Arg Phe Ala Ser Lys Phe Gln Thr 2020 2025 2030 Gln Pro Gly Leu Glu Leu Gly Ser Ala Ile Gly Cys Asp Pro Asp Val 2035 2040 2045 His Trp Thr Ala Phe Gly Val Ala Met Gln Gly Phe Glu Arg Val Tyr 2050 2055 2060 Asp Val Asp Tyr Ser Asn Phe Asp Ser Thr His Ser Val Ala Met Phe 2065 2070 2075 2080Arg Leu Leu Ala Glu Glu Phe Phe Thr Pro Glu Asn Gly Phe Asp Pro 2085 2090 2095 Leu Val Lys Glu Tyr Leu Glu Ser Leu Ala Ile Ser Thr His Ala Phe 2100 2105 2110 Glu Glu Lys Arg Tyr Leu Ile Thr Gly Gly Leu Pro Ser Gly Cys Ala 2115 2120 2125 Ala Thr Ser Met Leu Asn Thr Ile Met Asn Asn Ile Ile Ile Arg Ala 2130 2135 2140 Gly Leu Tyr Leu Thr Tyr Lys Asn Phe Glu Phe Asp Asp Val Lys Val 2145 2150 2155 2160Leu Ser Tyr Gly Asp Asp Leu Leu Val Ala Thr Asn Tyr Gln Leu Asn 2165 2170 2175 Phe Asp Lys Val Arg Ala Ser Leu Ala Lys Thr Gly Tyr Lys Ile Thr 2180 2185 2190 Pro Ala Asn Lys Thr Ser Thr Phe Pro Leu Asp Ser Thr Leu Glu Asp 2195 2200 2205 Val Val Phe Leu Lys Arg Lys Phe Lys Lys Glu Gly Pro Leu Tyr Arg 2210 2215 2220 Pro Val Met Asn Arg Glu Ala Leu Glu Ala Met Leu Ser Tyr Tyr Arg 2225 2230 2235 2240Pro Gly Thr Leu Ser Glu Lys Leu Thr Ser Ile Thr Met Leu Ala Val 2245 2250 2255 His Ser Gly Lys Pro Glu Tyr Asp Arg Leu Phe Ala Pro Phe Arg Glu 2260 2265 2270 Val Gly Val Val Val Pro Ser Phe Glu Ser Val Glu Tyr Arg Trp Arg 2275 2280 2285 Ser Leu Phe Trp 2290 262292PRTEncephalomyocarditis virus 26Met Ala Thr Thr Met Glu Gln Glu Ile Cys Ala His Ser Leu Thr Phe 1 5 10 15 Lys Gly Cys Pro Lys Cys Ser Ala Leu Gln Tyr Arg Asn Gly Phe Tyr 20 25 30 Leu Leu Lys Tyr Asp Glu Glu Trp Tyr Pro Glu Glu Leu Leu Thr Asp 35 40 45 Gly Glu Asp Asp Val Phe Asp Pro Glu Leu Asp Met Glu Val Val Phe 50 55 60 Glu Leu Gln Gly Asn Ser Thr Ser Ser Asp Lys Asn Asn Ser Ser Ser 65 70 75 80Asp Gly Asn Glu Gly Val Ile Ile Asn Asn Phe Tyr Ser Asn Gln Tyr 85 90 95 Gln Asn Ser Ile Asp Leu Ser Ala Asn Ala Thr Gly Ser Asp Pro Pro 100 105 110 Arg Thr Tyr Gly Gln Phe Ser Asn Leu Leu Ser Gly Ala Val Asn Ala 115 120 125 Phe Ser Asn Met Ile Pro Leu Leu Ala Asp Gln Asn Thr Glu Glu Met 130 135 140 Glu Asn Leu Ser Asp Arg Val Leu Gln Asp Thr Ala Gly Asn Thr Val 145 150 155 160Thr Asn Thr Gln Ser Thr Val Gly Arg Leu Val Gly Tyr Gly Ala Val 165 170 175 His Asp Gly Glu His Pro Ala Ser Cys Ala Asp Thr Ala Ser Glu Lys 180 185 190 Ile Leu Ala Val Glu Arg Tyr Tyr Thr Phe Lys Val Asn Asp Trp Thr 195 200 205 Ser Thr Gln Lys Pro Phe Glu Tyr Ile Arg Ile Pro Leu Pro His Val 210 215 220 Leu Ser Gly Glu Asp Gly Gly Val Phe Gly Ala Ala Leu Arg Arg His 225 230 235 240Tyr Leu Val Lys Thr Gly Trp Arg Val Gln Val Gln Cys Asn Ala Ser 245 250 255 Gln Phe His Ala Gly Ser Leu Leu Val Phe Met Ala Pro Glu Tyr Pro 260 265 270 Thr Leu Asp Ala Phe Ala Met Asp Asn Arg Trp Ser Lys Asp Asn Leu 275 280 285 Pro Asn Gly Thr Lys Thr Gln Thr Asn Arg Lys Gly Pro Phe Ala Met 290 295 300 Asp His Gln Asn Phe Trp Gln Trp Thr Leu Tyr Pro His Gln Phe Leu 305 310 315 320Asn Leu Arg Thr Asn Thr Thr Val Asp Leu Glu Val Pro Tyr Val Asn 325 330 335 Ile Ala Pro Thr Ser Ser Trp Thr Gln His Ala Ser Trp Thr Leu Val 340 345 350 Ile Ala Val Val Ala Pro Leu Thr Tyr Ser Thr Gly Ala Ser Thr Ser 355 360 365 Leu Asp Ile Thr Ala Ser Ile Gln Pro Val Arg Pro Val Phe Asn Gly 370 375 380 Leu Arg His Glu Thr Leu Ser Arg Gln Ser Pro Ile Pro Val Thr Ile 385 390 395 400Arg Glu His Ala Gly Thr Trp Tyr Ser Thr Leu Pro Asp Ser Thr Val 405 410 415 Pro Ile Tyr Gly Lys Thr Pro Val Ala Pro Ala Asn Tyr Met Val Gly 420 425 430 Glu Tyr Lys Asp Phe Leu Glu Ile Ala Gln Ile Pro Thr Phe Ile Gly 435 440 445 Asn Lys Ile Pro Asn Ala Val Pro Tyr Ile Glu Ala Ser Asn Thr Ala 450 455 460 Val Lys Thr Gln Pro Leu Ala Thr Tyr Gln Val Thr Leu Ser Cys Ser 465 470 475 480Cys Leu Ala Asn Thr Phe Leu Ala Ala Leu Ser Arg Asn Phe Ala Gln 485 490 495 Tyr Arg Gly Ser Leu Val Tyr Thr Phe Val Phe Thr Gly Thr Ala Met 500 505 510 Met Lys Gly Lys Phe Leu Ile Ala Tyr Thr Pro Pro Gly Ala Gly Lys 515 520 525 Pro Thr Ser Arg Asp Gln Ala Met Gln Ala Thr Tyr Ala Ile Trp Asp 530 535 540 Leu Gly Leu Asn Ser Ser Tyr Ser Phe Thr Val Pro Phe Ile Ser Pro 545 550 555 560Thr His Phe Arg Met Val Gly Thr Asp Gln Val Asn Ile Thr Asn Val 565 570 575 Asp Gly Trp Val Thr Val Trp Gln Leu Thr Pro Leu Thr Tyr Pro Pro 580 585 590 Gly Cys Pro Thr Ser Ala Lys Ile Leu Thr Met Val Ser Ala Gly Lys 595 600 605 Asp Phe Ser Leu Lys Met Pro Ile Ser Pro Ala Pro Trp Ser Pro Gln 610 615 620 Gly Val Glu Asn Ala Glu Arg Gly Val Thr Glu Asp Thr Asp Ala Thr 625 630 635 640Ala Asp Phe Val Ala Gln Pro Val Tyr Leu Pro Glu Asn Gln Thr Lys 645 650 655 Val Ala Phe Phe Tyr Asp Arg Ser Ser Pro Ile Gly Ala Phe Ala Val 660 665 670 Lys Ser Gly Ser Leu Glu Ser Gly Phe Ala Pro Phe Ser Asn Glu Thr 675 680 685 Cys Pro Asn Ser Val Ile Leu Thr Pro Gly Pro Gln Phe Asp Pro Ala 690 695 700 Tyr Asp Gln Leu Arg Pro Gln Arg Leu Thr Glu Ile Trp Gly Asn Gly 705 710 715 720Asn Glu Glu Thr Ser Lys Val Phe Pro Leu Lys Ser Lys Gln Asp Tyr 725 730 735 Ser Phe Cys Leu Phe Ser Pro Phe Val Tyr Tyr Lys Cys Asp Leu Glu 740 745 750 Val Thr Leu Ser Pro His Thr Ser Gly Asn His Gly Leu Leu Val Arg 755 760 765 Trp Cys Pro Thr Gly Thr Pro Ala Lys Pro Thr Thr Gln Val Leu His 770 775 780 Glu Val Ser Ser Leu Ser Glu Gly Arg Thr Pro Gln Val Tyr Ser Ala 785 790 795 800Gly Pro Gly Ile Ser Asn Gln Ile Ser Phe Val Val Pro Tyr Asn Ser 805 810 815 Pro Leu Ser Val Leu Pro Ala Val Trp Tyr Asn Gly His Lys Arg Phe 820 825 830 Asp Asn Thr Gly Ser Leu Gly Ile Ala Pro Asn Ser Asp Phe Gly Thr 835 840 845 Leu Phe Phe Ala Gly Thr Lys Pro Asp Ile Lys Phe Thr Val Tyr Leu 850 855 860 Arg Tyr Lys Asn Met Arg Val Phe Cys Pro Arg Pro Thr Val Phe Phe 865 870 875 880Pro Trp Pro Ser Ser Gly Asp Lys Ile Asp Met Thr Pro Arg Ala Gly 885 890 895 Val Leu Met Leu Glu Ser Pro Asn Ala Leu Asp Ile Ser Arg Thr Tyr 900 905 910 Pro Thr Leu His Ile Leu Ile Gln Phe Asn His Gly Gly Leu Glu Ile 915 920 925 Arg Leu Phe Arg His Gly Met Phe Trp Ala Glu Ala His Ala Asp Val 930 935 940 Ile Leu Arg Ser Arg Thr Lys Gln Ile Ser Phe Leu Asn Asn Gly Ser 945 950 955 960Phe Pro Ser Met Asp Ala Arg Ala Pro Trp Asn Pro Trp Lys Asn Thr 965 970 975 Tyr His Ala Val Leu Arg Ala Glu Pro Tyr Arg Val Thr Met Asp Val 980 985 990 Tyr His Lys Arg Ile Arg Pro Phe Arg Leu Pro Leu Val Gln Lys Glu 995 1000 1005 Trp Asn Val Arg Glu Glu Asn Val Phe Gly Leu Tyr Gly Ile Phe Asn 1010 1015 1020 Ala His Tyr Ala Gly Tyr Phe Ala Asp Leu Leu Ile His Asp Ile Glu 1025 1030 1035 1040Thr Asn Pro Gly Pro Phe Met Ala Lys Pro Lys Lys Gln Val Phe Gln 1045 1050 1055 Thr Gln Gly Ala Ala Val Ser Ser Met Ala Gln Thr Leu Leu Pro Asn 1060 1065 1070 Asp Leu Ala Ser Lys Val Met Gly Ser Ala Phe Thr Ala Leu Leu Asp 1075 1080 1085 Ala Asn Glu Asp Ala Gln Lys Ala Met Arg Ile Ile Lys Thr Leu Ser 1090 1095 1100 Ser Leu Ser Asp Ala Trp Glu Asn Val Lys Glu Thr Leu Asn Asn Pro 1105 1110 1115 1120Glu Phe Trp Lys Gln Leu Leu Ser Arg Cys Val Gln Leu Ile Ala Gly

1125 1130 1135 Met Thr Ile Ala Val Met His Pro Asp Pro Leu Thr Leu Leu Cys Leu 1140 1145 1150 Gly Thr Leu Thr Ala Ala Glu Ile Thr Ser Gln Thr Ser Leu Cys Glu 1155 1160 1165 Glu Ile Val Ala Lys Phe Lys Lys Ile Phe Thr Thr Pro Pro Pro Arg 1170 1175 1180 Phe Pro Thr Ile Ser Leu Phe Gln Gln Gln Ser Pro Leu Lys Gln Val 1185 1190 1195 1200Asn Asp Val Phe Ser Leu Ala Lys Asn Leu Asp Trp Ala Val Lys Thr 1205 1210 1215 Val Glu Lys Val Val Asp Trp Phe Gly Thr Trp Val Val Gln Glu Glu 1220 1225 1230 Lys Glu Gln Thr Leu Asp Gln Leu Leu Gln Arg Phe Pro Glu His Ala 1235 1240 1245 Lys Arg Ile Ser Asp Leu Arg Asn Gly Met Ser Ala Tyr Val Glu Cys 1250 1255 1260 Lys Glu Ser Phe Asp Phe Phe Glu Lys Leu Tyr Asn Gln Ala Val Lys 1265 1270 1275 1280Glu Lys Arg Thr Gly Ile Ala Ala Val Cys Glu Lys Phe Arg Gln Lys 1285 1290 1295 His Asp His Ala Thr Ala Arg Cys Glu Pro Val Val Ile Val Leu Arg 1300 1305 1310 Gly Asp Ala Gly Gln Gly Lys Ser Leu Ser Ser Gln Val Ile Ala Gln 1315 1320 1325 Ala Val Ser Lys Thr Ile Phe Gly Arg Gln Ser Val Tyr Ser Leu Pro 1330 1335 1340 Pro Asp Ser Asp Phe Phe Asp Gly Tyr Glu Asn Gln Phe Ala Ala Ile 1345 1350 1355 1360Met Asp Asp Leu Gly Gln Asn Pro Asp Gly Ser Asp Phe Thr Thr Phe 1365 1370 1375 Cys Gln Met Val Ser Thr Thr Asn Phe Leu Pro Asn Met Ala Ser Leu 1380 1385 1390 Glu Arg Asn Gly Thr Pro Phe Thr Ser Gln Ile Val Val Ala Thr Thr 1395 1400 1405 Asn Leu Pro Glu Phe Arg Pro Val Thr Ile Ala His Tyr Pro Ala Val 1410 1415 1420 Glu Arg Arg Ile Thr Phe Asp Tyr Ser Val Ser Ala Gly Pro Val Cys 1425 1430 1435 1440Ser Lys Thr Glu Ala Gly Tyr Lys Val Leu Asp Val Glu Arg Ala Phe 1445 1450 1455 Arg Pro Thr Gly Asp Ala Pro Leu Pro Cys Phe Gln Asn Asn Cys Leu 1460 1465 1470 Phe Leu Glu Lys Ala Gly Leu Gln Phe Arg Asp Asn Arg Thr Lys Glu 1475 1480 1485 Ile Leu Ser Leu Val Asp Val Ile Glu Arg Ala Val Ala Arg Ile Glu 1490 1495 1500 Arg Lys Lys Lys Val Leu Thr Thr Val Gln Thr Leu Val Ala Gln Ala 1505 1510 1515 1520Pro Val Ala Glu Val Ser Phe His Ser Val Val Gln Gln Leu Lys Ala 1525 1530 1535 Arg Gln Glu Ala Thr Asp Glu Gln Leu Glu Glu Leu Gln Glu Ala Phe 1540 1545 1550 Ala Lys Thr Gln Glu Arg Ser Ser Val Phe Ser Asp Trp Met Lys Ile 1555 1560 1565 Ser Ala Met Leu Cys Ala Ala Thr Leu Ala Leu Ser Gln Val Val Lys 1570 1575 1580 Met Ala Lys Thr Val Lys Gln Met Val Arg Pro Asp Leu Val Arg Val 1585 1590 1595 1600Gln Leu Asp Glu Gln Glu Gln Gly Pro Tyr Asn Glu Ala Val Arg Ala 1605 1610 1615 Lys Pro Lys Thr Leu Gln Leu Leu Asp Ile Gln Gly Pro Asn Pro Val 1620 1625 1630 Met Asp Phe Glu Lys Tyr Val Ala Lys Phe Val Thr Ala Pro Ile Asp 1635 1640 1645 Phe Val Tyr Pro Thr Gly Val Ser Thr Gln Thr Cys Leu Leu Val Lys 1650 1655 1660 Gly Arg Thr Leu Ala Val Asn Arg His Met Ala Glu Ser Asp Trp Ser 1665 1670 1675 1680Ser Ile Val Val Arg Gly Val Thr His Ala Arg Ser Thr Val Arg Ile 1685 1690 1695 Leu Ala Ile Ala Lys Ala Gly Lys Glu Thr Asp Val Ser Phe Ile Arg 1700 1705 1710 Leu Ser Ser Gly Pro Leu Phe Arg Asp Asn Thr Ser Lys Phe Val Lys 1715 1720 1725 Ala Asp Asp Val Leu Pro Ala Thr Ser Ala Pro Val Ile Gly Ile Met 1730 1735 1740 Asn Thr Asp Ile Pro Met Met Phe Thr Gly Thr Phe Leu Lys Ala Gly 1745 1750 1755 1760Val Ser Val Pro Val Glu Thr Gly Gln Thr Phe Asn His Cys Ile His 1765 1770 1775 Tyr Lys Ala Asn Thr Arg Lys Gly Trp Cys Gly Ser Ala Leu Leu Ala 1780 1785 1790 Asp Leu Gly Gly Lys Lys Lys Ile Leu Gly Met His Ser Ala Gly Ser 1795 1800 1805 Met Gly Arg Thr Ala Ala Ser Ile Val Ser Gln Glu Met Ile Cys Ala 1810 1815 1820 Val Val Ser Ala Phe Glu Pro Gln Gly Ala Leu Glu Arg Leu Pro Asp 1825 1830 1835 1840Gly Pro Arg Ile His Val Pro Arg Lys Thr Ala Leu Arg Pro Thr Val 1845 1850 1855 Ala Arg Arg Val Phe Gln Pro Ala Tyr Ala Pro Ala Val Leu Ser Lys 1860 1865 1870 Phe Asp Pro Arg Thr Glu Ala Asp Val Asp Glu Val Ala Phe Ser Lys 1875 1880 1885 His Thr Ser Asn Gln Glu Ser Leu Pro Pro Val Phe Arg Met Val Ala 1890 1895 1900 Lys Glu Tyr Ala Asn Arg Val Phe Thr Leu Leu Gly Arg Asp Asn Gly 1905 1910 1915 1920Arg Leu Thr Val Lys Gln Ala Leu Glu Gly Leu Glu Gly Met Asp Pro 1925 1930 1935 Met Asp Lys Asn Thr Ser Pro Gly Leu Pro Tyr Thr Ala Leu Gly Met 1940 1945 1950 Arg Arg Thr Asp Val Val Asp Trp Glu Ser Ala Thr Leu Ile Pro Tyr 1955 1960 1965 Ala Ala Asp Arg Leu Lys Lys Met Asn Glu Gly Asp Phe Ser Asp Ile 1970 1975 1980 Val Tyr Gln Thr Phe Leu Lys Asp Glu Leu Arg Pro Val Glu Lys Val 1985 1990 1995 2000Gln Ala Ala Lys Thr Arg Ile Val Asp Val Pro Pro Phe Glu His Cys 2005 2010 2015 Ile Leu Gly Arg Gln Leu Leu Gly Arg Phe Ala Ser Lys Phe Gln Thr 2020 2025 2030 Gln Pro Gly Leu Glu Leu Gly Ser Ala Ile Gly Cys Asp Pro Asp Val 2035 2040 2045 His Trp Thr Ala Phe Gly Val Ala Met Gln Gly Phe Glu Arg Val Tyr 2050 2055 2060 Asp Val Asp Tyr Ser Asn Phe Asp Ser Thr His Ser Val Ala Met Phe 2065 2070 2075 2080Arg Leu Leu Ala Glu Glu Phe Phe Thr Pro Glu Asn Gly Phe Asp Pro 2085 2090 2095 Leu Val Lys Glu Tyr Leu Glu Ser Leu Ala Ile Ser Thr His Ala Phe 2100 2105 2110 Glu Glu Lys Arg Tyr Leu Ile Thr Gly Gly Leu Pro Ser Gly Cys Ala 2115 2120 2125 Ala Thr Ser Met Leu Asn Thr Ile Met Asn Asn Ile Ile Ile Arg Ala 2130 2135 2140 Gly Leu Tyr Leu Thr Tyr Lys Asn Phe Glu Phe Asp Asp Val Lys Val 2145 2150 2155 2160Leu Ser Tyr Gly Asp Asp Leu Leu Val Ala Thr Asn Tyr Gln Leu Asn 2165 2170 2175 Phe Asp Lys Val Arg Ala Ser Leu Ala Lys Thr Gly Tyr Lys Ile Thr 2180 2185 2190 Pro Ala Asn Lys Thr Ser Thr Phe Pro Leu Asp Ser Thr Leu Glu Asp 2195 2200 2205 Val Val Phe Leu Lys Arg Lys Phe Lys Lys Glu Gly Pro Leu Tyr Arg 2210 2215 2220 Pro Val Met Asn Arg Glu Ala Leu Glu Ala Met Leu Ser Tyr Tyr Arg 2225 2230 2235 2240Pro Gly Thr Leu Ser Glu Lys Leu Thr Ser Ile Thr Met Leu Ala Val 2245 2250 2255 His Ser Gly Lys Pro Glu Tyr Asp Arg Leu Phe Ala Pro Phe Arg Glu 2260 2265 2270 Val Gly Val Val Val Pro Ser Phe Glu Ser Val Glu Tyr Arg Trp Arg 2275 2280 2285 Ser Leu Phe Trp 2290 272292PRTEncephalomyocarditis virus 27Met Ala Thr Thr Met Glu Gln Glu Ile Cys Ala His Ser Leu Thr Phe 1 5 10 15 Lys Gly Cys Pro Lys Cys Ser Ala Leu Gln Tyr Arg Asn Gly Phe Tyr 20 25 30 Leu Leu Lys Tyr Asp Glu Glu Trp Tyr Pro Glu Glu Leu Leu Thr Asp 35 40 45 Gly Glu Asp Asp Val Phe Asp Pro Glu Leu Asp Met Glu Val Val Phe 50 55 60 Glu Leu Gln Gly Asn Ser Thr Ser Ser Asp Lys Asn Asn Ser Ser Ser 65 70 75 80Asp Gly Asn Glu Gly Val Ile Ile Asn Asn Phe Tyr Ser Asn Gln Tyr 85 90 95 Gln Asn Ser Ile Asp Leu Ser Ala Asn Ala Thr Gly Ser Asp Pro Pro 100 105 110 Arg Thr Tyr Gly Gln Phe Ser Asn Leu Leu Ser Gly Ala Val Asn Ala 115 120 125 Phe Ser Asn Met Ile Pro Leu Leu Ala Asp Gln Asn Thr Glu Glu Met 130 135 140 Glu Asn Leu Ser Asp Arg Val Leu Gln Asp Thr Ala Gly Asn Thr Val 145 150 155 160Thr Asn Thr Gln Ser Thr Val Gly Arg Leu Val Gly Tyr Gly Ala Val 165 170 175 His Asp Gly Glu His Pro Ala Ser Cys Ala Asp Thr Ala Ser Glu Lys 180 185 190 Ile Leu Ala Val Glu Arg Tyr Tyr Thr Phe Lys Val Asn Asp Trp Thr 195 200 205 Ser Thr Gln Lys Pro Phe Glu Tyr Ile Arg Ile Pro Leu Pro His Val 210 215 220 Leu Ser Gly Glu Asp Gly Gly Val Phe Gly Ala Ala Leu Arg Arg His 225 230 235 240Tyr Leu Val Lys Thr Gly Trp Pro Val Gln Val Gln Cys Asn Ala Ser 245 250 255 Gln Phe His Ala Gly Ser Leu Leu Val Phe Met Ala Pro Glu Tyr Pro 260 265 270 Thr Leu Asp Ala Phe Ala Met Asp Asn Arg Trp Ser Lys Asp Asn Leu 275 280 285 Pro Asn Gly Thr Lys Thr Gln Thr Asn Arg Lys Gly Pro Phe Ala Met 290 295 300 Asp His Gln Asn Phe Trp Gln Trp Thr Leu Tyr Pro His Gln Phe Leu 305 310 315 320Asn Leu Arg Thr Asn Thr Thr Val Asp Leu Glu Val Pro Tyr Val Asn 325 330 335 Ile Ala Pro Thr Ser Ser Trp Thr Gln His Ala Ser Trp Thr Leu Val 340 345 350 Ile Ala Val Val Ala Pro Leu Thr Tyr Ser Thr Gly Ala Ser Thr Ser 355 360 365 Leu Asp Ile Thr Ala Ser Ile Gln Pro Val Arg Pro Val Phe Asn Gly 370 375 380 Leu Arg His Glu Thr Leu Ser Arg Gln Ser Pro Ile Pro Val Thr Ile 385 390 395 400Arg Glu His Ala Gly Thr Trp Tyr Ser Thr Leu Pro Asp Ser Thr Val 405 410 415 Pro Ile Tyr Gly Lys Thr Pro Val Ala Pro Ala Asn Tyr Met Val Gly 420 425 430 Glu Tyr Lys Asp Phe Leu Glu Ile Ala Gln Ile Pro Thr Phe Ile Gly 435 440 445 Asn Lys Ile Pro Asn Ala Val Pro Tyr Ile Glu Ala Ser Asn Thr Ala 450 455 460 Val Lys Thr Gln Pro Leu Ala Thr Tyr Gln Val Thr Leu Ser Cys Ser 465 470 475 480Cys Leu Ala Asn Thr Phe Leu Ala Ala Leu Ser Arg Asn Phe Ala Gln 485 490 495 Tyr Arg Gly Ser Leu Val Tyr Thr Phe Val Phe Thr Gly Thr Ala Met 500 505 510 Met Lys Gly Lys Phe Leu Ile Ala Tyr Thr Pro Pro Gly Ala Gly Lys 515 520 525 Pro Thr Ser Arg Asp Gln Ala Met Gln Ala Thr Tyr Ala Ile Trp Asp 530 535 540 Leu Gly Leu Asn Ser Ser Tyr Ser Phe Thr Val Pro Phe Ile Ser Pro 545 550 555 560Thr His Phe Arg Met Val Gly Thr Asp Gln Val Asn Ile Thr Asn Val 565 570 575 Asp Gly Trp Val Thr Val Trp Gln Leu Thr Pro Leu Thr Tyr Pro Pro 580 585 590 Gly Cys Pro Thr Ser Ala Lys Ile Leu Thr Met Val Ser Ala Gly Lys 595 600 605 Asp Phe Ser Leu Lys Met Pro Ile Ser Pro Ala Pro Trp Ser Pro Gln 610 615 620 Gly Val Glu Asn Ala Glu Arg Gly Val Thr Glu Asp Thr Asp Ala Thr 625 630 635 640Ala Asp Phe Val Ala Gln Pro Val Tyr Leu Pro Glu Asn Gln Thr Lys 645 650 655 Val Ala Phe Phe Tyr Asp Arg Ser Ser Pro Ile Gly Ala Phe Thr Val 660 665 670 Lys Ser Gly Ser Leu Glu Ser Gly Phe Ala Pro Phe Ser Asn Glu Thr 675 680 685 Cys Pro Asn Ser Val Ile Leu Thr Pro Gly Pro Gln Phe Asp Pro Ala 690 695 700 Tyr Asp Gln Leu Arg Pro Gln Arg Leu Thr Glu Ile Trp Gly Asn Gly 705 710 715 720Asn Glu Glu Thr Ser Lys Val Phe Pro Leu Lys Ser Lys Gln Asp Tyr 725 730 735 Ser Phe Cys Leu Phe Ser Pro Phe Val Tyr Tyr Lys Cys Asp Leu Glu 740 745 750 Val Thr Leu Ser Pro His Thr Ser Gly Asn His Gly Leu Leu Val Arg 755 760 765 Trp Cys Pro Thr Gly Thr Pro Ala Lys Pro Thr Thr Gln Val Leu His 770 775 780 Glu Val Ser Ser Leu Ser Glu Gly Arg Thr Pro Gln Val Tyr Ser Ala 785 790 795 800Gly Pro Gly Val Ser Asn Gln Ile Ser Phe Val Val Pro Tyr Asn Ser 805 810 815 Pro Leu Ser Val Leu Pro Ala Val Trp Tyr Asn Gly His Lys Arg Phe 820 825 830 Asp Asn Thr Gly Ser Leu Gly Ile Ala Pro Asn Ser Asp Phe Gly Thr 835 840 845 Leu Phe Phe Ala Gly Thr Lys Pro Asp Ile Lys Phe Thr Val Tyr Leu 850 855 860 Arg Tyr Lys Asn Met Arg Val Phe Cys Pro Arg Pro Thr Val Phe Phe 865 870 875 880Pro Trp Pro Ser Ser Gly Asp Lys Ile Asp Met Thr Pro Arg Ala Gly 885 890 895 Val Leu Met Leu Glu Ser Pro Asn Ala Leu Asp Ile Ser Arg Thr Tyr 900 905 910 Pro Thr Leu His Ile Leu Ile Gln Phe Asn His Gly Gly Leu Glu Ile 915 920 925 Arg Leu Phe Arg His Val Gln Phe Trp Ala Glu Ala His Ala Asp Val 930 935 940 Ile Leu Arg Ser Arg Thr Lys Gln Ile Ser Phe Leu Asn Asn Gly Ser 945 950 955 960Phe Pro Ser Met Asp Ala Arg Ala Pro Trp Asn Pro Trp Lys Asn Thr 965 970 975 Tyr His Ala Val Leu Arg Ala Glu Pro Tyr Arg Val Thr Met Asp Val 980 985 990 Tyr His Lys Arg Ile Arg Pro Phe Arg Leu Pro Leu Val Gln Lys Glu 995 1000 1005 Trp Asn Val Arg Glu Glu Asn Val Phe Gly Leu Tyr Gly Ile Phe Asn 1010 1015 1020 Ala His Tyr Ala Gly Tyr Phe Ala Asp Leu Leu Ile His Asp Ile Glu 1025 1030 1035 1040Thr Asn Pro Gly Pro Phe Met Ala Lys Pro Lys Lys Gln Val Phe Gln 1045 1050 1055 Thr Gln Gly Ala Ala Val Ser Ser Met Ala Gln Thr Leu Leu Pro Asn 1060 1065 1070 Asp Leu Ala Ser Lys Val Met Gly Ser Ala Phe Thr Ala Leu Leu Asp 1075 1080 1085 Ala Asn Glu Asp Ala Gln Lys Ala Met Arg Ile Ile Lys Thr Leu Ser 1090 1095 1100 Ser Leu Ser Asp Ala Trp Glu Asn Val Lys Glu Thr

Leu Asn Asn Pro 1105 1110 1115 1120Glu Phe Trp Lys Gln Leu Leu Ser Arg Cys Val Gln Leu Ile Ala Gly 1125 1130 1135 Met Thr Ile Ala Val Met His Pro Asp Pro Leu Thr Leu Leu Cys Leu 1140 1145 1150 Gly Thr Leu Thr Ala Ala Glu Ile Thr Ser Gln Thr Ser Leu Cys Glu 1155 1160 1165 Glu Ile Val Ala Lys Phe Lys Lys Ile Phe Thr Thr Pro Pro Pro Arg 1170 1175 1180 Phe Pro Thr Ile Ser Leu Phe Gln Gln Gln Ser Pro Leu Lys Gln Val 1185 1190 1195 1200Asn Asp Val Phe Ser Leu Ala Lys Asn Leu Asp Trp Ala Val Lys Thr 1205 1210 1215 Val Glu Lys Val Val Asp Trp Phe Gly Thr Trp Val Val Gln Glu Glu 1220 1225 1230 Lys Glu Gln Thr Leu Asp Gln Leu Leu Gln Arg Phe Pro Glu His Ala 1235 1240 1245 Lys Arg Ile Ser Asp Leu Arg Asn Gly Met Ser Ala Tyr Val Glu Cys 1250 1255 1260 Lys Glu Ser Phe Asp Phe Phe Glu Lys Leu Tyr Asn Gln Ala Val Lys 1265 1270 1275 1280Glu Lys Arg Thr Gly Ile Ala Ala Val Cys Glu Lys Phe Arg Gln Lys 1285 1290 1295 His Asp His Ala Thr Ala Arg Cys Glu Pro Val Val Ile Val Leu Arg 1300 1305 1310 Gly Asp Ala Gly Gln Gly Lys Ser Leu Ser Ser Gln Val Ile Ala Gln 1315 1320 1325 Ala Val Ser Lys Thr Ile Phe Gly Arg Gln Ser Val Tyr Ser Leu Pro 1330 1335 1340 Pro Asp Ser Asp Phe Phe Asp Gly Tyr Glu Asn Gln Phe Ala Ala Ile 1345 1350 1355 1360Met Asp Asp Leu Gly Gln Asn Pro Asp Gly Ser Asp Phe Thr Thr Phe 1365 1370 1375 Cys Gln Met Val Ser Thr Thr Asn Phe Leu Pro Asn Met Ala Ser Leu 1380 1385 1390 Glu Arg Asn Gly Thr Pro Phe Thr Ser Gln Leu Val Val Ala Thr Thr 1395 1400 1405 Asn Leu Pro Glu Phe Arg Pro Val Thr Ile Ala His Tyr Pro Ala Val 1410 1415 1420 Glu Arg Arg Ile Thr Phe Asp Tyr Ser Val Ser Ala Gly Pro Val Cys 1425 1430 1435 1440Ser Lys Thr Glu Ala Gly Tyr Lys Val Leu Asp Val Glu Arg Ala Phe 1445 1450 1455 Arg Pro Thr Gly Asp Ala Pro Leu Pro Cys Phe Gln Asn Asn Cys Leu 1460 1465 1470 Phe Leu Glu Lys Ala Gly Leu Gln Phe Arg Asp Asn Arg Thr Lys Glu 1475 1480 1485 Ile Leu Ser Leu Val Asp Val Ile Glu Arg Ala Val Ala Arg Ile Glu 1490 1495 1500 Arg Lys Lys Lys Val Leu Thr Thr Val Gln Thr Leu Val Ala Gln Ala 1505 1510 1515 1520Pro Val Ala Glu Val Ser Phe His Ser Val Val Gln Gln Leu Lys Ala 1525 1530 1535 Arg Gln Glu Ala Thr Asp Glu Gln Leu Glu Glu Leu Gln Glu Ala Phe 1540 1545 1550 Ala Lys Thr Gln Glu Arg Ser Ser Val Phe Ser Asp Trp Met Lys Ile 1555 1560 1565 Ser Ala Met Leu Cys Ala Ala Thr Leu Ala Leu Thr Gln Val Val Lys 1570 1575 1580 Met Ala Lys Thr Val Lys Gln Met Val Arg Pro Asp Leu Val Arg Val 1585 1590 1595 1600Gln Leu Asp Glu Gln Glu Gln Gly Pro Tyr Asn Glu Ala Val Arg Ala 1605 1610 1615 Lys Pro Lys Thr Leu Gln Leu Leu Asp Ile Gln Gly Pro Asn Pro Val 1620 1625 1630 Met Asp Phe Glu Lys Tyr Val Ala Lys Phe Val Thr Ala Pro Ile Asp 1635 1640 1645 Phe Val Tyr Pro Thr Gly Val Ser Thr Gln Thr Cys Leu Leu Val Lys 1650 1655 1660 Gly Arg Thr Leu Ala Val Asn Arg His Met Ala Glu Ser Asp Trp Ser 1665 1670 1675 1680Ser Ile Val Val Arg Gly Val Thr His Ala Arg Ser Thr Val Arg Ile 1685 1690 1695 Leu Ala Ile Ala Lys Ala Gly Lys Glu Thr Asp Val Ser Phe Ile Arg 1700 1705 1710 Leu Ser Ser Gly Pro Leu Phe Arg Asp Asn Thr Ser Lys Phe Val Lys 1715 1720 1725 Ala Asp Asp Val Leu Pro Ala Thr Ser Ala Pro Val Ile Gly Ile Met 1730 1735 1740 Asn Thr Asp Ile Pro Met Met Phe Thr Gly Thr Phe Leu Lys Ala Gly 1745 1750 1755 1760Val Ser Val Pro Val Glu Thr Gly Gln Thr Phe Asn His Cys Ile His 1765 1770 1775 Tyr Lys Ala Asn Thr Arg Lys Gly Trp Cys Gly Ser Ala Leu Leu Ala 1780 1785 1790 Asp Leu Gly Gly Lys Lys Lys Ile Leu Gly Met His Ser Ala Gly Ser 1795 1800 1805 Met Gly Val Ala Ala Ala Ser Ile Val Ser Gln Glu Met Ile Cys Ala 1810 1815 1820 Val Val Ser Ala Phe Glu Pro Gln Gly Ala Leu Glu Arg Leu Pro Asp 1825 1830 1835 1840Gly Pro Arg Ile His Val Pro Arg Lys Thr Ala Leu Arg Pro Thr Val 1845 1850 1855 Ala Arg Gln Val Phe Gln Pro Ala Tyr Ala Pro Ala Val Leu Ser Lys 1860 1865 1870 Phe Asp Pro Arg Thr Glu Ala Asp Val Asp Glu Val Ala Phe Ser Lys 1875 1880 1885 His Thr Ser Asn Gln Glu Ser Leu Pro Pro Val Phe Arg Met Val Ala 1890 1895 1900 Lys Glu Tyr Ala Asn Arg Val Phe Thr Leu Leu Gly Arg Asp Asn Gly 1905 1910 1915 1920Arg Leu Thr Val Lys Gln Ala Leu Glu Gly Leu Glu Gly Met Asp Pro 1925 1930 1935 Met Asp Lys Asn Thr Ser Pro Gly Leu Pro Tyr Thr Ala Leu Gly Met 1940 1945 1950 Arg Arg Thr Asp Val Val Asp Trp Glu Ser Ala Thr Leu Ile Pro Tyr 1955 1960 1965 Ala Ala Asp Arg Leu Lys Lys Met Asn Glu Gly Asp Phe Ser Asp Ile 1970 1975 1980 Val Tyr Gln Thr Phe Leu Lys Asp Glu Leu Arg Pro Val Glu Lys Val 1985 1990 1995 2000Gln Ala Ala Lys Thr Arg Ile Val Asp Val Pro Pro Phe Glu His Cys 2005 2010 2015 Ile Leu Gly Arg Gln Leu Leu Gly Arg Phe Ala Ser Lys Phe Gln Thr 2020 2025 2030 Gln Pro Gly Leu Glu Leu Gly Ser Ala Ile Gly Cys Asp Pro Asp Val 2035 2040 2045 His Trp Thr Ala Phe Gly Val Ala Met Gln Gly Phe Glu Arg Val Tyr 2050 2055 2060 Asp Val Asp Tyr Ser Asn Phe Asp Ser Thr His Ser Val Ala Met Phe 2065 2070 2075 2080Arg Leu Leu Ala Glu Glu Phe Phe Thr Pro Glu Asn Gly Phe Asp Pro 2085 2090 2095 Leu Val Lys Glu Tyr Leu Glu Ser Leu Ala Ile Ser Thr His Ala Phe 2100 2105 2110 Glu Glu Lys Arg Tyr Leu Ile Thr Gly Gly Leu Pro Ser Gly Cys Ala 2115 2120 2125 Ala Thr Ser Met Leu Asn Thr Ile Met Asn Asn Ile Ile Ile Arg Ala 2130 2135 2140 Gly Leu Tyr Leu Thr Tyr Lys Asn Phe Glu Phe Asp Asp Val Lys Val 2145 2150 2155 2160Leu Ser Tyr Gly Asp Asp Leu Leu Val Ala Thr Asn Tyr Gln Leu Asn 2165 2170 2175 Phe Asp Lys Val Arg Ala Ser Leu Ala Lys Thr Gly Tyr Lys Ile Thr 2180 2185 2190 Pro Ala Asn Lys Thr Ser Thr Phe Pro Leu Asp Ser Thr Leu Glu Asp 2195 2200 2205 Val Val Phe Leu Lys Arg Lys Phe Lys Lys Glu Gly Pro Leu Tyr Arg 2210 2215 2220 Pro Val Met Asn Arg Glu Ala Leu Glu Ala Met Leu Ser Tyr Tyr Arg 2225 2230 2235 2240Pro Gly Thr Leu Ser Glu Lys Leu Thr Ser Ile Thr Met Leu Ala Val 2245 2250 2255 His Ser Gly Lys Pro Glu Tyr Asp Arg Leu Phe Ala Pro Phe Arg Glu 2260 2265 2270 Val Gly Val Val Val Pro Ser Phe Glu Ser Val Glu Tyr Arg Trp Arg 2275 2280 2285 Ser Leu Phe Trp 2290 282292PRTEncephalomyocarditis virus 28Met Ala Thr Thr Met Glu Gln Glu Ile Cys Ala His Ser Leu Thr Phe 1 5 10 15 Lys Gly Cys Pro Lys Cys Ser Ala Leu Gln Tyr Arg Asn Gly Phe Tyr 20 25 30 Leu Leu Lys Tyr Asp Glu Glu Trp Tyr Pro Glu Glu Leu Leu Thr Asp 35 40 45 Gly Glu Asp Asp Val Phe Asp Pro Glu Leu Asp Met Glu Val Val Phe 50 55 60 Glu Leu Gln Gly Asn Ser Thr Ser Ser Asp Lys Asn Asn Ser Ser Ser 65 70 75 80Asp Gly Asn Glu Gly Val Ile Ile Asn Asn Phe Tyr Ser Asn Gln Tyr 85 90 95 Gln Asn Ser Ile Asp Leu Ser Ala Asn Ala Thr Gly Ser Asp Pro Pro 100 105 110 Arg Thr Tyr Gly Gln Phe Ser Asn Leu Leu Ser Gly Ala Val Asn Ala 115 120 125 Phe Ser Asn Met Ile Pro Leu Leu Ala Asp Gln Asn Thr Glu Glu Met 130 135 140 Glu Asn Leu Ser Asp Arg Val Leu Gln Asp Thr Ala Gly Asn Thr Val 145 150 155 160Thr Asn Thr Gln Ser Thr Val Gly Arg Leu Val Gly Tyr Gly Ala Val 165 170 175 His Asp Gly Glu His Pro Ala Ser Cys Ala Asp Thr Ala Ser Glu Lys 180 185 190 Ile Leu Ala Val Glu Arg Tyr Tyr Thr Phe Lys Val Asn Asp Trp Thr 195 200 205 Ser Thr Gln Lys Pro Phe Glu Tyr Ile Arg Ile Pro Leu Pro His Val 210 215 220 Leu Ser Gly Glu Asp Gly Gly Val Phe Gly Ala Ala Leu Arg Arg His 225 230 235 240Tyr Leu Val Lys Thr Gly Trp Arg Val Gln Val Gln Cys Asn Ala Ser 245 250 255 Gln Phe His Ala Gly Ser Leu Leu Val Phe Met Ala Pro Glu Tyr Pro 260 265 270 Thr Leu Asp Ala Phe Ala Met Asp Asn Arg Trp Ser Lys Asp Asn Leu 275 280 285 Pro Asn Gly Thr Lys Thr Gln Thr Asn Arg Lys Gly Pro Phe Ala Met 290 295 300 Asp His Gln Asn Phe Trp Gln Trp Thr Leu Tyr Pro His Gln Phe Leu 305 310 315 320Asn Leu Arg Thr Asn Thr Thr Val Asp Leu Glu Val Pro Tyr Val Asn 325 330 335 Ile Ala Pro Thr Ser Ser Trp Thr Gln His Ala Ser Trp Thr Leu Val 340 345 350 Ile Ala Val Val Ala Pro Leu Thr Tyr Ser Thr Gly Ala Ser Thr Ser 355 360 365 Leu Asp Ile Thr Ala Ser Ile Gln Pro Val Arg Pro Val Phe Asn Gly 370 375 380 Leu Arg His Glu Thr Leu Ser Arg Gln Ser Pro Ile Pro Val Thr Ile 385 390 395 400Arg Glu His Ala Gly Thr Trp Tyr Ser Thr Leu Pro Asp Ser Thr Val 405 410 415 Pro Ile Tyr Gly Lys Thr Pro Val Ala Pro Ala Asn Tyr Met Val Gly 420 425 430 Glu Tyr Lys Asp Phe Leu Glu Ile Ala Gln Ile Pro Thr Phe Ile Gly 435 440 445 Asn Lys Ile Pro Asn Ala Val Pro Tyr Ile Glu Ala Ser Asn Thr Ala 450 455 460 Val Lys Thr Gln Pro Leu Ala Thr Tyr Gln Val Thr Leu Ser Cys Ser 465 470 475 480Cys Leu Ala Asn Thr Phe Leu Ala Ala Leu Ser Arg Asn Phe Ala Gln 485 490 495 Tyr Arg Gly Ser Leu Val Tyr Thr Phe Val Phe Thr Gly Thr Ala Met 500 505 510 Met Lys Gly Lys Phe Leu Ile Ala Tyr Thr Pro Pro Gly Ala Gly Lys 515 520 525 Pro Thr Ser Arg Asp Gln Ala Met Gln Ala Thr Tyr Ala Ile Trp Asp 530 535 540 Leu Gly Leu Asn Ser Ser Tyr Ser Phe Thr Val Pro Phe Ile Ser Pro 545 550 555 560Thr His Phe Arg Met Val Gly Thr Asp Gln Val Asn Ile Thr Asn Val 565 570 575 Asp Gly Trp Val Thr Val Trp Gln Leu Thr Pro Leu Thr Tyr Pro Pro 580 585 590 Gly Cys Pro Thr Ser Ala Lys Ile Leu Thr Met Val Ser Ala Gly Lys 595 600 605 Asp Phe Ser Leu Lys Met Pro Ile Ser Pro Ala Pro Trp Ser Pro Gln 610 615 620 Gly Val Glu Asn Ala Glu Arg Gly Val Thr Glu Asp Thr Asp Ala Thr 625 630 635 640Ala Asp Phe Val Ala Gln Pro Val Tyr Leu Pro Glu Asn Gln Thr Lys 645 650 655 Val Ala Phe Phe Tyr Asp Arg Ser Ser Pro Ile Gly Ala Phe Thr Val 660 665 670 Lys Ser Gly Ser Leu Glu Ser Gly Phe Ala Pro Phe Ser Asn Lys Thr 675 680 685 Cys Pro Asn Ser Val Ile Leu Thr Pro Gly Pro Gln Phe Asp Pro Ala 690 695 700 Tyr Asp Gln Leu Arg Pro Gln Arg Leu Thr Glu Ile Trp Gly Asn Arg 705 710 715 720Asn Glu Glu Thr Ser Lys Val Phe Pro Leu Lys Ser Lys Gln Asp Tyr 725 730 735 Ser Phe Cys Leu Phe Ser Pro Phe Val Tyr Tyr Lys Cys Asp Leu Glu 740 745 750 Val Thr Leu Ser Pro His Thr Ser Gly Asn His Gly Leu Leu Val Arg 755 760 765 Trp Cys Pro Thr Gly Thr Pro Ala Lys Pro Thr Thr Gln Val Leu His 770 775 780 Glu Val Ser Ser Leu Ser Glu Gly Arg Thr Pro Gln Val Tyr Ser Ala 785 790 795 800Gly Pro Gly Ile Ser Asn Gln Ile Ser Phe Val Val Pro Tyr Asn Ser 805 810 815 Pro Leu Ser Val Leu Pro Ala Val Trp Tyr Asn Gly His Lys Arg Phe 820 825 830 Asp Asn Thr Gly Ser Leu Gly Ile Ala Pro Asn Ser Asp Phe Gly Thr 835 840 845 Leu Phe Phe Ala Gly Thr Lys Pro Asp Ile Lys Phe Thr Val Tyr Leu 850 855 860 Arg Tyr Lys Asn Met Arg Val Phe Cys Pro Arg Pro Thr Val Phe Phe 865 870 875 880Pro Trp Pro Ser Ser Gly Asp Lys Ile Asp Met Thr Pro Arg Ala Gly 885 890 895 Val Leu Met Leu Glu Ser Pro Asn Ala Leu Asp Ile Ser Arg Thr Tyr 900 905 910 Pro Thr Leu His Ile Leu Ile Gln Phe Asn His Gly Gly Leu Glu Ile 915 920 925 Arg Leu Phe Arg His Gly Gln Phe Trp Ala Glu Ala His Ala Asp Val 930 935 940 Ile Leu Arg Ser Arg Thr Lys Gln Ile Ser Phe Leu Asn Asn Gly Ser 945 950 955 960Phe Pro Ser Met Asp Ala Arg Ala Pro Trp Asn Pro Trp Lys Asn Thr 965 970 975 Tyr His Ala Val Leu Arg Ala Glu Pro Tyr Arg Val Thr Met Asp Val 980 985 990 Tyr His Lys Arg Ile Arg Pro Phe Arg Leu Pro Leu Val Gln Lys Glu 995 1000 1005 Trp Asn Val Arg Glu Glu Asn Val Phe Gly Leu Tyr Ser Ile Phe Asn 1010 1015 1020 Ala His Tyr Ala Gly Tyr Phe Ala Asp Leu Leu Ile His Asp Ile Glu 1025 1030 1035 1040Thr Asn Pro Gly Pro Phe Met Ala Lys Pro Lys Lys Gln Val Phe Gln 1045 1050 1055 Thr Gln Gly Ala Ala Val Ser Ser Met Ala Gln Thr Leu Leu Pro Asn 1060 1065 1070 Asp Leu Ala Ser Lys Val Met Gly Ser Ala Phe Thr Ala Leu Leu Asp 1075 1080 1085 Ala Asn Glu Asp Ala Gln Lys Ala

Met Arg Ile Ile Lys Thr Leu Ser 1090 1095 1100 Ser Leu Ser Asp Ala Trp Glu Asn Val Lys Glu Thr Leu Asn Asn Pro 1105 1110 1115 1120Glu Phe Trp Lys Gln Leu Leu Ser Arg Cys Val Gln Leu Ile Ala Gly 1125 1130 1135 Met Thr Ile Ala Val Met His Pro Asp Pro Leu Thr Leu Leu Cys Leu 1140 1145 1150 Gly Thr Leu Thr Ala Ala Glu Ile Thr Ser Gln Thr Ser Leu Cys Glu 1155 1160 1165 Glu Ile Val Ala Lys Phe Lys Lys Ile Phe Thr Thr Pro Pro Pro Arg 1170 1175 1180 Phe Pro Thr Ile Ser Leu Phe Gln Gln Gln Ser Pro Leu Lys Gln Val 1185 1190 1195 1200Asn Asp Val Phe Ser Leu Ala Lys Asn Leu Asp Trp Ala Val Lys Thr 1205 1210 1215 Val Glu Lys Val Val Asp Trp Phe Gly Thr Trp Val Val Gln Glu Glu 1220 1225 1230 Lys Glu Gln Thr Leu Asp Gln Leu Leu Gln Arg Phe Pro Glu His Ala 1235 1240 1245 Lys Arg Ile Ser Asp Leu Arg Asn Gly Met Ser Ala Tyr Val Glu Cys 1250 1255 1260 Lys Glu Ser Phe Asp Phe Phe Glu Lys Leu Tyr Asn Gln Ala Val Lys 1265 1270 1275 1280Glu Lys Arg Thr Gly Ile Ala Ala Val Cys Glu Lys Phe Arg Gln Lys 1285 1290 1295 His Asp His Ala Thr Ala Arg Cys Glu Pro Val Val Ile Val Leu Arg 1300 1305 1310 Gly Asp Ala Gly Gln Gly Lys Ser Leu Ser Ser Gln Val Ile Ala Gln 1315 1320 1325 Ala Val Ser Lys Thr Ile Phe Gly Arg Gln Ser Val Tyr Ser Leu Pro 1330 1335 1340 Pro Asp Ser Asp Phe Phe Asp Gly Tyr Glu Asn Gln Phe Ala Ala Ile 1345 1350 1355 1360Met Asp Asp Leu Gly Gln Asn Pro Asp Gly Ser Asp Phe Thr Thr Phe 1365 1370 1375 Cys Gln Met Val Ser Thr Thr Asn Phe Leu Pro Asn Met Ala Ser Leu 1380 1385 1390 Glu Arg Lys Gly Thr Pro Phe Thr Ser Gln Leu Val Val Ala Thr Thr 1395 1400 1405 Asn Leu Pro Glu Phe Arg Pro Val Thr Ile Ala His Tyr Pro Ala Val 1410 1415 1420 Glu Arg Arg Ile Thr Phe Asp Tyr Ser Val Ser Ala Gly Pro Val Cys 1425 1430 1435 1440Ser Lys Thr Glu Ala Gly Tyr Lys Val Leu Asp Val Glu Arg Ala Phe 1445 1450 1455 Arg Pro Thr Gly Asp Ala Pro Leu Pro Cys Phe Gln Asn Asn Cys Leu 1460 1465 1470 Phe Leu Glu Lys Ala Gly Leu Gln Phe Arg Asp Asn Arg Thr Lys Glu 1475 1480 1485 Ile Leu Ser Leu Val Asp Val Ile Glu Arg Ala Val Ala Arg Ile Glu 1490 1495 1500 Arg Lys Lys Lys Val Leu Thr Thr Val Gln Thr Leu Val Ala Gln Ala 1505 1510 1515 1520Pro Val Asp Glu Val Ser Phe His Ser Val Val Gln Gln Leu Lys Ala 1525 1530 1535 Arg Gln Glu Ala Thr Asp Glu Gln Leu Glu Glu Leu Gln Glu Ala Phe 1540 1545 1550 Ala Lys Thr Gln Glu Arg Ser Ser Val Phe Ser Asp Trp Met Lys Ile 1555 1560 1565 Ser Ala Met Leu Cys Ala Ala Thr Leu Ala Leu Thr Gln Val Val Lys 1570 1575 1580 Met Ala Lys Thr Val Lys Gln Met Val Arg Pro Asp Leu Val Arg Val 1585 1590 1595 1600Gln Leu Asp Glu Gln Glu Gln Gly Pro Tyr Asn Glu Ala Val Arg Ala 1605 1610 1615 Lys Pro Lys Thr Leu Gln Leu Leu Asp Ile Gln Gly Pro Asn Pro Val 1620 1625 1630 Met Asp Phe Glu Lys Tyr Val Ala Lys Phe Val Thr Ala Pro Ile Asp 1635 1640 1645 Phe Val Tyr Pro Thr Gly Val Ser Thr Gln Thr Cys Leu Leu Val Lys 1650 1655 1660 Gly Arg Thr Leu Ala Val Asn Arg His Met Ala Glu Ser Asp Trp Ser 1665 1670 1675 1680Ser Ile Val Val Arg Gly Val Thr His Ala Arg Ser Thr Val Arg Ile 1685 1690 1695 Leu Ala Ile Ala Lys Ala Gly Lys Glu Thr Asp Val Ser Phe Ile Arg 1700 1705 1710 Leu Ser Ser Gly Pro Leu Phe Arg Asp Asn Thr Ser Lys Phe Val Lys 1715 1720 1725 Ala Asp Asp Val Leu Pro Ala Thr Ser Ala Pro Val Ile Gly Ile Met 1730 1735 1740 Asn Thr Asp Ile Pro Met Met Phe Thr Gly Thr Phe Leu Lys Ala Gly 1745 1750 1755 1760Val Ser Val Pro Val Glu Thr Gly Gln Thr Phe Asn His Cys Ile His 1765 1770 1775 Tyr Lys Ala Asn Thr Arg Lys Gly Trp Cys Gly Ser Ala Leu Leu Ala 1780 1785 1790 Asp Leu Gly Gly Lys Lys Lys Ile Leu Gly Met His Ser Ala Gly Ser 1795 1800 1805 Met Gly Arg Thr Ala Ala Ser Ile Val Ser Gln Glu Met Ile Cys Ala 1810 1815 1820 Val Val Ser Ala Phe Glu Pro Gln Gly Ala Leu Glu Arg Leu Pro Asp 1825 1830 1835 1840Gly Pro Arg Ile His Val Pro Arg Lys Thr Ala Leu Arg Pro Thr Val 1845 1850 1855 Ala Arg Gln Val Phe Gln Pro Ala Tyr Ala Pro Ala Val Leu Ser Lys 1860 1865 1870 Phe Asp Pro Arg Thr Glu Ala Asp Val Asp Glu Val Ala Phe Ser Lys 1875 1880 1885 His Thr Ser Asn Gln Glu Ser Leu Pro Pro Val Phe Arg Met Val Ala 1890 1895 1900 Lys Glu Tyr Ala Asn Arg Val Phe Thr Leu Leu Gly Arg Asp Asn Gly 1905 1910 1915 1920Arg Leu Thr Val Lys Gln Ala Leu Glu Gly Leu Glu Gly Met Asp Pro 1925 1930 1935 Met Asp Lys Asn Thr Ser Pro Gly Leu Pro Tyr Thr Ala Leu Gly Met 1940 1945 1950 Arg Arg Thr Asp Val Val Asp Trp Glu Ser Ala Thr Leu Ile Pro Tyr 1955 1960 1965 Ala Ala Asp Arg Leu Lys Lys Met Asn Glu Gly Asp Phe Ser Asp Ile 1970 1975 1980 Val Tyr Gln Thr Phe Leu Lys Asp Glu Leu Arg Pro Val Glu Lys Val 1985 1990 1995 2000Gln Ala Ala Lys Thr Arg Ile Val Asp Val Pro Pro Phe Glu His Cys 2005 2010 2015 Ile Leu Gly Arg Gln Leu Leu Gly Arg Phe Ala Ser Lys Phe Gln Thr 2020 2025 2030 Gln Pro Gly Leu Glu Leu Gly Ser Ala Ile Gly Cys Asp Pro Asp Val 2035 2040 2045 His Trp Thr Ala Phe Gly Val Ala Met Gln Gly Phe Glu Arg Val Tyr 2050 2055 2060 Asp Val Asp Tyr Ser Asn Phe Asp Ser Thr His Ser Val Ala Met Phe 2065 2070 2075 2080Arg Leu Leu Ala Glu Glu Phe Phe Thr Pro Glu Asn Gly Phe Asp Pro 2085 2090 2095 Leu Val Lys Glu Tyr Leu Glu Ser Leu Ala Ile Ser Thr His Ala Phe 2100 2105 2110 Glu Glu Lys Arg Tyr Leu Ile Thr Gly Gly Leu Pro Ser Gly Cys Ala 2115 2120 2125 Ala Thr Ser Met Leu Asn Thr Ile Met Asn Asn Ile Ile Ile Arg Ala 2130 2135 2140 Gly Leu Tyr Leu Thr Tyr Lys Asn Phe Glu Phe Asp Asp Val Lys Val 2145 2150 2155 2160Leu Ser Tyr Gly Asp Asp Leu Leu Val Ala Thr Asn Tyr Gln Leu Asn 2165 2170 2175 Phe Asp Lys Val Arg Ala Ser Leu Ala Lys Thr Gly Tyr Lys Ile Thr 2180 2185 2190 Pro Ala Asn Lys Thr Ser Thr Phe Pro Leu Asp Ser Thr Leu Glu Asp 2195 2200 2205 Val Val Phe Leu Lys Arg Lys Phe Lys Lys Glu Gly Pro Leu Tyr Arg 2210 2215 2220 Pro Val Met Asn Arg Glu Ala Leu Glu Ala Met Leu Ser Tyr Tyr Arg 2225 2230 2235 2240Pro Gly Thr Leu Ser Glu Lys Leu Thr Ser Ile Thr Met Leu Ala Val 2245 2250 2255 His Ser Gly Lys Pro Glu Tyr Asp Arg Leu Phe Ala Pro Phe Arg Glu 2260 2265 2270 Val Gly Val Val Val Pro Ser Phe Glu Ser Val Glu Tyr Arg Trp Arg 2275 2280 2285 Ser Leu Phe Trp 2290 292293PRTEncephalomyocarditis virus 29Met Ala Thr Thr Met Glu Gln Glu Ile Cys Ala His Ser Met Thr Phe 1 5 10 15 Glu Glu Cys Pro Lys Cys Ser Ala Leu Gln Tyr Arg Asn Gly Phe Tyr 20 25 30 Leu Leu Lys Tyr Asp Glu Glu Trp Tyr Pro Glu Glu Ser Leu Thr Asp 35 40 45 Gly Glu Asp Asp Val Phe Asp Pro Asp Leu Asp Met Glu Val Val Phe 50 55 60 Glu Thr Gln Gly Asn Ser Thr Ser Ser Asp Lys Asn Asn Ser Ser Ser 65 70 75 80Glu Gly Asn Glu Gly Val Ile Ile Asn Asn Phe Tyr Ser Asn Gln Tyr 85 90 95 Gln Asn Ser Ile Asp Leu Ser Ala Asn Ala Thr Gly Ser Asp Pro Pro 100 105 110 Lys Thr Tyr Gly Gln Phe Ser Asn Leu Leu Ser Gly Ala Val Asn Ala 115 120 125 Phe Ser Asn Met Leu Pro Leu Leu Ala Asp Gln Asn Thr Glu Glu Met 130 135 140 Glu Asn Leu Ser Asp Arg Val Ser Gln Asp Thr Ala Gly Asn Thr Val 145 150 155 160Thr Asn Thr Gln Ser Thr Val Gly Arg Leu Val Gly Tyr Gly Thr Val 165 170 175 His Asp Gly Glu His Pro Ala Ser Cys Ala Asp Thr Ala Ser Glu Lys 180 185 190 Ile Leu Ala Val Glu Arg Tyr Tyr Thr Phe Lys Val Asn Asp Trp Thr 195 200 205 Ser Thr Gln Lys Pro Phe Glu Tyr Ile Arg Ile Pro Leu Pro His Val 210 215 220 Leu Ser Gly Glu Asp Gly Gly Val Phe Gly Ala Thr Leu Arg Arg His 225 230 235 240Tyr Leu Val Lys Thr Gly Trp Arg Val Gln Val Gln Cys Asn Ala Ser 245 250 255 Gln Phe His Ala Gly Ser Leu Leu Val Phe Met Ala Pro Glu Tyr Pro 260 265 270 Thr Leu Asp Val Phe Ala Met Asp Asn Arg Trp Ser Lys Asp Asn Leu 275 280 285 Pro Asn Gly Thr Arg Thr Gln Thr Asn Arg Lys Gly Pro Phe Ala Met 290 295 300 Asp His Gln Asn Phe Trp Gln Trp Thr Leu Tyr Pro His Gln Phe Leu 305 310 315 320Asn Leu Arg Thr Asn Thr Thr Val Asp Leu Glu Val Pro Tyr Val Asn 325 330 335 Ile Ala Pro Thr Ser Ser Trp Thr Gln His Ala Ser Trp Thr Leu Val 340 345 350 Ile Ala Val Val Ala Pro Leu Thr Tyr Ser Thr Gly Ala Ser Thr Ser 355 360 365 Leu Asp Ile Thr Ala Ser Ile Gln Pro Val Arg Pro Val Phe Asn Gly 370 375 380 Leu Arg His Glu Val Leu Ser Arg Gln Ser Pro Ile Pro Val Thr Ile 385 390 395 400Arg Glu His Ala Gly Thr Trp Tyr Ser Thr Leu Pro Asp Ser Thr Val 405 410 415 Pro Ile Tyr Gly Lys Thr Pro Val Ala Pro Ala Asn Tyr Met Val Gly 420 425 430 Glu Tyr Lys Asp Phe Leu Glu Ile Ala Gln Ile Pro Thr Phe Ile Gly 435 440 445 Asn Lys Val Pro Asn Ala Val Pro Tyr Ile Glu Ala Ser Asn Thr Ala 450 455 460 Val Lys Thr Gln Pro Leu Ala Val Tyr Gln Val Thr Leu Ser Cys Ser 465 470 475 480Cys Leu Ala Asn Thr Phe Leu Ala Ala Leu Ser Arg Asn Phe Ala Gln 485 490 495 Tyr Arg Gly Ser Leu Val Tyr Thr Phe Val Phe Thr Gly Thr Ala Met 500 505 510 Met Lys Gly Lys Phe Leu Ile Ala Tyr Thr Pro Pro Gly Ala Gly Lys 515 520 525 Pro Thr Ser Arg Asp Gln Ala Met Gln Ala Thr Tyr Ala Ile Trp Asp 530 535 540 Leu Gly Leu Asn Ser Ser Tyr Ser Phe Thr Val Pro Phe Ile Ser Pro 545 550 555 560Thr His Phe Arg Met Val Gly Thr Asp Gln Ala Asn Ile Thr Asn Val 565 570 575 Asp Gly Trp Val Thr Val Trp Gln Leu Thr Pro Leu Thr Tyr Pro Pro 580 585 590 Gly Cys Pro Thr Ser Ala Lys Ile Leu Thr Met Val Ser Ala Gly Lys 595 600 605 Asp Phe Ser Leu Lys Met Pro Ile Ser Pro Ala Pro Trp Ser Pro Gln 610 615 620 Gly Val Glu Asn Ala Glu Lys Gly Val Thr Glu Asn Thr Asp Ala Thr 625 630 635 640Ala Asp Phe Val Ala Gln Pro Val Tyr Leu Pro Glu Asn Gln Thr Lys 645 650 655 Val Ala Phe Phe Tyr Asp Arg Ser Ser Pro Ile Gly Ala Phe Ala Val 660 665 670 Lys Ser Gly Ser Leu Glu Ser Gly Phe Ala Pro Phe Ser Asn Lys Ala 675 680 685 Cys Pro Asn Ser Val Ile Leu Thr Pro Gly Pro Gln Phe Asp Pro Ala 690 695 700 Tyr Asp Gln Leu Arg Pro Gln Arg Leu Thr Glu Ile Trp Gly Asn Gly 705 710 715 720Asn Glu Glu Thr Ser Glu Val Phe Pro Leu Lys Thr Lys Gln Asp Tyr 725 730 735 Ser Phe Cys Leu Phe Ser Pro Phe Val Tyr Tyr Lys Cys Asp Leu Glu 740 745 750 Val Thr Leu Ser Pro His Thr Ser Gly Ala His Gly Leu Leu Val Arg 755 760 765 Trp Cys Pro Thr Gly Thr Pro Thr Lys Pro Thr Thr Gln Val Leu His 770 775 780 Glu Val Ser Ser Leu Ser Glu Gly Arg Thr Pro Gln Val Tyr Ser Ala 785 790 795 800Gly Pro Gly Thr Ser Asn Gln Ile Ser Phe Val Val Pro Tyr Asn Ser 805 810 815 Pro Leu Ser Val Leu Pro Ala Val Trp Tyr Asn Gly His Lys Arg Phe 820 825 830 Asp Asn Thr Gly Asp Leu Gly Ile Ala Pro Asn Ser Asp Phe Gly Thr 835 840 845 Leu Phe Phe Ala Gly Thr Lys Pro Asp Ile Lys Phe Thr Val Tyr Leu 850 855 860 Arg Tyr Lys Asn Met Arg Val Phe Cys Pro Arg Pro Thr Val Phe Phe 865 870 875 880Pro Trp Pro Thr Ser Gly Asp Lys Ile Asp Met Thr Pro Arg Ala Gly 885 890 895 Val Leu Met Leu Glu Ser Pro Asn Pro Leu Asp Val Ser Lys Thr Tyr 900 905 910 Pro Thr Leu His Ile Leu Leu Gln Phe Asn His Arg Gly Leu Glu Ala 915 920 925 Arg Ile Phe Arg His Gly Gln Leu Trp Ala Glu Thr His Ala Glu Val 930 935 940 Val Leu Arg Ser Lys Thr Lys Gln Ile Ser Phe Leu Ser Asn Gly Ser 945 950 955 960Tyr Pro Ser Met Asp Ala Thr Thr Pro Leu Asn Pro Trp Lys Ser Thr 965 970 975 Tyr Gln Ala Val Leu Arg Ala Glu Pro His Arg Val Thr Met Asp Val 980 985 990 Tyr His Lys Arg Ile Arg Pro Phe Arg Leu Pro Leu Val Gln Lys Glu 995 1000 1005 Trp Arg Thr Cys Glu Glu Asn Val Phe Gly Leu Tyr His Val Phe Glu 1010 1015 1020 Thr His Tyr Ala Gly Tyr Phe Ser Asp Leu Leu Ile His Asp Val Glu 1025 1030 1035 1040Thr Asn Pro Gly Pro Phe Thr Phe Lys Pro Arg Gln Arg Pro Val Phe 1045 1050 1055 Gln Thr Gln Gly Ala Ala Val Ser Ser Met Ala Gln Thr Leu Leu Pro 1060 1065 1070 Asn Asp Leu Ala Ser Lys

Ala Met Gly Ser Ala Phe Thr Ala Leu Leu 1075 1080 1085 Asp Ala Asn Glu Asp Ala Gln Lys Ala Met Lys Ile Ile Lys Thr Leu 1090 1095 1100 Ser Ser Leu Ser Asp Ala Trp Glu Asn Val Lys Gly Thr Leu Asn Asn 1105 1110 1115 1120Pro Glu Phe Trp Lys Gln Leu Leu Ser Arg Cys Val Gln Leu Ile Ala 1125 1130 1135 Gly Met Thr Ile Ala Val Met His Pro Asp Pro Leu Thr Leu Leu Cys 1140 1145 1150 Leu Gly Val Leu Thr Ala Ala Glu Ile Thr Ser Gln Thr Ser Leu Cys 1155 1160 1165 Glu Glu Ile Ala Ala Lys Phe Lys Thr Ile Phe Thr Thr Pro Pro Pro 1170 1175 1180 Arg Phe Pro Val Ile Ser Leu Phe Gln Gln Gln Ser Pro Leu Lys Gln 1185 1190 1195 1200Val Asn Asp Val Phe Ser Leu Ala Lys Asn Leu Asp Trp Ala Val Lys 1205 1210 1215 Thr Val Glu Lys Val Val Asp Trp Phe Gly Thr Trp Val Ala Gln Glu 1220 1225 1230 Glu Arg Glu Gln Thr Leu Asp Gln Leu Leu Gln Arg Phe Pro Glu His 1235 1240 1245 Ala Lys Arg Ile Ser Asp Leu Arg Asn Gly Met Ala Ala Tyr Val Glu 1250 1255 1260 Cys Lys Glu Ser Phe Asp Phe Phe Glu Lys Leu Tyr Asn Gln Ala Val 1265 1270 1275 1280Lys Glu Lys Arg Thr Gly Ile Ala Ala Val Cys Glu Lys Phe Arg Gln 1285 1290 1295 Lys His Asp His Ala Thr Ala Arg Cys Glu Pro Val Val Ile Val Leu 1300 1305 1310 Arg Gly Asp Ala Gly Gln Gly Lys Ser Leu Ser Ser Gln Ile Ile Ala 1315 1320 1325 Gln Ala Val Ser Lys Thr Ile Phe Gly Arg Gln Ser Val Tyr Ser Leu 1330 1335 1340 Pro Pro Asp Ser Asp Phe Phe Asp Gly Tyr Glu Asn Gln Phe Ala Ala 1345 1350 1355 1360Ile Met Asp Asp Leu Gly Gln Asn Pro Asp Gly Ser Asp Phe Thr Thr 1365 1370 1375 Phe Cys Gln Met Val Ser Thr Thr Asn Leu Leu Pro Asn Met Ala Ser 1380 1385 1390 Leu Glu Arg Lys Gly Thr Pro Phe Thr Ser Gln Leu Val Val Ala Thr 1395 1400 1405 Thr Asn Leu Pro Glu Phe Arg Pro Val Thr Ile Ala His Tyr Pro Ala 1410 1415 1420 Val Glu Arg Arg Ile Thr Phe Asp Tyr Ser Val Ser Ala Gly Pro Val 1425 1430 1435 1440Cys Ser Lys Thr Glu Ala Gly Cys Lys Val Leu Asp Val Glu Arg Ala 1445 1450 1455 Phe Arg Pro Thr Gly Asp Ala Pro Leu Pro Cys Phe Gln Asn Asn Cys 1460 1465 1470 Leu Phe Leu Glu Lys Ala Gly Leu Gln Phe Arg Asp Asn Arg Ser Lys 1475 1480 1485 Glu Ile Leu Ser Leu Val Asp Val Ile Glu Arg Ala Val Thr Arg Ile 1490 1495 1500 Glu Arg Lys Lys Lys Val Leu Thr Ala Val Gln Thr Leu Val Ala Gln 1505 1510 1515 1520Gly Pro Val Asp Glu Val Ser Phe Tyr Ser Val Val Gln Gln Leu Lys 1525 1530 1535 Ala Arg Gln Glu Ala Thr Asp Glu Gln Leu Glu Glu Leu Gln Glu Ala 1540 1545 1550 Phe Ala Arg Val Gln Glu Arg Ser Ser Val Phe Ser Asp Trp Met Lys 1555 1560 1565 Ile Ser Ala Met Leu Cys Ala Ala Thr Leu Ala Leu Thr Gln Val Val 1570 1575 1580 Lys Met Ala Lys Ala Val Lys Gln Met Val Arg Pro Asp Leu Val Arg 1585 1590 1595 1600Val Gln Leu Asp Glu Gln Glu Gln Gly Pro Tyr Asn Glu Thr Thr Arg 1605 1610 1615 Ile Lys Pro Lys Thr Leu Gln Leu Leu Asp Val Gln Gly Pro Asn Pro 1620 1625 1630 Thr Met Asp Phe Glu Lys Phe Val Ala Lys Phe Val Thr Ala Pro Ile 1635 1640 1645 Gly Phe Val Tyr Pro Thr Gly Val Ser Thr Gln Thr Cys Leu Leu Val 1650 1655 1660 Lys Gly Arg Thr Leu Ala Val Asn Arg His Met Ala Glu Ser Asp Trp 1665 1670 1675 1680Thr Ser Ile Val Val Arg Gly Val Ser His Thr Arg Ser Ser Val Lys 1685 1690 1695 Ile Ile Ala Ile Ala Lys Ala Gly Lys Glu Thr Asp Val Ser Phe Ile 1700 1705 1710 Arg Leu Ser Ser Gly Pro Leu Phe Arg Asp Asn Thr Ser Lys Phe Val 1715 1720 1725 Lys Ala Ser Asp Val Leu Pro His Ser Ser Ser Pro Leu Ile Gly Ile 1730 1735 1740 Met Asn Val Asp Ile Pro Met Met Tyr Thr Gly Thr Phe Leu Lys Ala 1745 1750 1755 1760Gly Val Ser Val Pro Val Glu Thr Gly Gln Thr Phe Asn His Cys Ile 1765 1770 1775 His Tyr Lys Ala Asn Thr Arg Lys Gly Trp Cys Gly Ser Ala Ile Leu 1780 1785 1790 Ala Asp Leu Gly Gly Ser Lys Lys Ile Leu Gly Phe His Ser Ala Gly 1795 1800 1805 Ser Met Gly Val Ala Ala Ala Ser Ile Ile Ser Gln Glu Met Ile Asp 1810 1815 1820 Ala Val Val Gln Ala Phe Glu Pro Gln Gly Ala Leu Glu Arg Leu Pro 1825 1830 1835 1840Asp Gly Pro Arg Ile His Val Pro Arg Lys Thr Ala Leu Arg Pro Thr 1845 1850 1855 Val Ala Arg Gln Val Phe Gln Pro Ala Phe Ala Pro Ala Val Leu Ser 1860 1865 1870 Lys Phe Asp Pro Arg Thr Asp Ala Asp Val Asp Glu Val Ala Phe Ser 1875 1880 1885 Lys His Thr Ser Asn Gln Glu Thr Leu Pro Pro Val Phe Arg Met Val 1890 1895 1900 Ala Arg Glu Tyr Ala Asn Arg Val Phe Ala Leu Leu Gly Arg Asp Asn 1905 1910 1915 1920Gly Arg Leu Ser Val Lys Gln Ala Leu Asp Gly Leu Glu Gly Met Asp 1925 1930 1935 Pro Met Asp Lys Asn Thr Ser Pro Gly Leu Pro Tyr Thr Thr Leu Gly 1940 1945 1950 Met Arg Arg Thr Asp Val Val Asp Trp Glu Thr Ala Thr Leu Ile Pro 1955 1960 1965 Phe Ala Ala Glu Arg Leu Glu Lys Met Asn Asn Lys Asp Phe Ser Asp 1970 1975 1980 Ile Val Tyr Gln Thr Phe Leu Lys Asp Glu Leu Arg Pro Ile Glu Lys 1985 1990 1995 2000Val Gln Ala Ala Lys Thr Arg Ile Val Asp Val Pro Pro Phe Glu His 2005 2010 2015 Cys Ile Leu Gly Arg Gln Leu Leu Gly Lys Phe Ala Ser Lys Phe Gln 2020 2025 2030 Thr Gln Pro Gly Leu Glu Leu Gly Ser Ala Ile Gly Cys Asp Pro Asp 2035 2040 2045 Val His Trp Thr Ala Phe Gly Val Ala Met Gln Gly Phe Glu Arg Val 2050 2055 2060 Tyr Asp Val Asp Tyr Ser Asn Phe Asp Ser Thr His Ser Val Ala Ile 2065 2070 2075 2080Phe Arg Leu Leu Ala Glu Glu Phe Phe Ser Glu Glu Asn Gly Phe Asp 2085 2090 2095 Pro Leu Val Lys Asp Tyr Leu Glu Ser Leu Ala Ile Ser Lys His Ala 2100 2105 2110 Tyr Glu Glu Lys Arg Tyr Leu Ile Thr Gly Gly Leu Pro Ser Gly Cys 2115 2120 2125 Ala Ala Thr Ser Met Leu Asn Thr Ile Met Asn Asn Ile Ile Ile Arg 2130 2135 2140 Ala Gly Leu Tyr Leu Thr Tyr Lys Asn Phe Glu Phe Asp Asp Val Lys 2145 2150 2155 2160Val Leu Ser Tyr Gly Asp Asp Leu Leu Val Ala Thr Asn Tyr Gln Leu 2165 2170 2175 Asn Phe Asp Arg Val Arg Thr Ser Leu Ala Lys Thr Gly Tyr Lys Ile 2180 2185 2190 Thr Pro Ala Asn Lys Thr Ser Thr Phe Pro Leu Glu Ser Thr Leu Glu 2195 2200 2205 Asp Val Val Phe Leu Lys Arg Lys Phe Lys Lys Glu Gly Pro Leu Tyr 2210 2215 2220 Arg Pro Val Met Asn Arg Glu Ala Leu Glu Ala Met Leu Ser Tyr Tyr 2225 2230 2235 2240Arg Pro Gly Thr Leu Ser Glu Lys Leu Thr Ser Ile Thr Met Leu Ala 2245 2250 2255 Val His Ser Gly Lys Gln Glu Tyr Asp Arg Leu Phe Ala Pro Phe Arg 2260 2265 2270 Glu Val Gly Val Ile Val Pro Thr Phe Glu Ser Val Glu Tyr Arg Trp 2275 2280 2285 Arg Ser Leu Phe Trp 2290 302301PRTTheiler's murine encephalomyelitis virus 30Met Ala Cys Lys His Gly Tyr Pro Asp Val Cys Pro Ile Cys Thr Ala 1 5 10 15 Val Asp Val Thr Pro Gly Phe Glu Tyr Leu Leu Leu Ala Asp Gly Glu 20 25 30 Trp Phe Pro Thr Asp Leu Leu Cys Val Asp Leu Asp Asp Asp Val Phe 35 40 45 Trp Pro Ser Asn Ser Ser Asn Gln Ser Glu Thr Met Glu Trp Thr Asp 50 55 60 Leu Pro Leu Val Arg Asp Ile Val Met Glu Pro Gln Gly Asn Ala Ser 65 70 75 80Ser Ser Asp Lys Ser Asn Ser Gln Ser Ser Gly Asn Glu Gly Val Ile 85 90 95 Ile Asn Asn Phe Tyr Ser Asn Gln Tyr Gln Asn Ser Ile Asp Leu Ser 100 105 110 Ala Ser Gly Gly Asn Ala Gly Asp Ala Pro Gln Asn Asn Gly Gln Leu 115 120 125 Ser Asn Ile Leu Gly Gly Ala Ala Asn Ala Phe Ala Thr Met Ala Pro 130 135 140 Leu Leu Leu Asp Gln Asn Thr Glu Glu Met Glu Asn Leu Ser Asp Arg 145 150 155 160Val Ala Ser Asp Lys Ala Gly Asn Ser Ala Thr Asn Thr Gln Ser Thr 165 170 175 Val Gly Arg Leu Cys Gly Tyr Gly Glu Ala His His Gly Glu His Pro 180 185 190 Ala Ser Cys Ala Asp Thr Ala Thr Asp Lys Val Leu Ala Ala Glu Arg 195 200 205 Tyr Tyr Thr Ile Asp Leu Ala Ser Trp Thr Thr Thr Gln Glu Ala Phe 210 215 220 Ser His Ile Arg Ile Pro Leu Pro His Val Leu Ala Gly Glu Asp Gly 225 230 235 240Gly Val Phe Gly Ala Thr Leu Arg Arg His Tyr Leu Cys Lys Thr Gly 245 250 255 Trp Arg Val Gln Val Gln Cys Asn Ala Ser Gln Phe His Ala Gly Ser 260 265 270 Leu Leu Val Phe Met Ala Pro Glu Phe Tyr Thr Gly Lys Gly Thr Lys 275 280 285 Thr Gly Asp Met Glu Pro Thr Asp Pro Phe Thr Met Asp Thr Thr Trp 290 295 300 Arg Ala Pro Gln Gly Ala Pro Thr Gly Tyr Arg Tyr Asp Ser Arg Thr 305 310 315 320Gly Phe Phe Ala Met Asn His Gln Asn Gln Trp Gln Trp Thr Val Tyr 325 330 335 Pro His Gln Ile Leu Asn Leu Arg Thr Asn Thr Thr Val Asp Leu Glu 340 345 350 Val Pro Tyr Val Asn Ile Ala Pro Thr Ser Ser Trp Thr Gln His Ala 355 360 365 Asn Trp Thr Leu Val Val Ala Val Phe Ser Pro Leu Gln Tyr Ala Ser 370 375 380 Gly Ser Ser Ser Asp Val Gln Ile Thr Ala Ser Ile Gln Pro Val Asn 385 390 395 400Pro Val Phe Asn Gly Leu Arg His Glu Thr Val Ile Ala Gln Ser Pro 405 410 415 Ile Ala Val Thr Val Arg Glu His Lys Gly Cys Phe Tyr Ser Thr Asn 420 425 430 Pro Asp Thr Thr Val Pro Ile Tyr Gly Lys Thr Ile Ser Thr Pro Asn 435 440 445 Asp Tyr Met Cys Gly Glu Phe Ser Asp Leu Leu Glu Leu Cys Lys Leu 450 455 460 Pro Thr Phe Leu Gly Asn Pro Asn Ser Asn Asn Lys Arg Tyr Pro Tyr 465 470 475 480Phe Ser Ala Thr Asn Ser Val Pro Thr Thr Ser Leu Val Asp Tyr Gln 485 490 495 Val Ala Leu Ser Cys Ser Cys Met Cys Asn Ser Met Leu Ala Ala Val 500 505 510 Ala Arg Asn Phe Asn Gln Tyr Arg Gly Ser Leu Asn Phe Leu Phe Val 515 520 525 Phe Thr Gly Ala Ala Met Val Lys Gly Lys Phe Leu Ile Ala Tyr Thr 530 535 540 Pro Pro Gly Ala Gly Lys Pro Thr Thr Arg Asp Gln Ala Met Gln Ala 545 550 555 560Thr Tyr Ala Ile Trp Asp Leu Gly Leu Asn Ser Ser Phe Val Phe Thr 565 570 575 Ala Pro Phe Ile Ser Pro Thr His Tyr Arg Gln Thr Ser Tyr Thr Ser 580 585 590 Ala Thr Ile Ala Ser Val Asp Gly Trp Val Thr Val Trp Gln Leu Thr 595 600 605 Pro Leu Thr Tyr Pro Ser Gly Ala Pro Val Asn Ser Asp Ile Leu Thr 610 615 620 Leu Val Ser Ala Gly Asp Asp Phe Thr Leu Arg Met Pro Ile Ser Pro 625 630 635 640Thr Lys Trp Ala Pro Gln Gly Ser Asp Asn Ala Glu Lys Gly Lys Val 645 650 655 Ser Asn Asp Asp Ala Ser Val Asp Phe Val Ala Glu Pro Val Lys Leu 660 665 670 Pro Glu Asn Gln Thr Arg Val Ala Phe Phe Tyr Asp Arg Ala Val Pro 675 680 685 Ile Gly Met Leu Arg Pro Gly Gln Asn Ile Glu Ser Thr Phe Val Tyr 690 695 700 Gln Glu Asn Asp Leu Arg Leu Asn Cys Leu Leu Leu Thr Pro Leu Pro 705 710 715 720Ser Phe Cys Pro Asp Ser Thr Ser Gly Pro Val Lys Thr Lys Ala Pro 725 730 735 Val Gln Trp Arg Trp Val Arg Ser Gly Gly Thr Thr Asn Phe Pro Leu 740 745 750 Met Thr Lys Gln Asp Tyr Ala Phe Leu Cys Phe Ser Pro Phe Thr Tyr 755 760 765 Tyr Lys Cys Asp Leu Glu Val Thr Val Ser Ala Leu Gly Thr Asp Thr 770 775 780 Val Ala Ser Val Leu Arg Trp Ala Pro Thr Gly Ala Pro Ala Asp Val 785 790 795 800Thr Asp Gln Leu Ile Gly Tyr Thr Pro Ser Leu Gly Glu Thr Arg Asn 805 810 815 Pro His Met Trp Leu Val Gly Ala Gly Asn Thr Gln Ile Ser Phe Val 820 825 830 Val Pro Tyr Asn Ser Pro Leu Ser Val Leu Pro Ala Ala Trp Phe Asn 835 840 845 Gly Trp Ser Asp Phe Gly Asn Thr Lys Asp Phe Gly Val Ala Pro Asn 850 855 860 Ala Asp Phe Gly Arg Leu Trp Ile Gln Gly Asn Thr Ser Ala Ser Val 865 870 875 880Arg Ile Arg Tyr Lys Lys Met Lys Val Phe Cys Pro Arg Pro Thr Leu 885 890 895 Phe Phe Pro Trp Pro Val Ser Thr Arg Ser Lys Ile Asn Ala Asp Asn 900 905 910 Pro Val Pro Ile Leu Glu Leu Glu Asn Pro Ala Ala Phe Tyr Arg Ile 915 920 925 Asp Leu Phe Ile Thr Phe Ile Asp Glu Phe Ile Thr Phe Asp Tyr Lys 930 935 940 Val His Gly Arg Pro Val Leu Thr Phe Arg Ile Pro Gly Phe Gly Leu 945 950 955 960Thr Pro Ala Gly Arg Met Leu Val Cys Met Gly Glu Lys Pro Ala His 965 970 975 Gly Pro Phe Thr Ser Ser Arg Ser Leu Tyr His Val Ile Phe Thr Ala 980 985 990 Thr Cys Ser Ser Phe Ser Phe Ser Ile Tyr Lys Gly Arg Tyr Arg Ser 995 1000 1005 Trp Lys Lys Pro Ile His Asp Glu Leu Val Asp Arg Gly Tyr Thr Thr 1010 1015 1020 Phe Gly Glu Phe Phe Arg Ala Val Arg Ala Tyr His Ala Asp Tyr Tyr 1025 1030 1035 1040Lys Gln Arg Leu Ile His Asp Val Glu Met Asn Pro Gly Pro Val Gln 1045 1050

1055 Ser Val Phe Gln Pro Gln Gly Ala Val Leu Thr Lys Ser Leu Ala Pro 1060 1065 1070 Gln Ala Gly Ile Gln Asn Leu Leu Leu Arg Leu Leu Gly Ile Asp Gly 1075 1080 1085 Asp Cys Ser Glu Val Ser Lys Ala Ile Thr Val Val Thr Asp Leu Phe 1090 1095 1100 Ala Ala Trp Glu Arg Ala Lys Thr Thr Leu Val Ser Pro Glu Phe Trp 1105 1110 1115 1120Ser Lys Leu Ile Leu Lys Thr Thr Lys Phe Ile Ala Ala Ser Val Leu 1125 1130 1135 Tyr Leu His Asn Pro Asp Phe Thr Thr Thr Val Cys Leu Ser Leu Met 1140 1145 1150 Thr Gly Val Asp Leu Leu Thr Asn Asp Ser Val Phe Asp Trp Leu Lys 1155 1160 1165 Asn Lys Leu Ser Ser Phe Phe Arg Thr Pro Pro Pro Val Cys Pro Asn 1170 1175 1180 Val Leu Gln Pro Gln Gly Pro Leu Arg Glu Ala Asn Glu Gly Phe Thr 1185 1190 1195 1200Phe Ala Lys Asn Ile Glu Trp Ala Met Lys Thr Ile Gln Ser Ile Val 1205 1210 1215 Asn Trp Leu Thr Ser Trp Phe Lys Gln Glu Glu Asp His Pro Gln Ser 1220 1225 1230 Lys Leu Asp Lys Phe Leu Met Glu Phe Pro Asp His Cys Arg Asn Ile 1235 1240 1245 Met Asp Met Arg Asn Gly Arg Lys Ala Tyr Cys Glu Cys Thr Ala Ser 1250 1255 1260 Phe Lys Tyr Phe Asp Glu Leu Tyr Asn Leu Ala Val Thr Cys Lys Arg 1265 1270 1275 1280Ile Pro Leu Ala Ser Leu Cys Glu Lys Phe Lys Asn Arg His Asp His 1285 1290 1295 Ser Val Thr Arg Pro Glu Pro Val Val Val Val Leu Arg Gly Ala Ala 1300 1305 1310 Gly Gln Gly Lys Ser Val Thr Ser Gln Ile Ile Ala Gln Ser Val Ser 1315 1320 1325 Lys Met Ala Phe Gly Arg Gln Ser Val Tyr Ser Met Pro Pro Asp Ser 1330 1335 1340 Glu Tyr Phe Asp Gly Tyr Glu Asn Gln Phe Ser Val Ile Met Asp Asp 1345 1350 1355 1360Leu Gly Gln Asn Pro Asp Gly Glu Asp Phe Thr Val Phe Cys Gln Met 1365 1370 1375 Val Ser Ser Thr Asn Phe Leu Pro Asn Met Ala His Leu Glu Arg Lys 1380 1385 1390 Gly Thr Pro Phe Thr Ser Ser Phe Ile Val Ala Thr Thr Asn Leu Pro 1395 1400 1405 Lys Phe Arg Pro Val Thr Val Ala His Tyr Pro Ala Val Asp Arg Arg 1410 1415 1420 Ile Thr Phe Asp Phe Thr Val Thr Ala Gly Pro His Cys Thr Thr Ser 1425 1430 1435 1440Asn Gly Met Leu Asp Ile Glu Lys Ala Phe Asp Glu Ile Pro Gly Ser 1445 1450 1455 Lys Pro Gln Leu Ala Cys Phe Ser Ala Asp Cys Pro Leu Leu His Lys 1460 1465 1470 Arg Gly Val Met Phe Thr Cys Asn Arg Thr Lys Ala Val Tyr Asn Leu 1475 1480 1485 Gln Gln Val Val Lys Met Val Asn Asp Thr Ile Thr Arg Lys Thr Glu 1490 1495 1500 Asn Val Lys Lys Met Asn Ser Leu Val Ala Gln Ser Pro Pro Asp Trp 1505 1510 1515 1520Glu His Phe Glu Asn Ile Leu Thr Cys Leu Arg Gln Asn Asn Ala Ala 1525 1530 1535 Leu Gln Asp Gln Leu Asp Glu Leu Gln Glu Ala Phe Ala Gln Ala Arg 1540 1545 1550 Glu Arg Ser Asp Phe Leu Ser Asp Trp Leu Lys Val Ser Ala Ile Ile 1555 1560 1565 Phe Ala Gly Ile Ala Ser Leu Ser Ala Val Ile Lys Leu Ala Ser Lys 1570 1575 1580 Phe Lys Glu Ser Ile Trp Pro Ser Pro Val Arg Val Glu Leu Ser Glu 1585 1590 1595 1600Gly Glu Gln Ala Ala Tyr Ala Gly Arg Ala Arg Ala Gln Lys Gln Ala 1605 1610 1615 Leu Gln Val Leu Asp Ile Gln Gly Gly Gly Lys Val Leu Ala Gln Ala 1620 1625 1630 Gly Asn Pro Val Met Asp Phe Glu Leu Phe Cys Ala Lys Asn Met Val 1635 1640 1645 Ala Pro Ile Thr Phe Tyr Tyr Pro Asp Lys Ala Glu Val Thr Gln Ser 1650 1655 1660 Cys Leu Leu Leu Arg Ala His Leu Phe Val Val Asn Arg His Val Ala 1665 1670 1675 1680Glu Thr Glu Trp Thr Ala Phe Lys Leu Lys Asp Val Arg His Glu Arg 1685 1690 1695 Asp Thr Val Val Thr Arg Ser Val Asn Arg Ser Gly Ala Glu Thr Asp 1700 1705 1710 Leu Thr Phe Ile Lys Val Thr Lys Gly Pro Leu Phe Lys Asp Asn Val 1715 1720 1725 Asn Lys Phe Cys Ser Asn Lys Asp Asp Phe Pro Ala Arg Asn Asp Ala 1730 1735 1740 Val Thr Gly Ile Met Asn Thr Gly Leu Ala Phe Val Tyr Ser Gly Asn 1745 1750 1755 1760Phe Leu Ile Gly Asn Gln Pro Val Asn Thr Thr Thr Gly Ala Cys Phe 1765 1770 1775 Asn His Cys Leu His Tyr Arg Ala Gln Thr Arg Arg Gly Trp Cys Gly 1780 1785 1790 Ser Ala Val Ile Cys Asn Val Asn Gly Lys Lys Ala Val Tyr Gly Met 1795 1800 1805 His Ser Ala Gly Gly Gly Gly Leu Ala Ala Ala Thr Ile Ile Thr Arg 1810 1815 1820 Glu Leu Ile Glu Ala Ala Glu Lys Ser Met Leu Ala Leu Glu Pro Gln 1825 1830 1835 1840Gly Ala Ile Val Asp Ile Ser Thr Gly Ser Val Val His Val Pro Arg 1845 1850 1855 Lys Thr Lys Leu Arg Arg Thr Val Ala His Asp Val Phe Gln Pro Lys 1860 1865 1870 Phe Glu Pro Ala Val Leu Ser Arg Tyr Asp Pro Arg Thr Asp Lys Asp 1875 1880 1885 Val Asp Val Val Ala Phe Ser Lys His Thr Thr Asn Met Glu Ser Leu 1890 1895 1900 Pro Pro Val Phe Asp Ile Val Cys Asp Glu Tyr Ala Asn Arg Val Phe 1905 1910 1915 1920Thr Ile Leu Gly Lys Asp Asn Gly Leu Leu Thr Val Glu Gln Ala Val 1925 1930 1935 Leu Gly Leu Pro Gly Met Asp Pro Met Glu Lys Asp Thr Ser Pro Gly 1940 1945 1950 Leu Pro Tyr Thr Gln Gln Gly Leu Arg Arg Thr Asp Leu Leu Asn Phe 1955 1960 1965 Asn Thr Ala Lys Met Thr Pro Gln Leu Asp Tyr Ala His Ser Lys Leu 1970 1975 1980 Val Leu Gly Val Tyr Asp Asp Val Val Tyr Gln Ser Phe Leu Lys Asp 1985 1990 1995 2000Glu Ile Arg Pro Leu Glu Lys Ile His Glu Ala Lys Thr Arg Ile Val 2005 2010 2015 Asp Val Pro Pro Phe Ala His Cys Ile Trp Gly Arg Gln Leu Leu Gly 2020 2025 2030 Arg Phe Ala Ser Lys Phe Gln Thr Lys Pro Gly Leu Glu Leu Gly Ser 2035 2040 2045 Ala Ile Gly Thr Asp Pro Asp Val Asp Trp Thr Pro Tyr Ala Ala Glu 2050 2055 2060 Leu Ser Gly Phe Asn Tyr Val Tyr Asp Val Asp Tyr Ser Asn Phe Asp 2065 2070 2075 2080Ala Ser His Ser Thr Ala Met Phe Glu Cys Leu Ile Lys Asn Phe Phe 2085 2090 2095 Thr Glu Gln Asn Gly Phe Asp Arg Arg Ile Ala Glu Tyr Leu Arg Ser 2100 2105 2110 Leu Ala Val Ser Arg His Ala Tyr Glu Asp Arg Arg Val Leu Ile Arg 2115 2120 2125 Gly Gly Leu Leu Ser Gly Cys Ala Ala Thr Ser Met Leu Asn Thr Ile 2130 2135 2140 Met Asn Asn Val Ile Ile Arg Ala Ala Leu Tyr Leu Thr Tyr Ser Asn 2145 2150 2155 2160Phe Glu Phe Asp Asp Ile Lys Val Leu Ser Tyr Gly Asp Asp Leu Leu 2165 2170 2175 Ile Gly Thr Asn Tyr Gln Ile Asp Phe Asn Leu Val Lys Glu Arg Leu 2180 2185 2190 Ala Pro Phe Gly Tyr Lys Ile Thr Pro Ala Asn Lys Thr Thr Thr Phe 2195 2200 2205 Pro Leu Thr Ser His Leu Gln Asp Val Thr Phe Leu Lys Arg Arg Phe 2210 2215 2220 Val Arg Phe Asn Ser Tyr Leu Phe Arg Pro Gln Met Asp Ala Val Asn 2225 2230 2235 2240Leu Lys Ala Met Val Ser Tyr Cys Lys Pro Gly Thr Leu Lys Glu Lys 2245 2250 2255 Leu Met Ser Ile Ala Leu Leu Ala Val His Ser Gly Pro Asp Ile Tyr 2260 2265 2270 Asp Glu Ile Phe Leu Pro Phe Arg Asn Val Gly Ile Val Val Pro Thr 2275 2280 2285 Tyr Ser Ser Met Leu Tyr Arg Trp Leu Ser Leu Phe Arg 2290 2295 2300 312303PRTTheiler's murine encephalomyelitis virus 31Met Ala Cys Lys His Gly Tyr Pro Asp Val Cys Pro Ile Cys Thr Ala 1 5 10 15 Val Asp Ala Thr Pro Asp Phe Glu Tyr Leu Leu Met Ala Asp Gly Glu 20 25 30 Trp Phe Pro Thr Asp Leu Leu Cys Val Asp Leu Asp Asp Asp Val Phe 35 40 45 Trp Pro Ser Asp Thr Ser Thr Gln Pro Gln Thr Met Glu Trp Thr Asp 50 55 60 Val Pro Leu Val Cys Asp Thr Val Met Glu Pro Gln Gly Asn Ala Ser 65 70 75 80Ser Ser Asp Lys Ser Asn Ser Gln Ser Ser Gly Asn Glu Gly Val Ile 85 90 95 Ile Asn Asn Phe Tyr Ser Asn Gln Tyr Gln Asn Ser Ile Asp Leu Ser 100 105 110 Ala Ser Gly Gly Asn Ala Gly Asp Ala Pro Gln Asn Asn Gly Gln Leu 115 120 125 Ser Ser Ile Leu Gly Gly Ala Ala Asn Ala Phe Ala Thr Met Ala Pro 130 135 140 Leu Leu Met Asp Gln Asn Thr Glu Glu Met Glu Asn Leu Ser Asp Arg 145 150 155 160Val Ala Ser Asp Lys Ala Gly Asn Ser Ala Thr Asn Thr Gln Ser Thr 165 170 175 Val Gly Arg Leu Cys Gly Tyr Gly Lys Ser His His Gly Glu His Pro 180 185 190 Thr Ser Cys Ala Asp Ala Ala Thr Asp Lys Val Leu Ala Ala Glu Arg 195 200 205 Tyr Tyr Thr Ile Asp Leu Ala Ser Trp Thr Thr Ser Gln Glu Ala Phe 210 215 220 Ser His Ile Arg Ile Pro Leu Pro His Val Leu Ala Gly Glu Asp Gly 225 230 235 240Gly Val Phe Gly Ala Thr Leu Arg Arg His Tyr Leu Cys Lys Thr Gly 245 250 255 Trp Arg Val Gln Val Gln Cys Asn Ala Ser Gln Phe His Ala Gly Ser 260 265 270 Leu Leu Val Phe Met Ala Pro Glu Phe Tyr Thr Gly Lys Gly Thr Lys 275 280 285 Ser Gly Thr Met Glu Pro Ser Asp Pro Phe Thr Met Asp Thr Thr Trp 290 295 300 Arg Ser Pro Gln Ser Ala Pro Thr Gly Tyr Arg Tyr Asp Arg Gln Ala 305 310 315 320Gly Phe Phe Ala Met Asn His Gln Asn Gln Trp Gln Trp Thr Val Tyr 325 330 335 Pro His Gln Ile Leu Asn Leu Arg Thr Asn Thr Thr Val Asp Leu Glu 340 345 350 Val Pro Tyr Val Asn Val Ala Pro Ser Ser Ser Trp Thr Gln His Ala 355 360 365 Asn Trp Thr Leu Val Val Ala Val Leu Ser Pro Leu Gln Tyr Ala Thr 370 375 380 Gly Ser Ser Pro Asp Val Gln Ile Thr Ala Ser Leu Gln Pro Val Asn 385 390 395 400Pro Val Phe Asn Gly Leu Arg His Glu Thr Val Leu Ala Gln Ser Pro 405 410 415 Ile Pro Val Thr Val Arg Glu His Gln Gly Cys Phe Tyr Ser Thr Asn 420 425 430 Pro Asp Thr Thr Val Pro Ile Tyr Gly Lys Thr Ile Ser Thr Pro Ser 435 440 445 Asp Tyr Met Cys Gly Glu Phe Ser Asp Leu Leu Glu Leu Cys Lys Leu 450 455 460 Pro Thr Phe Leu Gly Asn Pro Ser Thr Asp Asn Lys Arg Tyr Pro Tyr 465 470 475 480Phe Ser Ala Thr Asn Ser Val Pro Ala Thr Ser Leu Val Asp Tyr Gln 485 490 495 Val Ala Leu Ser Cys Ser Cys Thr Ala Asn Ser Met Leu Ala Ala Val 500 505 510 Ala Arg Asn Phe Asn Gln Tyr Arg Gly Ser Leu Asn Phe Leu Phe Val 515 520 525 Phe Thr Gly Ala Ala Met Val Lys Gly Lys Phe Arg Ile Ala Tyr Thr 530 535 540 Pro Pro Gly Ala Gly Lys Pro Thr Thr Arg Asp Gln Ala Met Gln Ala 545 550 555 560Thr Tyr Ala Ile Trp Asp Leu Gly Leu Asn Ser Ser Phe Asn Phe Thr 565 570 575 Ala Pro Phe Ile Ser Pro Thr His Tyr Arg Gln Thr Ser Tyr Thr Ser 580 585 590 Pro Thr Ile Thr Ser Val Asp Gly Trp Val Thr Val Trp Gln Leu Thr 595 600 605 Pro Leu Thr Tyr Pro Ser Gly Thr Pro Thr His Ser Asp Ile Leu Thr 610 615 620 Leu Val Ser Ala Gly Asp Asp Phe Thr Leu Arg Met Pro Ile Ser Pro 625 630 635 640Thr Lys Trp Val Pro Gln Gly Ile Asp Asn Ala Glu Lys Gly Lys Val 645 650 655 Ser Asn Asp Asp Ala Ser Val Asp Phe Val Ala Glu Pro Val Lys Leu 660 665 670 Pro Glu Asn Gln Thr Arg Val Ala Phe Phe Tyr Asp Arg Ala Val Pro 675 680 685 Ile Gly Met Leu Arg Pro Gly Gln Asn Met Glu Thr Thr Phe Ser Tyr 690 695 700 Gln Glu Asn Asp Phe Arg Leu Asn Cys Leu Leu Leu Thr Pro Leu Pro 705 710 715 720Ser Tyr Cys Pro Asp Ser Ser Ser Gly Pro Val Arg Thr Lys Ala Pro 725 730 735 Val Gln Trp Arg Trp Val Arg Ser Gly Gly Ala Asn Gly Ala Asn Phe 740 745 750 Pro Leu Met Thr Lys Gln Asp Tyr Ala Phe Leu Cys Phe Ser Pro Phe 755 760 765 Thr Tyr Tyr Lys Cys Asp Leu Glu Val Thr Val Ser Ala Met Gly Ala 770 775 780 Gly Thr Val Ser Ser Val Leu Arg Trp Ala Pro Thr Gly Ala Pro Ala 785 790 795 800Asp Val Thr Asp Gln Leu Ile Gly Tyr Thr Pro Ser Leu Gly Glu Thr 805 810 815 Arg Asn Pro His Met Trp Ile Val Gly Ser Gly Asn Ser Gln Ile Ser 820 825 830 Phe Val Val Pro Tyr Asn Ser Pro Leu Ser Val Leu Pro Ala Ala Trp 835 840 845 Phe Asn Gly Trp Ser Asp Phe Gly Asn Thr Lys Asp Phe Gly Val Ala 850 855 860 Pro Thr Ser Asp Phe Gly Arg Ile Trp Ile Gln Gly Asn Ser Ser Ala 865 870 875 880Ser Val Arg Ile Arg Tyr Lys Lys Met Lys Val Phe Cys Pro Arg Pro 885 890 895 Thr Leu Phe Phe Pro Trp Pro Thr Pro Thr Thr Thr Lys Ile Asn Ala 900 905 910 Asp Asn Pro Val Pro Ile Leu Glu Leu Glu Asn Pro Ala Ser Leu Tyr 915 920 925 Arg Ile Asp Leu Phe Ile Thr Phe Thr Asp Glu Leu Ile Thr Phe Asp 930 935 940 Tyr Lys Val His Gly Arg Pro Val Leu Thr Phe Arg Ile Pro Gly Phe 945 950 955 960Gly Leu Thr Pro Ala Gly Arg Met Leu Val Cys Met Gly Ala Lys Pro 965 970 975 Ala His Ser Pro Phe Thr Ser Ser Lys Ser Leu Tyr His Val Ile Phe 980 985 990 Thr Ser Thr Cys Asn Ser Phe Ser Phe Thr Ile Tyr Lys Gly Arg Tyr 995 1000 1005 Arg Ser Trp Lys Lys Pro Ile His Asp Glu Leu Val Asp Arg Gly Tyr 1010 1015 1020 Thr Thr Phe Arg Glu Phe Phe Lys Ala Val

Arg Gly Tyr His Ala Asp 1025 1030 1035 1040Tyr Tyr Lys Gln Arg Leu Ile His Asp Val Glu Met Asn Pro Gly Pro 1045 1050 1055 Val Gln Ser Val Phe Gln Pro Gln Gly Ala Val Leu Thr Lys Ser Leu 1060 1065 1070 Ala Pro Gln Ala Gly Ile Gln Asn Ile Leu Leu Arg Leu Leu Gly Ile 1075 1080 1085 Glu Gly Asp Cys Ser Glu Val Ser Lys Ala Ile Thr Val Val Thr Asp 1090 1095 1100 Leu Val Ala Ala Trp Glu Lys Ala Lys Thr Thr Leu Val Ser Pro Glu 1105 1110 1115 1120Phe Trp Ser Glu Leu Ile Leu Lys Thr Thr Lys Phe Ile Ala Ala Ser 1125 1130 1135 Val Leu Tyr Leu His Asn Pro Asp Phe Thr Thr Thr Val Cys Leu Ser 1140 1145 1150 Leu Met Thr Gly Val Asp Leu Leu Thr Asn Asp Ser Val Phe Asp Trp 1155 1160 1165 Leu Lys Ser Lys Leu Ser Ser Phe Phe Arg Thr Pro Pro Pro Ala Cys 1170 1175 1180 Pro Asn Val Met Gln Pro Gln Gly Pro Leu Arg Glu Ala Asn Glu Gly 1185 1190 1195 1200Phe Thr Phe Ala Lys Asn Ile Glu Trp Ala Thr Lys Thr Ile Gln Ser 1205 1210 1215 Ile Val Asn Trp Leu Thr Ser Trp Phe Lys Gln Glu Glu Asp His Pro 1220 1225 1230 Gln Ser Lys Leu Asp Lys Leu Leu Met Glu Phe Pro Asp His Cys Arg 1235 1240 1245 Asn Ile Met Asp Met Arg Asn Gly Arg Lys Ala Tyr Cys Glu Cys Thr 1250 1255 1260 Ala Ser Phe Lys Tyr Phe Asp Asp Leu Tyr Asn Leu Ala Val Thr Cys 1265 1270 1275 1280Lys Arg Ile Pro Leu Ala Ser Leu Cys Glu Lys Phe Lys Asn Arg His 1285 1290 1295 Asp His Ser Val Thr Arg Pro Glu Pro Val Val Ala Val Leu Arg Gly 1300 1305 1310 Ala Ala Gly Gln Gly Lys Ser Val Thr Ser Gln Ile Ile Ala Gln Ser 1315 1320 1325 Val Ser Lys Met Ala Phe Gly Arg Gln Ser Val Tyr Ser Met Pro Pro 1330 1335 1340 Asp Ser Glu Tyr Phe Asp Gly Tyr Glu Asn Gln Phe Ser Val Ile Met 1345 1350 1355 1360Asp Asp Leu Gly Gln Asn Pro Asp Gly Glu Asp Phe Thr Val Phe Cys 1365 1370 1375 Gln Met Val Ser Ser Thr Asn Phe Leu Pro Asn Met Ala His Leu Glu 1380 1385 1390 Arg Lys Gly Thr Pro Phe Thr Ser Ser Phe Ile Val Ala Thr Thr Asn 1395 1400 1405 Leu Pro Lys Phe Arg Pro Val Thr Val Ala His Tyr Pro Ala Val Asp 1410 1415 1420 Arg Arg Ile Thr Phe Asp Phe Thr Val Thr Ala Gly Pro His Cys Lys 1425 1430 1435 1440Thr Pro Ala Gly Met Leu Asp Ile Glu Lys Ala Phe Asp Glu Ile Pro 1445 1450 1455 Gly Ser Lys Pro Gln Leu Ala Cys Phe Ser Ala Asp Cys Pro Leu Leu 1460 1465 1470 His Lys Arg Gly Val Met Phe Thr Cys Asn Arg Thr Lys Thr Val Tyr 1475 1480 1485 Asn Leu Gln Gln Val Val Lys Met Val Asn Asp Thr Ile Thr Arg Lys 1490 1495 1500 Thr Glu Asn Val Lys Lys Met Asn Ser Leu Val Ala Gln Ser Pro Pro 1505 1510 1515 1520Asp Trp Gln His Phe Glu Asn Ile Leu Thr Cys Leu Arg Gln Asn Asn 1525 1530 1535 Ala Ala Leu Gln Asp Gln Val Asp Glu Leu Gln Glu Ala Phe Thr Gln 1540 1545 1550 Ala Arg Glu Arg Ser Asp Phe Leu Ser Asp Trp Leu Lys Val Ser Ala 1555 1560 1565 Ile Ile Phe Ala Gly Ile Val Ser Leu Ser Ala Val Ile Lys Leu Ala 1570 1575 1580 Ser Lys Phe Lys Glu Ser Ile Trp Pro Thr Pro Val Arg Val Glu Leu 1585 1590 1595 1600Ser Glu Gly Glu Gln Ala Ala Tyr Ala Gly Arg Ala Arg Ala Gln Lys 1605 1610 1615 Gln Ala Leu Gln Val Leu Asp Ile Gln Gly Gly Gly Lys Val Leu Ala 1620 1625 1630 Gln Ala Gly Asn Pro Val Met Asp Phe Glu Leu Phe Cys Ala Lys Asn 1635 1640 1645 Met Val Ser Pro Ile Thr Phe Tyr Tyr Pro Asp Lys Ala Glu Val Thr 1650 1655 1660 Gln Ser Cys Leu Leu Leu Arg Ala His Leu Phe Val Val Asn Arg His 1665 1670 1675 1680Val Ala Glu Thr Glu Trp Thr Ala Phe Lys Leu Arg Asp Val Arg His 1685 1690 1695 Glu Arg Asp Thr Val Val Met Arg Ser Val Asn Arg Ser Gly Ala Glu 1700 1705 1710 Thr Asp Leu Thr Phe Val Lys Val Thr Lys Gly Pro Leu Phe Lys Asp 1715 1720 1725 Asn Val Asn Lys Phe Cys Ser Asn Lys Asp Asp Phe Pro Ala Arg Asn 1730 1735 1740 Asp Thr Val Thr Gly Ile Met Asn Thr Gly Leu Ala Phe Val Tyr Ser 1745 1750 1755 1760Gly Asn Phe Leu Ile Gly Asn Gln Pro Val Asn Thr Thr Thr Gly Ala 1765 1770 1775 Cys Phe Asn His Cys Leu His Tyr Arg Ala Gln Thr Arg Arg Gly Trp 1780 1785 1790 Cys Gly Ser Ala Ile Ile Cys Asn Val Asn Gly Lys Lys Ala Val Tyr 1795 1800 1805 Gly Met His Ser Ala Gly Gly Gly Gly Leu Ala Ala Ala Thr Ile Ile 1810 1815 1820 Thr Arg Glu Leu Ile Glu Ala Ala Glu Lys Ser Met Leu Ala Leu Glu 1825 1830 1835 1840Pro Gln Gly Ala Ile Val Asp Ile Ser Thr Gly Ser Val Val His Val 1845 1850 1855 Pro Arg Lys Thr Lys Leu Arg Arg Thr Val Ala His Asp Val Phe Gln 1860 1865 1870 Pro Lys Phe Glu Pro Ala Val Leu Ser Arg Tyr Asp Pro Arg Thr Asp 1875 1880 1885 Lys Asp Val Asp Val Val Ala Phe Ser Lys His Thr Thr Asn Met Glu 1890 1895 1900 Ser Leu Pro Pro Ile Phe Asp Ile Val Cys Gly Glu Tyr Ala Asn Arg 1905 1910 1915 1920Val Phe Thr Ile Leu Gly Lys Asp Asn Gly Leu Leu Thr Val Glu Gln 1925 1930 1935 Ala Val Leu Gly Leu Ser Gly Met Asp Pro Met Glu Lys Asp Thr Ser 1940 1945 1950 Pro Gly Leu Pro Tyr Thr Gln Gln Gly Leu Arg Arg Thr Asp Leu Leu 1955 1960 1965 Asp Phe Asn Thr Ala Lys Met Thr Pro Gln Leu Asp Tyr Ala His Ser 1970 1975 1980 Lys Leu Val Leu Gly Val Tyr Asp Asp Val Val Tyr Gln Ser Phe Leu 1985 1990 1995 2000Lys Asp Glu Ile Arg Pro Leu Glu Lys Ile His Glu Ala Lys Thr Arg 2005 2010 2015 Ile Val Asp Val Pro Pro Phe Ala His Cys Ile Trp Gly Arg Gln Leu 2020 2025 2030 Leu Gly Arg Phe Ala Ser Lys Phe Gln Thr Lys Pro Gly Phe Glu Leu 2035 2040 2045 Gly Ser Ala Ile Gly Thr Asp Pro Asp Val Asp Trp Thr Arg Tyr Ala 2050 2055 2060 Ala Glu Leu Ser Gly Phe Asn Tyr Val Tyr Asp Val Asp Tyr Ser Asn 2065 2070 2075 2080Phe Asp Ala Ser His Ser Thr Ala Met Phe Glu Cys Leu Ile Asn Asn 2085 2090 2095 Phe Phe Thr Glu Gln Asn Gly Phe Asp Arg Arg Ile Ala Glu Tyr Leu 2100 2105 2110 Arg Ser Leu Ala Val Ser Arg His Ala Tyr Glu Asp Arg Arg Val Leu 2115 2120 2125 Ile Arg Gly Gly Leu Pro Ser Gly Cys Ala Ala Thr Ser Met Leu Asn 2130 2135 2140 Thr Ile Met Asn Asn Val Ile Ile Arg Ala Ala Leu Tyr Leu Thr Tyr 2145 2150 2155 2160Ser Asn Phe Glu Phe Asp Asp Ile Lys Val Leu Ser Tyr Gly Asp Asp 2165 2170 2175 Leu Leu Ile Gly Thr Asn Tyr Gln Ile Asp Phe Asn Leu Val Lys Glu 2180 2185 2190 Arg Leu Ala Pro Phe Gly Tyr Lys Ile Thr Pro Ala Asn Lys Thr Thr 2195 2200 2205 Thr Phe Pro Leu Thr Ser His Leu Gln Asp Val Thr Phe Leu Lys Arg 2210 2215 2220 Arg Phe Val Arg Phe Asn Ser Tyr Leu Phe Arg Pro Gln Met Asp Ala 2225 2230 2235 2240Val Asn Leu Lys Ala Met Val Ser Tyr Cys Lys Pro Gly Thr Leu Lys 2245 2250 2255 Glu Lys Leu Met Ser Ile Ala Leu Leu Ala Val His Ser Gly Pro Asp 2260 2265 2270 Ile Tyr Asp Glu Ile Phe Leu Pro Phe Arg Asn Val Gly Ile Val Val 2275 2280 2285 Pro Thr Tyr Asp Ser Met Leu Tyr Arg Trp Leu Ser Leu Phe Arg 2290 2295 2300 322303PRTTheiler's murine encephalomyelitis virus 32Met Ala Cys Lys His Gly Tyr Pro Asp Val Cys Pro Ile Cys Thr Ala 1 5 10 15 Val Asp Ala Thr Pro Gly Phe Glu Tyr Leu Leu Met Ala Asp Gly Glu 20 25 30 Trp Tyr Pro Thr Asp Leu Leu Cys Val Asp Leu Asp Asp Asp Val Phe 35 40 45 Trp Pro Ser Asp Thr Ser Asn Gln Ser Gln Thr Met Asp Trp Thr Asp 50 55 60 Val Pro Leu Ile Arg Asp Ile Val Met Glu Pro Gln Gly Asn Ser Ser 65 70 75 80Ser Ser Asp Lys Ser Asn Ser Gln Ser Ser Gly Asn Glu Gly Val Ile 85 90 95 Ile Asn Asn Phe Tyr Ser Asn Gln Tyr Gln Asn Ser Ile Asp Leu Ser 100 105 110 Ala Ser Gly Gly Asn Ala Gly Asp Ala Pro Gln Thr Asn Gly Gln Leu 115 120 125 Ser Asn Ile Leu Gly Gly Ala Ala Asn Ala Phe Ala Thr Met Ala Pro 130 135 140 Leu Leu Leu Asp Gln Asn Thr Glu Glu Met Glu Asn Leu Ser Asp Arg 145 150 155 160Val Ala Ser Asp Lys Ala Gly Asn Ser Ala Thr Asn Thr Gln Ser Thr 165 170 175 Val Gly Arg Leu Cys Gly Tyr Gly Lys Ser His His Gly Glu His Pro 180 185 190 Ala Ser Cys Ala Asp Thr Ala Thr Asp Lys Val Leu Ala Ala Glu Arg 195 200 205 Tyr Tyr Thr Ile Asp Leu Ala Ser Trp Thr Thr Ser Gln Glu Ala Phe 210 215 220 Ser His Ile Arg Ile Pro Leu Pro His Val Leu Ala Gly Glu Asp Gly 225 230 235 240Gly Val Phe Gly Ala Thr Leu Arg Arg His Tyr Leu Cys Lys Thr Gly 245 250 255 Trp Arg Val Gln Val Gln Cys Asn Ala Ser Gln Phe His Ala Gly Ser 260 265 270 Leu Leu Val Phe Met Ala Pro Glu Phe Tyr Thr Gly Lys Gly Thr Lys 275 280 285 Thr Gly Thr Met Glu Pro Ser Asp Pro Phe Thr Met Asp Thr Glu Trp 290 295 300 Arg Ser Pro Gln Gly Ala Pro Thr Gly Tyr Arg Tyr Asp Ser Arg Thr 305 310 315 320Gly Phe Phe Ala Thr Asn His Gln Asn Gln Trp Gln Trp Thr Val Tyr 325 330 335 Pro His Gln Ile Leu Asn Leu Arg Thr Asn Thr Thr Val Asp Leu Glu 340 345 350 Val Pro Tyr Val Asn Val Ala Pro Ser Ser Ser Trp Thr Gln His Ala 355 360 365 Asn Trp Thr Leu Val Val Ala Val Leu Ser Pro Leu Gln Tyr Ala Thr 370 375 380 Gly Ser Ser Pro Asp Val Gln Ile Thr Ala Ser Leu Gln Pro Val Asn 385 390 395 400Pro Val Phe Asn Gly Leu Arg His Glu Thr Val Ile Ala Gln Ser Pro 405 410 415 Ile Pro Val Thr Val Arg Glu His Lys Gly Cys Phe Tyr Ser Thr Asn 420 425 430 Pro Asp Thr Thr Val Pro Ile Tyr Gly Lys Thr Ile Ser Thr Pro Ser 435 440 445 Asp Tyr Met Cys Gly Glu Phe Ser Asp Leu Leu Glu Leu Cys Lys Leu 450 455 460 Pro Thr Phe Leu Gly Asn Pro Asn Thr Asn Asn Lys Arg Tyr Pro Tyr 465 470 475 480Phe Ser Ala Thr Asn Ser Val Pro Ala Thr Ser Met Val Asp Tyr Gln 485 490 495 Val Ala Leu Ser Cys Ser Cys Met Ala Asn Ser Met Leu Ala Ala Val 500 505 510 Ala Arg Asn Phe Asn Gln Tyr Arg Gly Ser Leu Asn Phe Leu Phe Val 515 520 525 Phe Thr Gly Ala Ala Met Val Lys Gly Lys Phe Leu Ile Ala Tyr Thr 530 535 540 Pro Pro Gly Ala Gly Lys Pro Thr Thr Arg Asp Gln Ala Met Gln Ser 545 550 555 560Thr Tyr Ala Ile Trp Asp Leu Gly Leu Asn Ser Ser Phe Asn Phe Thr 565 570 575 Ala Pro Phe Ile Ser Pro Thr His Tyr Arg Gln Thr Ser Tyr Thr Ser 580 585 590 Pro Thr Ile Thr Ser Val Asp Gly Trp Val Thr Val Trp Lys Leu Thr 595 600 605 Pro Leu Thr Tyr Pro Ser Gly Thr Pro Thr Asn Ser Asp Ile Leu Thr 610 615 620 Leu Val Ser Ala Gly Asp Asp Phe Thr Leu Arg Met Pro Ile Ser Pro 625 630 635 640Thr Lys Trp Val Pro Gln Gly Val Asp Asn Ala Glu Lys Gly Lys Val 645 650 655 Ser Asn Asp Asp Ala Ser Val Asp Phe Val Ala Glu Pro Val Lys Leu 660 665 670 Pro Glu Asn Gln Thr Arg Val Ala Phe Phe Tyr Asp Arg Ala Val Pro 675 680 685 Ile Gly Met Leu Arg Pro Gly Gln Asn Met Glu Thr Thr Phe Asn Tyr 690 695 700 Gln Glu Asn Asp Tyr Arg Leu Asn Cys Leu Leu Leu Thr Pro Leu Pro 705 710 715 720Ser Phe Cys Pro Asp Ser Ser Ser Gly Pro Gln Lys Thr Lys Ala Pro 725 730 735 Val Gln Trp Arg Trp Val Arg Ser Gly Gly Val Asn Gly Ala Asn Phe 740 745 750 Pro Leu Met Thr Lys Gln Asp Tyr Ala Phe Leu Cys Phe Ser Pro Phe 755 760 765 Thr Phe Tyr Lys Cys Asp Leu Glu Val Thr Val Ser Ala Leu Gly Met 770 775 780 Thr Arg Val Ala Ser Val Leu Arg Trp Ala Pro Thr Gly Ala Pro Ala 785 790 795 800Asp Val Thr Asp Gln Leu Ile Gly Tyr Thr Pro Ser Leu Gly Glu Thr 805 810 815 Arg Asn Pro His Met Trp Leu Val Gly Ala Gly Asn Ser Gln Val Ser 820 825 830 Phe Val Val Pro Tyr Asn Ser Pro Leu Ser Val Leu Pro Ala Ala Trp 835 840 845 Phe Asn Gly Trp Ser Asp Phe Gly Asn Thr Lys Asp Phe Gly Val Ala 850 855 860 Pro Asn Ala Asp Phe Gly Arg Leu Trp Ile Gln Gly Asn Thr Ser Ala 865 870 875 880Ser Val Arg Ile Arg Tyr Lys Lys Met Lys Val Phe Cys Pro Arg Pro 885 890 895 Thr Leu Phe Phe Pro Trp Pro Thr Pro Thr Thr Thr Lys Ile Asn Ala 900 905 910 Asp Asn Pro Val Pro Ile Leu Glu Leu Glu Asn Pro Ala Ala Leu Tyr 915 920 925 Arg Ile Asp Leu Phe Ile Thr Phe Thr Asp Glu Phe Ile Thr Phe Asp 930 935 940 Tyr Lys Val His Gly Arg Pro Val Leu Thr Phe Arg Ile Pro Gly Phe 945 950 955 960Gly Leu Thr Pro Ala Gly Arg Met Leu Val Cys Met Gly Glu Gln Pro 965 970 975 Ala His Gly Pro Phe Thr Ser Ser Arg Ser Leu Tyr His Val Ile Phe 980 985 990 Thr Ala Thr Cys Ser Ser Phe Ser Phe Ser Ile Tyr Lys Gly Arg Tyr 995

1000 1005 Arg Ser Trp Lys Lys Pro Ile His Asp Glu Leu Val Asp Arg Gly Tyr 1010 1015 1020 Thr Thr Phe Gly Glu Phe Phe Lys Ala Val Arg Gly Tyr His Ala Asp 1025 1030 1035 1040Tyr Tyr Arg Gln Arg Leu Ile His Asp Val Glu Thr Asn Pro Gly Pro 1045 1050 1055 Val Gln Ser Val Phe Gln Pro Gln Gly Ala Val Leu Thr Lys Ser Leu 1060 1065 1070 Ala Pro Gln Ala Gly Ile Gln Asn Leu Leu Leu Arg Leu Leu Gly Ile 1075 1080 1085 Asp Gly Asp Cys Ser Glu Val Ser Lys Ala Ile Thr Val Val Thr Asp 1090 1095 1100 Leu Val Ala Ala Trp Glu Lys Ala Lys Thr Thr Leu Val Ser Pro Glu 1105 1110 1115 1120Phe Trp Ser Lys Leu Ile Leu Lys Thr Thr Lys Phe Ile Ala Ala Ser 1125 1130 1135 Val Leu Tyr Leu His Asn Pro Asp Phe Thr Thr Thr Val Cys Leu Ser 1140 1145 1150 Leu Met Thr Gly Val Asp Leu Leu Thr Asn Asp Ser Val Phe Asp Trp 1155 1160 1165 Leu Lys Gln Lys Leu Ser Ser Phe Phe Arg Thr Pro Pro Pro Ala Cys 1170 1175 1180 Pro Asn Val Met Gln Pro Gln Gly Pro Leu Arg Glu Ala Asn Glu Gly 1185 1190 1195 1200Phe Thr Phe Ala Lys Asn Ile Glu Trp Ala Met Lys Thr Ile Gln Ser 1205 1210 1215 Val Val Asn Trp Leu Thr Ser Trp Phe Lys Gln Glu Glu Asp His Pro 1220 1225 1230 Gln Ser Lys Leu Asp Lys Leu Leu Met Glu Phe Pro Asp His Cys Arg 1235 1240 1245 Asn Ile Met Asp Met Arg Asn Gly Arg Lys Ala Tyr Cys Glu Cys Thr 1250 1255 1260 Ala Ser Phe Lys Tyr Phe Asp Glu Leu Tyr Asn Leu Ala Val Thr Cys 1265 1270 1275 1280Lys Arg Ile Pro Leu Ala Ser Leu Cys Glu Lys Phe Lys Asn Arg His 1285 1290 1295 Asp His Ser Val Thr Arg Pro Glu Pro Val Val Val Val Leu Arg Gly 1300 1305 1310 Ala Ala Gly Gln Gly Lys Ser Val Thr Ser Gln Ile Ile Ala Gln Ser 1315 1320 1325 Val Ser Lys Met Ala Phe Gly Arg Gln Ser Val Tyr Ser Met Pro Pro 1330 1335 1340 Asp Ser Glu Tyr Phe Asp Gly Tyr Glu Asn Gln Phe Ser Val Ile Met 1345 1350 1355 1360Asp Asp Leu Gly Gln Asn Pro Asp Gly Glu Asp Phe Thr Val Phe Cys 1365 1370 1375 Gln Met Val Ser Ser Thr Asn Phe Leu Pro Asn Met Ala His Leu Glu 1380 1385 1390 Arg Lys Gly Thr Pro Phe Thr Ser Ser Phe Ile Val Ala Thr Thr Asn 1395 1400 1405 Leu Pro Lys Phe Arg Pro Val Thr Val Ala His Tyr Pro Ala Val Asp 1410 1415 1420 Arg Arg Ile Thr Phe Asp Phe Thr Val Thr Ala Gly Pro His Cys Lys 1425 1430 1435 1440Thr Pro Ala Gly Met Leu Asp Val Glu Lys Ala Phe Asp Glu Ile Pro 1445 1450 1455 Gly Ser Lys Pro Gln Leu Ala Cys Phe Ser Ala Asp Cys Pro Leu Leu 1460 1465 1470 His Lys Arg Gly Val Met Phe Thr Cys Asn Arg Thr Gln Thr Val Tyr 1475 1480 1485 Asn Leu Gln Gln Val Val Lys Met Val Asn Asp Thr Ile Thr Arg Lys 1490 1495 1500 Thr Glu Asn Val Lys Lys Met Asn Ser Leu Val Ala Gln Ser Pro Pro 1505 1510 1515 1520Asp Trp Glu His Phe Glu Asn Ile Leu Thr Cys Leu Arg Gln Asn Asn 1525 1530 1535 Ala Ala Leu Gln Asp Gln Leu Asp Glu Leu Gln Glu Ala Phe Ala Gln 1540 1545 1550 Ala Arg Glu Arg Ser Asp Phe Leu Ser Asp Trp Leu Lys Val Ser Ala 1555 1560 1565 Ile Ile Phe Ala Gly Ile Ala Ser Leu Ser Ala Val Ile Lys Leu Ala 1570 1575 1580 Ser Lys Phe Lys Glu Ser Ile Trp Pro Thr Pro Val Arg Val Glu Leu 1585 1590 1595 1600Ser Glu Gly Glu Gln Ala Ala Tyr Ala Gly Arg Ala Arg Ala Gln Lys 1605 1610 1615 Gln Ala Leu Gln Val Leu Asp Ile Gln Gly Gly Gly Lys Val Leu Ala 1620 1625 1630 Gln Ala Gly Asn Pro Val Met Asp Phe Glu Leu Phe Cys Ala Lys Asn 1635 1640 1645 Ile Val Ala Pro Ile Thr Phe Tyr Tyr Pro Asp Lys Ala Glu Val Thr 1650 1655 1660 Gln Ser Cys Leu Leu Leu Arg Ala His Leu Phe Val Val Asn Arg His 1665 1670 1675 1680Val Ala Glu Thr Asp Trp Thr Ala Phe Lys Leu Lys Asp Val Arg His 1685 1690 1695 Glu Arg His Thr Val Ala Leu Arg Ser Val Asn Arg Ser Gly Ala Lys 1700 1705 1710 Thr Asp Leu Thr Phe Ile Lys Val Thr Lys Gly Pro Leu Phe Lys Asp 1715 1720 1725 Asn Val Asn Lys Phe Cys Ser Asn Lys Asp Asp Phe Pro Ala Arg Asn 1730 1735 1740 Asp Thr Val Thr Gly Ile Met Asn Thr Gly Leu Ala Phe Val Tyr Ser 1745 1750 1755 1760Gly Asn Phe Leu Ile Gly Asn Gln Pro Val Asn Thr Thr Thr Gly Ala 1765 1770 1775 Cys Phe Asn His Cys Leu His Tyr Arg Ala Gln Thr Arg Arg Gly Trp 1780 1785 1790 Cys Gly Ser Ala Ile Ile Cys Asn Val Asn Gly Lys Lys Ala Val Tyr 1795 1800 1805 Gly Met His Ser Ala Gly Gly Gly Gly Leu Ala Ala Ala Thr Ile Ile 1810 1815 1820 Thr Lys Glu Leu Ile Glu Ala Ala Glu Lys Ser Met Leu Ala Leu Glu 1825 1830 1835 1840Pro Gln Gly Ala Ile Val Asp Ile Ala Thr Gly Ser Val Val His Val 1845 1850 1855 Pro Arg Lys Thr Lys Leu Arg Arg Thr Val Ala His Asp Val Phe Gln 1860 1865 1870 Pro Lys Phe Glu Pro Ala Val Leu Ser Arg Tyr Asp Pro Arg Thr Asp 1875 1880 1885 Lys Asp Val Asp Val Val Ala Phe Ser Lys His Thr Thr Asn Met Glu 1890 1895 1900 Ser Leu Pro Pro Ile Phe Asp Val Val Cys Gly Glu Tyr Ala Asn Arg 1905 1910 1915 1920Val Phe Thr Ile Leu Gly Lys Glu Asn Gly Leu Leu Thr Val Glu Gln 1925 1930 1935 Ala Val Leu Gly Leu Pro Gly Met Asp Pro Met Glu Lys Asp Thr Ser 1940 1945 1950 Pro Gly Leu Pro Tyr Thr Gln Gln Gly Leu Arg Arg Thr Asp Leu Leu 1955 1960 1965 Asn Phe Ile Thr Ala Lys Met Thr Pro Gln Leu Asp Tyr Ala His Ser 1970 1975 1980 Lys Leu Val Ile Gly Val Tyr Asp Asp Val Val Tyr Gln Ser Phe Leu 1985 1990 1995 2000Lys Asp Glu Ile Arg Pro Ile Glu Lys Ile His Glu Ala Lys Thr Arg 2005 2010 2015 Ile Val Asp Val Pro Pro Phe Ala His Cys Ile Trp Gly Arg Gln Leu 2020 2025 2030 Leu Gly Arg Phe Ala Ser Lys Phe Gln Thr Lys Pro Gly Leu Glu Leu 2035 2040 2045 Gly Ser Ala Ile Gly Thr Asp Pro Asp Val Asp Trp Thr Arg Tyr Ala 2050 2055 2060 Val Glu Leu Ser Gly Phe Asn Tyr Val Tyr Asp Val Asp Tyr Ser Asn 2065 2070 2075 2080Phe Asp Ala Ser His Ser Thr Ala Met Phe Glu Cys Leu Ile Asn Asn 2085 2090 2095 Phe Phe Thr Glu Gln Asn Gly Phe Asp Arg Arg Ile Ala Glu Tyr Leu 2100 2105 2110 Arg Ser Leu Ala Val Ser Arg His Ala Tyr Glu Asp Arg Arg Val Leu 2115 2120 2125 Ile Arg Gly Gly Leu Pro Ser Gly Cys Ala Ala Thr Ser Met Leu Asn 2130 2135 2140 Thr Ile Met Asn Asn Val Ile Ile Arg Ala Ala Leu Tyr Leu Thr Tyr 2145 2150 2155 2160Ser Asn Phe Asp Phe Asp Asp Ile Lys Val Leu Ser Tyr Gly Asp Asp 2165 2170 2175 Leu Leu Ile Gly Thr Asn Tyr Gln Ile Asp Phe Asn Leu Val Lys Glu 2180 2185 2190 Arg Leu Ala Pro Phe Gly Tyr Lys Ile Thr Pro Ala Asn Lys Thr Thr 2195 2200 2205 Thr Phe Pro Leu Thr Ser His Leu Gln Asp Val Thr Phe Leu Lys Arg 2210 2215 2220 Arg Phe Val Arg Phe Asn Ser Tyr Leu Phe Arg Pro Gln Met Asp Ala 2225 2230 2235 2240Val Asn Leu Lys Ala Met Val Ser Tyr Cys Lys Pro Gly Thr Leu Lys 2245 2250 2255 Glu Lys Leu Met Ser Ile Ala Leu Leu Ala Val His Ser Gly Pro Asp 2260 2265 2270 Ile Tyr Asp Glu Ile Phe Leu Pro Phe Arg Asn Val Gly Ile Val Val 2275 2280 2285 Pro Thr Tyr Ser Ser Met Leu Tyr Arg Trp Leu Ser Leu Phe Arg 2290 2295 2300 332307PRTTheiler's murine encephalomyelitis virus 33Met Met Ala Cys Ile His Gly Tyr Pro Ser Val Cys Pro Ile Cys Thr 1 5 10 15 Ala Ile Asp Lys Ser Ser Asp Gly Met Tyr Leu Leu Leu Ala Asp Asn 20 25 30 Glu Trp Phe Pro Ala Asp Leu Leu Thr Met Asp Leu Asp Asp Asp Val 35 40 45 Phe Trp Pro Asn Asp Glu Ser Asp Val Ser Glu Thr Met Asp Trp Thr 50 55 60 Asp Leu Pro Phe Ile Leu Asp Thr Ile Met Glu Pro Gln Gly Asn Ser 65 70 75 80Thr Ser Ser Asp Lys Ser Asn Ser Gln Ser Ser Gly Asn Glu Gly Val 85 90 95 Ile Ile Asn Asn Phe Tyr Ser Asn Gln Tyr Gln Asn Ser Ile Asp Leu 100 105 110 Ser Ala Asn Gly Gly Asn Ala Gly Gly Ala Pro Lys Thr Glu Gly Gln 115 120 125 Leu Gly Asn Ile Leu Gly Asn Ala Ala Asn Ala Phe Ser Thr Met Ala 130 135 140 Pro Leu Leu Leu Asp Gln Asn Thr Glu Glu Met Glu Asn Leu Ser Asp 145 150 155 160Arg Val Asp Ser Asp Lys Ala Gly Asn Ser Ala Val Asn Thr Gln Ser 165 170 175 Ser Val Gly Arg Leu Cys Gly Tyr Gly Met His His Lys Gly Lys His 180 185 190 Pro Ala Ser Cys Ala Asp Thr Ala Thr Asp Lys Val Leu Ser Ala Glu 195 200 205 Arg Tyr Tyr Thr Ile Asp Leu Ala Thr Trp Thr Thr Thr Leu Gly Thr 210 215 220 Phe Ser His Ile Arg Ile Pro Leu Pro His Val Leu Ala Gly Glu Asp 225 230 235 240Gly Gly Val Phe Gly Ser Thr Leu Arg Arg His Tyr Leu Cys Lys Cys 245 250 255 Gly Trp Arg Ile Gln Val Gln Cys Asn Ala Ser Gln Phe His Ala Gly 260 265 270 Ser Leu Leu Val Phe Met Ala Pro Glu Phe Tyr Thr Gly His Thr Pro 275 280 285 Val Thr Gly Thr Thr Glu Pro Ala Thr Pro Phe Thr Met Asp Ser Ser 290 295 300 Trp Gln Thr Pro Gln Gln Asn Pro Val Gly Phe Arg Tyr Asp Gly Arg 305 310 315 320Thr Gly Tyr Phe Ala Leu Asn His Gln Asn Tyr Trp Gln Trp Met Val 325 330 335 Tyr Pro His Gln Ile Leu Asn Leu Arg Thr Asn Thr Ser Val Asp Leu 340 345 350 Glu Val Pro Phe Thr Asn Ile Ala Pro Thr Ser Ser Trp Thr Gln His 355 360 365 Ala Asn Trp Thr Leu Val Val Ala Val Leu Thr Pro Leu Gln Tyr Ala 370 375 380 Ala Gly Ser Ala Thr Asp Val Gln Ile Thr Ala Ser Ile Gln Pro Val 385 390 395 400Lys Pro Val Phe Asn Gly Leu Arg His Glu Ala Val Val Pro Gln Ser 405 410 415 Pro Ile Pro Val Thr Val Arg Glu His Gln Gly Thr Phe Tyr Ser Thr 420 425 430 Asn Pro Asp Thr Thr Val Pro Ile Tyr Gly Lys Thr Ile Ala Thr Pro 435 440 445 Ser Asp Tyr Met Cys Gly Glu Phe Ser Asp Leu Val Glu Leu Cys Lys 450 455 460 Leu Pro Thr Phe Leu Gly Asn Pro Ala Asn Thr Ser Pro Ala Gly Gly 465 470 475 480Arg Tyr Pro Tyr Phe Ser Ala Thr Asn Ser Val Pro Ala Thr Ala Leu 485 490 495 Ala Ser Tyr Gln Val Ala Leu Ser Cys Ser Cys Met Ser Asn Ser Met 500 505 510 Leu Ala Ala Val Ala Arg Asn Phe Asn Gln Tyr Arg Gly Ser Leu Asn 515 520 525 Phe Leu Phe Val Phe Thr Gly Thr Ala Met Thr Lys Gly Lys Phe Leu 530 535 540 Ile Ala Tyr Thr Pro Pro Gly Ala Gly Lys Pro Thr Thr Arg Glu Gln 545 550 555 560Ala Met Gln Ala Thr Tyr Ala Ile Trp Asp Leu Gly Leu Asn Ser Ser 565 570 575 Tyr Asn Phe Thr Val Pro Phe Ile Ser Pro Thr His Tyr Arg Gln Thr 580 585 590 Ser Tyr Thr Ser Thr Ser Ile Thr Ser Val Asp Gly Trp Leu Thr Val 595 600 605 Trp Gln Leu Thr Pro Leu Thr Tyr Pro Ala Asn Thr Pro Pro Asn Ala 610 615 620 Asp Ile Leu Thr Leu Val Ser Ala Gly Asp Asp Phe Thr Leu Arg Met 625 630 635 640Pro Ile Ser Pro Thr Lys Trp Ile Pro Gln Gly Val Asp Asn Ala Glu 645 650 655 Lys Gly Lys Val Ser Asn Asp Asp Ala Thr Val Asp Phe Val Ala Glu 660 665 670 Pro Val Lys Phe Pro Asp Asn Gln Thr Lys Val Ser Phe Phe Tyr Asp 675 680 685 Arg Ser Val Pro Leu Gly Leu Leu Arg Pro Ala Gln Gly Met Glu Gln 690 695 700 Asp Phe Ala Tyr Ala Ala Asn Asp Ser Arg Ala Asn Ser Ile Leu Leu 705 710 715 720Thr Pro Leu Pro Ser Tyr Ala Pro Asp Ser Thr Thr Gly Pro Thr Glu 725 730 735 Thr Gln Ala Pro Ile Gln Trp Arg Trp Leu Arg Gly Thr Ser Asp Gly 740 745 750 Ser Thr Thr Phe Pro Leu Met Thr Lys Gln Asp Tyr Ala Phe Leu Leu 755 760 765 Phe Ser Pro Phe Thr Tyr Tyr Lys Ala Asp Leu Glu Val Thr Leu Ser 770 775 780 Ala Ile Ser Asn Ser Asn Asn Val Thr Val Val Arg Trp Ala Pro Thr 785 790 795 800Gly Ala Pro Ala Asp Ile Ser Arg Gln Leu Ser Gly Tyr Thr Pro Ser 805 810 815 Ile Gly Asp Thr Arg Asp Pro His Leu Trp Phe Val Gly Ala Gly Asn 820 825 830 Ser Gln Thr Ser Phe Val Val Pro Tyr Asn Ser Pro Leu Ser Val Leu 835 840 845 Pro Ala Ala Trp Phe Asn Gly Trp Ser Asp Phe Gly Asn Thr Lys Asp 850 855 860 Phe Gly Val Ala Pro Asn Ala Asp Phe Gly Arg Leu Trp Ile Gln Gly 865 870 875 880Asn Thr Ser Val Ala Val Arg Val Arg Tyr Lys Lys Met Lys Val Phe 885 890 895 Cys Pro Arg Pro Thr Leu Phe Leu Pro Trp Pro Ser Thr Thr Thr Thr 900 905 910 Arg Ile His Ala Asp Asn Pro Val Ser Val Met Glu Leu Gln Asn Pro 915 920 925 Phe Ser Phe Tyr Arg Val Asp Leu Phe Ile Thr Phe Thr Asp Glu Leu 930 935 940 Ile Thr Phe Asp Tyr Lys Val His Gly Arg Pro Val Leu Gln Tyr Gln 945 950 955 960Val Pro Gly Leu Gly Leu Thr Cys Ala Gly Arg Met Leu Val Cys Met 965 970

975 Gly Gln Met Pro Asn His Ala Pro Phe Ser Thr Val Arg His Leu Tyr 980 985 990 His Val Val Phe Thr Gly Ser Arg Asn Ser Phe Gly Val Val Ile Tyr 995 1000 1005 Tyr Lys Arg His Arg Pro Trp Lys Lys Pro Leu His Glu Glu Leu His 1010 1015 1020 Asp Tyr Gly Phe Glu Cys Phe Ser Asp Phe Phe Lys His Val Arg Glu 1025 1030 1035 1040Tyr His Ala Ala Tyr Tyr Lys Gln Arg Leu Met His Asp Val Glu Thr 1045 1050 1055 Asn Pro Gly Pro Pro Val Gln Ser Val Phe Arg Pro Gln Gly Gly Val 1060 1065 1070 Leu Thr Lys Ser Gln Ala Pro Met Ser Gly Ile Gln Asn Leu Phe Leu 1075 1080 1085 Arg Ala Leu Gly Ile Asp Ala Asp His Gly Glu Phe Thr Arg Ala Val 1090 1095 1100 Thr Met Ile Thr Asp Leu Cys Asn Thr Trp Glu Lys Ala Lys Asn Thr 1105 1110 1115 1120Leu Val Ser Pro Glu Phe Trp Thr Val Leu Ile Met Lys Thr Val Lys 1125 1130 1135 Phe Ile Ala Ala Ser Val Leu Tyr Leu His Asn Pro Asp Leu Thr Ala 1140 1145 1150 Thr Ile Cys Leu Ser Leu Met Thr Gly Val Asp Val Leu Thr Asn Glu 1155 1160 1165 Ser Ile Phe Asn Trp Leu Ser Asn Lys Leu Ser Lys Leu Phe His Thr 1170 1175 1180 Pro Pro Pro Pro Thr Ser Pro Leu Leu Gln Ala Gln Ser Pro Leu Arg 1185 1190 1195 1200Glu Ala Asn Asp Gly Phe Asn Leu Ala Lys Asn Ile Glu Trp Ala Ile 1205 1210 1215 Lys Thr Val Gln Lys Ile Val Asp Trp Leu Met Ser Trp Phe Lys Gln 1220 1225 1230 Glu Glu Ala His Pro Gln Ala Lys Leu Asp Lys Met Leu Ala Asp Phe 1235 1240 1245 Pro Glu His Cys Ala Ser Ile Leu Ala Met Arg Asn Gly Arg Lys Ala 1250 1255 1260 Tyr Thr Asp Cys Ala Gly Ala Phe Lys Tyr Phe Glu Asp Leu Tyr Asn 1265 1270 1275 1280Leu Ala Val Gln Cys Lys Arg Ile Pro Leu Ala Thr Leu Cys Glu Lys 1285 1290 1295 Phe Lys Asn Lys His Asp His Ala Val Ala Arg Pro Glu Pro Val Val 1300 1305 1310 Val Val Leu Arg Gly Asn Ala Gly Gln Gly Lys Ser Val Thr Ser Gln 1315 1320 1325 Ile Ile Ala Gln Ala Val Ser Lys Leu Ala Phe Gly Arg Gln Ser Val 1330 1335 1340 Tyr Ser Ile Pro Pro Asp Ser Asp Tyr Leu Asp Gly Tyr Glu Asn Gln 1345 1350 1355 1360Phe Ser Val Ile Met Asp Asp Leu Gly Gln Asn Pro Asp Gly Glu Asp 1365 1370 1375 Phe Lys Val Phe Cys Gln Met Val Ser Ser Thr Asn Phe Leu Pro Asn 1380 1385 1390 Met Ala His Leu Glu Lys Lys Gly Thr Pro Phe Thr Ser Asn Phe Ile 1395 1400 1405 Val Ala Thr Thr Asn Leu Pro Lys Phe Arg Pro Val Thr Val Ala His 1410 1415 1420 Tyr Pro Ala Val Asp Arg Arg Ile Thr Phe Asp Leu Thr Val Glu Ala 1425 1430 1435 1440Gly Pro Ala Cys Lys Thr Pro Thr Gly Met Leu Asp Val Glu Lys Ala 1445 1450 1455 Phe Gln Glu Ile Pro Gly Glu Pro Gln Leu Asp Cys Phe Ser Ser Asp 1460 1465 1470 Cys Ala Leu Leu His Lys Arg Gly Val Gln Phe Ile Cys Asn Arg Thr 1475 1480 1485 Lys Lys Ile Tyr Asn Leu Gln Gln Ile Val Lys Met Val Lys Asp Thr 1490 1495 1500 Ile Asp Asn Lys Val Ala Asn Leu Lys Lys Met Asn Thr Leu Val Ala 1505 1510 1515 1520Gln Ser Pro Asn Asn Gly Asn Asp Met Glu His Ile Ile Thr Cys Leu 1525 1530 1535 Arg Gln Asn Asn Ala Ala Leu Gln Asp Gln Ile Asp Glu Leu Gln Glu 1540 1545 1550 Ala Phe Ala Gln Ala Gln Glu Arg Gln Asn Phe Leu Ser Asp Trp Met 1555 1560 1565 Lys Val Ser Ala Ile Ile Phe Ala Gly Ile Ala Ser Leu Ser Ala Val 1570 1575 1580 Cys Lys Leu Val Gly Arg Leu Lys Asn Leu Ile Trp Pro Ser Pro Val 1585 1590 1595 1600His Val Glu Leu Ser Glu Gly Glu Gln Ala Ala Tyr Ala Gly Ala Lys 1605 1610 1615 Arg Gly Ala Lys Gln Ala Leu Gln Val Leu Asp Leu Gln Gly Gly Gly 1620 1625 1630 Arg Ile Ile Ala Gln Ala Gly Asn Pro Val Met Asp Tyr Glu Val Cys 1635 1640 1645 Val Ala Lys Asn Met Val Ala Pro Ile Thr Phe Tyr Tyr Ala Asp Lys 1650 1655 1660 Ala Gln Val Thr Gln Ser Cys Leu Leu Val Lys Gly Arg Leu Phe Val 1665 1670 1675 1680Val Asn Arg His Val Ala Glu Thr Asp Trp Val Ser Phe Glu Leu Arg 1685 1690 1695 Asp Val Arg His Glu Arg Asp Thr Val Thr Met Arg Ser Val Asn Arg 1700 1705 1710 Ser Gly Met Glu Val Asp Leu Thr Phe Ile Lys Val Thr Lys Gly Pro 1715 1720 1725 Leu Phe Lys Asp Asn Thr Lys Lys Phe Cys Ser Asn Lys Asp Asp Phe 1730 1735 1740 Pro Gln Lys Asn Glu Thr Val Thr Gly Ile Met Asn Thr Gly Leu Pro 1745 1750 1755 1760Phe Val Phe Asn Gly Lys Phe Ile Ile Gly Asn His Pro Val Asn Thr 1765 1770 1775 Thr Thr Gly Ala Thr Phe Asn His Cys Leu His Tyr Arg Ala Asn Thr 1780 1785 1790 Arg Arg Gly Trp Cys Gly Ser Ala Val Ile Cys Gln Val Asn Gly Lys 1795 1800 1805 Lys Ala Val Tyr Gly Met His Ser Ala Gly Gly Gly Gly Leu Ala Ala 1810 1815 1820 Ala Thr Ile Ile Thr Gln Glu Leu Val Glu Ala Ala Glu Gln Asn Met 1825 1830 1835 1840Asp Arg Leu Val Pro Gln Gly Ala Ile Met Glu Ile Gly Thr Gly Ser 1845 1850 1855 Val Val His Val Pro Arg Lys Thr Lys Leu Arg Arg Thr Val Ala His 1860 1865 1870 Glu Ile Phe Leu Pro Lys Phe Glu Pro Ala Val Leu Ser Arg Tyr Asp 1875 1880 1885 Pro Arg Thr Glu Lys Asp Val Asp Gln Val Ala Phe Ser Lys His Thr 1890 1895 1900 Thr Asn Met Glu Glu Leu Pro Ala Val Phe Ser Met Val Ala Lys Glu 1905 1910 1915 1920Tyr Ala Asn Arg Val Phe Thr Lys Leu Gly Lys Glu Asn Gln Leu Leu 1925 1930 1935 Thr Thr Gln Gln Ala Ile Leu Gly Leu Pro Gly Met Asp Pro Met Glu 1940 1945 1950 Lys Asp Thr Ser Pro Gly Leu Pro Tyr Thr Gln Gln Gly Leu Arg Arg 1955 1960 1965 Thr Asp Leu Val Asn Phe Glu Thr Gly Lys Met Asp His Asn Leu Asp 1970 1975 1980 Tyr Ala His Ser Lys Leu Met Leu Gly His Tyr Glu Asp Val Val Tyr 1985 1990 1995 2000Gln Ser Phe Leu Lys Asp Glu Ile Arg Pro Ile Glu Lys Ile His Glu 2005 2010 2015 Ala Lys Thr Arg Ile Val Asp Val Pro Pro Phe His His Cys Ile Trp 2020 2025 2030 Gly Arg Gln Leu Leu Gly Arg Phe Ala Ser Arg Phe Gln Thr Asn Pro 2035 2040 2045 Gly Leu Asp Leu Gly Ser Ala Ile Gly Thr Asp Pro Asp Val Asp Trp 2050 2055 2060 Thr Val Phe Ala His Gln Leu Ala Glu Phe Lys Tyr Ile Tyr Asp Val 2065 2070 2075 2080Asp Tyr Ser Asn Phe Asp Ala Ser His Ser Thr Ala Ile Phe Glu Ile 2085 2090 2095 Leu Ile Gln Glu Phe Phe Thr Pro Gln Asn Gly Phe Asp Pro Arg Ile 2100 2105 2110 Gly Glu Tyr Leu Arg Ser Leu Ala Val Ser Arg His Ala Tyr Glu Asp 2115 2120 2125 Arg Arg Val Leu Ile Arg Gly Gly Leu Pro Ser Gly Cys Ala Ala Thr 2130 2135 2140 Ser Met Ile Asn Thr Ile Ile Asn Asn Ile Val Ile Arg Ala Ala Leu 2145 2150 2155 2160Tyr Met Thr Tyr Ala Asn Phe Glu Phe Asp Asp Ile Lys Val Leu Ser 2165 2170 2175 Tyr Gly Asp Asp Leu Leu Ile Ala Thr Asn Tyr Glu Ile Asn Phe Asn 2180 2185 2190 Leu Val Lys Glu Arg Leu Ala Pro Phe Asn Tyr Lys Ile Thr Pro Ala 2195 2200 2205 Asn Lys Thr Ser Thr Phe Pro Gln Thr Ser His Leu Gln Asp Val Val 2210 2215 2220 Phe Leu Lys Arg Arg Phe Val Gln Phe Asn Ser Phe Leu Phe Arg Pro 2225 2230 2235 2240Gln Met Glu Thr Glu Asn Leu Lys Ala Met Val Ser Tyr Cys Arg Pro 2245 2250 2255 Gly Val Leu Lys Glu Lys Leu Met Ser Ile Ala Leu Leu Ala Val His 2260 2265 2270 Ser Gly Pro Asp Val Tyr Asp Glu Ile Phe Met Pro Phe Arg Arg Ile 2275 2280 2285 Gly Val Val Val Pro Glu Tyr Ser Thr Met Leu Tyr Arg Trp Leu Asn 2290 2295 2300 Leu Phe Arg 2305 34930PRTTheiler's murine encephalomyelitis virusMOD_RES(557)variable amino acid 34Met Ala Cys Lys His Gly Tyr Pro Asp Val Cys Pro Ile Cys Thr Ala 1 5 10 15 Ile Asp Val Thr Pro Gly Phe Glu Tyr Leu Leu Leu Ala Asp Gly Glu 20 25 30 Trp Phe Pro Thr Asp Leu Leu Cys Val Asp Leu Asp Asp Asp Val Phe 35 40 45 Trp Pro Ser Asp Ser Ser Asn Gln Ser Gln Thr Met Glu Trp Thr Asp 50 55 60 Ile Pro Leu Ile Cys Asp Thr Val Met Glu Pro Gln Gly Asn Ser Thr 65 70 75 80Ser Ser Asp Lys Ser Asn Ser Gln Ser Ser Gly Asn Glu Gly Val Ile 85 90 95 Ile Asn Asn Phe Tyr Ser Asn Gln Tyr Gln Asn Ser Ile Asp Leu Ser 100 105 110 Ala Asn Gly Gly Asn Ala Gly Asp Gly Pro Lys Thr Glu Gly Gln Leu 115 120 125 Ser Asn Ile Leu Gly Gly Ala Ala Asn Ala Phe Ala Thr Met Ala Pro 130 135 140 Leu Leu Leu Asp Glu Asn Thr Glu Glu Met Glu Asn Leu Ser Asp Arg 145 150 155 160Val Asp Ser Asp Lys Ala Gly Asn Ser Ala Thr Asn Thr Gln Ser Ser 165 170 175 Val Gly Arg Leu His Gly Tyr Gly Ala Thr His Arg Gly Asp His Pro 180 185 190 Ala Ser Cys Ala Asp Thr Ala Thr Asp Lys Val Leu Ala Ala Glu Arg 195 200 205 Tyr Tyr Thr Ile Asp Leu Ala Thr Trp Thr Thr Ala Gln Thr Thr Phe 210 215 220 Ser His Ile Arg Val Pro Leu Pro His Ala Leu Ala Gly Glu His Gly 225 230 235 240Gly Val Phe Gly Ala Thr Leu Arg Arg His Tyr Leu Ala Lys Cys Gly 245 250 255 Trp Arg Val Gln Val Gln Cys Asn Ala Ser Gln Phe His Ala Gly Ser 260 265 270 Leu Leu Val Phe Leu Ala Pro Glu Phe Tyr Thr Gly Thr Gly Val Ala 275 280 285 Thr Ser Gly Gln Glu Pro Asn Lys Val Phe Leu Met Asp Thr Thr Trp 290 295 300 Gln Glu Pro Gln Ala Ala Pro Thr Gly Phe Arg Tyr Asp Gly Lys Asn 305 310 315 320Gly Phe Phe Thr Leu Asn His Gln Asn Tyr Trp Gln Trp Thr Val Tyr 325 330 335 Pro His Gln Ile Leu Asn Leu Arg Thr Asn Thr Ser Val Asp Leu Glu 340 345 350 Val Pro Tyr Val Asn Val Ala Pro Thr Ser Ser Trp Thr Gln His Ala 355 360 365 Asn Trp Ala Leu Val Val Ala Val Leu Thr Pro Leu Gln Tyr Ser Thr 370 375 380 Gly Ala Ala Thr Asp Val Ala Ile Thr Val Ser Leu Gln Pro Val Asn 385 390 395 400Pro Val Phe Asn Gly Leu Arg His Glu Ala Gln Val Pro Gln Ser Pro 405 410 415 Val Ala Val Thr Val Arg Glu His Gln Gly Ser Phe Tyr Ser Thr Asn 420 425 430 Pro Asp Thr Thr Val Pro Ile Tyr Gly Lys Thr Ile Val Thr Pro Ser 435 440 445 Asp Tyr Met Cys Gly Glu Phe Thr Asp Leu Leu Glu Leu Cys Lys Leu 450 455 460 Pro Thr Phe Leu Gly Asn Leu Ser Asn Asp Thr Arg Val Pro Phe Phe 465 470 475 480Thr Ala Thr Asn Ser Val Pro Thr Glu Ser Leu Val Glu Tyr Gln Val 485 490 495 Thr Leu Ser Cys Ser Cys Met Ser Asn Ser Met Leu Ala Ser Val Ala 500 505 510 Arg Asn Phe Asn Gln Tyr Arg Gly Ser Leu Asn Phe Leu Phe Val Phe 515 520 525 Thr Gly Ser Ala Met Thr Lys Gly Lys Phe Leu Ile Ala Tyr Thr Pro 530 535 540 Pro Gly Ala Gly Lys Pro Thr Thr Arg Asp Gln Ala Xaa Gln Ser Thr 545 550 555 560Tyr Ala Ile Trp Asp Leu Gly Leu Asn Ser Ser Tyr Asn Phe Thr Val 565 570 575 Pro Phe Ile Ser Pro Ser His Tyr Arg Gln Thr Ser Tyr Thr Ser Pro 580 585 590 Ser Ile Ala Ala Val Asp Gly Trp Leu Thr Val Trp Gln Leu Thr Pro 595 600 605 Leu Thr Phe Pro Ala Asn Val Pro Pro Ser Ser Asp Ile Leu Thr Leu 610 615 620 Val Ser Ala Gly Asn Asp Phe Thr Leu Arg Met Pro Ile Ser Pro Thr 625 630 635 640Lys Trp Ile Pro Gln Gly Val Asp Asn Ala Glu Lys Gly Lys Val Ser 645 650 655 Asp Asp Asn Ala Ser Val Asp Phe Val Ala Glu Pro Ile Lys Leu Pro 660 665 670 Glu Asn Gln Thr Arg Val Asn Phe Phe Tyr Asp Arg Ser Ser Pro Ile 675 680 685 Gly Leu Leu Arg Pro Asn Gln Ala Ile Glu Ser Asn Phe Ser Tyr Ser 690 695 700 Ala Asp Ser Asn Gly Ala Thr Asn Cys Ala Leu Leu Thr Pro Leu Pro 705 710 715 720Ser Tyr Ser Pro Asp Arg Pro Gly Gln Ser Pro Asp Thr Ser Lys Ala 725 730 735 Pro Ile Gln Trp Arg Trp Ile Ser Ala Val Thr Glu Ser Gly Thr Val 740 745 750 Ser Asn Thr Phe Pro Thr Arg Thr Arg Gln Asp Tyr Ala Phe Leu Leu 755 760 765 Phe Ser Pro Phe Thr Tyr Tyr Lys Cys Asp Leu Glu Val Thr Leu Ser 770 775 780 Ser Val Gly Asn Gly Val Val Ala Ser Leu Val Arg Trp Ala Pro Thr 785 790 795 800Gly Ala Pro Ala Asp Ile Thr Thr Gln Leu Thr Thr Ser Thr Pro Ser 805 810 815 Ile Gly Asp Thr Arg Asp Pro His Met Trp Leu Val Gly Ala Gly Asn 820 825 830 Ser Gln Thr Ser Phe Val Ile Pro Tyr Asn Ser Pro Leu Ser Val Leu 835 840 845 Pro Ala Ala Trp Phe Asn Gly Trp Ser Asn Phe Ser Asn Thr Tyr Asp 850 855 860 Phe Gly Ile Ala Pro Cys Ser Asp Phe Gly Arg Leu Trp Ile Gln Gly 865 870 875 880Asn Ala Pro Leu Ala Ile Arg Val Arg Tyr Lys Lys Met Arg Val Phe 885 890 895 Cys Pro Arg Pro Thr Leu Phe Phe Pro Trp Pro Thr Pro Thr Thr Thr 900 905 910 Lys Val Asn Ala Asp Asn Pro Val Pro Ile Leu Asp Leu Glu Asn Pro 915 920 925 Ala Ala

93035142PRTSeneca Valley Virus 35Leu Ala His His Gly Asn Lys Lys Ser Leu Gln Glu Leu Asn Glu Glu 1 5 10 15 Gln Trp Val Glu Met Ser Asp Asp Tyr Arg Thr Gly Lys Asn Met Pro 20 25 30 Phe Gln Ser Leu Gly Thr Tyr Tyr Arg Pro Pro Asn Trp Thr Trp Gly 35 40 45 Pro Asn Phe Ile Asn Pro Tyr Gln Val Thr Val Phe Pro His Gln Ile 50 55 60 Leu Asn Ala Arg Thr Ser Thr Ser Val Asp Ile Asn Val Pro Tyr Ile 65 70 75 80Gly Glu Thr Pro Thr Gln Ser Ser Glu Thr Gln Asn Ser Trp Thr Leu 85 90 95 Leu Val Met Val Leu Val Pro Leu Asp Tyr Lys Glu Gly Ala Thr Thr 100 105 110 Asp Pro Glu Ile Thr Phe Ser Val Arg Pro Thr Ser Pro Tyr Phe Asn 115 120 125 Gly Leu Arg Asn Arg Tyr Thr Ala Gly Thr Asp Glu Glu Gln 130 135 140 36239PRTSeneca Valley Virus 36Gly Pro Ile Pro Thr Ala Pro Arg Glu Asn Ser Leu Met Phe Leu Ser 1 5 10 15 Thr Leu Pro Asp Asp Thr Val Pro Ala Tyr Gly Asn Val Arg Thr Pro 20 25 30 Pro Val Asn Tyr Leu Pro Gly Glu Ile Thr Asp Leu Leu Gln Leu Ala 35 40 45 Arg Ile Pro Thr Leu Met Ala Phe Glu Arg Val Pro Glu Pro Val Pro 50 55 60 Ala Ser Asp Thr Tyr Val Pro Tyr Val Ala Val Pro Thr Gln Phe Asp 65 70 75 80Asp Arg Pro Leu Ile Ser Phe Pro Ile Thr Leu Ser Asp Pro Val Tyr 85 90 95 Gln Asn Thr Leu Val Gly Ala Ile Ser Ser Asn Phe Ala Asn Tyr Arg 100 105 110 Gly Cys Ile Gln Ile Thr Leu Thr Phe Cys Gly Pro Met Met Ala Arg 115 120 125 Gly Lys Phe Leu Leu Ser Tyr Ser Pro Pro Asn Gly Thr Gln Pro Gln 130 135 140 Thr Leu Ser Glu Ala Met Gln Cys Thr Tyr Ser Ile Trp Asp Ile Gly 145 150 155 160Leu Asn Ser Ser Trp Thr Phe Val Val Pro Tyr Ile Ser Pro Ser Asp 165 170 175 Tyr Arg Glu Thr Arg Ala Ile Thr Asn Ser Val Tyr Ser Ala Asp Gly 180 185 190 Trp Phe Ser Leu His Lys Leu Thr Lys Ile Thr Leu Pro Pro Asp Cys 195 200 205 Pro Gln Ser Pro Cys Ile Leu Phe Phe Ala Ser Ala Gly Glu Asp Tyr 210 215 220 Thr Leu Arg Leu Pro Val Asp Cys Asn Pro Ser Tyr Val Phe His 225 230 235 37259PRTSeneca Valley Virus 37Ser Thr Asp Asn Ala Glu Thr Gly Val Ile Glu Ala Gly Asn Thr Asp 1 5 10 15 Thr Asp Phe Ser Gly Glu Leu Ala Ala Pro Gly Pro Asn His Thr Asn 20 25 30 Val Lys Phe Leu Phe Asp Arg Ser Arg Leu Leu Asn Val Ile Lys Val 35 40 45 Leu Glu Lys Asp Ala Val Phe Pro Arg Pro Phe Pro Thr Gln Glu Gly 50 55 60 Ala Gln Gln Asp Asp Gly Tyr Phe Cys Leu Leu Thr Pro Arg Pro Thr 65 70 75 80Val Ala Ser Arg Pro Ala Thr Arg Phe Gly Leu Tyr Ala Asn Pro Ser 85 90 95 Gly Ser Gly Val Leu Ala Asn Thr Ser Leu Asp Phe Asn Phe Tyr Ser 100 105 110 Leu Ala Cys Phe Thr Tyr Phe Arg Ser Asp Leu Glu Val Thr Val Val 115 120 125 Ser Leu Glu Pro Asp Leu Glu Phe Ala Val Gly Trp Phe Pro Ser Gly 130 135 140 Ser Glu Tyr Gln Ala Ser Ser Phe Val Tyr Asp Gln Leu His Val Pro 145 150 155 160Phe His Phe Thr Gly Arg Thr Pro Arg Ala Phe Ala Ser Lys Gly Gly 165 170 175 Lys Val Ser Phe Val Leu Pro Trp Asn Ser Val Ser Ser Val Leu Pro 180 185 190 Val Arg Trp Gly Gly Ala Ser Lys Leu Ser Ser Ala Thr Arg Gly Leu 195 200 205 Pro Ala His Ala Asp Trp Gly Thr Ile Tyr Ala Phe Val Pro Arg Pro 210 215 220 Asn Glu Lys Lys Ser Thr Ala Val Lys His Val Ala Val Tyr Ile Arg 225 230 235 240Tyr Lys Asn Ala Arg Ala Trp Cys Pro Ser Met Leu Pro Phe Arg Ser 245 250 255 Tyr Lys Gln 3814PRTSeneca Valley Virus 38Lys Met Leu Met Gln Ser Gly Asp Ile Glu Thr Asn Pro Gly 1 5 10 39128PRTSeneca Valley Virus 39Pro Ala Ser Asp Asn Pro Ile Leu Glu Phe Leu Glu Ala Glu Asn Asp 1 5 10 15 Leu Val Thr Leu Ala Ser Leu Trp Lys Met Val His Ser Val Gln Gln 20 25 30 Thr Trp Arg Lys Tyr Val Lys Asn Asp Asp Phe Trp Pro Asn Leu Leu 35 40 45 Ser Glu Leu Val Gly Glu Gly Ser Val Ala Leu Ala Ala Thr Leu Ser 50 55 60 Asn Gln Ala Ser Val Lys Ala Leu Leu Gly Leu His Phe Leu Ser Arg 65 70 75 80Gly Leu Asn Tyr Thr Asp Phe Tyr Ser Leu Leu Ile Glu Lys Cys Ser 85 90 95 Ser Phe Phe Thr Val Glu Pro Pro Pro Pro Pro Ala Glu Asn Leu Met 100 105 110 Thr Lys Pro Ser Val Lys Ser Lys Phe Arg Lys Leu Phe Lys Met Gln 115 120 125 40322PRTSeneca Valley Virus 40Gly Pro Met Asp Lys Val Lys Asp Trp Asn Gln Ile Ala Ala Gly Leu 1 5 10 15 Lys Asn Phe Gln Phe Val Arg Asp Leu Val Lys Glu Val Val Asp Trp 20 25 30 Leu Gln Ala Trp Ile Asn Lys Glu Lys Ala Ser Pro Val Leu Gln Tyr 35 40 45 Gln Leu Glu Met Lys Lys Leu Gly Pro Val Ala Leu Ala His Asp Ala 50 55 60 Phe Met Ala Gly Ser Gly Pro Pro Leu Ser Asp Asp Gln Ile Glu Tyr 65 70 75 80Leu Gln Asn Leu Lys Ser Leu Ala Leu Thr Leu Gly Lys Thr Asn Leu 85 90 95 Ala Gln Ser Leu Thr Thr Met Ile Asn Ala Lys Gln Ser Ser Ala Gln 100 105 110 Arg Val Glu Pro Val Val Val Val Leu Arg Gly Lys Pro Gly Cys Gly 115 120 125 Lys Gly Leu Ala Ser Thr Leu Ile Ala Gln Ala Val Ser Lys Arg Leu 130 135 140 Tyr Gly Ser Gln Ser Val Tyr Ser Leu Pro Pro Asp Pro Asp Phe Phe 145 150 155 160Asp Gly Tyr Lys Gly Gln Phe Val Thr Leu Met Asp Asp Leu Gly Gln 165 170 175 Asn Pro Asp Gly Gln Asp Phe Ser Thr Phe Cys Gln Met Val Ser Thr 180 185 190 Ala Gln Phe Leu Pro Asn Met Ala Asp Leu Ala Glu Lys Gly Arg Pro 195 200 205 Phe Thr Ser Asn Leu Ile Ile Ala Thr Thr Asn Leu Pro His Phe Ser 210 215 220 Pro Val Thr Ile Ala Asp Pro Ser Ala Val Ser Arg Arg Ile Asn Tyr 225 230 235 240Asp Leu Thr Leu Glu Val Ser Glu Ala Tyr Lys Lys His Thr Arg Leu 245 250 255 Asn Phe Asp Leu Ala Phe Arg Arg Thr Asp Ala Pro Pro Ile Tyr Pro 260 265 270 Phe Ala Ala His Val Pro Phe Val Asp Val Ala Val Arg Phe Lys Asn 275 280 285 Gly His Gln Asn Phe Asn Leu Leu Glu Leu Val Asp Ser Ile Cys Thr 290 295 300 Asp Ile Arg Ala Lys Gln Gln Gly Ala Arg Asn Met Gln Thr Leu Val 305 310 315 320Leu Gln 4190PRTSeneca Valley Virus 41Ser Pro Asn Glu Asn Asp Asp Thr Pro Val Asp Glu Ala Leu Gly Arg 1 5 10 15 Val Leu Ser Pro Ala Ala Val Asp Glu Ala Leu Val Asp Leu Thr Pro 20 25 30 Glu Ala Asp Pro Val Gly Arg Leu Ala Ile Leu Ala Lys Leu Gly Leu 35 40 45 Ala Leu Ala Ala Val Thr Pro Gly Leu Ile Ile Leu Ala Val Gly Leu 50 55 60 Tyr Arg Tyr Phe Ser Gly Ser Asp Ala Asp Gln Glu Glu Thr Glu Ser 65 70 75 80Glu Gly Ser Val Lys Ala Pro Arg Ser Glu 85 904222PRTSeneca Valley Virus 42Asn Ala Tyr Asp Gly Pro Lys Lys Asn Ser Lys Pro Pro Gly Ala Leu 1 5 10 15 Ser Leu Met Glu Met Gln 20 43211PRTSeneca Valley Virus 43Gln Pro Asn Val Asp Met Gly Phe Glu Ala Ala Val Ala Lys Lys Val 1 5 10 15 Val Val Pro Ile Thr Phe Met Val Pro Asn Arg Pro Ser Gly Leu Thr 20 25 30 Gln Ser Ala Leu Leu Val Thr Gly Arg Thr Phe Leu Ile Asn Glu His 35 40 45 Thr Trp Ser Asn Pro Ser Trp Thr Ser Phe Thr Ile Arg Gly Glu Val 50 55 60 His Thr Arg Asp Glu Pro Phe Gln Thr Val His Phe Thr His His Gly 65 70 75 80Ile Pro Thr Asp Leu Met Met Val Arg Leu Gly Pro Gly Asn Ser Phe 85 90 95 Pro Asn Asn Leu Asp Lys Phe Gly Leu Asp Gln Met Pro Ala Arg Asn 100 105 110 Ser Arg Val Val Gly Val Ser Ser Ser Tyr Gly Asn Phe Phe Phe Ser 115 120 125 Gly Asn Phe Leu Gly Phe Val Asp Ser Val Thr Ser Glu Gln Gly Thr 130 135 140 Tyr Ala Arg Leu Phe Arg Tyr Arg Val Thr Thr Tyr Lys Gly Trp Cys 145 150 155 160Gly Ser Ala Leu Val Cys Glu Ala Gly Gly Val Arg Arg Ile Ile Gly 165 170 175 Leu His Ser Ala Gly Ala Ala Gly Ile Gly Ala Gly Thr Tyr Ile Ser 180 185 190 Lys Leu Gly Leu Ile Lys Ala Leu Lys His Leu Gly Glu Pro Leu Ala 195 200 205 Thr Met Gln 210 44462PRTSeneca Valley Virus 44Gly Leu Met Thr Glu Leu Glu Pro Gly Ile Thr Val His Val Pro Arg 1 5 10 15 Lys Ser Lys Leu Arg Lys Thr Thr Ala His Ala Val Tyr Lys Pro Glu 20 25 30 Phe Glu Pro Ala Val Leu Ser Lys Phe Asp Pro Arg Leu Asn Lys Asp 35 40 45 Val Asp Leu Asp Glu Val Ile Trp Ser Lys His Thr Ala Asn Val Pro 50 55 60 Tyr Gln Pro Pro Leu Phe Tyr Thr Tyr Met Ser Glu Tyr Ala His Arg 65 70 75 80Val Phe Ser Phe Leu Gly Lys Asp Asn Asp Ile Leu Thr Val Lys Glu 85 90 95 Ala Ile Leu Gly Ile Pro Gly Leu Asp Pro Met Asp Pro His Thr Ala 100 105 110 Pro Gly Leu Pro Tyr Ala Ile Asn Gly Leu Arg Arg Thr Asp Leu Val 115 120 125 Asp Phe Val Asn Gly Thr Val Asp Ala Ala Leu Ala Val Gln Ile Gln 130 135 140 Lys Phe Leu Asp Gly Asp Tyr Ser Asp His Val Phe Gln Thr Phe Leu 145 150 155 160Lys Asp Glu Ile Arg Pro Ser Glu Lys Val Arg Ala Gly Lys Thr Arg 165 170 175 Ile Val Asp Val Pro Ser Leu Ala His Cys Ile Val Gly Arg Met Leu 180 185 190 Leu Gly Arg Phe Ala Ala Lys Phe Gln Ser His Pro Gly Phe Leu Leu 195 200 205 Gly Ser Ala Ile Gly Ser Asp Pro Asp Val Phe Trp Thr Val Ile Gly 210 215 220 Ala Gln Leu Glu Gly Arg Lys Asn Thr Tyr Asp Val Asp Tyr Ser Ala 225 230 235 240Phe Asp Ser Ser His Gly Thr Gly Ser Phe Glu Ala Leu Ile Ser His 245 250 255 Phe Phe Thr Val Asp Asn Gly Phe Ser Pro Ala Leu Gly Pro Tyr Leu 260 265 270 Arg Ser Leu Ala Val Ser Val His Ala Tyr Gly Glu Arg Arg Ile Lys 275 280 285 Ile Thr Gly Gly Leu Pro Ser Gly Cys Ala Ala Thr Ser Leu Leu Asn 290 295 300 Thr Val Leu Asn Asn Val Ile Ile Arg Thr Ala Leu Ala Leu Thr Tyr 305 310 315 320Lys Glu Phe Glu Tyr Asp Thr Val Asp Ile Ile Ala Tyr Gly Asp Asp 325 330 335 Leu Leu Val Gly Thr Asp Tyr Asp Leu Asp Phe Asn Glu Val Ala Arg 340 345 350 Arg Ala Ala Lys Leu Gly Tyr Lys Met Thr Pro Ala Asn Lys Gly Ser 355 360 365 Val Phe Pro Pro Thr Ser Ser Leu Ser Asp Ala Val Phe Leu Lys Arg 370 375 380 Lys Phe Val Gln Asn Asn Asp Gly Leu Tyr Lys Pro Val Met Asp Leu 385 390 395 400Lys Asn Leu Glu Ala Met Leu Ser Tyr Phe Lys Pro Gly Thr Leu Leu 405 410 415 Glu Lys Leu Gln Ser Val Ser Met Leu Ala Gln His Ser Gly Lys Glu 420 425 430 Glu Tyr Asp Arg Leu Met His Pro Phe Ala Asp Tyr Gly Ala Val Pro 435 440 445 Ser His Glu Tyr Leu Gln Ala Arg Trp Arg Ala Leu Phe Asp 450 455 460 456PRTSeneca Valley Virus 45Ala Gly Phe Cys Thr Glu 1 5 466PRTSeneca Valley Virus 46Ala Gly Asp Cys Ala Glu 1 5 476PRTSeneca Valley Virus 47Thr Gly Phe Cys Ala Glu 1 5 486PRTSeneca Valley Virus 48Ala Gly Phe Cys Ala Glu 1 5 4922PRTPorcine teschovirus 49Gly Pro Gly Ala Thr Asn Phe Ser Leu Leu Lys Gln Ala Gly Asp Val 1 5 10 15 Glu Glu Asn Pro Gly Pro 20 5022PRTPorcine teschovirus 50Gly Pro Gly Ala Thr Asn Phe Ser Leu Leu Lys Gln Ala Gly Asp Val 1 5 10 15 Glu Glu Asn Pro Gly Pro 20 5122PRTPorcine teschovirus 51Gly Pro Gly Ala Ser Ser Phe Ser Leu Leu Lys Gln Ala Gly Asp Val 1 5 10 15 Glu Glu Asn Pro Gly Pro 20 5222PRTPorcine teschovirus 52Gly Pro Gly Ala Ser Asn Phe Ser Leu Leu Lys Gln Ala Gly Asp Val 1 5 10 15 Glu Glu Asn Pro Gly Pro 20 5322PRTPorcine teschovirus 53Gly Pro Gly Ala Thr Asn Phe Ser Leu Leu Lys Gln Ala Gly Asp Val 1 5 10 15 Glu Glu Asn Pro Gly Pro 20 5422PRTPorcine teschovirus 54Gly Pro Gly Ala Ala Asn Phe Ser Leu Leu Arg Gln Ala Gly Asp Val 1 5 10 15 Glu Glu Asn Pro Gly Pro 20 5522PRTPorcine teschovirus 55Gly Pro Gly Ala Thr Asn Phe Ser Leu Leu Lys Gln Ala Gly Asp Val 1 5 10 15 Glu Glu Asn Pro Gly Pro 20 5622PRTPorcine teschovirus 56Gly Pro Gly Ala Thr Asn Phe Ser Leu Leu Lys Gln Ala Gly Asp Val 1 5 10 15 Glu Glu Asn Pro Gly Pro 20 5722PRTPorcine teschovirus 57Gly Pro Gly Ala Thr Asn Phe Ser Leu Leu Lys Gln Ala Gly Asp Ile 1 5 10 15 Glu Glu Asn Pro Gly Pro 20 5822PRTPorcine teschovirus 58Gly Pro Gly Ala Thr Asn Phe Ser Leu Leu Lys Gln Ala Gly Asp Val 1 5

10 15 Glu Glu Asn Pro Gly Pro 20 5922PRTPorcine teschovirus 59Gly Pro Gly Ala Thr Asn Phe Ser Leu Leu Lys Gln Ala Gly Asp Val 1 5 10 15 Glu Glu Asn Pro Gly Pro 20 6022PRTPorcine teschovirus 60Gly Pro Gly Ala Thr Asn Phe Ser Leu Leu Lys Arg Ala Gly Asp Val 1 5 10 15 Glu Glu Asn Pro Gly Pro 20 6119PRTFoot and mouth disease virus 61Thr Leu Asn Phe Asp Leu Leu Lys Leu Ala Gly Asp Val Glu Ser Asn 1 5 10 15 Pro Gly Pro 6219PRTFoot and mouth disease virus 62Leu Leu Asn Phe Asp Leu Leu Lys Leu Ala Gly Asp Val Glu Ser Asn 1 5 10 15 Pro Gly Pro 6319PRTFoot and mouth disease virus 63Leu Leu Asn Phe Asp Leu Leu Gln Leu Ala Gly Asp Val Glu Ser Asn 1 5 10 15 Pro Gly Pro 6419PRTFoot and mouth disease virus 64Leu Leu Asn Phe Asp Leu Leu Lys Leu Ala Gly Asp Val Glu Pro Asn 1 5 10 15 Pro Gly Pro 6519PRTFoot and mouth disease virus 65Leu Leu Asn Phe Asp Leu Leu Lys Leu Ala Gly Asp Val Glu Ser Asn 1 5 10 15 Pro Gly Pro 6619PRTFoot and mouth disease virus 66Leu Ser Asn Phe Asp Leu Leu Lys Leu Ala Gly Asp Val Glu Ser Asn 1 5 10 15 Pro Gly Pro 6719PRTFoot and mouth disease virus 67Leu Leu Asn Phe Asp Leu Leu Lys Leu Ala Gly Asp Val Glu Ser Asn 1 5 10 15 Pro Gly Pro 6819PRTFoot and mouth disease virus 68Thr Leu Asn Phe Asp Leu Leu Lys Leu Ala Gly Asp Val Glu Ser Asn 1 5 10 15 Pro Gly Pro 6919PRTFoot and mouth disease virus 69Ala Leu Asn Phe Asp Leu Leu Lys Leu Ala Gly Asp Val Glu Ser Asn 1 5 10 15 Pro Gly Pro 7019PRTFoot and mouth disease virus 70Val Leu Asn Phe Asp Leu Leu Lys Leu Ala Gly Asp Val Glu Ser Asn 1 5 10 15 Pro Gly Pro 7119PRTFoot and mouth disease virus 71Met Cys Ser Phe Asp Leu Leu Lys Leu Ala Gly Asp Val Glu Ser Asn 1 5 10 15 Pro Gly Pro 7219PRTFoot and mouth disease virus 72Leu Cys Asn Phe Asp Leu Leu Lys Leu Ala Gly Asp Val Glu Ser Asn 1 5 10 15 Pro Gly Pro 7319PRTFoot and mouth disease virus 73Leu Cys Asn Phe Asp Leu Leu Met Leu Ala Gly Asp Val Glu Ser Asn 1 5 10 15 Pro Gly Pro 7419PRTFoot and mouth disease virus 74Met Ala Asn Phe Ala Leu Leu Lys Leu Ala Gly Asp Val Glu Ser Asn 1 5 10 15 Pro Gly Pro 7519PRTFoot and mouth disease virus 75Leu Ser Asn Phe Asp Leu Leu Lys Leu Ala Gly Asp Val Glu Ser Asn 1 5 10 15 Pro Gly Pro 7619PRTFoot and mouth disease virus 76Leu Phe Asn Phe Asp Leu Leu Lys Leu Ala Gly Asp Val Glu Ser Asn 1 5 10 15 Pro Gly Pro 7719PRTFoot and mouth disease virus 77Leu Cys Asn Cys Asp Leu Leu Lys Leu Ala Gly Asp Val Glu Ser Asn 1 5 10 15 Pro Gly Pro 7819PRTFoot and mouth disease virus 78Leu Leu Asn Phe Asp Leu Leu Lys Leu Ala Gly Asp Val Glu Ser Asn 1 5 10 15 Pro Gly Pro 7919PRTFoot and mouth disease virus 79Leu Cys Asn Phe Asp Leu Leu Lys Leu Ala Gly Asp Val Glu Ser Asn 1 5 10 15 Pro Gly Pro 8019PRTFoot and mouth disease virus 80Met Cys Asn Phe Asp Leu Leu Lys Leu Ala Gly Asp Val Glu Ser Asn 1 5 10 15 Pro Gly Pro 8122PRTEquine rhinitis virus 81Asn Lys Gln Cys Thr Asn Tyr Ala Leu Leu Lys Leu Ala Gly Asp Val 1 5 10 15 Glu Ser Asn Pro Gly Pro 20 8222PRTEquine rhinitis virus 82Ser Glu Gly Ala Thr Asn Phe Ser Leu Leu Lys Leu Ala Gly Asp Val 1 5 10 15 Glu Leu Asn Pro Gly Pro 20 8322PRTEquine rhinitis virus 83Ser Gln Gly Ala Thr Asn Phe Asp Leu Leu Lys Leu Ala Gly Asp Val 1 5 10 15 Glu Ser Asn Pro Gly Pro 20 8422PRTEncephalomyocarditis virus 84Asn Ala His Tyr Ala Gly Tyr Phe Ala Asp Leu Leu Ile His Asp Ile 1 5 10 15 Glu Thr Asn Pro Gly Pro 20 8522PRTEncephalomyocarditis virus 85Asn Ala His Tyr Ala Gly Tyr Phe Ala Asp Leu Leu Ile His Asp Ile 1 5 10 15 Glu Thr Asn Pro Gly Pro 20 8622PRTMengo virus 86Glu Thr His Tyr Ala Gly Tyr Phe Ser Asp Leu Leu Ile His Asp Val 1 5 10 15 Glu Thr Asn Pro Gly Pro 20 8722PRTSeneca Valley Virus 87Pro Phe Arg Ser Tyr Lys Gln Lys Met Leu Met Gln Ser Gly Asp Ile 1 5 10 15 Glu Thr Asn Pro Gly Pro 20 8822PRTTheiler's murine encephalomyelitis virus 88Arg Gly Tyr His Ala Asp Tyr Tyr Arg Gln Arg Leu Ile His Asp Val 1 5 10 15 Glu Thr Asn Pro Gly Pro 20 8922PRTTheiler's murine encephalomyelitis virus 89Arg Gly Tyr His Ala Asp Tyr Tyr Lys Gln Arg Leu Ile His Asp Val 1 5 10 15 Glu Met Asn Pro Gly Pro 20 9022PRTTheiler's murine encephalomyelitis virus 90Arg Ala Tyr His Ala Asp Tyr Tyr Lys Gln Arg Leu Ile His Asp Val 1 5 10 15 Glu Met Asn Pro Gly Pro 20 9122PRTRat theiler's like virus 91Arg Glu Tyr His Ala Ala Tyr Tyr Lys Gln Arg Leu Met His Asp Val 1 5 10 15 Glu Thr Asn Pro Gly Pro 20 9222PRTLjungan virus 92Glu Met Asp Phe Ala Gly Gly Lys Phe Leu Asn Gln Cys Gly Asp Val 1 5 10 15 Glu Thr Asn Pro Gly Pro 20 9322PRTLjungan virus 93Glu Met Asp Phe Ala Gly Gly Lys Leu Phe Asn Gln Cys Gly Asp Val 1 5 10 15 Glu Thr Asn Pro Gly Pro 20 9422PRTLjungan virus 94Glu Met Asp Tyr Ser Gly Gly Lys Phe Leu Asn Gln Cys Gly Asp Val 1 5 10 15 Glu Ser Asn Pro Gly Pro 20 9522PRTLjungan virus 95Asp Met Asp Tyr Ala Gly Gly Lys Leu Phe Asn Gln Cys Gly Asp Val 1 5 10 15 Glu Thr Asn Pro Gly Pro 20 9622PRTTrypanosoma brucei 96Ala Ile Ser Ser Ile Ile Arg Thr Lys Met Leu Leu Ser Gly Asp Val 1 5 10 15 Glu Glu Asn Pro Gly Pro 20 9722PRTTrypanosoma cruzi 97Ala Val Cys Asp Ala Gln Arg Gln Lys Leu Leu Leu Ser Gly Asp Ile 1 5 10 15 Glu Gln Asn Pro Gly Pro 20 9822PRTBovine rotavirus 98Ala Asn Ser Lys Phe Gln Ile Asp Arg Ile Leu Ile Ser Gly Asp Ile 1 5 10 15 Glu Leu Asn Pro Gly Pro 20 9922PRTHuman rotavirus 99Ala Asn Ser Lys Phe Gln Ile Asp Lys Ile Leu Ile Ser Gly Asp Ile 1 5 10 15 Glu Leu Asn Pro Gly Pro 20 10023PRTPorcine rotavirus 100Ala Asn Ala Lys Phe Gly Gln Ile Asp Lys Ile Leu Ile Ser Gly Asp 1 5 10 15 Val Glu Leu Asn Pro Gly Pro 20 10122PRTDrosophila C virus 101Lys Gln Glu Ala Ala Arg Gln Met Leu Leu Leu Leu Ser Gly Asp Val 1 5 10 15 Glu Thr Asn Pro Gly Pro 20 10222PRTCricket paralysis virus 102Arg Ala Phe Leu Arg Lys Arg Thr Gln Leu Leu Met Ser Gly Asp Val 1 5 10 15 Glu Ser Asn Pro Gly Pro 20 10322PRTAcute bee paralysis virus 103His Cys Gly Ser Trp Thr Asp Ile Leu Leu Leu Leu Ser Gly Asp Val 1 5 10 15 Glu Thr Asn Pro Gly Pro 20 10422PRTCricket paralysis virus 104Thr Leu Thr Arg Ala Glu Ile Glu Asp Glu Leu Ile Arg Ala Gly Ile 1 5 10 15 Glu Ser Asn Pro Gly Pro 20 10522PRTPerina nuda picorna like virus 105Val Thr Ala Gln Gly Trp Val Pro Asp Leu Thr Val Asp Gly Asp Val 1 5 10 15 Glu Ser Asn Pro Gly Pro 20 10622PRTEctropis obliqua picorna like virus 106Val Thr Ala Gln Gly Trp Ala Pro Asp Leu Thr Gln Asp Gly Asp Val 1 5 10 15 Glu Ser Asn Pro Gly Pro 20 10722PRTPerina nuda picorna like virus 107Asn Ile Ile Gly Gly Gly Gln Lys Asp Leu Thr Gln Asp Gly Asp Ile 1 5 10 15 Glu Ser Asn Pro Gly Pro 20 10822PRTEctropis obliqua picorna like virus 108Asn Ile Ile Gly Gly Gly Gln Arg Asp Leu Thr Gln Asp Gly Asp Ile 1 5 10 15 Glu Ser Asn Pro Gly Pro 20 10922PRTDeformed wing virus 109Asn Leu Leu Gln Leu Ser Asn Pro Val Gln Ala Lys Pro Glu Met Asp 1 5 10 15 Asn Pro Asn Pro Gly Pro 20 11022PRTKakugo virus 110Asn Leu Leu Gln Leu Ser Asn Pro Val Gln Ala Lys Pro Glu Met Asp 1 5 10 15 Asn Pro Asn Pro Gly Pro 20 1114PRTSeneca Valley Virus 111Asn Pro Gly Pro 1 1129PRTArtificial SequenceDescription of Artificial Sequence Illustrative conserved motif 112Gly Xaa Xaa Gly Xaa Gly Lys Ser Thr 1 5 1136PRTSeneca Valley Virus 113Lys Asp Glu Leu Ile Arg 1 5 1144PRTSeneca Valley Virus 114Tyr Gly Asp Asp 1 1154PRTSeneca Valley Virus 115Phe Leu Lys Arg 1 1166PRTArtificial SequenceDescription of Artificial Sequence synthetic 6-His tag peptide 116His His His His His His 1 5 1174PRTSeneca Valley Virus 117Glu Gln Gly Pro 1 1184PRTSeneca Valley Virus 118Phe His Ser Thr 1 1194PRTSeneca Valley Virus 119Lys Gln Lys Met 1 1204PRTSeneca Valley Virus 120Met Gln Gly Pro 1 1214PRTSeneca Valley Virus 121Leu Gln Ser Pro 1 1224PRTSeneca Valley Virus 122Ser Glu Asn Ala 1 1234PRTSeneca Valley Virus 123Met Gln Gln Pro 1 1244PRTSeneca Valley Virus 124Met Gln Gly Leu 1 1254PRTEncephalomyocarditis virus 125Leu Gln Gly Asn 1 1264PRTEncephalomyocarditis virus 126Leu Ala Asp Gln 1 1274PRTEncephalomyocarditis virus 127Arg Gln Ser Pro 1 1284PRTEncephalomyocarditis virus 128Pro Gln Gly Val 1 1294PRTEncephalomyocarditis virus 129Leu Glu Ser Pro 1 1304PRTEncephalomyocarditis virus 130Asn Pro Gly Pro 1 1314PRTEncephalomyocarditis virus 131Gln Gln Ser Pro 1 1324PRTEncephalomyocarditis virus 132Ala Gln Gly Pro 1 1334PRTEncephalomyocarditis virus 133Ala Gln Ala Pro 1 1344PRTEncephalomyocarditis virus 134Glu Gln Gly Pro 1 1354PRTEncephalomyocarditis virus 135Ile Gln Gly Pro 1 1364PRTEncephalomyocarditis virus 136Val Gln Gly Pro 1 1374PRTEncephalomyocarditis virus 137Pro Gln Gly Ala 1 1384PRTTheiler's murine encephalomyelitis virus 138Pro Gln Gly Asn 1 1394PRTTheiler's murine encephalomyelitis virus 139Leu Leu Asp Gln 1 1404PRTTheiler's murine encephalomyelitis virus 140Leu Met Asp Gln 1 1414PRTTheiler's murine encephalomyelitis virus 141Ala Gln Ser Pro 1 1424PRTTheiler's murine encephalomyelitis virus 142Pro Gln Gly Val 1 1434PRTTheiler's murine encephalomyelitis virus 143Pro Gln Gly Ile 1 1444PRTTheiler's murine encephalomyelitis virus 144Pro Gln Gly Ser 1 1454PRTTheiler's murine encephalomyelitis virus 145Leu Glu Asn Pro 1 1464PRTTheiler's murine encephalomyelitis virus 146Asn Pro Gly Pro 1 1474PRTTheiler's murine encephalomyelitis virus 147Pro Gln Gly Pro 1 1484PRTTheiler's murine encephalomyelitis virus 148Ala Gln Ser Pro 1 1494PRTTheiler's murine encephalomyelitis virus 149Glu Gln Ala Ala 1 1504PRTTheiler's murine encephalomyelitis virus 150Ile Gln Gly Gly 1 1514PRTTheiler's murine encephalomyelitis virus 151Pro Gln Gly Ala 1 1524PRTRat Theilovirus 152Pro Gln Gly Asn 1 1534PRTRat Theilovirus 153Leu Leu Asp Gln 1 1544PRTRat Theilovirus 154Pro Gln Ser Pro 1 1554PRTRat Theilovirus 155Pro Gln Gly Val 1 1564PRTRat Theilovirus 156Leu Gln Asn Pro 1

1574PRTRat Theilovirus 157Asn Pro Gly Pro 1 1584PRTRat Theilovirus 158Ala Gln Ser Pro 1 1594PRTRat Theilovirus 159Ala Gln Ser Pro 1 1604PRTRat Theilovirus 160Glu Gln Ala Ala 1 1614PRTRat Theilovirus 161Ile Gln Gly Gly 1 1624PRTRat Theilovirus 162Pro Gln Gly Ala 1 1634PRTVilyuisk human encephalomyelitis virus 163Pro Gln Gly Asn 1 1644PRTVilyuisk human encephalomyelitis virus 164Leu Leu Asp Glu 1 1654PRTVilyuisk human encephalomyelitis virus 165Pro Gln Ser Pro 1 1664PRTVilyuisk human encephalomyelitis virus 166Pro Gln Gly Val 1 1674PRTVilyuisk human encephalomyelitis virus 167Leu Glu Asn Pro 1 1687310DNASeneca Valley Virus 168tttgaaatgg ggggctgggc cctgatgccc agtccttcct ttccccttcc ggggggttaa 60ccggctgtgt ttgctagagg cacagagggg caacatccaa cctgcttttg cggggaacgg 120tgcggctccg attcctgcgt cgccaaaggt gttagcgcac ccaaacggcg cacctaccaa 180tgttattggt gtggtctgcg agttctagcc tactcgtttc tcccccgacc attcactcac 240ccacgaaaag tgtgttgtaa ccataagatt taacccccgc acgggatgtg cgataaccgt 300aagactggct caagcgcgga aagcgctgta accacatgct gttagtccct ttatggctgc 360aagatggcta cccacctcgg atcactgaac tggagctcga ccctccttag taagggaacc 420gagaggcctt cgtgcaacaa gctccgacac agagtccacg tgactgctac caccatgagt 480acatggttct cccctctcga cccaggactt ctttttgaat atccacggct cgatccagag 540ggtggggcat gacccctagc atagcgagct acagcgggaa ctgtagctag gccttagcgt 600gccttggata ctgcctgata gggcgacggc ctagtcgtgt cggttctata ggtagcacat 660acaaatatgc agaactctca tttttctttc gatacagcct ctggcacctt tgaagatgta 720accggaacaa aagtcaagat cgttgaatac cccagatcgg tgaacaatgg tgtttacgat 780tcgtctactc atttggagat actgaaccta cagggtgaaa ttgaaatttt aaggtctttc 840aatgaatacc aaattcgcgc cgccaaacaa caactcggac tggacatcgt gtacgaacta 900cagggtaatg ttcagacaac gtcaaagaat gattttgatt cccgtggcaa taatggtaac 960atgaccttca attactacgc aaacacttat cagaattcag tagacttctc gacctcctcg 1020tcggcgtcag gcgccggacc cgggaactcc cggggcggat tagcgggtct cctcacaaat 1080ttcagtggaa tcttgaaccc tcttggctac ctcaaagatc acaacaccga agaaatggaa 1140aactctgctg atcgagtcac aacgcaaacg gcgggcaaca ctgccataaa cacgcaatca 1200tcattgggtg tgttgtgtgc ctacgttgaa gacccgacca aatctgatcc tccgtccagc 1260agcacagatc aacccaccac cactttcact gccatcgaca ggtggtacac tggacgtctc 1320aattcttgga caaaagctgt aaaaaccttc tcttttcagg ccgtcccgct tcccggggcc 1380tttctgtcta ggcagggagg cctcaacgga ggggccttca cagctaccct acatagacac 1440tttttgatga agtgcgggtg gcaggtgcag gtccaatgta atttgacaca attccaccaa 1500ggcgctcttc ttgttgccat ggttcctgaa accacccttg atgtcaagcc cgacggtaag 1560gcaaagagct tacaggagct gaatgaagaa cagtgggtgg aaatgtctga cgattaccgg 1620accgggaaaa acatgccttt tcagtctctt ggcacatact atcggccccc taactggact 1680tggggtccca atttcatcaa cccctatcaa gtaacggttt tcccacacca aattctgaac 1740gcgagaacct ctacctcggt agacataaac gtcccataca tcggggagac ccccacgcaa 1800tcctcagaga cacagaactc ctggaccctc ctcgttatgg tgctcgttcc cctagactat 1860aaggaaggag ccacaactga cccagaaatt acattttctg taaggcctac aagtccctac 1920ttcaatgggc ttcgcaaccg ctacacggcc gggacggacg aagaacaggg gcccattcct 1980acggcaccca gagaaaattc gcttatgttt ctctcaaccc tccctgacga cactgtccct 2040gcttacggga atgtgcgtac ccctcctgtc aattacctcc ctggtgaaat aaccgacctt 2100ttgcaactgg cccgcatacc cactctcatg gcatttgagc gggtgcctga acccgtgcct 2160gcctcagaca catatgtgcc ctacgttgcc gttcccaccc agttcgatga caggcctctc 2220atctccttcc cgatcaccct ttcagatccc gtctatcaga acaccctggt tggcgccatc 2280agttcaaatt tcgccaatta ccgtgggtgt atccaaatca ctctgacatt ttgtggaccc 2340atgatggcga gagggaaatt cctgctctcg tattctcccc caaatggaac gcaaccacag 2400actctttccg aagctatgca gtgcacatac tctatttggg acataggctt gaactctagt 2460tggaccttcg tcgtccccta catctcgccc agtgactacc gtgaaactcg agccattacc 2520aactcggttt actccgctga tggttggttt agcctgcaca agttgaccaa aattactcta 2580ccacctgact gtccgcaaag tccctgcatt ctctttttcg cttctgctgg tgaggattac 2640actctccgtc tccccgttga ttgtaatcct tcctatgtgt tccactccac cgacaacgcc 2700gagaccgggg ttattgaggc gggtaacact gacaccgatt tctctggtga actggcggct 2760cctggctcta accacactaa tgtcaagttc ctgtttgatc gatctcgatt attgaatgta 2820atcaaggtac tggagaagga cgccgttttc ccccgccctt tccctacaca agaaggtgcg 2880cagcaggatg atggttactt ttgtcttctg accccccgcc caacagtcgc ttcccgaccc 2940gccactcgtt tcggcctgta cgccaatccg tccggcagtg gtgttcttgc taacacttca 3000ctggacttca atttttatag cttggcctgt ttcacttact ttagatcgga ccttgaggtt 3060acggtggtct cactagagcc ggatctggaa tttgctgtag ggtggtttcc ttctggcagt 3120gaataccagg cttccagctt tgtctacgac cagctgcatg tgcccttcca ctttactggg 3180cgcactcccc gcgctttcgc tagcaagggt gggaaggtat ctttcgtgct cccttggaac 3240tctgtctcgt ctgtgctccc cgtgcgctgg gggggggctt ccaagctctc ttctgctacg 3300cggggtctac cggcgcatgc tgattggggg actatttacg cctttgtccc ccgtcctaat 3360gagaagaaaa gcaccgctgt aaaacacgtg gccgtgtaca ttcggtacaa gaacgcacgt 3420gcctggtgcc ccagcatgct tccctttcgc agctacaagc agaagatgct gatgcaatct 3480ggcgatatcg agaccaatcc tggtcctgct tctgacaacc caattttgga gtttcttgaa 3540gcagaaaatg atctagtcac tctggcctct ctctggaaga tggtgcactc tgttcaacag 3600acctggagaa agtatgtgaa gaacgatgat ttttggccca atttactcag cgagctagtg 3660ggggaaggct ctgtcgcctt ggccgccacg ctatccaacc aagcttcagt aaaggctctt 3720ttgggcctgc actttctctc tcgggggctc aattacactg acttttactc tttactgata 3780gagaaatgct ctagtttctt taccgtagaa ccacctcctc caccagctga aaacctgatg 3840accaagccct cagtgaagtc gaaattccga aaactgttta agatgcaagg acccatggac 3900aaagtcaaag actggaacca aatagctgcc ggcttgaaga attttcaatt tgttcgtgac 3960ctagtcaaag aggtggtcga ttggctgcag gcctggatca acaaagagaa agccagccct 4020gtcctccagt accagttgga gatgaagaag ctcgggcctg tggccttggc tcatgacgct 4080ttcatggctg gttccgggcc ccctcttagc gacgaccaga ttgaatacct ccagaacctc 4140aaatctcttg ccctaacact ggggaagact aatttggccc aaagtctcac cactatgatc 4200aatgccaaac aaagttcagc ccaacgagtt gaacccgttg tggtggtcct tagaggcaag 4260ccgggatgcg gcaagagctt ggcctctacg ttgattgccc aggctgtgtc caagcgcctc 4320tatggctccc aaagtgtata ttctcttccc ccagatccag atttcttcga tggatacaaa 4380ggacagttcg tgaccttgat ggatgatttg ggacaaaacc cggatggaca agatttctcc 4440accttttgtc agatggtgtc gaccgcccaa tttctcccca acatggcgga ccttgcagag 4500aaagggcgtc cctttacctc caatctcatc attgcaacta caaatctccc ccacttcagt 4560cctgtcacca ttgctgatcc ttctgcagtc tctcgccgta tcaactacga tctgactcta 4620gaagtatctg aggcctacaa gaaacacaca cggctgaatt ttgacttggc tttcaggcgc 4680acagacgccc cccccattta tccttttgct gcccatgtgc cctttgtgga cgtagctgtg 4740cgcttcaaaa atggtcacca gaattttaat ctcctagagt tggtcgattc catttgtaca 4800gacattcgag ccaagcaaca aggtgcccga aacatgcaga ctctggttct acagagcccc 4860aacgagaatg atgacacccc cgtcgacgag gcgttgggta gagttctctc ccccgctgcg 4920gtcgatgagg cgcttgtcga cctcactcca gaggccgacc cggttggccg tttggctatt 4980cttgccaagc taggtcttgc cctagctgcg gtcacccctg gtctgataat cttggcagtg 5040ggactctaca ggtacttctc tggctctgat gcagaccaag aagaaacaga aagtgaggga 5100tctgtcaagg cacccaggag cgaaaatgct tatgacggcc cgaagaaaaa ctctaagccc 5160cctggagcac tctctctcat ggaaatgcaa cagcccaacg tggacatggg ctttgaggct 5220gcggtcgcta agaaagtggt cgtccccatt accttcatgg ttcccaacag accttctggg 5280cttacacagt ccgctcttct ggtgaccggc cggaccttcc taatcaatga acatacatgg 5340tccaatccct cctggaccag cttcacaatc cgcggtgagg tacacactcg tgatgagccc 5400ttccaaacgg ttcatttcac tcaccacggt attcccacag atctgatgat ggtacgtctc 5460ggaccgggca attctttccc taacaatcta gacaagtttg gacttgacca gatgccggca 5520cgcaactccc gtgtggttgg cgtttcgtcc agttacggaa acttcttctt ctctggaaat 5580ttcctcggat ttgttgattc catcacctct gaacaaggaa cttacgcaag actctttagg 5640tacagggtga cgacctacaa aggatggtgc ggctcggccc tggtctgtga ggccggtggc 5700gtccgacgca tcattggcct gcattctgct ggcgccgccg gtatcggcgc cgggacctat 5760atctcaaaat taggactaat caaagccctg aaacacctcg gtgaaccttt ggccacaatg 5820caaggactga tgactgaatt agagcctgga atcaccgtac atgtaccccg gaaatccaaa 5880ttgagaaaga cgaccgcaca cgcggtgtac aaaccggagt ttgagcctgc tgtgttgtca 5940aaatttgatc ccagactgaa caaggatgtt gacttggatg aagtaatttg gtctaaacac 6000actgccaatg tcccttacca acctcctttg ttctacacat acatgtcaga gtacgctcat 6060cgagtcttct ccttcttggg gaaagacaat gacattctga ccgtcaaaga agcaattctg 6120ggcatccccg gactagaccc catggatccc cacacagctc cgggtctgcc ttacgccatc 6180aacggccttc gacgtactga tctcgtcgat tttgtgaacg gtacagtaga tgcggcgctg 6240gctgtacaaa tccagaaatt cttagacggt gactactctg accatgtctt ccaaactttt 6300ctgaaagatg agatcagacc ctcagagaaa gtccgagcgg gaaaaacccg cattgttgat 6360gtgccctccc tggcgcattg cattgtgggc agaatgttgc ttgggcgctt tgctgccaag 6420tttcaatccc atcctggctt tctcctcggc tctgctatcg ggtctgaccc tgatgttttc 6480tggaccgtca taggggctca actcgagggg agaaagaaca cgtatgacgt ggactacagt 6540gcctttgact cttcacacgg cactggctcc ttcgaggctc tcatctctca ctttttcacc 6600gtggacaatg gttttagccc tgcgctggga ccgtatctca gatccctggc tgtctcggtg 6660cacgcttacg gcgagcgtcg catcaagatt accggtggcc tcccctccgg ttgtgccgcg 6720accagcctgc tgaacacagt gctcaacaat gtgatcatca ggactgctct ggcattgact 6780tacaaggaat ttgaatatga catggttgat atcatcgcct acggtgacga ccttctggtt 6840ggcacggatt acgatctgga cttcaatgag gtggcacgac gcgctgccaa gttggggtat 6900aagatgactc ctgccaacaa gggttctgtc ttccctccga cttcctctct ttccgatgct 6960gtttttctaa agcgcaaatt cgtccaaaac aacgacggct tatacaaacc agttatggat 7020ttaaagaatt tggaagccat gctctcctac ttcaaaccag gaacactact cgagaagctg 7080caatctgttt ctatgttggc tcaacattct ggaaaagaag aatatgatag attgatgcac 7140cccttcgctg actacggtgc cgtaccgagt cacgagtacc tgcaggcaag atggagggcc 7200ttgttcgact gacccagata gcccaaggcg cttcggtgct gccggcgatt ctgggagaac 7260tcagtcggaa cagaaaaggg aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 73101692181PRTSeneca Valley Virus 169Met Gln Asn Ser His Phe Ser Phe Asp Thr Ala Ser Gly Thr Phe Glu 1 5 10 15 Asp Val Thr Gly Thr Lys Val Lys Ile Val Glu Tyr Pro Arg Ser Val 20 25 30 Asn Asn Gly Val Tyr Asp Ser Ser Thr His Leu Glu Ile Leu Asn Leu 35 40 45 Gln Gly Glu Ile Glu Ile Leu Arg Ser Phe Asn Glu Tyr Gln Ile Arg 50 55 60 Ala Ala Lys Gln Gln Leu Gly Leu Asp Ile Val Tyr Glu Leu Gln Gly 65 70 75 80 Asn Val Gln Thr Thr Ser Lys Asn Asp Phe Asp Ser Arg Gly Asn Asn 85 90 95 Gly Asn Met Thr Phe Asn Tyr Tyr Ala Asn Thr Tyr Gln Asn Ser Val 100 105 110 Asp Phe Ser Thr Ser Ser Ser Ala Ser Gly Ala Gly Pro Gly Asn Ser 115 120 125 Arg Gly Gly Leu Ala Gly Leu Leu Thr Asn Phe Ser Gly Ile Leu Asn 130 135 140 Pro Leu Gly Tyr Leu Lys Asp His Asn Thr Glu Glu Met Glu Asn Ser 145 150 155 160 Ala Asp Arg Val Thr Thr Gln Thr Ala Gly Asn Thr Ala Ile Asn Thr 165 170 175 Gln Ser Ser Leu Gly Val Leu Cys Ala Tyr Val Glu Asp Pro Thr Lys 180 185 190 Ser Asp Pro Pro Ser Ser Ser Thr Asp Gln Pro Thr Thr Thr Phe Thr 195 200 205 Ala Ile Asp Arg Trp Tyr Thr Gly Arg Leu Asn Ser Trp Thr Lys Ala 210 215 220 Val Lys Thr Phe Ser Phe Gln Ala Val Pro Leu Pro Gly Ala Phe Leu 225 230 235 240 Ser Arg Gln Gly Gly Leu Asn Gly Gly Ala Phe Thr Ala Thr Leu His 245 250 255 Arg His Phe Leu Met Lys Cys Gly Trp Gln Val Gln Val Gln Cys Asn 260 265 270 Leu Thr Gln Phe His Gln Gly Ala Leu Leu Val Ala Met Val Pro Glu 275 280 285 Thr Thr Leu Asp Val Lys Pro Asp Gly Lys Ala Lys Ser Leu Gln Glu 290 295 300 Leu Asn Glu Glu Gln Trp Val Glu Met Ser Asp Asp Tyr Arg Thr Gly 305 310 315 320 Lys Asn Met Pro Phe Gln Ser Leu Gly Thr Tyr Tyr Arg Pro Pro Asn 325 330 335 Trp Thr Trp Gly Pro Asn Phe Ile Asn Pro Tyr Gln Val Thr Val Phe 340 345 350 Pro His Gln Ile Leu Asn Ala Arg Thr Ser Thr Ser Val Asp Ile Asn 355 360 365 Val Pro Tyr Ile Gly Glu Thr Pro Thr Gln Ser Ser Glu Thr Gln Asn 370 375 380 Ser Trp Thr Leu Leu Val Met Val Leu Val Pro Leu Asp Tyr Lys Glu 385 390 395 400 Gly Ala Thr Thr Asp Pro Glu Ile Thr Phe Ser Val Arg Pro Thr Ser 405 410 415 Pro Tyr Phe Asn Gly Leu Arg Asn Arg Tyr Thr Ala Gly Thr Asp Glu 420 425 430 Glu Gln Gly Pro Ile Pro Thr Ala Pro Arg Glu Asn Ser Leu Met Phe 435 440 445 Leu Ser Thr Leu Pro Asp Asp Thr Val Pro Ala Tyr Gly Asn Val Arg 450 455 460 Thr Pro Pro Val Asn Tyr Leu Pro Gly Glu Ile Thr Asp Leu Leu Gln 465 470 475 480 Leu Ala Arg Ile Pro Thr Leu Met Ala Phe Glu Arg Val Pro Glu Pro 485 490 495 Val Pro Ala Ser Asp Thr Tyr Val Pro Tyr Val Ala Val Pro Thr Gln 500 505 510 Phe Asp Asp Arg Pro Leu Ile Ser Phe Pro Ile Thr Leu Ser Asp Pro 515 520 525 Val Tyr Gln Asn Thr Leu Val Gly Ala Ile Ser Ser Asn Phe Ala Asn 530 535 540 Tyr Arg Gly Cys Ile Gln Ile Thr Leu Thr Phe Cys Gly Pro Met Met 545 550 555 560 Ala Arg Gly Lys Phe Leu Leu Ser Tyr Ser Pro Pro Asn Gly Thr Gln 565 570 575 Pro Gln Thr Leu Ser Glu Ala Met Gln Cys Thr Tyr Ser Ile Trp Asp 580 585 590 Ile Gly Leu Asn Ser Ser Trp Thr Phe Val Val Pro Tyr Ile Ser Pro 595 600 605 Ser Asp Tyr Arg Glu Thr Arg Ala Ile Thr Asn Ser Val Tyr Ser Ala 610 615 620 Asp Gly Trp Phe Ser Leu His Lys Leu Thr Lys Ile Thr Leu Pro Pro 625 630 635 640 Asp Cys Pro Gln Ser Pro Cys Ile Leu Phe Phe Ala Ser Ala Gly Glu 645 650 655 Asp Tyr Thr Leu Arg Leu Pro Val Asp Cys Asn Pro Ser Tyr Val Phe 660 665 670 His Ser Thr Asp Asn Ala Glu Thr Gly Val Ile Glu Ala Gly Asn Thr 675 680 685 Asp Thr Asp Phe Ser Gly Glu Leu Ala Ala Pro Gly Ser Asn His Thr 690 695 700 Asn Val Lys Phe Leu Phe Asp Arg Ser Arg Leu Leu Asn Val Ile Lys 705 710 715 720 Val Leu Glu Lys Asp Ala Val Phe Pro Arg Pro Phe Pro Thr Gln Glu 725 730 735 Gly Ala Gln Gln Asp Asp Gly Tyr Phe Cys Leu Leu Thr Pro Arg Pro 740 745 750 Thr Val Ala Ser Arg Pro Ala Thr Arg Phe Gly Leu Tyr Ala Asn Pro 755 760 765 Ser Gly Ser Gly Val Leu Ala Asn Thr Ser Leu Asp Phe Asn Phe Tyr 770 775 780 Ser Leu Ala Cys Phe Thr Tyr Phe Arg Ser Asp Leu Glu Val Thr Val 785 790 795 800 Val Ser Leu Glu Pro Asp Leu Glu Phe Ala Val Gly Trp Phe Pro Ser 805 810 815 Gly Ser Glu Tyr Gln Ala Ser Ser Phe Val Tyr Asp Gln Leu His Val 820 825 830 Pro Phe His Phe Thr Gly Arg Thr Pro Arg Ala Phe Ala Ser Lys Gly 835 840 845 Gly Lys Val Ser Phe Val Leu Pro Trp Asn Ser Val Ser Ser Val Leu 850 855 860 Pro Val Arg Trp Gly Gly Ala Ser Lys Leu Ser Ser Ala Thr Arg Gly 865 870 875 880 Leu Pro Ala His Ala Asp Trp Gly Thr Ile Tyr Ala Phe Val Pro Arg 885 890 895 Pro Asn Glu Lys Lys Ser Thr Ala Val Lys His Val Ala Val Tyr Ile 900 905 910 Arg Tyr Lys Asn Ala Arg Ala Trp Cys Pro Ser Met Leu Pro Phe Arg 915 920 925 Ser Tyr Lys Gln Lys Met Leu Met Gln Ser Gly Asp Ile Glu Thr Asn 930 935 940 Pro Gly Pro Ala Ser Asp Asn Pro Ile Leu Glu Phe Leu Glu Ala Glu 945 950 955 960 Asn Asp Leu Val Thr Leu Ala Ser Leu Trp Lys Met Val His Ser Val 965 970 975 Gln Gln Thr Trp Arg Lys Tyr Val Lys Asn Asp Asp Phe Trp Pro Asn 980 985 990 Leu Leu Ser Glu Leu Val Gly Glu Gly Ser Val Ala Leu Ala Ala Thr 995 1000 1005 Leu Ser Asn Gln Ala Ser Val Lys Ala Leu Leu

Gly Leu His Phe 1010 1015 1020 Leu Ser Arg Gly Leu Asn Tyr Thr Asp Phe Tyr Ser Leu Leu Ile 1025 1030 1035 Glu Lys Cys Ser Ser Phe Phe Thr Val Glu Pro Pro Pro Pro Pro 1040 1045 1050 Ala Glu Asn Leu Met Thr Lys Pro Ser Val Lys Ser Lys Phe Arg 1055 1060 1065 Lys Leu Phe Lys Met Gln Gly Pro Met Asp Lys Val Lys Asp Trp 1070 1075 1080 Asn Gln Ile Ala Ala Gly Leu Lys Asn Phe Gln Phe Val Arg Asp 1085 1090 1095 Leu Val Lys Glu Val Val Asp Trp Leu Gln Ala Trp Ile Asn Lys 1100 1105 1110 Glu Lys Ala Ser Pro Val Leu Gln Tyr Gln Leu Glu Met Lys Lys 1115 1120 1125 Leu Gly Pro Val Ala Leu Ala His Asp Ala Phe Met Ala Gly Ser 1130 1135 1140 Gly Pro Pro Leu Ser Asp Asp Gln Ile Glu Tyr Leu Gln Asn Leu 1145 1150 1155 Lys Ser Leu Ala Leu Thr Leu Gly Lys Thr Asn Leu Ala Gln Ser 1160 1165 1170 Leu Thr Thr Met Ile Asn Ala Lys Gln Ser Ser Ala Gln Arg Val 1175 1180 1185 Glu Pro Val Val Val Val Leu Arg Gly Lys Pro Gly Cys Gly Lys 1190 1195 1200 Ser Leu Ala Ser Thr Leu Ile Ala Gln Ala Val Ser Lys Arg Leu 1205 1210 1215 Tyr Gly Ser Gln Ser Val Tyr Ser Leu Pro Pro Asp Pro Asp Phe 1220 1225 1230 Phe Asp Gly Tyr Lys Gly Gln Phe Val Thr Leu Met Asp Asp Leu 1235 1240 1245 Gly Gln Asn Pro Asp Gly Gln Asp Phe Ser Thr Phe Cys Gln Met 1250 1255 1260 Val Ser Thr Ala Gln Phe Leu Pro Asn Met Ala Asp Leu Ala Glu 1265 1270 1275 Lys Gly Arg Pro Phe Thr Ser Asn Leu Ile Ile Ala Thr Thr Asn 1280 1285 1290 Leu Pro His Phe Ser Pro Val Thr Ile Ala Asp Pro Ser Ala Val 1295 1300 1305 Ser Arg Arg Ile Asn Tyr Asp Leu Thr Leu Glu Val Ser Glu Ala 1310 1315 1320 Tyr Lys Lys His Thr Arg Leu Asn Phe Asp Leu Ala Phe Arg Arg 1325 1330 1335 Thr Asp Ala Pro Pro Ile Tyr Pro Phe Ala Ala His Val Pro Phe 1340 1345 1350 Val Asp Val Ala Val Arg Phe Lys Asn Gly His Gln Asn Phe Asn 1355 1360 1365 Leu Leu Glu Leu Val Asp Ser Ile Cys Thr Asp Ile Arg Ala Lys 1370 1375 1380 Gln Gln Gly Ala Arg Asn Met Gln Thr Leu Val Leu Gln Ser Pro 1385 1390 1395 Asn Glu Asn Asp Asp Thr Pro Val Asp Glu Ala Leu Gly Arg Val 1400 1405 1410 Leu Ser Pro Ala Ala Val Asp Glu Ala Leu Val Asp Leu Thr Pro 1415 1420 1425 Glu Ala Asp Pro Val Gly Arg Leu Ala Ile Leu Ala Lys Leu Gly 1430 1435 1440 Leu Ala Leu Ala Ala Val Thr Pro Gly Leu Ile Ile Leu Ala Val 1445 1450 1455 Gly Leu Tyr Arg Tyr Phe Ser Gly Ser Asp Ala Asp Gln Glu Glu 1460 1465 1470 Thr Glu Ser Glu Gly Ser Val Lys Ala Pro Arg Ser Glu Asn Ala 1475 1480 1485 Tyr Asp Gly Pro Lys Lys Asn Ser Lys Pro Pro Gly Ala Leu Ser 1490 1495 1500 Leu Met Glu Met Gln Gln Pro Asn Val Asp Met Gly Phe Glu Ala 1505 1510 1515 Ala Val Ala Lys Lys Val Val Val Pro Ile Thr Phe Met Val Pro 1520 1525 1530 Asn Arg Pro Ser Gly Leu Thr Gln Ser Ala Leu Leu Val Thr Gly 1535 1540 1545 Arg Thr Phe Leu Ile Asn Glu His Thr Trp Ser Asn Pro Ser Trp 1550 1555 1560 Thr Ser Phe Thr Ile Arg Gly Glu Val His Thr Arg Asp Glu Pro 1565 1570 1575 Phe Gln Thr Val His Phe Thr His His Gly Ile Pro Thr Asp Leu 1580 1585 1590 Met Met Val Arg Leu Gly Pro Gly Asn Ser Phe Pro Asn Asn Leu 1595 1600 1605 Asp Lys Phe Gly Leu Asp Gln Met Pro Ala Arg Asn Ser Arg Val 1610 1615 1620 Val Gly Val Ser Ser Ser Tyr Gly Asn Phe Phe Phe Ser Gly Asn 1625 1630 1635 Phe Leu Gly Phe Val Asp Ser Ile Thr Ser Glu Gln Gly Thr Tyr 1640 1645 1650 Ala Arg Leu Phe Arg Tyr Arg Val Thr Thr Tyr Lys Gly Trp Cys 1655 1660 1665 Gly Ser Ala Leu Val Cys Glu Ala Gly Gly Val Arg Arg Ile Ile 1670 1675 1680 Gly Leu His Ser Ala Gly Ala Ala Gly Ile Gly Ala Gly Thr Tyr 1685 1690 1695 Ile Ser Lys Leu Gly Leu Ile Lys Ala Leu Lys His Leu Gly Glu 1700 1705 1710 Pro Leu Ala Thr Met Gln Gly Leu Met Thr Glu Leu Glu Pro Gly 1715 1720 1725 Ile Thr Val His Val Pro Arg Lys Ser Lys Leu Arg Lys Thr Thr 1730 1735 1740 Ala His Ala Val Tyr Lys Pro Glu Phe Glu Pro Ala Val Leu Ser 1745 1750 1755 Lys Phe Asp Pro Arg Leu Asn Lys Asp Val Asp Leu Asp Glu Val 1760 1765 1770 Ile Trp Ser Lys His Thr Ala Asn Val Pro Tyr Gln Pro Pro Leu 1775 1780 1785 Phe Tyr Thr Tyr Met Ser Glu Tyr Ala His Arg Val Phe Ser Phe 1790 1795 1800 Leu Gly Lys Asp Asn Asp Ile Leu Thr Val Lys Glu Ala Ile Leu 1805 1810 1815 Gly Ile Pro Gly Leu Asp Pro Met Asp Pro His Thr Ala Pro Gly 1820 1825 1830 Leu Pro Tyr Ala Ile Asn Gly Leu Arg Arg Thr Asp Leu Val Asp 1835 1840 1845 Phe Val Asn Gly Thr Val Asp Ala Ala Leu Ala Val Gln Ile Gln 1850 1855 1860 Lys Phe Leu Asp Gly Asp Tyr Ser Asp His Val Phe Gln Thr Phe 1865 1870 1875 Leu Lys Asp Glu Ile Arg Pro Ser Glu Lys Val Arg Ala Gly Lys 1880 1885 1890 Thr Arg Ile Val Asp Val Pro Ser Leu Ala His Cys Ile Val Gly 1895 1900 1905 Arg Met Leu Leu Gly Arg Phe Ala Ala Lys Phe Gln Ser His Pro 1910 1915 1920 Gly Phe Leu Leu Gly Ser Ala Ile Gly Ser Asp Pro Asp Val Phe 1925 1930 1935 Trp Thr Val Ile Gly Ala Gln Leu Glu Gly Arg Lys Asn Thr Tyr 1940 1945 1950 Asp Val Asp Tyr Ser Ala Phe Asp Ser Ser His Gly Thr Gly Ser 1955 1960 1965 Phe Glu Ala Leu Ile Ser His Phe Phe Thr Val Asp Asn Gly Phe 1970 1975 1980 Ser Pro Ala Leu Gly Pro Tyr Leu Arg Ser Leu Ala Val Ser Val 1985 1990 1995 His Ala Tyr Gly Glu Arg Arg Ile Lys Ile Thr Gly Gly Leu Pro 2000 2005 2010 Ser Gly Cys Ala Ala Thr Ser Leu Leu Asn Thr Val Leu Asn Asn 2015 2020 2025 Val Ile Ile Arg Thr Ala Leu Ala Leu Thr Tyr Lys Glu Phe Glu 2030 2035 2040 Tyr Asp Met Val Asp Ile Ile Ala Tyr Gly Asp Asp Leu Leu Val 2045 2050 2055 Gly Thr Asp Tyr Asp Leu Asp Phe Asn Glu Val Ala Arg Arg Ala 2060 2065 2070 Ala Lys Leu Gly Tyr Lys Met Thr Pro Ala Asn Lys Gly Ser Val 2075 2080 2085 Phe Pro Pro Thr Ser Ser Leu Ser Asp Ala Val Phe Leu Lys Arg 2090 2095 2100 Lys Phe Val Gln Asn Asn Asp Gly Leu Tyr Lys Pro Val Met Asp 2105 2110 2115 Leu Lys Asn Leu Glu Ala Met Leu Ser Tyr Phe Lys Pro Gly Thr 2120 2125 2130 Leu Leu Glu Lys Leu Gln Ser Val Ser Met Leu Ala Gln His Ser 2135 2140 2145 Gly Lys Glu Glu Tyr Asp Arg Leu Met His Pro Phe Ala Asp Tyr 2150 2155 2160 Gly Ala Val Pro Ser His Glu Tyr Leu Gln Ala Arg Trp Arg Ala 2165 2170 2175 Leu Phe Asp 2180 1701560DNASeneca Valley Virus 170aatgggcttc gcaaccgcta cacggccggg acggacgaag aacaggggcc cattcctacg 60gcacccagag aaaattcgct tatgtttctc tcaaccctcc ctgacgacac tgtccctgct 120tacgggaatg tgcgtacccc tcctgtcaat tacctccctg gtgaaataac cgaccttttg 180caactggccc gcatacccac tctcatggca tttgagcggg tgcctgaacc cgtgcctgcc 240tcagacacat atgtgcccta cgttgccgtt cccacccagt tcgatgacag gcctctcatc 300tccttcccga tcaccctttc agatcccgtc tatcagaaca ccctggttgg cgccatcagt 360tcaaatttcg ccaattaccg tgggtgtatc caaatcactc tgacattttg tggacccatg 420atggcgagag ggaaattcct gctctcgtat tctcccccaa atggaacgca accacagact 480ctttccgaag ctatgcagtg cacatactct atttgggaca taggcttgaa ctctagttgg 540accttcgtcg tcccctacat ctcgcccagt gactaccgtg aaactcgagc cattaccaac 600tcggtttact ccgctgatgg ttggtttagc ctgcacaagt tgaccaaaat tactctacca 660cctgactgtc cgcaaagtcc ctgcattctc tttttcgctt ctgctggtga ggattacact 720ctccgtctcc ccgttgattg taatccttcc tatgtgttcc actccaccga caacgccgag 780accggggtta ttgaggcggg taacactgac accgatttct ctggtgaact ggcggctcct 840ggctctaacc acactaatgt caagttcctg tttgatcgat ctcgattatt gaatgtaatc 900aaggtactgg agaaggacgc cgttttcccc cgccctttcc ctacacaaga aggtgcgcag 960caggatgatg gttacttttg tcttctgacc ccccgcccaa cagtcgcttc ccgacccgcc 1020actcgtttcg gcctgtacgc caatccgtcc ggcagtggtg ttcttgctaa cacttcactg 1080gacttcaatt tttatagctt ggcctgtttc acttacttta gatcggacct tgaggttacg 1140gtggtctcac tagagccgga tctggaattt gctgtagggt ggtttccttc tggcagtgaa 1200taccaggctt ccagctttgt ctacgaccag ctgcatgtgc ccttccactt tactgggcgc 1260actccccgcg ctttcgctag caagggtggg aaggtatctt tcgtgctccc ttggaactct 1320gtctcgtctg tgctccccgt gcgctggggg ggggcttcca agctctcttc tgctacgcgg 1380ggtctaccgg cgcatgctga ttgggggact atttacgcct ttgtcccccg tcctaatgag 1440aagaaaagca ccgctgtaaa acacgtggcc gtgtacattc ggtacaagaa cgcacgtgcc 1500tggtgcccca gcatgcttcc ctttcgcagc tacaagcaga agatgctgat gcaatctggc 15601711560DNAPicornavirusmisc_feature(8)..(8)n is a, c, g, or t 171aatgggcntc gcaaccgcta catggccggg acggacgaag aacaggggcc nattcccacg 60gcacccagag agaattcgct tatgtttctt tcaactctgc ctgacgacac agttcctgct 120tacgggaatg tgcgcacccc tcctgtcaat tacctccctg gtgaaataac cgacctcttg 180caactggccc gcatacccac tctcatggca tttgagcggg tgcccgaacc cgtgcctgcc 240tcagatacgt atgtgcccta cgttgccgtt cccacccaat tcgctgacag gcctctcatc 300tccttcccga tcaccctttc agaccctgtc tatcaaaaca ccctggttgg cgccatcagt 360tcttactttg ccaattaccg tgggtgtatc cagatcactc tgacgttttg tggacccatg 420atggcgagag ggaaattcct actgttatat tctcccccaa atggaacgca accacagact 480ctttccgaag ctatgcagtg cacatactct atttgggaca taggcttgaa ctcgagttgg 540accttcgtcg tcccctacat ctcgcccagt gactaccgtg aaacccgggc cattaccaac 600tcggtttact ccgctgatgg ttggtttagc ctgcacaagt tgaccaaaat tactctccca 660cctgactgtc cgcaaagtcc ctgcgttctc tttttcgctt ctgctggtga ggattacact 720ctccgtctcc ccattgattg taatccttcc tacgtgttcc actccaccga caacgccgaa 780actggggttg ttgaggcggg taacactgac accgatttct ctggtgaact agcggctcct 840ggctccaacc acactaatgt caagttcctg tttgatcgat ctcgattatt gaatgtaatt 900aaggtactgg agaaagacgc cgttttcccc caccctttcc ctacgctaga aggtgcgcaa 960caggatgatg gttacttttg tcttctgacc ccccgcccaa cagtcgcttc ccgacccgcc 1020actcgtttcg gcctgtacgc caatccgtcc ggcagcggtg ttcttgctaa tacttcattg 1080gactttaact tttatagctt ggcctgtttc acttacttta gatcggacct cgaggttacg 1140gtggtctcac tggagccaga tctggaattt gctgtagggt ggtttccttc cggcagtgaa 1200tatcaggctt ccagctttgt ctacgaccag ctgcacgtgc ccttccactt cactggacgc 1260accccccgcg cttttgctag caagggtggg aaggtatcct ttgtgctccc ttggaactct 1320gtctcatctg tgctccccgt gcgctggggg ggggcttcca agctctcttc tgccacgcgg 1380ggtctaccgg cgcatgctga ctgggggact atctacgcct ttgtcccccg tcccaatgaa 1440aagaaaagca ccgctgcaaa acacgtggcc gtgtacattc ggtacaagaa cgcacgcgcc 1500tggtgcccca gcatgcttcc ctttcgcagc tataagcaga agatgctgat gcaatctggc 15601721560DNAPicornavirus 172aatgggcttc gcaaccgcta cacgaccggg acggacgagg aacaggggcc cattcccacg 60gcacccagag aacattcgct tatgtttctc tcaaccctcc ctgacgacac tgtccctgct 120tacgggaatg tgcgtacccc tcctgtcaat tacctccctg gtgaaataac cgaccttttg 180caactggccc gcatacccac tctcatggcg tttgagcggg tgcctgaacc cgtgcctgcc 240tcagacacat atgtgcccta cgttgccgtt cccacccagt ttgatgacaa gcctctcatc 300tccttcccga tcaccctttc agatcctgtc tatcagaaca ccctggtagg cgccatcagt 360tcaaatttcg ccaactaccg tgggtgtatc caaatcactc tgacattttg tggacccatg 420atggcaagag ggaaattcct actctcgtat tctcccccaa atggaacgca accacagact 480ctttccgaag ctatgcagtg cacatactct atttgggaca taggcttgaa ctctagttgg 540accttcgtcg tcccctacat ctcgcccagt gactaccgtg aaactcgggc cattaccaac 600tcggtttact ccgctgatgg ttggtttagt ctgcacaagt tgaccaaaat tactctacca 660cctgactgcc cgcaaagtcc ctgtattctc tttttcgctt ctgctggtga ggattacacc 720ctccgtctcc ccgttgattg taatccttcc tatgtgttcc actccaccga caacgccgag 780actggggtta ttgaggcggg taacactgac accgatttct ctggtgaact ggcggctcct 840ggctctaacc acactaatgt caagttcctg tttgatcgat ctcgattact gaatgtaatt 900aaggtactgg agaaggacgc cgttttcccc cgtcctttcc ctacaaaaga aggtgcgcag 960caggacgatg gttacttttg tcttctgaca ccccgcccaa cagtcgcctc ccgacccgcc 1020actcgtttcg gcctgtacgt caatccgtcc ggcagtggtg ttctcgccaa cacttcactg 1080gacttcaatt tttatagctt ggcctgtttc acttacttta gatcggacct tgaagttacg 1140gtggtctcac tggagccaga tctggaattt gctgtagggt ggtttccttc tggcagtgaa 1200taccaggctt ccagctttgt ctacggccag ctgcatgtac ccttccactt tactgggcgc 1260actccccgcg ctttcgccag caagggtggg aaggtatctt tcgtgctccc ttggaactct 1320gtctcatctg tgctccccgt gcgctggggg ggcgcttcca agctctcttc tgccacgcgg 1380ggtctaccgg cgcatgctga ctgggggact atttacgcct ttgtcccccg tcctaatgag 1440aagaaaagca ccgctgtaaa gcacgtggcc gtgtacgttc ggtacaagaa cgcacgtgcc 1500tggtgcccca gcatgcttcc ttttcgcagc tacaagcaga agatgctgat gcaatctggc 1560173936DNAPicornavirus 173tttagcctgc acaagttgac caaaattact ctaccacctg actgcccgca aaatccctgc 60attctctttt tcgcttctgc tggtgaggat tacactctcc gtctccccat tgattgcaat 120ccttcctacg tgttccactc caccgacaac gccgaaactg gggttgtcga ggcgggtaac 180actgacaccg atttctctgg tgaactagcg gctcctggct ccaaccacac taatgtcaag 240ttcctgtttg atcgatctcg gttattgaat gtaattaagg tactggagaa agacgctgtt 300ttccctcacc ctttccctac gctagaaggt gtgcaacagg atgatggcta cttttgtctt 360ctgacccccc gcccaacagt cgcttcccgg cctgccactc gtttcggcct gtacgccaat 420ccgtccggca gcggtgttct tgctaatact tcattggact ttaactttta tagcttggcc 480tgtttcactt actttagatc ggacctcgag gttacggtgg tctcactaga gccggatctg 540gaatttgctg tggggtggtt tccttccggc agtgaatatc aggcttccag ctttgtctac 600gaccagctgc acgtgccctt ccacttcact ggacgcaccc cccgcgcttt tgctagcaag 660ggtgggaagg tatcctttgt gctcccttgg aactctgtct catctgtgct ccccgtgcgc 720tgggggggag cttccaagct ctcttctgcc acgcggggtc taccggtgca cgctgactgg 780gggactatct acgcctttgt cccccgtccc aatgaaaaga aaagcaccgc tgcaaaacat 840gtggccgtgt acattcggta caagaacgca cgcgcctggt gtcccaacat gctccccttt 900cgcagctata agcagaagat gctgatgcaa tctggc 936174936DNAPicornavirus 174tttagcctgc acaagttgac caaaataact ctaccacctg actgcccgca aaatccctgc 60attctctttt tcgcttctgc tggtgaggat tacactctcc gtctccccat tgattgcaat 120ccttcctacg tgttccactc caccgacaac gccgaaactg gggttgtcga ggcgggtaac 180actgacaccg atttctctgg tgaactagcg gctcctggct ccaaccacac taatgtcaag 240ttcctgtttg atcgatctcg gttattgaat gtaattaagg tactggagaa agacgctgtt 300ttccctcacc ctttccctac gctagaaggt gtgcaacagg atgatggcta cttttgtctt 360ctgacccccc gcccaacagt cgcttcccgg cctgccactc gtttcggcct gtacgccaat 420ccgtccggca gcggtgttct tgctaatact tcattggact ttaactttta tagcttggcc 480tgtttcactt actttagatc ggacctcgag gttacggtgg tctcactaga gccggatctg 540gaatttgctg tggggtggtt tccttccggc agtgaatatc aggcttccag ctttgtctac 600gaccagctgc acgtgccctt ccacttcact ggacgcaccc cccgcgcttt tgctagcaag 660ggtgggaagg tatcctttgt gctcccttgg aactctgtct catctgtgct ccccgtgcgc 720tgggggggag cttccaagct ctcttctgcc acgcggggtc taccggtgca cgctgactgg 780gggactatct acgcctttgt cccccgtccc aatgaaaaga aaagcaccgc tgcaaaacat 840gtggccgtgt acattcggta caagaacgca cgcgcctggt gtcccaacat gctccccttt 900cgcagctata agcagaagat gctgatgcaa tctggc 936175936DNAPicornavirus 175tttagcctgc acaagttgac caaaattact ytaccacctg actgtccgca aagtccctgc 60attctctttt tcgcttctgc tggtgaggat tacactctcc gtctccccat tgattgtaat 120ccttcctacg tgttccactc caccgacaac gccgaaactg gggttgttga ggcgggtaac 180actgacaccg atttctctgg tgaactagcg gctcctggct ccaaccacac taatgtcaag 240ttcctgtttg atcgatctcg

attattgaat gtaattaagg tactggagaa agacgccgtt 300ttcccccacc ctttccctac gctagaaggt gcgcaacagg atgatggtta cttttgtctt 360ctgacccccc gcccaacagt cgcttcccga cccgccactc gtttcggcct gtacgccaat 420ccgtccggca gcggtgttct tgctaatact tcattggact ttaactttta tagcttggcc 480tgcttcactt actttagatc ggacctcgag gttacggtgg tctcactgga gccagatctg 540gaatttgctg tagggtggtt tccttccggc agtgaatatc aggcttccag ctttgtctac 600gaccagctgc acgtgccctt ccacttcact ggacgcaccc cccgcgcttt tgctagcaag 660ggtgggaagg tatcctttgt gctcccttgg aactctgtct catctgtgct ccccgtgcgc 720tggggggggg cttccaagct ctcttctgcc acgcggggtc taccggcgca tgctgactgg 780gggactatct acgcctttgt cccccgtccc aatgaaaaga aaagcaccgc tgcaaaacac 840gtggccgtgt acattcggta caagaacgca cgcgcctggt gccccagcat gcttcccttt 900cgcagctata agcagaagat gctgatgcaa tctggc 936176936DNAPicornavirus 176tttagcctgc acaagttgac aaaaattact ctaccacctg actgtccgca aagtccctgc 60attctctttt tcgcttctgc tggtgaggat tacactctcc gtctccccgt tgattgtaat 120ccttcttacg tgttccactc caccgacaac gccgaaactg gggttgttga ggcgggtaac 180actgacaccg atttctctgg tgaactagcg gctcctggtt ccaaccacac taacgtcaag 240ttcctgtttg atcgatctcg attattgaat gtaattaagg tactggagaa agacgccgtt 300ttccctcacc ctttccctac gctagaaggt gcgcaacagg atgatggtta cttttgtctt 360ctgacccccc gcccaacagt cgcttcccga cccgccactc gtttcggcct gtacgccaat 420ccgtccggca gtggtgttct tgctaatact tcattggact ttaactttta tagcttggcc 480tgtttcactt actttagatc ggacctcgag gttacggtgg tctcactgga gccagatctg 540gaatttgctg taggatggtt tccttccggc agtgaatacc aggcttccag ctttgtctac 600gaccagctgc acgtgccttt ccacttcact gggcgcactc cccgcgcttt tgctagcaag 660ggtgggaagg tatccttcgt gctcccttgg aactctgttt cgtctgtgct ccccgtgcgc 720tggggggggg cttccaagct ctcttctgcc acgcggggtc taccggcgca tgctgactgg 780gggactatct acgcctttgt cccccgtccc aatgaaaaga aaagcaccgc tgcaaaacac 840gtggccgtgt acattcggta caagaacgca cgcgcctggt gccccagcat gcttcccttt 900cgcagctata agcagaagat gctgatgcaa tctggc 936177936DNAPicornavirus 177tttagcctgc acaagttgac caaaattact ctaccacctg actgtccgca aagtccctgc 60attctctttt tcgcttctgc tggtgaggat tacactctcc gtctccccgt tgattgtaat 120ccttcctatg tgttccactc caccgacaac gccgagactg gggttattga ggcgggtaac 180actgacaccg atttctctgg tgaactggcg gctcctggct ctaaccacac taatgtcaag 240ttcctgtttg atcgatctcg attattgaat gtaattaagg tactggagaa ggacgccgtt 300ttcccccgcc ctttccctac acaagaaggt gcgcagcagg acgatggtta cttttgtctt 360ctgacccccc gcccaacagt cgcttcccga cccgccactc gtttcggcct gtacgccaat 420ccgtccggca gtggtgttct cgccaacact tcactggact tcaattttta tagcttggcc 480tgtttcactt actttagatc ggaccttgag gttacggtgg tctcactgga gccaaatctc 540gaatttgctg tagggtggtt tccttccggt agtgaatacc aggcttccag ttttgtctac 600gaccagctgc acgtgccctt ccacttcact gggcgcactc cccgcgcttt cgctagcaag 660ggtgggaagg tgtccttcgt gctcccttgg aactctgtct cgtctgtgct ccccgtgcgc 720tggggggggg cttccaagct ctcttctgcc acacggggtc taccagtgca tgctgactgg 780gggactattt acgcctttgt cccccgtccc aatgaaaaga aaagcactgc tgtaaaacac 840gtggccgtgt acattcggta caagaacgca cgcgcctggt gccccagcat gcttcctttt 900cgcagctaca agcagaagat gctgatgcaa tctggc 9361781421DNAArtificial SequenceConsensus Sequence from SVV and SVV-like Picornaviruses in a coding region sequence for VP2(partial), VP3, VP1, and 2A (partial) 178aatgggctcg caaccgctac agccgggacg gacgagaaca ggggccattc cacggcaccc 60agagaattcg cttatgtttc ttcaacctcc tgacgacacg tcctgcttac gggaatgtgc 120gacccctcct gtcaattacc tccctggtga aataaccgac ctttgcaact ggcccgcata 180cccactctca tggctttgag cgggtgccga acccgtgcct gcctcagaac tatgtgccct 240acgttgccgt tcccacccat tgtgacagcc tctcatctcc ttcccgatca ccctttcaga 300ccgtctatca aacaccctgg tggcgccatc agttcattgc caataccgtg ggtgtatcca 360atcactctga cttttgtgga cccatgatgg cagagggaaa ttcctcttta ttctccccca 420aatggaacgc aaccacagac tctttccgaa gctatgcagt gcacatactc tatttgggac 480ataggcttga actcagttgg accttcgtcg tcccctacat ctcgcccagt gactaccgtg 540aaaccggcca ttaccaactc ggtttactcc gctgatggtt ggtttagctg cacaagttga 600caaaatactt ccacctgact gccgcaaatc cctgttctct ttttcgcttc tgctggtgag 660gattacacct ccgtctcccc ttgattgaat ccttctagtg ttccactcca ccgacaacgc 720cgaacggggt ttgaggcggg taacactgac accgatttct ctggtgaact gcggctcctg 780gtcaaccaca ctaagtcaag ttcctgtttg atcgatctcg ttatgaatgt aataaggtac 840tggagaagac gcgttttccc ccctttccct acagaaggtg gcacaggaga tggtactttt 900gtcttctgac ccccgcccaa cagtcgctcc cgccgccact cgtttcggcc tgtacgcaat 960ccgtccggca gggtgttctg caaacttcat ggacttaatt ttatagcttg gcctgttcac 1020ttactttaga tcggacctga gttacggtgg tctcactgag ccatctgaat ttgctgtggt 1080ggtttccttc ggagtgaata caggcttcca gtttgtctac gccagctgca gtccttccac 1140ttactggcgc acccccgcgc tttgcagcaa gggtgggaag gttcttgtgc tcccttggaa 1200ctctgttctc tgtgctcccc gtgcgctggg gggggcttcc aagctctctt ctgcaccggg 1260gtctaccggc agctgatggg ggactattac gcctttgtcc cccgtccaat gaaagaaaag 1320cacgctgaaa cagtggccgt gtacttcggt acaagaacgc acggcctggt gcccacatgc 1380tcctttcgca gctaaagcag aagatgctga tgcaatctgg c 1421179551DNASeneca Valley Virus 179aaactgttta agatgcaagg acccatggac aaagtcaaag actggaacca aatagctgcc 60ggcttgaaga attttcaatt tgttcgtgac ctagtcaaag aggtggtcga ttggctgcag 120gcctggatca acaaagagaa agccagccct gtcctccagt accagttgga gatgaagaag 180ctcgggcctg tggccttggc tcatgacgct ttcatggctg gttccgggcc ccctcttagc 240gacgaccaga ttgaatacct ccagaacctc aaatctcttg ccctaacact ggggaagact 300aatttggccc aaagtctcac cactatgatc aatgccaaac aaagttcagc ccaacgagtt 360gaacccgttg tggtggtcct tagaggcaag ccgggatgcg gcaagagctt ggcctctacg 420ttgattgccc aggctgtgtc caagcgcctc tatggctccc aaagtgtata ttctcttccc 480ccagatccag atttcttcga tggatacaaa ggacagttcg tgaccttgat ggatgatttg 540ggacaaaacc c 551180551DNAPicornavirus 180aaactgttta agatgcaagt acccatggac aaagtcaaag actggaacca aatagccgcc 60ggcttgaaga actttcaatt tgttcgtgac ctagtcaaag aggtggtcga ctggctgcag 120gcctggatca acaaggagaa agccagccct gtcctccaat accagttgga gatgaagaag 180ctcgggcctg tggctttggc tcatgacgct tttatggctg gttccgggcc ccctcttagc 240gacgaccaga ttgagtatct ccagaacctc aaatctcttg ccctaacact agggaagact 300aatttggccc aaagtctcac cactatgatc aatgccaaac aaagttccgc ccaacgagtt 360gaacccgttg tggtggtcct tagaggtaag cctggatgtg gcaagagctt ggcctctacg 420ctgattgctc aggctgtgtc caagcgcctc tatggctccc aaagtgtata ttccctcccc 480ccagacccag atttctttga tggatacaaa ggacaattcg tgaccttgat ggatgatttg 540ggacaaaacc c 551181551DNAPicornavirus 181aaactgttta agatgcaagt acccatggac aaagtcaaag actggaacca aatagccgcc 60ggcttgaaga attttcaatt tgttcgtgac ctagtcaaag aggtggtcga ctggctgcag 120gcctggatca acaaagagaa agccagccct gtcctccagt accagttgga gatgaagaag 180ctcgggcccg tggctttggc tcatgacgct ttcatggctg gttccgggcc ccctcttagc 240gacgaccaga ttgaatacct ccagaacctc aaatctcttg ccctaacact ggggaagact 300aatttggccc aaagtctcac cactatgatc aatgccaaac aaagttccgc ccaacgagtt 360gaacccgttg tggtggtcct tagaggcaag ccgggatgcg gcaagagctt ggcctccacg 420ttgattgccc aggctgtgtc caagcgtctc tatggctccc aaagtgtgta ttctcttccc 480ccggatccag atttcttcga tggatacaaa ggacagtttg tgaccttgat ggatgatttg 540ggacaaaacc c 551182523DNAArtificial SequenceConsensus Sequence from SVV and SVV-like Picornaviruses in a partial coding region sequence for 2C 182aaactgttta agatgcaaga cccatggaca aagtcaaaga ctggaaccaa atagcgccgg 60cttgaagaat ttcaatttgt tcgtgaccta gtcaaagagg tggtcgatgg ctgcaggcct 120ggatcaacaa gagaaagcca gccctgtcct ccataccagt tggagatgaa gaagctcggg 180ccgtggcttg gctcatgacg ctttatggct ggttccgggc cccctcttag cgacgaccag 240attgatactc cagaacctca aatctcttgc cctaacactg ggaagactaa tttggcccaa 300agtctcacca ctatgatcaa tgccaaacaa agttcgccca acgagttgaa cccgttgtgg 360tggtccttag aggaagccgg atgggcaaga gcttggcctc acgtgattgc caggctgtgt 420ccaagcgctc tatggctccc aaagtgttat tcctcccccg accagatttc ttgatggata 480caaaggacat tgtgaccttg atggatgatt tgggacaaaa ccc 523183460DNASeneca Valley Virus 183cttctggttg gcacggatta cgatctggac ttcaatgagg tggcacgacg cgctgccaag 60ttggggtata agatgactcc tgccaacaag ggttctgtct tccctccgac ttcctctctt 120tccgatgctg tttttctaaa gcgcaaattc gtccaaaaca acgacggctt atacaaacca 180gttatggatt taaagaattt ggaagccatg ctctcctact tcaaaccagg aacactactc 240gagaagctgc aatctgtttc tatgttggct caacattctg gaaaagaaga atatgataga 300ttgatgcacc ccttcgctga ctacggtgcc gtaccgagtc acgagtacct gcaggcaaga 360tggagggcct tgttcgactg acccagatag cccaaggcgc ttcggtgctg ccggcgattc 420tgggagaact cagtcggaac agaaaaggga aaaaaaaaaa 460184460DNAPicornavirusmisc_feature(19)..(19)n is a, c, g, or t 184cttctggttg gcacggatna cnatctggac ttcaatgagg tggcgcggcg cgctgccaaa 60ttggggtata agatgacgcc tgccaacaag ggttccgtct tccctccgac ttcctctctt 120tccaatgctg tttttctaaa acgtaaattc gtccaaaaca atgacggctt gtacaagcca 180gttatggatt caaagaattt ggaagccatg ctctcctact tcaaaccagg aacactactc 240gagaagctgc aatctgtttc tatgttggct caacattctg gaaaagaaga atacgataga 300ttgatgcatc ccttcgctga ctacggtgcc gtaccgagtc acgagtacct gcaggcaaga 360tggagggcct tgttcgattg acccagatag cccaaggcgc ttcggtgctg acggtgattc 420tgggagaact cagtcggaac aaaaagggga aaaaaaaaaa 460185460DNAPicornavirusmisc_feature(40)..(40)n is a, c, g, or t 185cttctggttg gcacggatta cgatctggac ttcaatgagn tggcgcggcg cgctgccaaa 60ttggggtata agatgacgcc tgccaacaag ggttccgtct tccctccgac ttcctctctt 120tccaatgctg tttttctaaa acgtaaattc gtccaaaaca atgacggctt gtacaagcca 180gttatggatt caaagaattt ggaagccatg ctctcctact tcaaaccagg aacactactc 240gagaagctgc aatctgtttc tatgttggct caacattctg gaaaagaaga atacgataga 300ttgatgcatc ccttcgctga ctacggtgcc gtaccgagtc acgagtacct gcaggcaaga 360tggagggcct tgttcgattg acccaganag cccaaggcgc ttnggtgctg acggtgattc 420tgggagaact cagtcggaac aaaaagggga aaaaaaaaaa 460186460DNAPicornavirus 186cttctggttg gcacggatga cgatctggac ttcaatgagg tggcgcggcg cgctgccaaa 60ttggggtata agatgacgcc tgccaacaag ggttccgtct tccctccgac ttcctctctt 120tccgatgctg tttttctaaa acgcaaattc gtccaaaaca acgacggctt gtacaaacca 180gttatggatt caaagaattt ggaagccatg ctctcctact tcaaaccagg aacactactc 240gagaagctgc aatctgtttc tatgttggct caacattctg gaaaagaaga atatgataga 300ttgatgcatc ccttcgctga ctacggtgcc gtaccgagtc acgagtacct gcaggcaaga 360tggagggcct tgttcgactg acccagatag cccaaggcgc ttcggtgctg acggtgattc 420tgggagaact cagtcggaac agaaaagggg aaaaaaaaaa 460187460DNAPicornavirusmisc_feature(5)..(5)n is a, c, g, or t 187cttcnggttg gcacggatga cgatctggac ttcaatgagg tggcgcggcg cgctgccaaa 60ttggggtata agatgacgcc tgccaacaag ggttccgtct tccctccgac ttcctctctt 120tccgatgctg tttttctaaa acgcaaattc gtccaaaaca acgacggctt gtacaaacca 180gttatggatt caaagaattt ggaagccatg ctctcctact tcaaaccagg aacactactc 240gagaagctgc aatctgtctc tatgttggct caacattctg gaaaagaaga atatgataga 300ttgatgcatc ccttcgctga ctacggtgcc gtaccgagtc acgagtacct gcaggcaaga 360tggagggcct tgttcgactg acccagatag cccaaggcgc ttcggtgctg acggtgattc 420tgggagaact cagtcggaac agaaaaggga aaaaaaaaaa 460188460DNAPicornavirusmisc_feature(11)..(11)n is a, c, g, or t 188cttctggttg ncacggatna cgatctgnac ttcaatgagg tggcgcggcg cgctgccaaa 60ttggggtata agatgacgcc tgccaacaag ggttccgtct tccctccgac ttcctctctt 120tccgatgctg tttttctaaa acgcaaattc gtccaaaaca acgacggctt gtacaaacca 180gttatggatt caaagaattt ggaagccatg ctctcctact tcaaaccagg aacactactc 240gagaagctgc aatctgtttc tatgttggct caacattctg gaaaagaaga atatgataga 300ctgatgcacc ccttcgctga ctacggtgcc gtaccgagtc acgagtacct gcaggcaaga 360tggagggcct tgttcgactg acccagatag cccaaggcgc ctcggtgctg ccggtgattn 420tnggagaact cagtcggaac agaaaaggga gaaaaaaaaa 460189460DNAPicornavirus 189cttctggttg gcacggatta cgatctggac ttcaatgagg tggcgcgacg cgctgccaaa 60ttggggtata agatgactcc tgccaacaag ggttccgtct tcccttcgac ttcctctctt 120tccgacgctg tttttctaaa acgcaaattc gtccaaaaca acgacggctt atacaaacca 180gttatggatt taaagaattt ggaagccatg ctctcctact tcaaaccagg aacactactc 240gagaagctgc aatctgtttc tatgttggct caacattctg gaaaagaaga atatgataga 300ttgatgcacc ccttcgctga ctacggtgcc gtaccgagtc acgagtacct gcaggcaaga 360tggagggcct tgttcgactg acccagatag cccaaggcgc ttcggtgctg ccggcgattc 420tgggagaact cagtcggaac agaaagggga aaaaaaaaaa 460190460DNAPicornavirus 190cttctggttg gtacggatta cgatctggac ttcaatgagg tggcgcgacg cgctgccaag 60ctggggtata agatgactcc tgccaacaag ggttccgtct tccctccgac ttcctctctc 120tccgatgctg ttttcctaaa acgcaaattc gtccaaaaca acgacggctt atacaaacca 180gttatggatt taaagaattt ggaagccatg ctctcctact tcaaaccagg aacactactc 240gagaagctgc aatctgtttc tatgttggct caacattctg gaaaagaaga atatgataga 300ttgatgcacc ccttcgctga ctacggtgcc gtaccgagtc acgagtacct gcaggcaaga 360tggagggcct tgttcgactg acccagatag cccaaggcgc ttcggtgctg ccggcgattc 420tgggagaact cagtcggaac agaaaagggg aaaaaaaaaa 460191420DNAArtificial SequenceConsensus Sequence from SVV and SVV-like Picornaviruses in a partial coding region sequence for the 3D polymerase and the 3'UTR region 191cttcggttga cggatacatc tgacttcaat gagtggccgc gcgctgccaa tggggtataa 60gatgaccctg ccaacaaggg ttcgtcttcc ctcgacttcc tctcttccag ctgttttcta 120aacgaaattc gtccaaaaca agacggcttt acaaccagtt atggattaaa gaatttggaa 180gccatgctct cctacttcaa accaggaaca ctactcgaga agctgcaatc tgttctatgt 240tggctcaaca ttctggaaaa gaagaataga tagatgatgc acccttcgct gactacggtg 300ccgtaccgag tcacgagtac ctgcaggcaa gatggagggc cttgttcgat gacccagaag 360cccaaggcgc tggtgctgcg ggatttggag aactcagtcg gaacaaaagg gaaaaaaaaa 4201924PRTPicornavirus 192Leu Gln Gly Asn 1 1934PRTPicornavirus 193Pro Gln Gly Asn 1 1944PRTPicornavirus 194Leu Lys Asp His 1 1954PRTPicornavirus 195Leu Ala Asp Gln 1 1964PRTPicornavirus 196Leu Leu Asp Gln 1 1974PRTPicornavirus 197Leu Met Asp Gln 1 1984PRTPicornavirus 198Leu Leu Asp Glu 1 1994PRTPicornavirus 199Arg Gln Ser Pro 1 2004PRTPicornavirus 200Ala Gln Ser Pro 1 2014PRTPicornavirus 201Pro Gln Ser Pro 1 2024PRTPicornavirus 202Pro Gln Gly Val 1 2034PRTPicornavirus 203Pro Gln Gly Ile 1 2044PRTPicornavirus 204Pro Gln Gly Ser 1 2054PRTPicornavirus 205Met Gln Ser Gly 1 2064PRTPicornavirus 206Leu Glu Ser Pro 1 2074PRTPicornavirus 207Leu Glu Asn Pro 1 2084PRTPicornavirus 208Leu Gln Asn Pro 1 2094PRTPicornavirus 209Gln Gln Ser Pro 1 2104PRTPicornavirus 210Pro Gln Gly Pro 1 2114PRTPicornavirus 211Ala Gln Gly Pro 1 2124PRTPicornavirus 212Ala Gln Ala Pro 1 2134PRTPicornavirus 213Glu Gln Gly Pro 1 2144PRTPicornavirus 214Glu Gln Ala Ala 1 2154PRTPicornavirus 215Ile Gln Gly Pro 1 2164PRTPicornavirus 216Val Gln Gly Pro 1 2174PRTPicornavirus 217Ile Gln Gly Gly 1 2184PRTPicornavirus 218Pro Gln Gly Ala 1 21920DNAArtificial SequencePrimer sequence for Seneca Valley Virus 219tttgaaatgg ggggctgggc 2022019DNAArtificial SequencePrimer Sequence for Seneca Valley Virus 220gaggagaccc gctaatccg 1922154DNAArtificial SequencePrimer Sequence for generating an infectious plasmid clone of Seneca Valley Virus 221tatgggtacc tgtaatacga ctcactatag ggctttgaaa tggggggctg ggcc 5422224DNAArtificial SequencePrimer sequence for generating an infectious plasmid clone of Seneca Valley Virus 222ccgtcaaaga agcaattctg ggca 2422360DNAArtificial SequencePrimer sequence for generating an infectious plasmid clone of Seneca Valley Virus 223gcatgcattt tttttttttt tttttttttt tttttttccc ttttctgttc cgactgagtt 6022430DNAArtificial SequencePrimer sequence for generating an infectious clone of Seneca Valley Virus 224ggtaacatga ccttcaatta ctacgcaaac 3022523DNAArtificial SequencePrimer sequence for generating an infectious plasmid clone of Seneca Valley Virus 225gatcagtacg tcgaaggccg ttg 2322676DNAArtificial SequencePrimer sequence for generating an infectious plasmid clone of Seneca Valley Virus 226gcttgcatgc atttaaattt tttttttttt tttttttttt ttttttttcc cttttctgtt 60ccgactgagt tctccc 7622747DNAArtificial SequencePrimer sequence for generating an infectious plasmid clone of Seneca Valley Virus 227caattgtgta atacgactca ctatagtttg aaatgggggg ctgggcc 47

Claims

1. An isolated nucleic acid comprising a nucleic acid sequence having at least 75% sequence identity to: (i) SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, or 168, or (ii) a contiguous portion of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, or 168, that is at least 20 nucleotides in length.

2. The isolated nucleic acid of claim 1, wherein the nucleic acid is RNA or DNA.

3. An isolated polypeptide comprising an amino acid sequence having at least 75% sequence identity to: (i) SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, or 169, or (ii) a contiguous portion of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, or 169 that is at least 10 amino acids in length.

4. An isolated Seneca Valley virus or derivative thereof, comprising identifying characteristics of ATCC Patent Deposit number PTA-5343.

5. An isolated Seneca Valley virus or derivative or relative thereof, having a genome comprising a sequence that is at least 95%, 90%, 85%, 80%, 75%, 70%, or 65% identical to SEQ ID NO:1 or SEQ ID NO:168.

6. The virus of claim 5 comprising the following characteristics: replication competence in tumor cells, tumor-cell tropism, and lack of cytolysis in normal cells.

7. The virus of claim 6, wherein said virus is replication competent in tumor cell types having neuroendocrine properties.

8. A pharmaceutical composition comprising an effective amount of the virus of claim 5 and a pharmaceutically acceptable carrier.

9. An isolated antibody that specifically binds to the epitope of the isolated virus of claim 5.

10. A method for treating cancer comprising administering an effective amount of a virus or derivative thereof, so as to treat the cancer, wherein the virus has a genome that comprises a sequence that is at least 75% identical to a contiguous sequence of SEQ ID NO:1 or SEQ ID NO:168 that is at least 100 nucleotides in length.

11. The method of claim 10, wherein the virus is a picornavirus.

12. The method of claim 11, wherein the picornavirus is a cardiovirus.

13. The method of claim 12, wherein the cardiovirus is selected from the group consisting of: vilyuisk human encephalomyelitis virus, Theiler's murine encephalomyelitis virus, and encephalomyocarditis virus.

14. The method of claim 11, wherein the picornavirus is a member of a genus to which Seneca Valley virus belongs.

15. The method of claim 11, wherein the picornavirus is Seneca Valley virus.

16. The method of claim 15, wherein the Seneca Valley virus has an ATCC deposit number PTA-5343.

17. The method of claim 11, wherein the picornavirus is a Seneca Valley virus-like picornavirus.

18. The method of claim 17, wherein the Seneca Valley virus-like picornavirus is selected from the group of isolates consisting of: MN 88-36695, NC 88-23626, IA 89-47552, NJ 90-10324, IL 92-48963, CA 131395, LA 1278, IL 66289, IL 94-9356, MN/GA 99-29256, MN 99197, and SC 363649.

19. A method of killing an abnormally proliferative cell comprising contacting the cell with the virus of claim 5.

20. A method for making an oncolytic virus, the method comprising: (a) comparing a Seneca Valley virus genomic sequence with a test virus genomic sequence; (b) identifying at least a first amino acid difference between a polypeptide encoded by the Seneca Valley virus genomic sequence and a polypeptide encoded by the test virus genomic sequence; (c) mutating the test virus genomic sequence such that the polypeptide encoded by the test virus genomic sequence has at least one less amino acid difference to the polypeptide encoded by the Seneca Valley virus genomic sequence; (d) transfecting the mutated test virus genomic sequence into a tumor cell; and (e) determining whether the tumor cell is lytically infected by the mutated test virus genomic sequence.

21. The method of claim 20, wherein the Seneca Valley virus genome comprises a sequence that is at least 95% identical to SEQ ID NO:1 or SEQ ID NO:168.

22. The method of claim 20, wherein the test virus is a picornavirus.

23. The method of claim 22, wherein the test virus is a Seneca Valley virus-like picornavirus.

24. The method of claim 20, wherein the amino acid differences are between a Seneca Valley virus capsid protein and a test virus capsid protein.

25. The method of claim 20, wherein mutating the test virus genomic sequence comprises mutating a cDNA having the test virus genomic sequence.

26. The method of claim 20, wherein transfecting the mutated test virus genomic sequence comprises transfecting RNA, wherein the RNA is generated from the cDNA having the mutated test virus genomic sequence.

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