Patent application title: EXPRESSION AND ASSEMBLY OF HUMAN GROUP C ROTAVIRUS-LIKE PARTICLES AND USES THEREOF
Inventors:
Baoming Jiang (Duluth, GA, US)
Assignees:
The Government of the US, as Represented by the Secretary, Department of Health and Human Services
IPC8 Class: AA61K3915FI
USPC Class:
424499
Class name: Preparations characterized by special physical form particulate form (e.g., powders, granules, beads, microcapsules, and pellets) contains proteins or derivative or polysaccharides or derivative
Publication date: 2011-07-14
Patent application number: 20110171316
Abstract:
Group C rotaviruses are a cause of acute gastroenteritis in children and
adults that is distinct from group A RV. However, human group C
rotaviruses cannot be grown in culture, resulting in a lack of tools for
detection and treatment of GrpC RV disease. Consequently, the burden of
GpC RV disease has not been clearly established. Isolated recombinant
human rotavirus group C virus-like particles are provided according to
embodiments of the present invention along with methods of their
production and use in, inter alia, detection of Grp C RV infection,
diagnostic assays and immunogenic compositions.Claims:
1. An isolated recombinant human rotavirus group C virus-like particle
comprising human rotavirus group C VP6 protein and a human rotavirus
group C VP7 protein.
2. The isolated recombinant human rotavirus group C virus-like particle of claim 1 further comprising human rotavirus group C VP2 protein.
3. The isolated recombinant human rotavirus group C virus-like particle of claim 1 wherein the virus-like particle is free of human rotavirus group C VP1, VP3, VP4 and human rotavirus group C non-structural proteins NSP1, NSP2, NPS3, NSP4, NSP5, NSP6 and NSP7.
4. The isolated recombinant human rotavirus group C virus-like particle of claim 1 wherein the human rotavirus group C VP6 protein comprises the amino acid sequence of SEQ ID No. 32.
5. The isolated recombinant human rotavirus group C virus-like particle of claim 1 wherein the human rotavirus group C VP7 protein comprises the amino acid sequence of SEQ ID No. 34.
6. The isolated recombinant human rotavirus group C virus-like particle of claim 2 wherein the human rotavirus group C VP2 protein comprises the amino acid sequence of SEQ ID No. 1.
7. (canceled)
8. The isolated recombinant human rotavirus group C virus-like particle according to claim 1 admixed with a pharmaceutically acceptable carrier.
9. The isolated recombinant human rotavirus group C virus-like particle of claim 1, further comprising a cargo moiety in an internal space defined by the isolated recombinant human rotavirus group C virus-like particle.
10. The isolated recombinant human rotavirus group C virus-like particle according to claim 9, wherein the cargo moiety is selected from the group consisting of: a label, an antigen, a nucleic acid sequence encoding a protein or peptide, and a therapeutic agent.
11. (canceled)
12. The isolated recombinant human rotavirus group C virus-like particle of claim 1 attached to a solid substrate.
13. (canceled)
14. The isolated recombinant human rotavirus group C virus-like particle of claim 1, further comprising an immunological adjuvant.
15. A process of generating an immunological response in a human comprising administering an isolated recombinant human rotavirus group C virus-like particle comprising human rotavirus group C VP6 protein and a human rotavirus group C VP7 protein. to a human.
16. The process of claim 15, wherein the step of administering the immunological composition is administered to a mucosal surface.
17-25. (canceled)
26. A process of forming a human group C rotavirus-like particle comprising: constructing a first vector comprising a nucleic acid molecule comprising a sequence encoding a human group C rotavirus VP6 capsid protein operably linked to a promoter that drives expression of said protein in an insect cell; constructing a second vector comprising a nucleic acid molecule comprising a sequence encoding a human group C rotavirus VP7 capsid protein operably linked to a promoter that drives expression of said protein in an insect cell; infecting an insect cell culture with said first and second baculovirus vector under conditions that promote expression of the VP6 capsid protein and VP7 capsid protein and association to form the human group C rotavirus-like particle.
27. The process of claim 26 further comprising: constructing a third vector comprising a nucleic acid molecule comprising a sequence encoding a human group C rotavirus VP2 core protein operably linked to a promoter that drives expression of said protein in an insect cell; and infecting an insect cell culture with said first baculovirus vector, second baculovirus vector, and third baculovirus vector under conditions that promote expression of the VP6 capsid protein, and VP7 capsid protein and said VP2 core protein.
28. The process of claim 26 further comprising determining that human group C rotavirus-like particle is present in said culture.
29. The process of claim 26 further comprising isolating the human group C rotavirus-like particle from said culture.
30. The process of claim 26, wherein said first vector and said second vector both have baculovirus promoters.
Description:
REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority from U.S. Provisional Patent Application Ser. No. 61/130,615 filed May 29, 2008 and U.S. Provisional Patent Application Ser. No. 61/111,425 filed Nov. 5, 2008, the entire content of both are incorporated herein by reference.
FIELD OF THE INVENTION
[0003] The present invention relates to group C rotaviruses (GpC RV) and rotavirus-like particles, methods of producing immunogenic rotavirus-like particles, immunogenic compositions inclusive of rotavirus-like particles and methods for eliciting an immune response using compositions inclusive of rotavirus-like particles, as well as producing a diagnostic for rotavirus infection.
BACKGROUND OF THE INVENTION
[0004] Rotaviruses are a diverse set of pathogens classified into groups A through G based on the distinct characteristics of the inner capsid protein, VP6. Among these, GpC RV has been identified as a pathogen in humans and attributed to outbreaks and sporadic cases of gastroenteritis worldwide in young children <3 years of age (8, 13, 15, 25, 26, 29, 30) and in older children and adults (2, 15, 20, 21, 25, 26, 30, 32). While some studies have reported low detection rates in children with diarrhea (1, 2, 4, 26, 29), seroprevalence studies have demonstrated that GpC RV is a commonplace pathogen with a much higher occurrence in adults (4, 6, 10, 20, 27, 31).
[0005] One possible cause of low GpC RV detection is the unavailability of adequate diagnostic tools. While PCR is a frequently employed technique, it is often insensitive for diagnosis of GpC RV due to the instability of its capsid proteins and the degradation of its RNA genome. It is also not an accessible technique to many clinical laboratories that are involved in diagnostics of samples from patients with gastroenteritis. If a more practical and economical tool, like a GpC RV-specific enzyme immunoassay (EIA), was available, testing of large numbers of samples could be performed to better estimate GpC RV disease burden. Propagation of the Cowden strain, a prototype porcine GpC RV, has been successful and antibodies to this virus have been employed for GpC RV diagnostics (13, 28, 30, 33, 34). However their specificity and sensitivity to human GpC RV is questionable. Progress in the development of a sensitive and specific EIA for human GpC RV has been stunted by its fastidious growth in cell culture. To circumvent the prior art problems of GpC RV fastidious growth problem, VP6 from a human GpC RV was expressed in insect cells using the Baculovirus System and antibody to this recombinant protein was employed in seroprevalence studies (6, 11, 31). To the best of our knowledge, these reagents have not been utilized for viral detection in human specimens and their specificity to GpC RV remains questionable.
[0006] GpC RV are a cause of acute gastroenteritis in children and adults that is distinct from group A RV. Human group A RV detection methods are well established and widely available while group C RV diagnostics are only available in a few reference laboratories. Since native human group C RV are unstable and cannot be grown in cell culture, reagents from animal group C RV have been used for diagnostics. However these diagnostic tools may not be sensitive or specific enough for human strains. Thus, sensitive and specific detection methods and reagents for human group C RV are not readily available. Consequently, the burden of GpC RV disease has not been clearly established.
[0007] Thus, there exists a need for a human specific group C rotavirus diagnostic. There also exists a need for a human group C RV-like particle for use in such a diagnostic and for eliciting an immune response as a vaccine.
SUMMARY OF THE INVENTION
[0008] An isolated recombinant human rotavirus group C virus-like particle including human rotavirus group C VP6 protein and a human rotavirus group C VP7 protein is provided according to embodiments of the present invention. In further embodiments, an isolated recombinant human rotavirus group C virus-like particle including human rotavirus group C VP6 protein, a human rotavirus group C VP7 protein and a human rotavirus group C VP2 protein is provided. According to certain embodiments, the isolated recombinant human rotavirus group C virus-like particles of the present invention are free of other human rotavirus group C proteins such as VP1, VP3, VP4, NSP1, NSP2, NPS3, NSP4, NSP5, NSP6 and NSP7.
[0009] Isolated recombinant human rotavirus group C virus-like particles are provided according to embodiments of the present invention which include human rotavirus group C VP6 protein including the amino acid sequence of SEQ ID No. 32. Isolated recombinant human rotavirus group C virus-like particles are provided according to embodiments of the present invention which include human rotavirus group C VP7 protein including the amino acid sequence of SEQ ID No. 34. In particular embodiments, isolated recombinant human rotavirus group C virus-like particles are provided according to embodiments of the present invention which include human rotavirus group C VP6 protein including the amino acid sequence of SEQ ID No. 32 and human rotavirus group C VP7 protein including the amino acid sequence of SEQ ID No. 34.
[0010] Isolated recombinant human rotavirus group C virus-like particles are provided according to embodiments of the present invention which include the human rotavirus group C VP2 protein including the amino acid sequence of SEQ ID No. 1. In further embodiments, isolated recombinant human rotavirus group C virus-like particles are provided according to embodiments of the present invention which include human rotavirus group C VP6 protein including the amino acid sequence of SEQ ID No. 32, human rotavirus group C VP7 protein including the amino acid sequence of SEQ ID No. 34 and human rotavirus group C VP2 protein including the amino acid sequence of SEQ ID No. 1.
[0011] Processes for detection of a human rotavirus group C antibody in a biological sample are provided according to embodiments of the present invention which include contacting a first biological sample with a plurality of isolated recombinant human rotavirus group C virus-like particles and detecting the formation of a complex between an anti-human rotavirus group C antibody present in the first biological sample and the plurality of isolated recombinant human rotavirus group C virus-like particles, to obtain a first signal indicative of the presence of an anti-human rotavirus group C antibody.
[0012] Anti-human rotavirus group C vaccines are provided according to embodiments of the present invention which includes isolated recombinant human rotavirus group C virus-like particles admixed with a pharmaceutically acceptable carrier.
[0013] Processes of delivering a cargo moiety to a cell are provided according to embodiments of the present invention which include introducing a cargo moiety into an internal space defined by an isolated recombinant human rotavirus group C virus-like particle and contacting the isolated recombinant human rotavirus group C virus-like particle and a cell.
[0014] Exemplary cargo moieties are a label, an antigen, a nucleic acid sequence encoding a protein or peptide, and a therapeutic agent.
[0015] Anti-human rotavirus group C antibody assay kits are provided according to embodiments of the present invention which include isolated recombinant human rotavirus group C virus-like particles and at least one ancillary reagent. Optionally, the virus-like particles are attached to a solid substrate.
[0016] Immunogenic compositions are provided according to embodiments of the present invention which include an isolated recombinant human rotavirus group C virus-like particle described herein and a pharmaceutically acceptable carrier. Optionally, an inventive immunogenic composition includes an immunological adjuvant.
[0017] Processes of generating an immunological response in a human including administering an immunogenic composition including an inventive human rotavirus group C virus-like particle to a human are provided according to embodiments of the present invention. Optionally, an inventive process includes administering the immunological composition to a mucosal surface.
[0018] An isolated polypeptide including an amino acid sequence of: a) an amino acid sequence having at least 98% to the amino acid sequence set forth in SEQ ID NO: 1 (ASP88 VP2 amino acid sequence); b) an amino acid sequence having at least 99% to the amino acid sequence set forth in SEQ ID NO: 1 (ASP88 VP2 amino acid sequence); c) an amino acid sequence set forth in SEQ ID NO: 1 (ASP88 VP2 amino acid sequence); or d) an amino acid sequence set forth in SEQ ID NO: 32 (S 1 VP6 amino acid sequence) is provided according to embodiments of the invention. An isolated nucleic acid molecule including a nucleotide sequence encoding the isolated polypeptide including an amino acid sequence of: a) an amino acid sequence having at least 98% to the amino acid sequence set forth in SEQ ID NO: 1 (ASP88 VP2 amino acid sequence); b) an amino acid sequence having at least 99% to the amino acid sequence set forth in SEQ ID NO: 1 (ASP88 VP2 amino acid sequence); c) an amino acid sequence set forth in SEQ ID NO: 1 (ASP88 VP2 amino acid sequence); or d) an amino acid sequence set forth in SEQ ID NO: 32 (S 1 VP6 amino acid sequence) is provided according to embodiments of the invention.
[0019] Immunogenic compositions are provided according to embodiments of the present invention including a polypeptide including at least one amino acid sequence of any of SEQ ID NOS: 1-13 wherein said amino sequence is an antigenic epitope recognized by an antibody. Optionally, such an immunogenic composition further includes a rotavirus-like particle described herein.
[0020] An antibody is provided according to embodiments of the present invention that is specific for a group C rotavirus and which does not recognize a group A rotavirus. In particular embodiments, an antibody according to embodiments of the present invention is specific for an amino acid sequence of any of SEQ ID NOS: 3-13 and does not recognize a group A rotavirus.
[0021] An isolated polypeptide is provided according to embodiments of the present invention that includes at least one amino acid sequence of SEQ ID NO: 3 or 8. An isolated nucleic acid molecule including a nucleotide sequence encoding the isolated polypeptide that includes at least one amino acid sequence of SEQ ID NO: 3 or 8 is provided according to embodiments of the present invention.
[0022] Vectors including an isolated nucleic acid molecule including a nucleotide sequence encoding the isolated polypeptide that includes at least one amino acid sequence of SEQ ID NO: 3 or 8 are provided according to embodiments of the present invention. Isolated host cells including a vector of the present invention are provided according to particular embodiments.
[0023] Processes of forming a human group C rotavirus-like particle are provided according to embodiments of the present invention which include constructing a first vector comprising a nucleic acid molecule comprising a sequence encoding a human group C rotavirus VP6 capsid protein operably linked to a promoter that drives expression of said protein in an insect cell; constructing a second vector comprising a nucleic acid molecule comprising a sequence encoding a human group C rotavirus VP7 capsid protein operably linked to a promoter that drives expression of said protein in an insect cell; and infecting an insect cell culture with said first and second baculovirus vector under conditions that promote expression of the VP6 capsid protein and VP7 capsid protein and association to form the human group C rotavirus-like particle. In further embodiments, processes of forming a human group C rotavirus-like particle include constructing a third vector comprising a nucleic acid molecule comprising a sequence encoding a human group C rotavirus VP2 core protein operably linked to a promoter that drives expression of said protein in an insect cell; and infecting an insect cell culture with said first baculovirus vector, second baculovirus vector, and third baculovirus vector under conditions that promote expression of the VP6 capsid protein, and VP7 capsid protein and said VP2 core protein.
BRIEF DESCRIPTION OF THE DRAWINGS
[0024] FIG. 1A is an image of an electrophoretic gel showing the kinetics of GpC RV VP6 and VP7 expression in Sf9 cells in different media;
[0025] FIG. 1B is an image of an electrophoretic gel showing the kinetics of GpC RV VP6 and VP7 expression in Hi5 (B) cells in different media;
[0026] FIG. 2A is an image of an electron micrograph of human GpC RV VLPs formed by self-assembly of recombinant human rotavirus C VP2, VP6, and VP7 proteins expressed in Sf9 cells infected with recombinant baculoviruses at an MOI of 1 each;
[0027] FIG. 2B is an image of an electron micrograph of human GpC RV VLPs formed by self-assembly of recombinant human rotavirus C VP6 and VP7 proteins expressed in Sf9 cells infected with recombinant baculoviruses at an MOI of 1.4 each;
[0028] FIG. 3A is an image showing Coomassie blue staining of major structural viral proteins from GpA RV YK-1 and human rotavirus GpC VLPs;
[0029] FIG. 3B is an image of an immunoblot showing the comparison of major structural viral proteins from GpA RV YK-1 and human rotavirus GpC VLPs;
[0030] FIG. 4A is an image of an immunoelectron micrograph showing human GpC RV VLPs immunostained with GpC-specific rabbit hyperimmune serum;
[0031] FIG. 4B is an image of an immunoelectron micrograph showing human GpC RV VLPs immunostained with GpA-specific rabbit hyperimmune serum;
[0032] FIG. 4C is an image of an immunoelectron micrograph showing GpA RV immunostained with GpC-specific rabbit hyperimmune serum;
[0033] FIG. 4D is an image of an immunoelectron micrograph showing GpA RV immunostained with GpA-specific rabbit hyperimmune serum;
[0034] FIG. 5 is an amino acid sequence alignment for Group C rotavirus VP2 proteins from human strain ASP88 (SEQ ID NO: 1); "Bristol" human strain (SEQ ID NO: 16, Accession CAC 44890) and "Cowden" porcine strain (SEQ ID NO: 17, Accession M74217);
[0035] FIG. 6 is a nucleotide sequence alignment of sequences encoding human Group C VP-2 for inventive strain ASP88 (SEQ ID NO: 18), "Cowden" porcine strain (SEQ ID No. 44) and Bristol (SEQ ID NO: 19, Accession AJ303139);
[0036] FIG. 7 is a nucleotide sequence alignment of sequences encoding human Group C VP-6 for inventive strain S-1 (SEQ ID NO: 31) relative to conventional strains Bristol (SEQ ID NO: 25, Accession CAA42504); Jajeri (SEQ ID NO: 26, Accession AAK26534); CMH004 (SEQ ID NO: 27, Accession ABR31794); V508 (SEQ ID NO: 28, Accession AAX13496); China (SEQ ID NO: 29, Accession BAB83829); and BCN6 (SEQ ID NO: 30, Accession CAJ41549);
[0037] FIG. 8 is an amino acid sequence alignment of sequences encoding human Group C VP-6 for inventive strain S-1 (SEQ ID NO: 32) relative to conventional strains Bristol (SEQ ID NO: 35, Accession CAA42504); Jajeri (SEQ ID NO: 36, Accession AAK26534); CMH004 (SEQ ID NO: 37, Accession ABR31794); V508 (SEQ ID NO: 38, Accession AAX13496); China (SEQ ID NO: 39, Accession BAB83829); and BCN6 (SEQ ID NO: 40, Accession CAJ41549);
[0038] FIG. 9 is a nucleotide sequence of human rotavirus VP6 protein from S-1 strain (SEQ ID No. 46) including an open reading frame, 5' and 3' non-coding sequences; and
[0039] FIG. 10 is a nucleotide sequence alignment of sequences encoding human Group C VP-6 and including 5' and 3' non-coding sequences for inventive strain S-1 (SEQ ID NO: 46) and Bristol VP6 (SEQ ID No. 47).
DETAILED DESCRIPTION OF THE INVENTION
[0040] Group C rotavirus (GpC RV) is a causative agent of acute gastroenteritis in children and adults. Characterization of GpC RV has only been accomplished to date with porcine and bovine strains that can be grown in cell culture. Because human GpC RVs are unstable and cannot be cultivated in cell culture, reagents and sensitive and specific detection methods are not available. Consequently, the impact of GpC RV on diarrheal disease has not been clearly established.
[0041] Demonstrated herein is the expression of the major inner and outer capsid human GpC proteins VP6 and VP7 and the human GpC core protein VP2 and the self-assembly of human GpC VP6/7 virus-like particles (VLPs) or human GpC VP2/6/7 VLPs. Antibodies to these human GpC RV VLPs show highly specific reactivities with the corresponding GpC but not GpA RV.
[0042] The ability to produce large amounts of human GpC RV antigenic materials, such as human GpC RV proteins and VLPs, and the availability of high quality antibody reagents provide sensitive and specific diagnostic assays and provide tools for investigation of the epidemiology and disease burden of GpC RV in humans.
[0043] The instant invention has numerous uses including, but not limited to, detection of human rotavirus C antibodies in biological samples, diagnosis of human rotavirus C infection, identification of individuals previously or currently infected with human rotavirus C, as an antigen for generation of antibodies and for the development of therapeutics for prophylaxis or treatment of disease associated with human rotavirus C infection.
[0044] In accordance with the present invention, various techniques and terms including, but not limited to, conventional molecular biology, microbiology, immunology and recombinant DNA techniques and terms, may be used which are known by those of skill in the art. Such techniques and terms are described and/or defined in detail in standard references such as J. Sambrook and D. W. Russell, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 3rd Ed., 2001; F. M. Ausubel et al., Eds., Short Protocols in Molecular Biology, Current Protocols, Wiley, 2002; Wild, D., The Immunoassay Handbook, 3rd Ed., Elsevier Science, 2005; Gosling, J. P., Immunoassays: A Practical Approach, Practical Approach Series, Oxford University Press, 2005; and Harlow, E. and Lane, D., Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 1988; F. Breitling and S. Diibel, Recombinant Antibodies, John Wiley & Sons, New York, 1999; H. Zola, Monoclonal Antibodies: Preparation and Use of Monoclonal Antibodies and Engineered Antibody Derivatives, Basics: From Background to Bench, BIOS Scientific Publishers, 2000; B.K.C. Lo, Antibody Engineering: Methods and Protocols, Methods in Molecular Biology, Humana Press, 2003; Crowther, J. R., The ELISA Guidebook (Methods in Molecular Biology), Humana Press, 2000; and other references described herein.
[0045] Human Rotavirus C Virus-Like Particles
[0046] Human rotavirus C virus-like particles (VLPs) are provided according to the present invention. The term "virus-like particle" refers to a capsid defining an internal space. The internal space defined by the capsid is "empty" of an intact human rotavirus C genome and the human rotavirus C VLPs of the present invention are therefore non-replicating and incapable of causing human rotavirus C-associated disease.
[0047] Human rotavirus C VLPs include human rotavirus C VP6 and VP7 proteins according to embodiments of the present invention. In further embodiments of the present invention, human rotavirus C VLPs include human rotavirus C VP2, VP6 and VP7 proteins.
[0048] Genes encoding human rotavirus C proteins VP2, VP6 and VP7 have been identified and sequenced.
[0049] Any human Group C RV VP6 protein can be included in human rotavirus C VLPs of the present invention. Examples of human Group C RV VP6 proteins that can be included in human rotavirus C VLPs of the present invention include VP6 protein of human Group C strain S-1 VP6 (SEQ ID NO: 32); Bristol strain (SEQ ID NO: 35, Accession CAA42504); Jajeri strain (SEQ ID NO: 36, Accession AAK26534); CMH004 strain (SEQ ID NO: 37, Accession ABR31794); V508 strain (SEQ ID NO: 38, Accession AAX13496); China strain (SEQ ID NO: 39, Accession BAB83829); and BCN6 strain (SEQ ID NO: 40, Accession CAJ41549).
[0050] Human Group C RV VP6 proteins that can be included in human GpC RV VLPs include those known by NCBI Accession numbers BAB83829, AAK26535, AAK26534, AAX13496, AAX13492, AAX13491, CAJ41551, CAJ41550, CAJ41549, AAW82662, AAW82661, ABD96606, ABD96605, ABD96604, AAA47340, AAA47339, CAA42504, AAX08120, ABR31794, YP--392512, P69481, P69483 and P69482.
[0051] Any human Group C RV VP7 protein can be included in human rotavirus C VLPs of the present invention. Examples of human Group C RV VP7 proteins that can be included in human GpC RV VLPs include those known by NCRI Accession numbers BAB83828, AAX16188, AAX16187, AAX16186, CAJ41554, CAJ41553, CAJ41552, AAW82659, AAD25388, BAA20340, BAA20339, AAQ93808, AAQ93807, AAA47352, BAF73591, BAF73590, BAF73589, BAF73588, BAF73587, BAD20702, BAD20701, BAD20700, BAD20699, AAK26533, AAK26530, ABR31795, BAC53881, BAC53880, BAC53879, BAC53878, BAC53877, BAC53876, BAC53875, BAC53874, ABE01860, ABE01859, ABE01858, AAF33400, AAF33399, AAF33398, AAF33397, AAF33396, AAF33395, AAF33394, AAF33393, AAF33392, AAF33391, AAF33390, AAF33389, BAA33952, P30216, ABO25864 and 2209225A.
[0052] Any human Group C RV VP2 protein can be included in human rotavirus C VLPs of the present invention. Examples of human Group C RV VP2 proteins that can be included in human rotavirus C VLPs of the present invention include VP2 protein of human Group C strain ASP88 VP2 (SEQ ID NO: 1); and Bristol strain (SEQ ID NO: 16, Accession CAB52753).
[0053] In addition to these VP2, VP6 and VP7 amino acid sequences, the term VP2, VP6 or VP7 amino acid sequence encompasses variants. In particular embodiments, a VP2, amino acid sequence included in a VLP composition of the present invention is a variant of ASP88 VP2 (SEQ ID No. 1). In further embodiments, a VP6, amino acid sequence included in a VLP composition of the present invention is a variant of S-1 VP6 (SEQ ID No. 32). In further embodiments, a VP7, amino acid sequence included in a VLP composition of the present invention is a variant of S-1 VP7 (SEQ ID No. 34).
[0054] In another aspect, the invention provides a rotavirus-like particle having a core VP2 structural protein of human group C RV of strain ASP88 protein (SEQ ID NO: 1) or a fragment or variant thereof.
[0055] In another aspect, the invention provides a rotavirus-like particle comprising VP6 capsid protein and VP7 capsid protein, or fragments or variants thereof, with the proviso that said particle does not comprise an amino acid sequence set forth in (SEQ ID NO: 16; Bristol).
[0056] In another aspect, the invention provides an isolated polypeptide comprising an amino acid sequence selected from the group consisting of: a) an amino acid sequence having at least 98% identity to the amino acid sequence set forth in SEQ ID NO: 1 (ASP88 VP2 amino acid sequence); b) an amino acid sequence having at least 99% identity to the amino acid sequence set forth in SEQ ID NO: 1 (ASP88 VP2 amino acid sequence); c) an amino acid sequence set forth in SEQ ID NO: 1 (ASP88 VP2 amino acid sequence); and d) an amino acid sequence set forth in SEQ ID NO: 32 (S-1 VP6 amino acid sequence).
[0057] In another aspect, the invention provides an isolated nucleic acid molecule comprising a nucleotide sequence encoding one or more of the inventive polypeptides described herein. In certain embodiments the invention provides an isolated nucleic acid molecule encoding VP2 selected from the group consisting of: a) an isolated nucleic acid molecule encoding an inventive polypeptide having at least 98% identity to the amino acid sequence set forth in SEQ ID NO: 1 (ASP88 VP2 amino acid sequence); b) an isolated nucleic acid molecule encoding an inventive polypeptide having at least 99% identity to the amino acid sequence set forth in SEQ ID NO: 1 (ASP88 VP2 amino acid sequence); c) an isolated nucleic acid molecule encoding an inventive polypeptide set forth in SEQ ID NO: 1 (ASP88 VP2 amino acid sequence); and d) an isolated nucleic acid molecule encoding an inventive amino acid sequence set forth in SEQ ID NO: 32 (S-1 VP6 amino acid sequence).
[0058] In another aspect, the invention provides an immunogenic composition that includes a polypeptide containing at least one amino acid sequence of:
TABLE-US-00001 (SEQ ID NO: 2) LETIIDKEVK ENKDSTKDEK LVVTEESNGD VTA, (SEQ ID NO: 3) LETIINKEVK ENKDSMKEDK LVVTEESNGD VTT, (SEQ ID NO: 4) TENVEEKEIK EAKEQVKDEK QVITEENVDS PKD, (SEQ ID NO: 5) KLTEIQESSA KTYNTLFRLF TP, (SEQ ID NO: 6) NYRNSRIKCQ TYNKLFRL, (SEQ ID NO: 7) LNVLEG MPDYIMLRDM AV, (SEQ ID NO: 8) LNVLEE MPDYIMLRDM AV, (SEQ ID NO: 9) LNVLDE MPDYVMLRDM AV, (SEQ ID NO: 10) AAHLQLE AITVQVESQF LAGISAAAAN EA, (SEQ ID NO: 11) LQCKLNH NSWQELVHGR NE, (SEQ ID NO: 12) LSACIVMNMH LVG, and (SEQ ID NO: 13) IPPDQMYRLR NRLRNIP;
wherein said amino sequence is an antigenic epitope recognized by an antibody.
[0059] In another aspect, the invention provides a antibody preparation that recognizes an amino acid sequence of:
TABLE-US-00002 (SEQ ID NO: 2) LETIIDKEVK ENKDSTKDEK LVVTEESNGD VTA, (SEQ ID NO: 3) LETIINKEVK ENKDSMKEDK LVVTEESNGD VTT, (SEQ ID NO: 4) TENVEEKEIK EAKEQVKDEK QVITEENVDS PKD, (SEQ ID NO: 5) KLTEIQESSA KTYNTLFRLF TP, (SEQ ID NO: 6) NYRNSRIKCQ TYNKLFRL, (SEQ ID NO: 7) LNVLEG MPDYIMLRDM AV, (SEQ ID NO: 8) LNVLEE MPDYIMLRDM AV, (SEQ ID NO: 9) LNVLDE MPDYVMLRDM AV, (SEQ ID NO: 10) AAHLQLE AITVQVESQF LAGISAAAAN EA, (SEQ ID NO: 11) LQCKLNH NSWQELVHGR NE, (SEQ ID NO: 12) LSACIVMNMH LVG, and (SEQ ID NO: 13) IPPDQMYRLR NRLRNIP.
[0060] In another aspect, the invention provides a vector comprising the inventive nucleic acid molecules described herein.
[0061] In another aspect, the invention provides an isolated host cell comprising one or more of the inventive vectors described herein.
[0062] The inventive methods and compositions are not limited to the VP proteins and polypeptides having the amino acid sequence described herein in detail. Where appropriate, variants, such as homologs from other strains and groups, may be used.
[0063] As used herein, the term "variant" defines either a naturally occurring genetic mutant of a human rotavirus C virus or a recombinantly prepared variation of a human rotavirus C virus, each of which contain one or more mutations in its genome compared to a reference human rotavirus C VP2, VP6 or VP7. The term "variant" may also refer to either a naturally occurring variation of a given peptide or a recombinantly prepared variation of a given peptide or protein in which one or more amino acid residues have been modified by amino acid substitution, addition, or deletion.
[0064] Preferred are human rotavirus C proteins having at least 95%, 96%, 97%, 98% or 99% identity to SEQ ID No. 1, SEQ ID No.32 or SEQ ID No. 34. Further preferred are human rotavirus C proteins having 99% or greater identity to SEQ ID No. 1, SEQ ID No.32 or SEQ ID No.34.
[0065] Mutations can be introduced using standard molecular biology techniques, such as site-directed mutagenesis and PCR-mediated mutagenesis. One of skill in the art will recognize that one or more amino acid mutations can be introduced without altering the functional properties of the human rotavirus C VP2, VP6 or VP7 proteins.
[0066] It is also recognized by one of ordinary skilled in the art that VP protein and polypeptide variants encompass conservative amino acid substitutions in the amino acid sequences of the VP proteins and polypeptides set forth in detail herein. Conservative amino acid substitutions can be made in human rotavirus C VP2, VP6 or VP7 proteins to produce human rotavirus C VP2, VP6 or VP7 protein variants. Conservative amino acid substitutions are art recognized substitutions of one amino acid for another amino acid having similar characteristics. For example, each amino acid may be described as having one or more of the following characteristics: electropositive, electronegative, aliphatic, aromatic, polar, hydrophobic and hydrophilic. A conservative substitution is a substitution of one amino acid having a specified structural or functional characteristic for another amino acid having the same characteristic. Acidic amino acids include aspartate, glutamate; basic amino acids include histidine, lysine, arginine; aliphatic amino acids include isoleucine, leucine and valine; aromatic amino acids include phenylalanine, glycine, tyrosine and tryptophan; polar amino acids include aspartate, glutamate, histidine, lysine, asparagine, glutamine, arginine, serine, threonine and tyrosine; and hydrophobic amino acids include alanine, cysteine, phenylalanine, glycine, isoleucine, leucine, methionine, proline, valine and tryptophan; and conservative substitutions include substitution among amino acids within each group. Amino acids may also be described in terms of relative size, alanine, cysteine, aspartate, glycine, asparagine, proline, threonine, serine, valine, all typically considered to be small.
[0067] Human rotavirus C VP2, VP6 or VP7 variants can include synthetic amino acid analogs, amino acid derivatives and/or non-standard amino acids, illustratively including, without limitation, alpha-aminobutyric acid, citrulline, canavanine, cyanoalanine, diaminobutyric acid, diaminopimelic acid, dihydroxy-phenylalanine, djenkolic acid, homoarginine, hydroxyproline, norleucine, norvaline, 3-phosphoserine, homoserine, 5-hydroxytryptophan, 1-methylhistidine, 3-methylhistidine, and ornithine.
[0068] In addition, as will be appreciated by one of skill in the art, due to the degeneracy of the genetic code, more than one nucleic acid will encode an identical protein. Thus, nucleic acids encoding the VP proteins and polypeptides or a variant thereof are not limited to those nucleic acids described herein in detail.
[0069] Variants of VP proteins and polypeptides having 95%, 96%, 97%, 98%, or 99% homology to the amino acid sequence described herein in detail are operable in the described methods and compositions. Variants of nucleic acids having 95%, 96%, 97%, 98%, or 99% homology to the nucleotide sequence described herein in detail are operable in the described methods and compositions.
[0070] "Homology" refers to sequence similarity or, alternatively, sequence identity, between two or more polynucleotide sequences or two or more polypeptide sequences.
[0071] The terms "percent identity" and "% identity", as applied to polynucleotide sequences, refer to the percentage of identical nucleotide matches between at least two polynucleotide sequences aligned using a standardized algorithm. Such an algorithm may insert, in a standardized and reproducible way, gaps in the sequences being compared in order to optimize alignment between two sequences, and therefore achieve a more meaningful comparison of the two sequences.
[0072] Percent identity between polynucleotide sequences may be determined using one or more computer algorithms or programs known in the art or described herein. For example, percent identity can be determined using the default parameters of the CLUSTAL V algorithm as incorporated into the MEGALIGN version 3.12e sequence alignment program. This program is part of the LASERGENE software package, a suite of molecular biological analysis programs (DNASTAR, Madison Wis.). CLUSTAL V is described in Higgins, D. G. and P. M. Sharp (1989; CABIOS 5:151-153) and in Higgins, D. G. et al. (1992; CABIOS 8:189-191). For pairwise alignments of polynucleotide sequences, the default parameters are set as follows: Ktuple=2, gap penalty=5, window=4, and "diagonals saved"=4. The "weighted" residue weight table is selected as the default.
[0073] Alternatively, a suite of commonly used and freely available sequence comparison algorithms which can be used is provided by the National Center for Biotechnology Information (NCBI) Basic Local Alignment Search Tool (BLAST) (Altschul, S. F. et al. (1990) J. Mol. Biol. 215:403-410), which is available from several sources, including the NCBI, Bethesda, Md., and on the NCBI World Wide Web site available on the Internet. The BLAST software suite includes various sequence analysis programs including "blastn," that is used to align a known polynucleotide sequence with other polynucleotide sequences from a variety of databases. Also available is a tool called "BLAST 2 Sequences" that is used for direct pairwise comparison of two nucleotide sequences. "BLAST 2 Sequences" can be accessed and used interactively on the Internet via the NCBI World Wide Web site as well. The "BLAST 2 Sequences" tool can be used for both blastn and blastp (discussed below). BLAST programs are commonly used with gap and other parameters set to default settings. For example, to compare two nucleotide sequences, one may use blastn with the "BLAST 2 Sequences" tool Version 2.0.12 (Apr. 21, 2000) set at default parameters. Such default parameters may be, for example: Matrix:BLOSUM62; Reward for match: 1; Penalty for mismatch: -2; Open Gap: 5 and Extension Gap: 2 penalties; Gap x drop-off: 50; Expect: 10; Word Size: 11; Filter: on.
[0074] Percent identity may be measured over the length of an entire defined sequence, for example, as defined by a particular SEQ ID number, or may be measured over a shorter length, for example, over the length of a fragment taken from a larger, defined sequence, for instance, a fragment of at least 20, at least 30, at least 40, at least 50, at least 70, at least 100, or at least 200 contiguous nucleotides. Such lengths are exemplary only, and it is understood that any fragment length supported by the sequences shown herein, in the tables, figures, or sequence listing, may be used to describe a length over which percentage identity may be measured.
[0075] The phrases "percent identity" and "% identity", as applied to polypeptide sequences, refer to the percentage of identical residue matches between at least two polypeptide sequences aligned using a standardized algorithm. Methods of polypeptide sequence alignment are well known. Some alignment methods take into account conservative amino acid substitutions. Such conservative substitutions, explained in more detail above, generally preserve the charge and hydrophobicity at the site of substitution, thus preserving the structure (and therefore function) of the polypeptide. The phrases "percent similarity" and "% similarity", as applied to polypeptide sequences, refer to the percentage of residue matches, including identical residue matches and conservative substitutions, between at least two polypeptide sequences aligned using a standardized algorithm. In contrast, conservative substitutions are not included in the calculation of percent identity between polypeptide sequences.
[0076] Percent identity between polypeptide sequences may be determined using the default parameters of the CLUSTAL V algorithm as incorporated into the MEGALIGN version 3.12e sequence alignment program (described and referenced above). For pairwise alignments of polypeptide sequences using CLUSTAL V, the default parameters are set as follows: Ktuple=1, gap penalty=3, window=5, and "diagonals saved"=5. The PAM250 matrix is selected as the default residue weight table.
[0077] Alternatively the NCBI BLAST software suite may be used. For example, for a pairwise comparison of two polypeptide sequences, one may use the "BLAST 2 Sequences" tool Version 2.0.12 (Apr. 21, 2000) with blastp set at default parameters. Such default parameters may be, for example: Matrix: BLOSUM62; Open Gap: 11 and Extension Gap: 1 penalties; Gap x drop-off: 50; Expect: 10; Word Size: 3; Filter: on.
[0078] Percent identity may be measured over the length of an entire defined polypeptide sequence, for example, as defined by a particular SEQ ID number, or may be measured over a shorter length, for example, over the length of a fragment taken from a larger, defined polypeptide sequence, for instance, a fragment of at least 15, at least 20, at least 30, at least 40, at least 50, at least 70 or at least 150 contiguous residues. Such lengths are exemplary only, and it is understood that any fragment length supported by the sequences shown herein, in the tables, figures or sequence listing, may be used to describe a length over which percentage identity may be measured.
[0079] Furthermore, fragments of the proteins and polypeptides and variants thereof are encompassed in the methods and compositions of the invention, so long as the fragment is operable in effecting the relevant biological activity as understood by the ordinarily skilled artisan. Thus, for example, fragments include fragments of VP2 proteins and polypeptides; where the fragment is contained in a virus-like particle when expressed in an insect cell culture along with a VP6 and VP7 protein or polypeptide, or a variant or fragment thereof. Thus, for example, fragments include fragments of VP6 proteins and polypeptides; where the fragment is contained in a virus-like particle when expressed in an insect cell culture along with a VP7 protein or polypeptide, or a variant or fragment thereof. Thus, for example, fragments include fragments of VP7 proteins and polypeptides; where the fragment is contained in a virus-like particle when expressed in an insect cell culture along with a VP6 protein or polypeptide, or a variant or fragment thereof. Fragments also encompass those fragments which effect an immunogenic response as described herein for a VP protein, polypeptide or a variant there.
[0080] Processes for Making VLPs
[0081] VP2 core protein and the VP6 and VP7 capsid proteins and polypeptides (VP proteins and polypeptides) described in the compositions and methods described herein can be generated by recombinant methods, such as the inventive methods described herein, or by suitable expression methods known to the ordinarily skilled artisan where appropriate. Nucleic acid sequences encoding the VP proteins and polypeptides are isolated as exemplified by nucleic acid sequences described herein.
[0082] VLPs are produced using recombinant nucleic acid technology according to embodiments of the present invention. VLP production includes introducing a recombinant expression vector encompassing a DNA sequence encoding human rotavirus C VP2, VP6 and/or VP7 into a host cell.
[0083] Specific nucleic acid sequences encoding human rotavirus C VP2, VP6 or VP7 introduced into a host cell to produce human rotavirus C VLPs are
[0084] It is appreciated that due to the degenerate nature of the genetic code, alternate nucleic acid sequences encode human rotavirus C VP2, VP6 or VP7 and variants thereof, and that such alternate nucleic acids may be included in an expression vector and expressed to produce human rotavirus C VLPs of the present invention.
[0085] In embodiments of the present invention, a nucleic acid sequence which is substantially identical to SEQ ID No. 31, SEQ ID NO: 46, or SEQ ID No. 48 encoding human rotavirus GpC VP6, is included in an expression vector and expressed to produce human rotavirus C VLPs of the present invention. In further embodiments of the present invention, a nucleic acid sequence which is substantially identical to SEQ ID No. 33 encoding human rotavirus GpC VP7, is included in an expression vector and expressed to produce human rotavirus C VLPs of the present invention. In further embodiments of the present invention, a nucleic acid sequence which is substantially identical to SEQ ID No. 18, SEQ ID No. 42 or SEQ ID No. 43 encoding human rotavirus GpC VP2, is included in an expression vector and expressed to produce human rotavirus C VLPs of the present invention.
[0086] A nucleic acid sequence which is substantially identical to SEQ ID No. 31 or SEQ ID NO: 46 is characterized as having a complementary nucleic acid sequence capable of hybridizing to SEQ ID No. 31, SEQ ID NO: 46, or SEQ ID No. 48 under high stringency hybridization conditions. Similarly, a nucleic acid sequence which is substantially identical to SEQ ID No. 33, SEQ ID No. 18, SEQ ID No. 42 or SEQ ID No. 43, is characterized as having a complementary nucleic acid sequence capable of hybridizing to SEQ ID No. 33 or SEQ ID No. 18, SEQ ID No. 42 or SEQ ID No. 43, respectively, under high stringency hybridization conditions.
[0087] The term "nucleic acid" refers to RNA or DNA molecules having more than one nucleotide in any form including single-stranded, double-stranded, oligonucleotide or polynucleotide. The term "nucleotide sequence" refers to the ordering of nucleotides in an oligonucleotide or polynucleotide in a single-stranded form of nucleic acid.
[0088] The term "complementary" refers to Watson-Crick base pairing between nucleotides and specifically refers to nucleotides hydrogen bonded to one another with thymine or uracil residues linked to adenine residues by two hydrogen bonds and cytosine and guanine residues linked by three hydrogen bonds. In general, a nucleic acid includes a nucleotide sequence described as having a "percent complementarity" to a specified second nucleotide sequence. For example, a nucleotide sequence may have 80%, 90%, or 100% complementarity to a specified second nucleotide sequence, indicating that 8 of 10, 9 of 10 or 10 of 10 nucleotides of a sequence are complementary to the specified second nucleotide sequence. For instance, the nucleotide sequence 3'-TCGA-5' is 100% complementary to the nucleotide sequence 5'-AGCT-3'. Further, the nucleotide sequence 3'-TCGA- is 100% complementary to a region of the nucleotide sequence 5'-TTAGCTGG-3'.
[0089] The terms "hybridization" and "hybridizes" refer to pairing and binding of complementary nucleic acids. Hybridization occurs to varying extents between two nucleic acids depending on factors such as the degree of complementarity of the nucleic acids, the melting temperature, Tm, of the nucleic acids and the stringency of hybridization conditions, as is well known in the art. The term "stringency of hybridization conditions" refers to conditions of temperature, ionic strength, and composition of a hybridization medium with respect to particular common additives such as formamide and Denhardt's solution. Determination of particular hybridization conditions relating to a specified nucleic acid is routine and is well known in the art, for instance, as described in J. Sambrook and D. W. Russell, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press; 3rd Ed., 2001; and F. M. Ausubel, Ed., Short Protocols in Molecular Biology, Current Protocols; 5th Ed., 2002. High stringency hybridization conditions are those which only allow hybridization of substantially complementary nucleic acids. Typically, nucleic acids having about 85-100% complementarity are considered highly complementary and hybridize under high stringency conditions. Intermediate stringency conditions are exemplified by conditions under which nucleic acids having intermediate complementarity, about 50-84% complementarity, as well as those having a high degree of complementarity, hybridize. In contrast, low stringency hybridization conditions are those in which nucleic acids having a low degree of complementarity hybridize.
[0090] The terms "specific hybridization" and "specifically hybridizes" refer to hybridization of a particular nucleic acid to a target nucleic acid without substantial hybridization to nucleic acids other than the target nucleic acid in a sample.
[0091] Stringency of hybridization and washing conditions depends on several factors, including the Tm of the probe and target and ionic strength of the hybridization and wash conditions, as is well-known to the skilled artisan. Hybridization and conditions to achieve a desired hybridization stringency are described, for example, in Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 2001; and Ausubel, F. et al., (Eds.), Short Protocols in Molecular Biology, Wiley, 2002.
[0092] An example of high stringency hybridization conditions is hybridization of nucleic acids over about 100 nucleotides in length in a solution containing 6×SSC, 5×Denhardt's solution, 30% formamide, and 100 micrograms/ml denatured salmon sperm at 37° C. overnight followed by washing in a solution of 0.1×SSC and 0.1% SDS at 60° C. for 15 minutes. SSC is 0.15M NaCl/0.015M Na citrate. Denhardt's solution is 0.02% bovine serum albumin/0.02% FICOLL/0.02% polyvinylpyrrolidone. Under highly stringent conditions, SEQ ID No. 31, SEQ ID No. 33 and SEQ ID No. 18 will hybridize to the complement of substantially identical targets and not to unrelated sequences.
[0093] The term "expression vector" refers to a recombinant vehicle for introducing a DNA sequence encoding one or more human rotavirus C proteins into a host cell where the DNA sequence is expressed to produce the one or more human rotavirus C proteins.
[0094] In particular embodiments, a DNA sequence encoding one or more human rotavirus C proteins includes an open reading frame encoding the one or more human rotavirus C proteins.
[0095] In further embodiments, 5' non-coding sequence and/or 3' non-coding sequence is present in addition to the open reading frame encoding the one or more human rotavirus C proteins. Preferably, where 5' non-coding sequence and/or 3' non-coding sequence is present, it is contiguous with the DNA sequence encoding one or more human rotavirus C proteins, 5' non-coding sequence, if any, is positioned upstream (5') of the start codon and 3' non-coding sequence, if any, is positioned downstream (3') of the stop codon.
[0096] In particular embodiments, an expression vector including SEQ ID No. 42 or a substantially identical nucleic acid sequence is expressed to produce human rotavirus C VP2 and self-assembled VLPs in cells containing the expression vector. SEQ ID No. 42 includes an open reading frame encoding human rotavirus C VP2 from strain Asp88. Optionally, 5' non-coding sequence and/or 3' non-coding sequence of human rotavirus C VP2 from strain Asp88 is included in the expression vector. For example, 1-36 nucleotides of 5' non-coding sequence can be included contiguous with the 5' end of the nucleotide sequence encoding human rotavirus C VP2 from strain Asp88. In particular embodiments, an expression vector including SEQ ID No. 18 or a substantially identical nucleic acid sequence is expressed to produce human rotavirus C VP2 and self-assembled VLPs in cells containing the expression vector. In further embodiments, an expression vector including SEQ ID No. 43 or a substantially identical nucleic acid sequence is expressed to produce human rotavirus C VP2 and self-assembled VLPs in cells containing the expression vector.
[0097] In additional embodiments, an expression vector including SEQ ID No. 31, SEQ ID NO: 46, SEQ ID No. 48 or a substantially identical nucleic acid sequence is expressed to produce human rotavirus C VP6 and self-assembled VLPs in cells containing the expression vector.
[0098] In additional embodiments, an expression vector including SEQ ID No. 33, SEQ ID No. 45, or a substantially identical nucleic acid sequence is expressed to produce human rotavirus C VP7 and self-assembled VLPs in cells containing the expression vector.
[0099] In further embodiments, an expression vector including SEQ ID No. 31, SEQ ID NO: 46, SEQ ID No. 48 or a substantially identical nucleic acid sequence and SEQ ID No. 33, SEQ ID No. 45, or a substantially identical nucleic acid sequence is expressed to produce human rotavirus C VP6, human rotavirus C VP7 and self-assembled VLPs, in cells containing the expression vector.
[0100] In further embodiments, an expression vector including SEQ ID No. 31, SEQ ID NO: 46, SEQ ID No. 48 or a substantially identical nucleic acid sequence, SEQ ID No. 33, SEQ ID No. 45, or a substantially identical nucleic acid sequence and SEQ ID No. 18, SEQ ID No. 42, SEQ ID No. 43, or a substantially identical nucleic acid sequence is expressed to produce human rotavirus C VP6, human rotavirus C VP7, human rotavirus C VP2 and self-assembled VLPs in cells containing the expression vector.
[0101] In still further embodiments, a first expression vector including SEQ ID No. 31, SEQ ID NO: 46, SEQ ID No. 48 or a substantially identical nucleic acid sequence and a second expression vector including SEQ ID No. 33, SEQ ID No. 45, or a substantially identical nucleic acid sequence are both expressed to produce human rotavirus C VP6, human rotavirus C VP7 and self-assembled VLPs in cells containing the expression vectors.
[0102] In still further embodiments, a first expression vector including SEQ ID No.31, SEQ ID NO: 46, SEQ ID No. 48 or a substantially identical nucleic acid sequence, a second expression vector including SEQ ID No. 33, SEQ ID No. 45, or a substantially identical nucleic acid sequence and a third expression vector including SEQ ID No. 18, SEQ ID No. 42, SEQ ID No. 43, or a substantially identical nucleic acid sequence are expressed to produce human rotavirus C VP6, human rotavirus C VP7, human rotavirus C VP2, and self-assembled VLPs in cells containing the expression vectors.
[0103] In addition to one or more DNA sequences encoding proteins of human rotavirus C, one or more DNA sequences encoding additional proteins can be included in an expression vector. For example, such additional proteins include non-human rotavirus C proteins such as reporters, including, but not limited to, beta-galactosidase, green fluorescent protein and antibiotic resistance reporters; and antigens.
[0104] Expression vectors are known in the art and include plasmids and viruses, for example. An expression vector contains a DNA molecule that includes segment encoding a polypeptide of interest operably linked to one or more regulatory elements that provide for transcription of the segment encoding the polypeptide of interest. Such regulatory elements include, but are not limited to, promoters, terminators, enhancers, origins of replication and polyadenylation signals.
[0105] In particular embodiments, the recombinant expression vector encodes human rotavirus C VP2 of SEQ ID No. 1, a protein having at least 95% identity to SEQ ID No. 1, a protein encoded by SEQ ID No. 42, or a protein encoded by a nucleic acid sequence substantially identical to SEQ ID No. 42.
[0106] In particular embodiments, the recombinant expression vector encodes human rotavirus C VP6 of SEQ ID No. 32, a protein having at least 95% identity to SEQ ID No. 32, a protein encoded by SEQ ID No. 31, or a protein encoded by a nucleic acid sequence substantially identical to SEQ ID No. 31.
[0107] In particular embodiments, the recombinant expression vector encodes human rotavirus C VP7 of SEQ ID No. 34, a protein having at least 95% identity to SEQ ID No. 34, a protein encoded by SEQ ID No. 33 or SEQ ID No. 45, or a protein encoded by a nucleic acid sequence substantially identical to SEQ ID No. 33 or SEQ ID No. 45.
[0108] In further embodiments, the recombinant expression vector encodes human rotavirus C VP6 of SEQ ID No. 32, a protein having at least 95% identity to SEQ ID No. 32, a protein encoded by SEQ ID No. 31, or a protein encoded by a nucleic acid sequence substantially identical to SEQ ID No. 31; and human rotavirus C VP7 of SEQ ID No. 34, a protein having at least 95% identity to SEQ ID No. 34, a protein encoded by SEQ ID No. 33 or SEQ ID No. 45, or a protein encoded by a nucleic acid sequence substantially identical to SEQ ID No. 33 or SEQ ID No. 45.
[0109] A preferred expression vector of the present invention is a baculovirus.
[0110] Expression of human rotavirus C VP2, VP6 and/or VP7 encoded by a recombinant expression vector is accomplished by introduction of the expression vector into a eukaryotic or prokaryotic host cell expression system such as an insect cell, mammalian cell, yeast cell, bacterial cell or any other single or multicellular organism recognized in the art. In preferred embodiments, a eukaryotic host cell is used. Host cells are optionally primary cells or immortalized derivative cells. Immortalized cells are those which can be maintained in-vitro for at least 5 replication passages.
[0111] Host cells containing the recombinant expression vector are maintained under conditions where human rotavirus C proteins are produced. The human rotavirus C proteins self-associate to produce VLPs of the present invention in the host cell.
[0112] The invention provides a host cell containing a nucleic acid sequence according to the invention. Host cells may be cultured and maintained using known cell culture techniques such as described in Celis, Julio, ed., 1994, Cell Biology Laboratory Handbook, Academic Press, N.Y. Various culturing conditions for these cells, including media formulations with regard to specific nutrients, oxygen, tension, carbon dioxide and reduced serum levels, can be selected and optimized by one of skill in the art.
[0113] A preferred cell line of the present invention is a eukaryotic cell line, preferably an insect cell line, such as Sf9 or Hi5, transiently or stably expressing one or more full-length or partial human rotavirus C proteins. Such cells can be made by transfection (proteins or nucleic acid vectors), infection (viral vectors) or transduction (viral vectors). The cell lines for use in the present invention are cloned using known cell culture techniques familiar to one skilled in the art. The cells are cultured and expanded from a single cell using commercially available culture media under known conditions suitable for propagating cells.
[0114] In a preferred embodiment human rotavirus C VLPs are produced by infection of a host cell with at least one recombinant baculovirus encoding human rotavirus C protein(s).
[0115] It is appreciated that a single baculovirus may encode either a single human rotavirus C protein or multiple human rotavirus C proteins. The resulting infected cells are then cultured under conditions whereby the encoded human rotavirus C proteins from the respective recombinant baculoviruses are produced and self assemble to form the capsids. The resulting human rotavirus C VLPs are then optionally and preferably isolated.
[0116] In further preferred embodiments, the recombinant baculovirus encodes at least human rotavirus C VP6 of SEQ ID No. 32, a protein having at least 95% identity to SEQ ID No. 32, a protein encoded by SEQ ID No. 31, or a protein encoded by a nucleic acid sequence substantially identical to SEQ ID No. 31, SEQ ID NO: 46 or SEQ ID No. 48; and human rotavirus C VP7 of SEQ ID No. 34, a protein having at least 95% identity to SEQ ID No. 34, a protein encoded by SEQ ID No. 33 or SEQ ID No. 45, or a protein encoded by a nucleic acid sequence substantially identical to SEQ ID No. 33 or SEQ ID No. 45.
[0117] In a further option, the recombinant baculovirus encodes human rotavirus C VP2 of SEQ ID No. 1, a protein having at least 95% identity to SEQ ID No. 1, a protein encoded by SEQ ID No. 42, or a protein encoded by a nucleic acid sequence substantially identical to SEQ ID No. 42.
[0118] Any suitable baculovirus known in the art is operable in the instant inventive process. Preferably, the baculovirus is Autographa california nuclear polyhedrosis virus.
[0119] Processes for infecting cells with baculovirus are known in the art. Following infection of a host cell the inventive process proceeds by culturing the host cells under conditions such that protein(s) produced self assemble to form VLPs.
[0120] A VLP of the present invention optionally includes a non-human rotavirus C protein or peptide in contact with or bonded to at least one of the human rotavirus C proteins VP2, VP6 or VP7. Bonding of the non-human rotavirus C protein or peptide is achieved, for example, by expression of a fusion construct including a nucleic acid sequence encoding VP2, VP6 or VP7 and the non-human rotavirus C protein or peptide. Thus, the non-human rotavirus C protein or peptide is optionally a fusion protein or peptide wherein the non-human rotavirus C protein is synthesized as a single polypeptide chain with a human rotavirus C structural protein.
[0121] The non-human rotavirus C protein is optionally fused with glutathione-S-transferase (GST) for rapid isolation. A human rotavirus C protein is also optionally fused to GST.
[0122] Chemical bonding methods are optionally used to bond a VLP and a non-human rotavirus C protein or peptide, illustratively including reaction using a cross-linking agent such as carbodiimide or glutaraldehyde.
[0123] In particular embodiments, the non-human rotavirus C protein or peptide included in the VLP includes one or more antigenic epitopes such that the VLP serves to present the one or more antigenic epitopes to the immune system of a subject to induce antibody generation.
[0124] In a further option, the non-human rotavirus C protein or peptide is a targeting moiety such as a receptor ligand or receptor. A targeting moiety is included in the VLP to direct the VLP to a target, such as to a particular cell type.
[0125] Human rotavirus C VLPs produced in a host cell are optionally isolated. The term "isolated" in reference to a human rotavirus C VLP describes a human rotavirus C VLP which is separated from a cell in which the human rotavirus C VLP is produced and which is substantially free of host cell components not intended to be associated with the human rotavirus C VLP. Generally, human rotavirus C VLPs are separated from whole cell extracts of host cells. Numerous processes of isolating VLPs are known in the art and are applicable to isolation of human rotavirus C VLPs illustratively including sucrose continuous and discontinuous gradients, cesium chloride single and multi-density gradient centrifugation, size-exclusion chromatography, antigen capture chromatography, affinity chromatography, or other suitable process known in the art. An exemplary method for isolating human rotavirus C VLPs of the present invention is described in Gillock, ET. et al, 1997. J. Virol., 71:2857-2865.
[0126] Human rotavirus C VLPs having different compositions, that is, different "types" of human rotavirus C VLPs are optionally present in a composition of the present invention. For example, human rotavirus C VLPs including human rotavirus C VP2 are optionally included in a composition with antigen presenting human rotavirus C VLPs including a non-human rotavirus C protein or peptide and/or human rotavirus C VLPs containing a cargo moiety.
[0127] In one aspect, the invention provides a method of making a human group C rotavirus-like particle comprising: constructing a first baculovirus vector comprising a nucleic acid molecule comprising a sequence encoding a human group C RV VP6 capsid protein operably linked to a baculovirus promoter that drives expression of said protein in an insect cell; constructing a second baculovirus vector comprising a nucleic acid molecule comprising a sequence encoding a human group C RV VP7 capsid protein operably linked to a baculovirus promoter that drives expression of said protein in an insect cell; and infecting an insect cell culture with said first and second baculovirus vector under conditions that promote expression of the VP6- and VP7 capsid proteins.
[0128] In one embodiment of the present invention, the method further comprises constructing a third baculovirus vector comprising a nucleic acid molecule comprising a sequence encoding a human group C RV VP2 core protein operably linked to a baculovirus promoter that drives expression of said protein in an insect cell; and infecting an insect cell culture with said first, second, and third baculovirus vector under conditions that promote expression of the VP6 capsid protein and VP7 capsid protein and said VP2 core protein.
[0129] In another aspect, the invention provides a rotavirus-like particle made by the herein described inventive method.
[0130] A virus-like particle containing a fragment of the VP proteins described herein can be formed by any of the above described methods for making a human group C rotavirus-like particle, and also includes: constructing a third baculovirus vector comprising a nucleic acid molecule comprising a sequence encoding a core protein operably linked to a baculovirus promoter that drives expression of said protein in an insect cell; infecting an insect cell culture with said first, second, and third baculovirus vector under conditions that promote expression of the VP6- and VP7 capsid proteins, and the VP2 core protein.
[0131] In one embodiment of the present invention, the core protein is a group C VP2 protein of ASP 88 strain.
[0132] Rotavirus particles are harvested, typically from cell culture supernatant for inclusion in an immunogenic composition including a vaccine composition. The rotavirus particles may be isolated from the cell culture supernatant, for example by filtration and/or centrifugation. The isolated rotavirus particles are optionally lyophilized, such as for later resuspension in a pharmaceutically acceptable carrier.
Pharmaceutical Compositions and Processes
[0133] Pharmaceutical Compositions and Processes
[0134] Vaccines and methods for their use to induce active immunity and protection against human rotavirus C-induced illness in a subject are provided according to the present invention.
[0135] In particular embodiments, human rotavirus C VLPs are administered as antigens for prevention or treatment of human rotavirus C infection such as by serving as an active vaccine component, or by eliciting an immune response in a host organism. Vaccine delivery may occur prior to or following human rotavirus C infection of a host organism or patient. A vaccine optionally contains one or more adjuvants and preservatives or other pharmaceutically acceptable carrier.
[0136] In particular embodiments, vaccine compositions include one or more types of human rotavirus C VLP admixed with a pharmaceutically acceptable carrier.
[0137] The term "pharmaceutically acceptable carrier" refers to a carrier which is substantially non-toxic to a subject and substantially inert to the human rotavirus C VLPs included in a vaccine composition. A pharmaceutically acceptable carrier is a solid, liquid or gel in form and is typically sterile and pyrogen free.
[0138] An immunogenic composition of the present invention may be in any form suitable for administration to a subject.
[0139] An immunogenic composition is administered by any suitable route of administration including oral and parenteral such as intravenous, intradermal, intramuscular, intraperitoneal, mucosal, nasal, or subcutaneous routes of administration.
[0140] For example, an immunogenic composition for parenteral administration may be formulated as an injectable liquid including a rotavirus and a pharmaceutically acceptable carrier. Examples of suitable aqueous and nonaqueous carriers include water, ethanol, polyols such as propylene glycol, polyethylene glycol, glycerol, and the like, suitable mixtures thereof; vegetable oils such as olive oil; and injectable organic esters such as ethyloleate. Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of a desirable particle size in the case of dispersions, and/or by the use of a surfactant, such as sodium lauryl sulfate. A stabilizer is optionally included such as, for example, EDTA, EGTA, and an antioxidant.
[0141] A solid dosage form for administration or for suspension in a liquid prior to administration illustratively includes capsules, tablets, powders, and granules. In such solid dosage forms, rotavirus particles are admixed with at least one carrier illustratively including a buffer such as, for example, sodium citrate or an alkali metal phosphate illustratively including sodium phosphates, potassium phosphates and calcium phosphates; a filler such as, for example, starch, lactose, sucrose, glucose, mannitol, and silicic acid; a binder such as, for example, carboxymethylcellulose, alignates, gelatin, polyvinylpyrrolidone, sucrose, and acacia; a humectant such as, for example, glycerol; a disintegrating agent such as, for example, agar-agar, calcium carbonate, plant starches such as potato or tapioca starch, alginic acid, certain complex silicates, and sodium carbonate; a solution retarder such as, for example, paraffin; an absorption accelerator such as, for example, a quaternary ammonium compound; a wetting agent such as, for example, cetyl alcohol, glycerol monostearate, and a glycol; an adsorbent such as, for example, kaolin and bentonite; a lubricant such as, for example, talc, calcium stearate, magnesium stearate, a solid polyethylene glycol or sodium lauryl sulfate; a preservative such as an antibacterial agent and an antifungal agent, including for example, thimerosal, sorbic acid, gentamycin and phenol; and a stabilizer such as, for example, EDTA, EGTA, and an antioxidant.
[0142] Solid dosage forms optionally include a coating such as an enteric coating. The enteric coating is typically a polymeric material. Preferred enteric coating materials have the characteristics of being bioerodible, gradually hydrolyzable and/or gradually water-soluble polymers. The amount of coating material applied to a solid dosage generally dictates the time interval between ingestion and drug release. A coating is applied having a thickness such that the entire coating does not dissolve in the gastrointestinal fluids at pH below 3 associated with stomach acids, yet dissolves above pH 3 in the small intestine environment. It is expected that any anionic polymer exhibiting a pH-dependent solubility profile is readily used as an enteric coating in the practice of the present invention to achieve delivery of the active agent to the lower gastrointestinal tract. The selection of the specific enteric coating material depends on properties such as resistance to disintegration in the stomach; impermeability to gastric fluids and active agent diffusion while in the stomach; ability to dissipate at the target intestine site; physical and chemical stability during storage; non-toxicity; and ease of application.
[0143] Suitable enteric coating materials illustratively include cellulosic polymers such as hydroxypropyl cellulose, hydroxyethyl cellulose, hydroxypropyl methyl cellulose, methyl cellulose, ethyl cellulose, cellulose acetate, cellulose acetate phthalate, cellulose acetate trimellitate, hydroxypropylmethyl cellulose phthalate, hydroxypropylmethyl cellulose succinate and carboxymethylcellulose sodium; acrylic acid polymers and copolymers, preferably formed from acrylic acid, methacrylic acid, methyl acrylate, ammonium methylacrylate, ethyl acrylate, methyl methacrylate and/or ethyl; vinyl polymers and copolymers such as polyvinyl pyrrolidone, polyvinyl acetate, polyvinylacetate phthalate, vinylacetate crotonic acid copolymer, and ethylene-vinyl acetate copolymers; shellac; and combinations thereof. A particular enteric coating material includes acrylic acid polymers and copolymers described for example U.S. Pat. No. 6,136,345.
[0144] The enteric coating optionally contains a plasticizer to prevent the formation of pores and cracks that allow the penetration of the gastric fluids into the solid dosage form. Suitable plasticizers illustratively include triethyl citrate (Citroflex 2), triacetin (glyceryl triacetate), acetyl triethyl citrate (Citroflec A2), Carbowax 400 (polyethylene glycol 400), diethyl phthalate, tributyl citrate, acetylated monoglycerides, glycerol, fatty acid esters, propylene glycol, and dibutyl phthalate. In particular, a coating composed of an anionic carboxylic acrylic polymer typically contains approximately 10% to 25% by weight of a plasticizer, particularly dibutyl phthalate, polyethylene glycol, triethyl citrate and triacetin. The coating can also contain other coating excipients such as detackifiers, antifoaming agents, lubricants (e.g., magnesium stearate), and stabilizers (e.g. hydroxypropylcellulose, acids or bases) to solubilize or disperse the coating material, and to improve coating performance and the coated product.
[0145] Liquid dosage forms for oral administration include rotavirus and a pharmaceutically acceptable carrier formulated as an emulsion, solution, suspension, syrup, or elixir. A liquid dosage form of a vaccine composition of the present invention may include a wetting agent, an emulsifying agent, a suspending agent, a sweetener, a flavoring, or a perfuming agent.
[0146] Detailed information concerning customary ingredients, equipment and processes for preparing dosage forms is found in Pharmaceutical Dosage Forms: Tablets, eds. H. A. Lieberman et al., New York: Marcel Dekker, Inc., 1989; and in L. V. Allen, Jr. et al., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, 8th Ed., Philadelphia, Pa.: Lippincott, Williams & Wilkins, 2004; A. R. Gennaro, Remington: The Science and Practice of Pharmacy, Lippincott Williams & Wilkins, 20th ed., 2003; and J. G. Hardman et al., Goodman & Gilman's The Pharmacological Basis of Therapeutics, McGraw-Hill Professional, 10th ed., 2001.
[0147] An adjuvant is optionally included in a virus composition according to embodiments of the present invention. Adjuvants are known in the art and illustratively include Freund's adjuvant, aluminum hydroxide, aluminum phosphate, aluminum oxide, saponin, dextrans such as DEAE-dextran, vegetable oils such as peanut oil, olive oil, and/or vitamin E acetate, mineral oil, bacterial lipopolysaccharides, peptidoglycans, and proteoglycans.
[0148] The term "subject" is used herein to refer to a human. Non-human animals, illustratively including cows, horses, sheep, goats, pigs, dogs, cats, birds, poultry, and rodents, are also referred to by the term subject in particular embodiments of the present invention.
[0149] A vaccine composition of the present invention may be in any form suitable for administration to a subject.
[0150] A vaccine composition is administered by any suitable route of administration including oral and parenteral such as intravenous, intradermal, intramuscular, intraperitoneal, mucosal, nasal, or subcutaneous routes of administration.
[0151] The phrase "therapeutically effective amount" refers to an amount effective to induce an immunological response and prevent or ameliorate signs or symptoms of human rotavirus C-mediated disease. Induction of an immunological response in a subject can be determined by any of various techniques known in the art, illustratively including detection of anti-human rotavirus C antibodies, measurement of anti-human rotavirus C antibody titer and/or lymphocyte proliferation assay. Signs and symptoms of human rotavirus C-mediated disease may be monitored to detect induction of an immunological response to administration of a vaccine composition of the present invention in a subject.
[0152] Administration of a vaccine composition according to a method of the present invention includes administration of one or more doses of a vaccine composition to a subject at one time in particular embodiments. Alternatively, two or more doses of a vaccine composition are administered at time intervals of weeks--years. A suitable schedule for administration of vaccine composition doses depends on several factors including age and health status of the subject, type of vaccine composition used and route of administration, for example. One of skill in the art is able to readily determine a dose and schedule of administration to be administered to a particular subject.
[0153] Immunogenicity of human rotavirus C VLPs is tested by any of various assays known in the art. In a particular example, purified human rotavirus C VLPs are administered intramuscularly to mice with or without an adjuvant. Immunogenicity is assayed by measuring immunoglobulin titers including IgM, IgA and/or IgG in blood samples obtained at various times after administration.
[0154] Neutralizing antibody titers are measured by neutralization assays known in the art, such as those generally described in Kuby, J., Immunology, 3rd ed. W.H. Freeman and Co., New York, N.Y., 1997. Since human rotavirus C does not grow in culture, sera from mice injected with human rotavirus C VLPs are serially diluted two-fold in duplicate wells and incubated with trypsin-activated porcine rotavirus C. Activated porcine rotavirus C or serum-free MEM medium is incubated in the absence of mouse serum and serve as positive and negative controls, respectively. MA104 cells in MEM medium supplemented with trypsin are added to each well. After incubation at 37° C. for 18 hours, cells are fixed with formalin. Porcine rotavirus C antigens in the fixed MA104 cells are detected by incubating cells with HRP-labeled rabbit IgG against human rotavirus VLPs, and then tetramethyl benzidine. Neutralizing antibody titer in a serum is defined as the reciprocal of the highest dilution giving a 70% reduction in absorbance value compared to that in the virus control.
[0155] Optionally, antibodies raised to immunogenic human rotavirus C VLPs are administered to a subject for prevention or therapeutic treatment relating to human rotavirus C-mediated disease.
[0156] Additional therapeutics that are optionally administered with the vaccine composition or antibodies raised to human rotavirus C VLPs include antivirals such as amantadine, rimantadine, gancyclovir, acyclovir, ribavirin, penciclovir, oseltamivir, foscarnet zidovudine (AZT), didanosine (ddI), lamivudine (3TC), zalcitabine (ddC), stavudine (d4T), nevirapine, delavirdine, indinavir, ritonavir, vidarabine, nelfinavir, saquinavir, relenza, tamiflu, pleconaril, interferons; steroids and corticosteroids such as prednisone, cortisone, fluticasone and glucocorticoid; antibiotics; analgesics; antidiarrheals, fluid replacement; or other treatments for rotavirus infection.
[0157] The invention also provides a pharmaceutical kit that includes one or more receptacles containing one or more of the ingredients of the pharmaceutical compositions of the invention. Optionally associated with such container(s) can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, use or sale for human administration.
[0158] In a preferred embodiment, the kit contains an antibody specific for human rotavirus C VP2, human rotavirus C VP6, human rotavirus C VP7, the polypeptide of SEQ ID NO:1, an epitope or a variant thereof, the polypeptide of SEQ ID NO:32, an epitope or a variant thereof, the polypeptide of SEQ ID NO:34, an epitope or a variant thereof, or any human rotavirus C epitope, a polypeptide or protein of the present invention, or a nucleic acid molecule of the invention, alone or in combination with adjuvants, antivirals, antibiotics, analgesic, bronchodilators, or other pharmaceutically acceptable excipients. The present invention further encompasses kits comprising a container containing a pharmaceutical composition of the present invention and instructions for use.
[0159] Also provided is a diagnostic kit for detecting human rotavirus C infection that contains human rotavirus C VLPs as reagents for the detection of human rotavirus C antibodies. It is further appreciated that a diagnostic kit optionally includes ancillary reagents such as buffers, solvents, a detectable label and other reagents necessary and recognized in the art for detection of an antibody in a biological sample.
[0160] Detection of Anti-Human Rotavirus C Antibodies
[0161] Human rotavirus C VLPs are used to detect anti-human rotavirus C antibodies in a biological sample according to embodiments of a process of the present invention.
[0162] The term "biological sample" refers to a sample obtained from a biological organism, a tissue, cell, cell culture medium, or any medium suitable for mimicking biological conditions, or from the environment. Non-limiting examples include, saliva, gingival secretions, cerebrospinal fluid, gastrointestinal fluid, mucous, urogenital secretions, synovial fluid, blood, serum, plasma, urine, cystic fluid, lymph fluid, ascites, pleural effusion, interstitial fluid, intracellular fluid, ocular fluids, seminal fluid, mammary secretions, and vitreal fluid, feces, and nasal secretions. Environmental samples such as sewage or water samples can be used. In a preferred embodiment, the sample is serum, plasma or whole blood.
[0163] A process of detecting anti-human rotavirus C antibodies in a biological sample according to the present invention includes contacting a biological sample with recombinant human rotavirus C VLPs and detecting formation of a complex between anti-human rotavirus C antibodies present in the biological sample and the human rotavirus C VLPs. Formation of the complex between anti-human rotavirus C antibodies present in the biological sample and the human rotavirus C VLPs is indicative of exposure of the subject to human rotavirus C sufficient to activate the immune system of the subject to produce anti-human rotavirus C antibodies. Formation of the complex specifically indicates presence of anti-human rotavirus C antibodies since other enteric virus antibodies, particularly anti-human rotavirus A antibodies, do not form a complex with the human rotavirus C VLPs.
[0164] In a preferred embodiment, human rotavirus C VLPs are used to detect anti-human rotavirus C antibodies in a biological sample to diagnose current and recent human rotavirus C infection in a subject.
[0165] In a further preferred embodiment human rotavirus C VLPs are used in a process of assessing the immune status of an individual with respect to past or present exposure to a human rotavirus C antigen in human rotavirus C infection susceptible organisms, particularly in a human subject.
[0166] Detecting formation of a complex between anti-human rotavirus C antibodies present in a biological sample and human rotavirus C VLPs is achieved by any of various methods known in the art, illustratively including detection of a label attached to human rotavirus C VLPs or attached to the anti-human rotavirus C antibodies. The term "label" or "labeled" refers to any composition which can be used to detect, qualitatively or quantitatively, a substance attached to the label. Suitable labels include a fluorescent moiety, a radioisotope, a chromophore, a bioluminescent moiety, an enzyme, a magnetic particle, an electron dense particle, and the like. The term "label" or "labeled" is intended to encompass direct labeling of human rotavirus C VLPs or an antibody by coupling (i.e., physically linking) a detectable substance to the human rotavirus C VLPs or antibody, as well as indirect labeling of the human rotavirus C VLPs or antibody by interaction with another reagent that is directly labeled. An example of indirect labeling of a primary antibody includes detection of a primary antibody using a fluorescently labeled secondary antibody.
[0167] Labels used in detection of complex formation depend on the detection process used. Such detection processes are incorporated in particular assay formats illustratively including ELISA, western blot, immunoprecipitation, immunocytochemistry, immuno-fluorescence assay, liquid chromatography, flow cytometry, other detection processes known in the art, or combinations thereof.
[0168] In one embodiment, an ELISA is used to detect the presence of human rotavirus C antibodies in a biological sample.
[0169] In one configuration of an ELISA for human rotavirus C antibodies, human rotavirus C VLPs are coated on a support such as a microtiter plate, beads, slide, silicon chip or other solid support such as a nitrocellulose or PVDF membrane. A biological sample is incubated with the human rotavirus C VLPs on the support and the presence of complex between antibodies to human rotavirus C and human rotavirus C VLPs is detected by standard ELISA protocols. For example, a complex between human rotavirus C VLPs and human rotavirus C antibodies is detected by reaction of a labeled secondary antibody with the anti-human rotavirus C antibodies and detection of the label.
[0170] Another example of an ELISA for human rotavirus C antibodies is a sandwich ELISA. One embodiment of a sandwich ELISA includes depositing a binding antibody onto a solid support. The binding antibody is optionally a non-competing antibody that recognizes human rotavirus C VLPs. The binding antibody is incubated with human rotavirus C VLPs. The complex is washed to remove any unbound material and a detectable label, such as a fluorescently labeled antibody directed to human rotavirus C VLPs, is applied. The detectable label is detected, if present, indicating the presence of anti-human rotavirus C antibody in the biological sample.
[0171] Further details of ELISA assays in general are found in Crowther, J. R., The ELISA Guidebook (Methods in Molecular Biology), Humana Press, 2000; and Wild, D., The Immunoassay Handbook, 3rd Edition, Elsevier Science, 2005.
[0172] A human rotavirus C antibody detection kit is provided including one or more types of human rotavirus C VLPs and ancillary reagents for use in detecting anti-human rotavirus C antibodies in a biological sample. Ancillary reagents are any signal producing system materials for detection of a complex between an anti-human rotavirus C antibody and a human rotavirus C VLP in any suitable detection process such as ELISA, western blot, immunoprecipitation, immunocytochemistry, immuno-fluorescence, mass spectrometry, or other assay known in the art.
[0173] Optionally, an anti-human human rotavirus C antibody assay kit according to embodiments of the present invention includes human rotavirus C VLPs attached to a solid substrate. Suitable solid substrates include, but are not limited to, microtiter plates, chips, tubes, membranes, such as nylon or nitrocellulose membranes, and particles, such as beads. Attachment of protein-containing materials to solid substrates is well-known in the art and includes, but is not limited to, adsorption.
[0174] In a preferred embodiment, a human rotavirus C antibody detection kit of the present invention illustratively includes one or more types of human rotavirus C VLPs; and one or more ancillary reagents such as a high binding microtiter plate or other support, blocking agent, washing buffer such as phosphate buffered saline, a labeled anti-immunoglobulin antibody, and matching detection agents, swab or other sample collection devices, control reagents such as labeled non-competing or unlabelled reagents, control nucleotide sequence and relevant primers and probes, and other materials and reagents for detection. The kit optionally includes instructions printed or in electronically accessible form and/or customer support contact information.
[0175] Anti-immunoglobulin antibodies in a signal producing system or otherwise are optionally labeled with a fluorophore, biotin, peroxidase, or other enzymatic or non-enzymatic detection label. It is appreciated that a signal producing system may employ an unlabeled primary antibody and a labeled secondary antibody derived from the same or a different organism. It is further appreciated that non-antibody signal producing systems are similarly operable.
[0176] It is further appreciated that a kit optionally includes ancillary reagents such as buffers, solvents, a detectable label and other reagents necessary and recognized in the art for detection of an antibody in a biological sample.
[0177] VLPs Containing a Cargo
[0178] Optionally, the VLP contains a cargo in the internal space defined by the VLP. In particular embodiments, a cargo moiety is a substance to be delivered to a subject or cell. Exemplary cargo moieties include an antigen, a nucleic acid which is not an intact human rotavirus C genome and a therapeutic agent.
[0179] Particularly provided is a process of delivery of genetic information whereby genetic material is encapsulated in a human rotavirus C capsid which is then introduced into a host cell. The genetic material is optionally DNA or RNA, or modifications thereof. The genetic information is optionally derived from a human rotavirus C or other viral or nonviral organism, or is synthetic.
[0180] A cargo is incorporated in the internal space defined by a human rotavirus C VLP by any of various methods including introducing the cargo into a host cell such that human rotavirus C VLPs are produced in the presence of the cargo and thereby include the cargo in the internal space. Alternatively or additionally, a cargo is incorporated in the internal space by incubating produced human rotavirus C VLPs with the cargo such that the cargo enters the internal space, e.g. by diffusion.
[0181] VLP Antibodies
[0182] Human rotavirus C VLPs are used as antigens for production of monoclonal or polyclonal antibodies to human rotavirus C for clinical use such as in therapy, analysis or diagnosis; or laboratory research.
[0183] In a preferred embodiment, human rotavirus C VLPs are used for eliciting human rotavirus C specific antibody or T cell responses to the VP2, VP6, VP7 or any antigen included in the human rotavirus C VLPs, in vivo (e.g., for protective or therapeutic purposes or for providing diagnostic antibodies) and in vitro (e.g., by phage display technology or another technique useful for generating synthetic antibodies).
[0184] As used herein, the terms "antibody" and "antibodies" relate to monoclonal antibodies, polyclonal antibodies, bispecific antibodies, multispecific antibodies, human antibodies, humanized antibodies, chimeric antibodies, camelized antibodies, single domain antibodies, single-chain Fvs (scFv), single chain antibodies, disulfide-linked Fvs (sdFv), and anti-idiotypic (anti-Id) antibodies (including, e.g., anti-Id antibodies to antibodies of the invention), and epitope-binding fragments of any of the above. In particular, antibodies include immunoglobulin molecules and immunologically active fragments of immunoglobulin molecules, i.e., molecules that contain an antigen binding site. Immunoglobulin molecules are of any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2), or subclass.
[0185] As used herein, the term "antibody fragment" defines a fragment of an antibody that immunospecifically binds to a human rotavirus C virus, any epitope of the human rotavirus C virus or human rotavirus C VLP. Antibody fragments may be generated by any technique known to one of skill in the art. For example, Fab and F(ab')2 fragments may be produced by proteolytic cleavage of immunoglobulin molecules, using enzymes such as papain (to produce Fab fragments) or pepsin (to produce F(ab') 2 fragments). F(ab') 2 fragments contain the complete light chain, and the variable region, the CH 1 region and the hinge region of the heavy chain. Antibody fragments are also produced by recombinant DNA technologies. Antibody fragments may be one or more complementarity determining regions (CDRs) of antibodies.
[0186] Human rotavirus C-specific antibodies are provided according to the present invention which specifically bind to human rotavirus C and do not specifically bind to other rotavirus types such as rotavirus A, B, D, E, F and G.
[0187] A hybridoma cell line expressing monoclonal antibody raised against human rotavirus C VLPs of the present invention specifically binds to human rotavirus C and does not specifically bind to other rotavirus types such as rotavirus A, B, D, E, F and G.
[0188] An antibody raised to human rotavirus C VLPs by any of the methods known in the art, is optionally purified by any method known in the art for purification of an immunoglobulin molecule, for example, by ion exchange chromatography, affinity, particularly by affinity for the specific antigen or size exclusion; centrifugation; differential solubility; or by any other standard techniques for the purification of proteins. It is also appreciated thatan inventive antibody or fragments thereof may be fused to heterologous polypeptide sequences known in the art to facilitate purification.
[0189] For some uses, including in vivo use of antibodies in humans and in vitro detection assays, it may be preferable to use chimeric, humanized, or human antibodies. A chimeric antibody is a molecule in which different portions of the antibody are derived from different animal species, such as antibodies having a variable region derived from a murine monoclonal antibody and a constant region derived from a human immunoglobulin. Methods for producing chimeric antibodies are known in the art. (Morrison, 1985, Science, 229:1202; U.S. Pat. Nos. 5,807,715; 4,816,567; and 4,816,397). Humanized antibodies are antibody molecules from non-human species that bind the desired antigen having one or more complementarity determining regions (CDRs) from the non-human species and framework regions from a human immunoglobulin molecule. Often, framework residues in the human framework regions are substituted with the corresponding residue from the CDR donor antibody to alter, preferably improve, antigen binding. These framework substitutions are identified by methods well known in the art, such as by modeling of the interactions of the CDR and framework residues to identify framework residues important for antigen binding and sequence comparison to identify unusual framework residues at particular positions. (U.S. Pat. No. 5,585,089; Riechmann et al., 1988, Nature 332:323). Antibodies can be humanized using a variety of techniques known in the art including, for example, CDR-grafting (PCT publication WO 91/09967; U.S. Pat. Nos. 5,225,539; 5,530,101 and 5,585,089), veneering or resurfacing (Studnicka et al., 1994, Protein Engineering 7(6):805 814; Roguska et al., 1994, PNAS. 91:969 973), and chain shuffling (U.S. Pat. No. 5,565,332).
[0190] Completely human antibodies are particularly desirable for therapeutic treatment of human patients. Human antibodies can be made by a variety of methods known in the art including phage display methods described above using antibody libraries derived from human immunoglobulin sequences. (U.S. Pat. Nos. 4,444,887 and 4,716,111).
[0191] Human antibodies are readily produced using transgenic mice which are incapable of expressing functional endogenous immunoglobulins, but which can express human immunoglobulin genes.
[0192] An inventive antibody is optionally fused or conjugated to heterologous polypeptides may be used in vitro immunoassays and in purification methods such as affinity chromatography. (PCT publication Number WO 93/21232; U.S. Pat. No. 5,474,981).
[0193] An inventive antibody is optionally attached to solid supports, which are particularly useful for immunoassays or purification of the polypeptides of the invention or fragments, derivatives, analogs, or variants thereof, or similar molecules having the similar enzymatic activities as the polypeptide of the invention. Such solid supports include, but are not limited to, glass, cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride or polypropylene.
[0194] Assays for Human Rotavirus C
[0195] Anti-human rotavirus C VLP antibodies of the present invention are used to detect human rotavirus C in a biological sample in embodiments of the present invention.
[0196] An assay for human rotavirus C in a biological sample of the present invention includes contacting a biological sample with an anti-human rotavirus C antibody and detecting formation of a complex between anti-human rotavirus C antibody and the human rotavirus C present in the biological sample. Formation of the complex is indicative of current infection by human rotavirus C in a subject from which a biological sample is obtained. Formation of the complex specifically indicates presence of human rotavirus C since other rotavirus types such as rotavirus A, B, D, E, F and G, do not form a complex with an anti-human rotavirus C antibody of the present invention.
[0197] In a specific embodiment, the processes further involve obtaining a biological sample from a subject, contacting the sample with a compound or agent capable of detecting the presence of human rotavirus C nucleic acid in the sample in order to confirm presence of human rotavirus C in the sample.
[0198] In further embodiments, a control sample is assayed for presence of human rotavirus C and/or anti-human rotavirus C antibodies and results are compared with a test sample to ascertain a difference in presence or amount of human rotavirus C or anti-human rotavirus C antibodies.
[0199] In another aspect, the invention provides a method of determining exposure of a human or animal to a group C rotavirus comprising: contacting a biological sample of said human or animal with the inventive rotavirus-like particle described herein, under conditions which promote binding of antibodies in said biological sample to said rotavirus-like particles; and detecting binding of antibodies within the biological sample with the rotavirus-like particles. For the purposes of determining exposure of a human or animal to a group C rotavirus, biological sample typically is blood and/or feces; however, biological sample also includes a sample from other tissues; e.g. an intestinal biopsy.
[0200] The invention also encompasses kits for detecting the presence of human rotavirus C in a test sample. The kit, for example, includes an anti-human rotavirus C antibody and optionally includes a reagent such as a labeled secondary antibody or agent capable of detecting an antibody in a complex with a human rotavirus C and, in certain embodiments, for determining the titer in the sample.
[0201] Embodiments of inventive compositions and methods are illustrated in the following examples. These examples are provided for illustrative purposes and are not considered limitations on the scope of inventive compositions and methods.
Example 1
[0202] Cloning and construction of baculovirus recombinants. Segment 5, encoding VP6, from human group C RV strain S-1 was amplified by RT-PCR using BMJ44 (5'-AGC-CAC-ATA-GTT-CAC-ATT-TC-3') (SEQ ID NO: 14) and BMJ141 (5'-ATC-TCA-TTC-ACA-ATG-GAT-G-3') (SEQ ID NO: 15) (28). Segment 8, encoding VP7, from strain S-1 was amplified by RT-PCR using primers BMJ13 (5'-AGC-CAC-ATG-ATC-TTG-TTT-3') (SEQ ID NO: 20) and BMJ14 (5'-GGC-ATT-TAA-AAA-AGA-AGA-3') (SEQ ID NO: 21) (13, 28). Segment 2, encoding VP2, from strain ASP88 was amplified by RT-PCR using BMJ197 (5'-TCG-AGG-ACA-AAT-CGT-CCA-AG-3') (SEQ ID NO: 22) and BMJ180 (5'-AGC-CAC-AGA-GTT-TGA-GGT-C-3') (SEQ ID NO: 23). Cloning and construction of recombinant baculovirus expressing S-1 VP7 was previously described (14). DNA fragments of segment 2 and 5 were cloned into vector pVL1393 and transfections were performed with the Bac-N-Blue transfection kit (Gibco, Grand Island, N.Y.). Baculovirus constructs were amplified in Spodoptera frugiperda 9 (Sf9) cell culture for 2 passages, plaque purified, and then amplified for two more passages in Sf9 cells in serum-free HyQ SFX-Insect media (Hyclone, Logan, Utah).
[0203] FIG. 5 provides an amino acid sequence alignment for VP2 from strain ASP88 described above (SEQ ID NO: 1); human group C VP2 strain referred to as "Bristol" with protein (SEQ ID NO: 16) has NCBI Accession CAC 44890, version CAC 44890.1 GI: 15027005; as well as the porcine VP2 referred to as "Cowden" (SEQ ID NO: 17).
[0204] FIG. 6 is a nucleotide sequence alignment of sequences encoding human Group C VP-2 for inventive strain ASP88 (SEQ ID NO: 18), Cowden porcine strain (SEQ ID No. 44) and Bristol (SEQ ID NO: 19, Accession AJ303139). The start and stop codons are underlined.
[0205] FIG. 7 is a nucleotide sequence alignment of sequences encoding human Group C VP-6 for inventive strain S-1 relative to conventional strains Bristol (SEQ ID NO: 25, Accession CAA42504); Jajeri (SEQ ID NO: 26, Accession AAK26534); CMH004 (SEQ ID NO: 27, Accession ABR31794); V508 (SEQ ID NO: 28, Accession AAX13496); China (SEQ ID NO: 29, Accession BAB83829); and BCN6 (SEQ ID NO: 30, Accession CAJ41549). It is noted that FIG. 7 provides the sequence comparison in a format standard in the art wherein a "dot" indicates identity with a reference sequence. In FIG. 7, the reference sequence is a consensus sequence (SEQ ID No. 41).
[0206] FIG. 8 is an amino acid sequence alignment of sequences encoding human Group C VP-6 for inventive strain S-1 (SEQ ID NO: 32) relative to conventional strains Bristol (SEQ ID NO: 34, Accession CAA42504); Jajeri (SEQ ID NO: 35, Accession AAK26534); CMH004 (SEQ ID NO: 36, Accession ABR31794); V508 (SEQ ID NO: 37, Accession AAX13496); China (SEQ ID NO: 38, Accession BAB83829); and BCN6 (SEQ ID NO: 39, Accession CAJ41549). It is noted that FIG. 8 provides the sequence comparison in a format standard in the art wherein a "dot" indicates identity with a reference sequence. In FIG. 8, the reference sequence is a consensus sequence (SEQ ID No. 24).
TABLE-US-00003 TABLE I Comparison of VP2 Genes of Group C Rotaviruses Asp88 Bristol3 Cowden2 ORF 2652 2652 2652 Size (aa) 884 884 884 MW (kDa)4 101.57 101.67 101.68 Nucleotide and amino acid homology Asp88 -- 97.2 83.2 Bristol 98.5 -- 82.9 Cowden 92.8 92.6 --
[0207] Table I shows results of a comparison of VP2 Genes of Group C rotaviruses. MEGA version 4 program was used for the sequence analysis of the VP2 genes containing a single ORF extending from nt 37-2688. Results indicate that the nucleotide sequence encoding Asp88 VP2 has 97.2% homology to the nucleotide sequence encoding Bristol VP2, the nucleotide sequence encoding Asp88 VP2 has 83.2% homology to the nucleotide sequence encoding Cowden (porcine) VP2, and the nucleotide sequence encoding Bristol VP2 has 82.9% homology to the nucleotide sequence encoding Cowden VP2. Further, the amino acid sequence of Asp88 VP2 has 98.5% homology to the amino acid sequence of Bristol VP2, the amino acid sequence of Asp88 VP2 has 92.8% homology to the amino acid sequence of Cowden (porcine) VP2, and the amino acid sequence of Bristol VP2 has 92.6% homology to the amino acid sequence of Cowden VP2. Accession numbers of group C rotavirus Bristol strain is AJ303139. 2Cowden VP2 sequence was resequenced. 3 Bristol sequence is found in Chen, Z. et al, 2002.
Example 2
[0208] Cells and superinfections. Sf9 or High Five (Hi5) insect cells were grown and maintained in EX-CELL 420 or 405 media (Sigma, Lenexa, Kans.) or HyQ SFX-INSECT media in shaker flasks at 27° C. Sf9 and Hi5 cells were subcultured every 3 or 4 days at a concentration of 1×106 cells/ml and 5×105 cells/ml, respectively. Stationary superinfections were performed by seeding Sf9 cells in HyQ or EX-CELL 420 and Hi5 cells in HyQ or EX-CELL 405 into a T150 flask at a concentration of 3×105 cells/ml. Baculovirus constructs (rVP2, rVP6 and rVP7) were added at a multiplicity of infection (MOI) of 1 each. Infections were carried out without proteinase inhibitors and infected cultures were harvested at day 5. Large scale VLP production was performed in suspension culture by seeding Sf9 cells in EX-CELL 420 into fernbach flasks at a concentration of 1×106 cells/ml. Baculovirus recombinants were added one day later at an MOI of 1.4 each and harvested on day 4.
Example 3
[0209] Western blot. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was carried out with 12% separating and 5% stacking gels using the Laemmli discontinuous buffer system (16). Samples were heated at 97° C. for 5 min with 10% β-mecaptoethanol prior to loading and then electrophoresed. Proteins were transferred to a PVDF membrane in transfer buffer (25 mM Tris, 192 mM glycine, 10% methanol). After blocking with 10% (for unpurified GpC RV proteins) blotto in PBS-T for 1-2 hrs at room temperature or 15% (for purified GpC RV proteins) blotto in PBS-T overnight at 4° C., membranes were incubated with porcine hyperimmune serum (1:2,000) to Cowden in 5% blotto in PBS-T overnight at 4° C. or rabbit hyperimmune serum (1:20,000) to human GpC VLPs in 10% blotto in PBS-T for 1 h. Membranes were washed in PBS-T, incubated with horseradish peroxidase (HRP) goat anti-pig (1:142,000) (KPL, Gaithersburg, Md.) in 5% blotto or HRP-goat anti rabbit (1:20,000) (Pierce, Rockford, Ill.) in 10% blotto. GpC RV proteins were visualized with Supersignal West Femto Maximum Sensitivity Substrate Kit (Pierce, Rockford, Ill.) by exposing membranes to film and processing with medical film processor SRX-101A (Konica Minolta, Jakarta, Indonesia).
Example 4
[0210] Purification of VLPs. Infected cell cultures were clarified twice at 15,344×g for 30 min at 4° C. with a JA-14 rotor in J2-MC centrifuge. Clarified supernatants were layered over 35% sucrose cushions and centrifuged for two hrs at 107,000×g (4° C.) with the SW32Ti rotor in an Optima L-80 XP Ultracentrifuge (Beckman Coulter, Fullerton, Calif.). Pellets were resuspended in TNC buffer (10 mM Tris pH 7.4, 140 mM NaC1, 5 mM CaC12), re-clarified in a microcentrifuge and treated twice sequentially with equal volume Vertrel (Miller-Stephenson, Danbury, Conn.). Samples were centrifuged 10 min at 2,095×g (4° C.) with a SX4750A rotor in the Allegra X-12R tabletop centrifuge (Beckman Coulter, Fullerton, Calif.). The aqueous layer was overlaid on top of a CsCl solution (1.2738 g/ml) and centrifuged 17-18 hrs at 111,000×g (4° C.) with the SW40Ti rotor. Fractions that contained VLPs were collected, diluted in TNC, and pelleted out by centrifugation at 107,000×g (4° C.) for 1 hr in the SW32Ti rotor. Particles were resuspended in Hanks balanced salt solution (Gibco, Grand Island, N.Y.) supplemented with 10% sorbitol.
Example 5
[0211] Electron microscopy and immunoelectron microscopy. GpC RV VLPs were examined by electron microscopy (EM) and immunoelectron microscopy (IEM) as previously described with modifications (19). Briefly, 1% ammonium molybdate-1% trehalose in water (pH 6.95) was used to provide negative contrast on specimens adsorbed to nickel formvar-carbon filmed grids (9). Each grid was pretreated with 1% alcian blue 8GX in water to enhance hydrophilicity and provide cationic charges to the film surface prior to applying specimens. IEM was done by mixing 1 μl of purified GpC RV VLPs or GpA RV RRV with 1 μl of rabbit antibody to GpA or GpC RV diluted 1:500 and applying to nickel formvar-carbon coated grids. After incubation for 0.5-1 hr, the grids were blotted with filter paper, rinsed with 0.1 M Tris buffer supplemented with 0.4% acetylated bovine serum albumin (BSA) (Aurion, Hatfield, Pa.), and incubated for 30 min with goat anti-rabbit secondary antibody conjugated to 6 nm colloidal gold (1:20). Grids were rinsed twice with Tris buffer without BSA and with deionized water, blotted, stained with ammonium molybdate-trehalose, and viewed within an FEI Technai BioTwin transmission electron microscope at 120 KV accelerating voltage. Images were captured digitally with a 2K×2K AMT digital camera.
Example 6
[0212] Production of antisera to GpC VLPs. Rabbits (Covance, Denver, Pa.) were screened for the presence of GpA and C RV antibodies by antigen capture EIA prior to immunization. Rabbit CD94, which tested negative for GpA and C antigens, was selected for antibody production. CD94 was injected subcutaneously with 50 μg GpC VLPs formulated in Freud's complete adjuvant. Subsequent doses formulated with Freud's incomplete adjuvant were administered three weeks after the previous injection. Five injections in total were administered. The first bleed was 10 days after dose two and all subsequent bleeds were scheduled three weeks after the previous bleed. All bleeds tested positive for GpC RV antibody and were pooled.
Example 7
[0213] Enzyme Immunoassays (EIAs). 96-well plates were coated with 100 μl supernatant from GpA RRV infected MA104 cell cultures or GpC VLPs in recombinant baculovirus-infected Sf9 cells diluted (1:100) in coating buffer and incubated overnight at 37° C. Plates were washed with PBS-T and then blocked with 150 μl 5% blotto for 1 hr at 37° C. Plates were washed and then incubated with 1000 of serially diluted hyperimmune serum from rabbits CD94, CD8, and 8807A in diluent (1% blotto, 0.5% polyoxethylene ether W1 in PBS) for 2 hrs at 37° C. Rabbit CD8 was immunized with GpA RV RRV, whereas rabbit 8807A was naturally infected with GpA RV and also immunized with GpC RV Cowden. Plates were washed and then incubated with HRP goat anti-rabbit IgG (KPL, Gaithersburg, Md.) diluted (1:3,000) in diluent for 1 hr at 37° C. Plates were washed 6 times with PBS-T and then reacted with 100 μl of tetramethyl benzidine (TMB) for 10 min. Reactions were stopped with 100 μl 1N HC1 and plates were read at Abs450. Antibody titers were defined as the reciprocal of the highest dilution of serum giving a net optical density (OD) value (OD with virus minus OD with blotto) above 0.1.
Example 8
[0214] Kinetics of GpC RV protein synthesis in Sf9 and Hi5 cells. Sf9 and Hi5 cells in EX-CELL or HyQ media were infected with GpC RV VP2, VP6, and VP7 baculovirus recombinants at an MOI of 1 each and infected cultures were harvested on days 3, 4 and 5. Infected cultures were clarified and analyzed by Western blot using Cowden specific porcine hyperimmune serum to determine the expression profiles of proteins secreted into the supernatant. FIGS. 1A and 1B are electrophoretic gels showing the kinetics of GpC RV VP6 and VP7 expression in Sf9, FIG. 1A, or Hi5, FIG. 1B, cells in different media. In both FIGS. 1A and 1B, lane 1, Cowden strain; lanes 2-5, infected cultures in HyQ harvested 0, 3-5 dpi; and lanes 6-9, infected cultures in EX-CELL harvested at 0, 3-5 dpi. GpC RV VP6 and VP7 are indicated on the right. Molecular markers 54 kDa and 37.5 kDa are indicated by arrows on the left.
[0215] Expression of GpC RV VP6 and VP7 increased with time, with the highest levels seen at 4 or 5 days post infection (dpi) in Sf9 and Hi5 cells. Use of EX-CELL media resulted in higher rotavirus protein yields in both cell lines. Higher protein expression was achieved in EX-CELL media than in HyQ medium and similar levels of protein expression were observed in Sf9 and Hi5 cells. The cell line Sf9 and EX-CELL 420 were used in further examples of GpC RV protein production described herein. Human GpC RV VP2 was not detectable with the serum used.
Example 9
[0216] Self-assembly and characterization of GpC RV VLPs. Superinfections of Sf9 cells with rVP2, rVP6 and rVP7 at an MOI of 1 each resulted in the formation of intact GpC RV VLPs that have the structural order of typical rotavirus.
[0217] FIG. 2A is an image of an electron micrograph showing VLPs purified from cultures of Sf9 cells in EX-CELL 420 medium that were infected with recombinant baculoviruses encoding human rotavirus C VP2, VP6, and VP7 at an MOI of 1 each. FIG. 2B is an image of an electron micrograph showing VLPs purified from cultures of Sf9 cells in EX-CELL 420 medium that were infected with recombinant baculoviruses encoding human rotavirus C VP6 and VP7 at an MOI of 1.4 each. VLPs shown in FIGS. 2A and 2B were stained with 5% ammonium molybdate-1% trehalose. The bar in FIGS. 2A and 2B represents 100 nm.
[0218] Sf9 cells were also superinfected with all three recombinant viruses at various MOIs (0.1, 0.2, 1 and 2 each) in order to optimize conditions for VLP production. EM analysis of supernatants demonstrated better VLP formation in superinfections performed at the higher MOIs of 1 and 2. To determine if VP2 is essential for VLP formation, superinfections were performed with and without rVP2 and analysis was performed by EM. Because robust VLP formation was demonstrated without rVP2, all subsequent experiments were performed excluding this recombinant.
[0219] Biochemical composition and antigenicity of purified VP6/7 VLPs were compared with a GpA RV strain, YK-1, by SDS-PAGE and Western blot. Images comparing major structural viral proteins from GpA RV YK-1 and GpC VLPs by SDS-PAGE and Western blot are shown in FIGS. 3A and 3B, respectively.
[0220] Proteins from purified YK-1 (lane 1) and VLPs (lane 2) were separated on a 12% SDS-PAGE and stained by coomassie blue, FIG. 3A, or analyzed by Western blot, FIG. 3B. For western blot, proteins were transferred to a PVDF membrane and incubated with rabbit hyperimmune serum to GpC VLPs. GpC RV VP6 and VP7 are indicated on the right. Arrows indicate molecular weight markers 54 kDa and 37.5 kDa.
[0221] Human GpC recombinant proteins VP6 and VP7 migrate at similar molecular weights and are present at similar ratios as seen in GpA YK-1, indicating a proper molar ratio in assembled VLPs, illustrate in FIG. 3A. Western blot performed with human GpC VLP specific antibody demonstrated that these human rotavirus C VLPs were antigenic and of good quality, illustrated in FIG. 3B.
Example 10
[0222] Antigenic reactivity of GpC RV antibody. Rabbit polyclonal antibody was produced to purified human rotavirus C VLPs. Prior to immunization, rabbit CD94 had no antibody to GpA or C RV and after inoculations developed a high antibody titer to GpC RV (Table II). In controls, rabbit CD8, immunized with RRV, gave strong positive response to GpA RV, whereas rabbit 8807A, which was naturally infected with GpA RV and immunized with GpC RV Cowden strain, had similar antibody titers to GpA and GpC RV antigens. Hyperimmune serum from rabbit CD94 was utilized to enhance EIA that employs only porcine hyperimmune sera (8). This assay proved to specifically detect GpC RV antigens (VLPs and Cowden) but did not react with other control samples, such as MA104, Sf9 cells, and GpA RV RRV.
TABLE-US-00004 TABLE II Antibody Titers in Hyperimmune Sera to GpA and C Rotaviruses Reciprocal of IgG Titer Rotavirus Group A (RRV) Group C (VLPs) Rabbit Antigen Pre Post Pre Post CD94 GpC VLPs <100 <100 <100 51,200 CD8 GpA RRV ND 25,600 ND <100 8807A GpC Cowden ND 1,600 ND 3,200 & GpA Hyperimmune sera were tested and antibody titers were determined as described in the text. Pre = pre-immunization serum. Post = post-immunization serum. ND = not determined.
[0223] Human GpC RV VLPs were further characterized by examining their antigenic properties with immunoelectron microscopy using rabbit hyperimmune sera to GpA and C RV
[0224] FIG. 4A shows GpC RV VLPs immunostained with GpC-specific rabbit hyperimmune serum. FIG. 4B shows GpC RV VLPs immunostained with GpA-specific rabbit hyperimmune serum. FIG. 4C shows GpA RV immunostained with GpC-specific rabbit hyperimmune serum. FIG. 4D shows GpA RV immunostained with GpA-specific rabbit hyperimmune serum. GpC RV VLPs were specifically labeled with GpC-specific hyperimmune serum and GpA RVs were heavily coated with GpA-specific hyperimmune serum. The bar in FIGS. 4A, 4B, 4C and 4D represents 100 nm.
[0225] GpC RV antibody specifically reacted with human GpC VLPs but not with GpA RV, RRV. Correspondingly, GpA antibody exhibited specific reactivity with RRV and not with GpC VLPs. These results indicate the occurrence of group-specific interactions between RV antigen and antibody and confirm the absence of cross-reactivities between GpA and GpC RV reagents.
TABLE-US-00005 [VP2 from human strain Asp88-amino acid sequence] (SEQ ID NO: 1) MISRNRRRNNQQKDIGKEKQLETIIDKEVKENKDSTKEDKLVVTEESNGDVTAVKEQSN NINLQKNDLVKEVMNIQNQTLNTVVAENKVEIEEIVKKYIPSYNTDSLIVKKLTEIQESSA KTYNTLFRLFTPVKSYLYDINGEKKLSTRWYWKLLKDDLPAGDYSVRQFFLSLYLNVLE GMPDYIMLRDMAVDNPYSAEAGKIVDGKSKEILVELYQDQMTEGYIRRYMSELRHKIS GETNTAKYPAILHPVDNELNQYFLEHQLIQPLTTRNIAELIPTQLYHDPNYVFNIDAAFLT NSRFVPPYLTQDRIGLHDGFESIWDSKTHADYVSARRFIPDLTELVDAEKQIKEMAAHLQ LEAITVQVESQFLAGISAAAANEAFKFIIGSVLSTRTIAVEFITSNYMSLASCMYLMTIMPS EIFLRESLVAMRLAIINTLIYPALGLAQMHYQAGEVRTPFELAEMRVANRSIRQWLHHC NTLQFGRQITEGIIHLRFTNDIMTGRIVNLFSTMLVALSSQPFATYPLDYKRSVQRALQLL SNRTAQIADLTRLIVYNYTTLSACIVMNMHLVGTLTVERIQATSLTSLMMLISNKTVIPEP SSLFSYFSSNINFLTNYNEQIDNVVAEIMAAYRLNLYQQKMLMLVTRFVSRLYIFDAPKI PPDQMYRLRNRLRNIPVERRRADVFRIIMNNRDLIEKTSERICQGVLLSYTPMPLTYVED VGLTNVINDTNNFQIINIEEIEKTGDYSAITNALLRDTPIILKGAIPYVTNSSVIDVLSKVDT TVFASIVKDRDISKLKPIKFIINSDSSEYYLVHNNKWTPTTTTAVYKARSQQFDIQHSVSM LESNLFFVVYNDLFKYIKTTTVLPINAVSYDGARIMQET [VP2 from human strain ASP88-nucleotide sequence] (SEQ ID NO: 18) TCGAGGACAAATCGTCCAAGATGATAAGCAGAAACAGGCGCAGAAATAAC CAACAAAAAGATATAGGAAAAGAGAAACAATTAGAGACTATAATTGACAA AGAAGTAAAGGAAAACAAAGATTCTACAAAAGAAGATAAGCTAGTAGTTA CGGAAGAAAGTAATGGTGACGTCACAGCTGTTAAAGAACAATCGAATAAT ATTAATTTACAAAAGAATGATTTGGTTAAAGAAGTCATGAATATACAGAA TCAAACATTAAATACAGTAGTTGCTGAGAATAAAGTTGAAATAGAAGAAA TAGTTAAAAAATACATTCCCTCATATAATACTGACAGCCTTATTGTTAAA AAGTTAACTGAAATCCAGGAATCAAGTGCTAAAACATATAATACATTATT CAGATTATTTACTCCAGTTAAAAGTTATTTATATGACATAAATGGTGAGA AAAAATTATCGACTAGATGGTATTGGAAATTGCTCAAAGATGATTTACCT GCTGGTGATTACTCAGTTAGACAATTCTTCCTGTCACTATATTTAAATGT TTTAGAGGGAATGCCCGATTACATAATGCTTCGTGATATGGCAGTGGATA ACCCATATTCAGCAGAAGCAGGTAAAATCGTAGATGGAAAGTCTAAAGAA ATTTTAGTTGAACTATATCAAGACCAAATGACAGAAGGGTATATTAGAAG ATATATGTCTGAATTAAGACATAAAATATCTGGAGAAACAAATACTGCAA AATATCCAGCTATTCTACATCCCGTGGATAATGAGCTTAATCAATACTTT CTTGAGCATCAGTTAATTCAACCATTAACTACAAGAAATATTGCAGAATT GATTCCAACTCAATTATATCATGATCCAAATTACGTTTTTAATATTGATG CAGCCTTTTTAACAAATTCAAGATTTGTTCCACCATACTTAACACAGGAT AGGATTGGATTACATGATGGATTCGAATCAATATGGGATTCAAAAACCCA TGCTGATTACGTTTCAGCTAGAAGATTTATACCTGATTTAACTGAACTGG TAGATGCTGAAAAGCAAATAAAAGAAATGGCTGCACATTTACAACTAGAG GCTATTACAGTACAGGTTGAATCACAATTTTTAGCGGGAATTAGTGCTGC TGCAGCTAATGAAGCGTTCAAATTTATAATTGGCTCAGTTTTATCTACCA GAACAATAGCTGTAGAATTCATAACCTCAAACTATATGTCGTTAGCATCA TGTATGTATTTAATGACTATTATGCCATCAGAGATTTTCTTGAGAGAATC ATTAGTTGCTATGCGATTAGCAATAATAAATACCCTTATTTATCCAGCTC TAGGTTTAGCGCAAATGCATTATCAAGCAGGTGAAGTGAGGACCCCATTC GAATTAGCTGAGATGCGAGTAGCTAATAGATCTATTAGACAATGGTTACA TCATTGTAATACACTTCAATTTGGTAGACAGATAACGGAAGGGATAATTC ATCTACGATTTACTAATGATATCATGACAGGTAGGATAGTGAACTTATTT TCAACAATGCTAGTAGCTTTATCATCTCAGCCATTCGCTACATATCCTTT AGACTATAAAAGATCTGTACAAAGAGCATTACAACTTTTATCAAATAGAA CAGCCCAAATAGCAGATTTAACCAGATTAATAGTATACAATTATACTACA TTATCTGCATGTATAGTCATGAATATGCATTTAGTAGGAACTCTTACTGT TGAACGTATACAGGCCACTTCTCTAACTTCTTTAATGATGTTAATTTCTA ATAAGACAGTTATTCCAGAACCATCGTCTCTTTTTTCATATTTCTCTAGT AACATTAATTTTCTTACAAATTATAATGAGCAAATTGATAATGTGGTAGC AGAAATAATGGCCGCATATAGATTGAATTTATATCAACAGAAAATGTTGA TGCTCGTTACCAGGTTTGTGTCAAGGTTGTACATATTTGATGCTCCTAAA ATACCGCCAGATCAGATGTATAGATTAAGAAACCGATTAAGAAATATTCC AGTTGAAAGAAGAAGAGCTGATGTGTTCAGAATTATTATGAATAATAGAG ATTTAATCGAAAAAACATCAGAACGTATATGTCAGGGTGTGTTGTTATCT TATACACCAATGCCTTTAACTTACGTTGAAGATGTCGGGTTAACAAATGT AATTAATGACACTAATAACTTCCAAATAATTAATATAGAAGAAATTGAGA AGACCGGTGACTATTCAGCCATAACGAATGCATTACTTCGGGATACTCCA ATTATATTGAAAGGTGCGATTCCATATGTTACTAACTCATCAGTAATTGA TGTTTTATCTAAAGTGGACACCACAGTGTTCGCAAGCATCGTAAAAGATA GGGATATTTCAAAGTTAAAACCAATAAAATTCATAATTAATTCAGATTCA TCCGAATATTATTTAGTACACAATAATAAATGGACACCAACAACAACTAC AGCAGTATATAAAGCTAGATCTCAGCAATTTGATATACAACATTCAGTAT CAATGCTAGAGTCAAACTTATTTTTTGTGGTATATAATGATTTATTTAAA TACATTAAAACCACTACAGTTCTGCCGATAAATGCTGTCTCTTATGATGG TGCAAGAATTATGCAAGAAACATAAATGATTGTATAGTATCATCTTGTAA CGACCTCAAACTCTGTGGCT [VP2 open reading frame from human strain ASP88-nucleotide sequence] (SEQ ID NO: 42) ATGATAAGCAGAAACAGGCGCAGAAATAAC CAACAAAAAGATATAGGAAAAGAGAAACAATTAGAGACTATAATTGACAA AGAAGTAAAGGAAAACAAAGATTCTACAAAAGAAGATAAGCTAGTAGTTA CGGAAGAAAGTAATGGTGACGTCACAGCTGTTAAAGAACAATCGAATAAT ATTAATTTACAAAAGAATGATTTGGTTAAAGAAGTCATGAATATACAGAA TCAAACATTAAATACAGTAGTTGCTGAGAATAAAGTTGAAATAGAAGAAA TAGTTAAAAAATACATTCCCTCATATAATACTGACAGCCTTATTGTTAAA AAGTTAACTGAAATCCAGGAATCAAGTGCTAAAACATATAATACATTATT CAGATTATTTACTCCAGTTAAAAGTTATTTATATGACATAAATGGTGAGA AAAAATTATCGACTAGATGGTATTGGAAATTGCTCAAAGATGATTTACCT GCTGGTGATTACTCAGTTAGACAATTCTTCCTGTCACTATATTTAAATGT TTTAGAGGGAATGCCCGATTACATAATGCTTCGTGATATGGCAGTGGATA ACCCATATTCAGCAGAAGCAGGTAAAATCGTAGATGGAAAGTCTAAAGAA ATTTTAGTTGAACTATATCAAGACCAAATGACAGAAGGGTATATTAGAAG ATATATGTCTGAATTAAGACATAAAATATCTGGAGAAACAAATACTGCAA AATATCCAGCTATTCTACATCCCGTGGATAATGAGCTTAATCAATACTTT CTTGAGCATCAGTTAATTCAACCATTAACTACAAGAAATATTGCAGAATT GATTCCAACTCAATTATATCATGATCCAAATTACGTTTTTAATATTGATG CAGCCTTTTTAACAAATTCAAGATTTGTTCCACCATACTTAACACAGGAT AGGATTGGATTACATGATGGATTCGAATCAATATGGGATTCAAAAACCCA TGCTGATTACGTTTCAGCTAGAAGATTTATACCTGATTTAACTGAACTGG TAGATGCTGAAAAGCAAATAAAAGAAATGGCTGCACATTTACAACTAGAG GCTATTACAGTACAGGTTGAATCACAATTTTTAGCGGGAATTAGTGCTGC TGCAGCTAATGAAGCGTTCAAATTTATAATTGGCTCAGTTTTATCTACCA GAACAATAGCTGTAGAATTCATAACCTCAAACTATATGTCGTTAGCATCA TGTATGTATTTAATGACTATTATGCCATCAGAGATTTTCTTGAGAGAATC ATTAGTTGCTATGCGATTAGCAATAATAAATACCCTTATTTATCCAGCTC TAGGTTTAGCGCAAATGCATTATCAAGCAGGTGAAGTGAGGACCCCATTC GAATTAGCTGAGATGCGAGTAGCTAATAGATCTATTAGACAATGGTTACA TCATTGTAATACACTTCAATTTGGTAGACAGATAACGGAAGGGATAATTC ATCTACGATTTACTAATGATATCATGACAGGTAGGATAGTGAACTTATTT TCAACAATGCTAGTAGCTTTATCATCTCAGCCATTCGCTACATATCCTTT AGACTATAAAAGATCTGTACAAAGAGCATTACAACTTTTATCAAATAGAA CAGCCCAAATAGCAGATTTAACCAGATTAATAGTATACAATTATACTACA TTATCTGCATGTATAGTCATGAATATGCATTTAGTAGGAACTCTTACTGT TGAACGTATACAGGCCACTTCTCTAACTTCTTTAATGATGTTAATTTCTA ATAAGACAGTTATTCCAGAACCATCGTCTCTTTTTTCATATTTCTCTAGT AACATTAATTTTCTTACAAATTATAATGAGCAAATTGATAATGTGGTAGC AGAAATAATGGCCGCATATAGATTGAATTTATATCAACAGAAAATGTTGA TGCTCGTTACCAGGTTTGTGTCAAGGTTGTACATATTTGATGCTCCTAAA ATACCGCCAGATCAGATGTATAGATTAAGAAACCGATTAAGAAATATTCC AGTTGAAAGAAGAAGAGCTGATGTGTTCAGAATTATTATGAATAATAGAG ATTTAATCGAAAAAACATCAGAACGTATATGTCAGGGTGTGTTGTTATCT TATACACCAATGCCTTTAACTTACGTTGAAGATGTCGGGTTAACAAATGT AATTAATGACACTAATAACTTCCAAATAATTAATATAGAAGAAATTGAGA AGACCGGTGACTATTCAGCCATAACGAATGCATTACTTCGGGATACTCCA ATTATATTGAAAGGTGCGATTCCATATGTTACTAACTCATCAGTAATTGA TGTTTTATCTAAAGTGGACACCACAGTGTTCGCAAGCATCGTAAAAGATA GGGATATTTCAAAGTTAAAACCAATAAAATTCATAATTAATTCAGATTCA TCCGAATATTATTTAGTACACAATAATAAATGGACACCAACAACAACTAC AGCAGTATATAAAGCTAGATCTCAGCAATTTGATATACAACATTCAGTAT CAATGCTAGAGTCAAACTTATTTTTTGTGGTATATAATGATTTATTTAAA TACATTAAAACCACTACAGTTCTGCCGATAAATGCTGTCTCTTATGATGG TGCAAGAATTATGCAAGAAACATAA VP2 from human strain ASP88-nucleotide sequence including 36 5' non-coding bases (SEQ ID NO: 43) GGCTTAAAAAGATCAG TCGAGGACAAATCGTCCAAGATGATAAGCAGAAACAGGCGCAGAAATAAC CAACAAAAAGATATAGGAAAAGAGAAACAATTAGAGACTATAATTGACAA AGAAGTAAAGGAAAACAAAGATTCTACAAAAGAAGATAAGCTAGTAGTTA CGGAAGAAAGTAATGGTGACGTCACAGCTGTTAAAGAACAATCGAATAAT ATTAATTTACAAAAGAATGATTTGGTTAAAGAAGTCATGAATATACAGAA TCAAACATTAAATACAGTAGTTGCTGAGAATAAAGTTGAAATAGAAGAAA TAGTTAAAAAATACATTCCCTCATATAATACTGACAGCCTTATTGTTAAA AAGTTAACTGAAATCCAGGAATCAAGTGCTAAAACATATAATACATTATT CAGATTATTTACTCCAGTTAAAAGTTATTTATATGACATAAATGGTGAGA AAAAATTATCGACTAGATGGTATTGGAAATTGCTCAAAGATGATTTACCT GCTGGTGATTACTCAGTTAGACAATTCTTCCTGTCACTATATTTAAATGT TTTAGAGGGAATGCCCGATTACATAATGCTTCGTGATATGGCAGTGGATA ACCCATATTCAGCAGAAGCAGGTAAAATCGTAGATGGAAAGTCTAAAGAA ATTTTAGTTGAACTATATCAAGACCAAATGACAGAAGGGTATATTAGAAG ATATATGTCTGAATTAAGACATAAAATATCTGGAGAAACAAATACTGCAA AATATCCAGCTATTCTACATCCCGTGGATAATGAGCTTAATCAATACTTT CTTGAGCATCAGTTAATTCAACCATTAACTACAAGAAATATTGCAGAATT GATTCCAACTCAATTATATCATGATCCAAATTACGTTTTTAATATTGATG CAGCCTTTTTAACAAATTCAAGATTTGTTCCACCATACTTAACACAGGAT AGGATTGGATTACATGATGGATTCGAATCAATATGGGATTCAAAAACCCA TGCTGATTACGTTTCAGCTAGAAGATTTATACCTGATTTAACTGAACTGG TAGATGCTGAAAAGCAAATAAAAGAAATGGCTGCACATTTACAACTAGAG GCTATTACAGTACAGGTTGAATCACAATTTTTAGCGGGAATTAGTGCTGC TGCAGCTAATGAAGCGTTCAAATTTATAATTGGCTCAGTTTTATCTACCA GAACAATAGCTGTAGAATTCATAACCTCAAACTATATGTCGTTAGCATCA TGTATGTATTTAATGACTATTATGCCATCAGAGATTTTCTTGAGAGAATC ATTAGTTGCTATGCGATTAGCAATAATAAATACCCTTATTTATCCAGCTC TAGGTTTAGCGCAAATGCATTATCAAGCAGGTGAAGTGAGGACCCCATTC GAATTAGCTGAGATGCGAGTAGCTAATAGATCTATTAGACAATGGTTACA TCATTGTAATACACTTCAATTTGGTAGACAGATAACGGAAGGGATAATTC ATCTACGATTTACTAATGATATCATGACAGGTAGGATAGTGAACTTATTT TCAACAATGCTAGTAGCTTTATCATCTCAGCCATTCGCTACATATCCTTT AGACTATAAAAGATCTGTACAAAGAGCATTACAACTTTTATCAAATAGAA CAGCCCAAATAGCAGATTTAACCAGATTAATAGTATACAATTATACTACA TTATCTGCATGTATAGTCATGAATATGCATTTAGTAGGAACTCTTACTGT TGAACGTATACAGGCCACTTCTCTAACTTCTTTAATGATGTTAATTTCTA ATAAGACAGTTATTCCAGAACCATCGTCTCTTTTTTCATATTTCTCTAGT AACATTAATTTTCTTACAAATTATAATGAGCAAATTGATAATGTGGTAGC AGAAATAATGGCCGCATATAGATTGAATTTATATCAACAGAAAATGTTGA TGCTCGTTACCAGGTTTGTGTCAAGGTTGTACATATTTGATGCTCCTAAA ATACCGCCAGATCAGATGTATAGATTAAGAAACCGATTAAGAAATATTCC AGTTGAAAGAAGAAGAGCTGATGTGTTCAGAATTATTATGAATAATAGAG ATTTAATCGAAAAAACATCAGAACGTATATGTCAGGGTGTGTTGTTATCT TATACACCAATGCCTTTAACTTACGTTGAAGATGTCGGGTTAACAAATGT AATTAATGACACTAATAACTTCCAAATAATTAATATAGAAGAAATTGAGA AGACCGGTGACTATTCAGCCATAACGAATGCATTACTTCGGGATACTCCA ATTATATTGAAAGGTGCGATTCCATATGTTACTAACTCATCAGTAATTGA TGTTTTATCTAAAGTGGACACCACAGTGTTCGCAAGCATCGTAAAAGATA GGGATATTTCAAAGTTAAAACCAATAAAATTCATAATTAATTCAGATTCA TCCGAATATTATTTAGTACACAATAATAAATGGACACCAACAACAACTAC AGCAGTATATAAAGCTAGATCTCAGCAATTTGATATACAACATTCAGTAT CAATGCTAGAGTCAAACTTATTTTTTGTGGTATATAATGATTTATTTAAA TACATTAAAACCACTACAGTTCTGCCGATAAATGCTGTCTCTTATGATGG TGCAAGAATTATGCAAGAAACATAAATGATTGTATAGTATCATCTTGTAA CGACCTCAAACTCTGTGGCT [VP6 from human strain S-1-nucleotide sequence] (SEQ ID NO: 31) * 20 * 40 * 60 * 80 ATGGATGTACTTTTTTCTATAGCGAAAACCGTGTCAGATCTTAAAGAGAAAGTTGTAGTTGGAACAATTTATAC- TAATGT * 100 * 120 * 140 * 160 AGAAGATGTTGTACAACAGACGAATGAATTGATTAGAACTTTGAATGGAAATATTTTTCATACTGGTGGCATTG- GAACAC * 180 * 200 * 220 * 240 AGCCTCAGAAAGAGTGGAATTTTCAGCTCCCACAATTGGGTACCACTTTATTAAATTTAGATGATAATTATGTT- CAATCA * 260 * 280 * 300 * 320 ACTAGAGGCATAATTGATTTTTTATCATCTTTTATAGAAGCTGTATGTGATGATGAAATTGTTAGAGAAGCTTC- AAGAAA * 340 * 360 * 380 * 400 TGGTATGCAACCTCAATCACCAGCTCTTATATTATTATCTTCATCAAAATTTAAAACAATTAATTTTAATAATA- GTTCTC * 420 * 440 * 460 * 480 AATCTATCAAAAATTGGAATGCTCAATCAAGACGTGAGAATCCTGTATATGAGTACAAAAATCCAATGTTGTTT- GAATAT * 500 * 520 * 540 * 560 AAAAATTCTTATATTTTACAACGCGCAAATCCACAATTTGGAAGCGTCATGGGTTTAAGATATTATACAACAAG- TAATAT * 580 * 600 * 620 * 640 TTGTCAAATTGCAGCATTTGATTCCACCCTAGCTGAAAATGCACCAAATAATACGCAACGCTTCGTTTATAATG- GCAGAC * 660 * 680 * 700 * 720 TAAAAAGACCCATATCAAATGTTTTAATGAAAATAGAAGCTGGTGCTCCAAATATAAGCAACCCAACTATTTTA- CCTGAT * 740 * 760 * 780 * 800 CCTAATAATCAAACAACTTGGCTTTTTAATCCGGTACAATTAATGAATGGAACATTTACCATTGAATTCTATAA- TAATGG * 820 * 840 * 860 * 880 TCAACTAATTGATATGGTTCGAAATATGGGAATAGTTACTGTAAGAACTTTTGATTCTTATAGAATAACAATTG- ACATGA * 900 * 920 * 940 * 960 TTAGACCAGCTGCTATGACTCAATACGTTCAACGAATTTTTCCACAAGGTGGACCTTATCATTTTCAGGCTACA- TATATG * 980 * 1000 * 1020 * 1040 TTAACATTAAGTATATTAGATGCTACCACAGAGTCCGTTCTATGTGATTCTCATTCAGTAGAATATTCAATAGT- AGCAAA * 1060 * 1080 * 1100 * 1120 CGTCAGAAGAGATTCAGCAATGCCAGCTGGAACTGTTTTTCAACCGGGATTTCCATGGGAACACACACTATCCA-
ATTACA * 1140 * 1160 * 1180 CTGTTGCTCAAGAAGATAATTTAGAAAGATTATTGTTAATCGCATCTGTGAAGAGAATGGTAATG [VP6 from human strain S-1-amino acid sequence] (SEQ ID NO: 32) MDVLFSIAKTVSDLKEKVVVGTIYTNVEDVVQQTNELIRTLNGNIFHTGG [50] IGTQPQKEWNFQLPQLGTTLLNLDDNYVQSTRGIIDFLSSFIEAVCDDEI [100] VREASRNGMQPQSPALILLSSSKFKTINFNNSSQSIKNWNAQSRRENPVY [150] EYKNPMLFEYKNSYILQRANPQFGSVMGLRYYTTSNICQIAAFDSTLAEN [200] APNNTQRFVYNGRLKRPISNVLMKIEAGAPNISNPTILPDPNNQTTWLFN [250] PVQLMNGTFTIEFYNNGQLIDMVRNMGIVTVRTFDSYRITIDMIRPAAMT [300] QYVQRIFPQGGPYHFQATYMLTLSILDATTESVLCDSHSVEYSIVANVRR [350] DSAMPAGTVFQPGFPWEHTLSNYTVAQEDNLERLLLIASVKRMVM [395] [VP7 from human strain S-1-nucleotide sequence] (SEQ ID NO: 33) >gi|1314237|gb|U20995.1|RGU20995 Human rotavirus group C isolate S-1 outer capsid glycoprotein (VP7) gene, complete cds GGCATTTAAAAAAGAAGAAGCTGTCTGACAAACTGGTCTTCTTTTTAAATGGTTTGTACAACATTGTACA CTGTTTGCGCCATTCTCTTCATTCTTTTCATTTATATATTATTATTTAGAAAAATGTTCCACCTAATAAC TGATACTTTAATAGTGATGCTTATTTTATCTAATTGTGTAGAGTGGTCACAAGGTCAGATGTTTATTGAT GATATACATTATAATGGTAACGTTGAGACTATCATAAATTCTACTGATCCTTTTAATGTTGAATCTTTAT GTATTTATTTTCCAAATGCAGTTGTAGGATCACAGGGACCAGGTAAATCCGATGGACATTTGAATGATGG TAATTATGCACAGACTATCGCCACTTTGTTTGAAACAAAAGGATTCCCAAAAGGTTCAATAATAATTAAA ACATATACACAGACATCAGACTTTATAAATTCAGTAGAAATGACATGCTCTTATAATATAGTTATCATTC CTGATAGCCCAAATGATTCAGAATCTATTGAACAGATAGCAGAATGGATTTTAAATGTTTGGAGATGTGA TGACATGAATTTGGAAATTTATACTTATGAACAAATTGGAATAAACAATTTATGGGCTGCATTTGGTAGT GACTGTGATATATCTGTCTGTCCATTAGATACTACAAGTAATGGAATCGGATGTTCACCAGCTAGTACAG AAACTTATGAAGTTGTATCAAATGACACCCAATTGGCCTTAATTAATGTTGTGGATAATGTTAGACATAG AATACAGATGAACACTGCTCAATGTAAATTGAAAAATTGTATTAAGGGTGAAGCTCGACTGAATACTGCA CTAATAAGAATTTCAACATCATCAAGTTTTGATAATTCATTGTCACCATTAAATAACGGCCAAACAACAA GATCGTTTAAAATAAATGCAAAGAAATGGTGGACTATATTTTATACAATAATTGATTATATTAATACAAT TGTACAATCAATGACTCCCAGACATCGGGCGATTTATCCAGAAGGGTGGATGTTGAGGTATGCGTAAACA AGATCATGTGGCT [VP7 from human strain S-1-nucleotide sequence of open reading frame] (SEQ ID NO: 45) ATGGTTTGTACAACATTGTACA CTGTTTGCGCCATTCTCTTCATTCTTTTCATTTATATATTATTATTTAGAAAAATGTTCCACCTAATAAC TGATACTTTAATAGTGATGCTTATTTTATCTAATTGTGTAGAGTGGTCACAAGGTCAGATGTTTATTGAT GATATACATTATAATGGTAACGTTGAGACTATCATAAATTCTACTGATCCTTTTAATGTTGAATCTTTAT GTATTTATTTTCCAAATGCAGTTGTAGGATCACAGGGACCAGGTAAATCCGATGGACATTTGAATGATGG TAATTATGCACAGACTATCGCCACTTTGTTTGAAACAAAAGGATTCCCAAAAGGTTCAATAATAATTAAA ACATATACACAGACATCAGACTTTATAAATTCAGTAGAAATGACATGCTCTTATAATATAGTTATCATTC CTGATAGCCCAAATGATTCAGAATCTATTGAACAGATAGCAGAATGGATTTTAAATGTTTGGAGATGTGA TGACATGAATTTGGAAATTTATACTTATGAACAAATTGGAATAAACAATTTATGGGCTGCATTTGGTAGT GACTGTGATATATCTGTCTGTCCATTAGATACTACAAGTAATGGAATCGGATGTTCACCAGCTAGTACAG AAACTTATGAAGTTGTATCAAATGACACCCAATTGGCCTTAATTAATGTTGTGGATAATGTTAGACATAG AATACAGATGAACACTGCTCAATGTAAATTGAAAAATTGTATTAAGGGTGAAGCTCGACTGAATACTGCA CTAATAAGAATTTCAACATCATCAAGTTTTGATAATTCATTGTCACCATTAAATAACGGCCAAACAACAA GATCGTTTAAAATAAATGCAAAGAAATGGTGGACTATATTTTATACAATAATTGATTATATTAATACAAT TGTACAATCAATGACTCCCAGACATCGGGCGATTTATCCAGAAGGGTGGATGTTGAGGTATGCGTAA [VP7 from human strain S-1-amino acid sequence] (SEQ ID NO: 34) MVCTTLYTVCAILFILFIYILLFRKMFHLITDTLIVMLILSNCVEWSQGQMFIDDIHYNG NVETIINSTDPFNVESLCIYFPNAVVGSQGPGKSDGHLNDGNYAQTIATLFLTKGFPKGS IIIKTYTQTSDFINSVEMTCSYNIVIIPDSPNDSESIEQIAEWILNVWRCDDMNLEIYTY EQIGINNLWAAFGSDCDISVCPLDTTSNGIGCSPASTETYEVVSNDTQLALINVVDNVRH RIQMNTAQCKLKNCIKGEARLNTALIRISTSSSFDNSLSPLNNGQTTRSFKINAKKWWTI FYTIIDYINTIVQSMTPRHRAIYPEGWMLRYA [VP6 from human strain S-1-nucleotide sequence including 5' and 3' non-coding; start and stop codons of ORF underlined] (SEQ ID No. 48) GGCATTTAAAATCTCATTCACAATGGATGTACTTTTTTCTATAGCGAAAACCGTGTCAGATCTTAAAGAGAAAG- TTGTAG TTGGAACAATTTATACTAATGTAGAAGATGTTGTACAACAGACGAATGAATTGATTAGAACTTTGAATGGAAAT- ATTTTT CATACTGGTGGCATTGGAACACAGCCTCAGAAAGAGTGGAATTTTCAGCTCCCACAATTGGGTACCACTTTATT- AAATTT AGATGATAATTATGTTCAATCAACTAGAGGCATAATTGATTTTTTATCATCTTTTATAGAAGCTGTATGTGATG- ATGAAA TTGTTAGAGAAGCTTCAAGAAATGGTATGCAACCTCAATCACCAGCTCTTATATTATTATCTTCATCAAAATTT- AAAACA ATTAATTTTAATAATAGTTCTCAATCTATCAAAAATTGGAATGCTCAATCAAGACGTGAGAATCCTGTATATGA- GTACAA AAATCCAATGTTGTTTGAATATAAAAATTCTTATATTTTACAACGCGCAAATCCACAATTTGGAAGCGTCATGG- GTTTAA GATATTATACAACAAGTAATATTTGTCAAATTGCAGCATTTGATTCCACCCTAGCTGAAAATGCACCAAATAAT- ACGCAA CGCTTCGTTTATAATGGCAGACTAAAAAGACCCATATCAAATGTTTTAATGAAAATAGAAGCTGGTGCTCCAAA- TATAAG CAACCCAACTATTTTACCTGATCCTAATAATCAAACAACTTGGCTTTTTAATCCGGTACAATTAATGAATGGAA- CATTTA CCATTGAATTCTATAATAATGGTCAACTAATTGATATGGTTCGAAATATGGGAATAGTTACTGTAAGAACTTTT- GATTCT TATAGAATAACAATTGACATGATTAGACCAGCTGCTATGACTCAATACGTTCAACGAATTTTTCCACAAGGTGG- ACCTTA TCATTTTCAGGCTACATATATGTTAACATTAAGTATATTAGATGCTACCACAGAGTCCGTTCTATGTGATTCTC- ATTCAG TAGAATATTCAATAGTAGCAAACGTCAGAAGAGATTCAGCAATGCCAGCTGGAACTGTTTTTCAACCGGGATTT- CCATGG GAACACACACTATCCAATTACACTGTTGCTCAAGAAGATAATTTAGAAAGATTATTGTTAATCGCATCTGTGAA- GAGAAT GGTAATGTAGATAAGCTAGAAGACTAAACATCTTCTATGCGGCCTACATACCATGTAGCATGAATCACGACTGG- GTTTAG TCCATGCTCGCATAGGGGCAAATATGCATGATATGGATGATCCCCAGAAGGATGAAATGTGAACTATGTGGCT
REFERENCES
[0226] 1. Abid, I., et al. 2007. Journal of Clinical Virology 38:78-82. [0227] 2. Banyai, K., B. et al. 2006. 37:317-22. [0228] 3. Berois, M., C. et al. 2003. 77:1757-63. [0229] 4. Castello, A. et al. 2002. 67:106-12. [0230] 5. Charpilienne, A., et al. 2002. Journal of Virology 76:7822-31. [0231] 6. Cox, M. J. et al. 1998. Tropical Medicine & International Health 3:891-5. [0232] 7. Crawford, S. E. et al. 1994. Journal of Virology 68:5945-52. [0233] 8. Gabbay, Y. B., et al. 1999. Journal of Diarrhoeal Diseases Research 17:69-74. [0234] 9. Harris, J. R., et al. 2006. Microscopy and Analysis 20:17-21. [0235] 10. Iturriza-Gomara, et al. 2004. European Journal of Epidemiology 19:589-95. [0236] 11. James, V. L., et al. 1997. UK. Journal of Medical Virology 52:86-91. [0237] 12. Jiang, B., et al. 1998 Biotechnology & Bioengineering 60:369-74. [0238] 13. Jiang, B., et al. 1995. Journal of Infectious Diseases 172:45-50. [0239] 14. Jiang, B., H. et al. 1996. Archives of Virology 141:381-90. [0240] 15. Kuzuya, M., et al. 1998. Journal of Clinical Microbiology 36:6-10. [0241] 16. Laemmli, U. K. 1970. Nature 227:680-5. [0242] 17. Mena, J. A., et al. 2006. J Biotechnol 122:443-52. [0243] 18. Mena, J. A., et al. 2007. BMC Biotechnol 7:39. [0244] 19. Milne, R. G. 1993. Solid Phase Immune Electron Microscopy of Virus Preparations, p. 25-70. In A. D. Hyatt and B. T. Eaton (ed.), Immuno-Gold Electron Microscopy in Virus Diagnosis and Research; CRC Press, Boca Raton, Fla. [0245] 20. Nilsson, M., et al. 2000. Journal of Infectious Diseases 182:678-84. [0246] 21. Otsu, R. 1998. Comparative Immunology, Microbiology & Infectious Diseases 21:75-80. [0247] 22. Palomares, L. A., et al. 2002. Biotechnol Bioeng 78:635-44. [0248] 23. Patton, J. T., and D. Chen. 1999. Journal of Virology 73:1382-91. [0249] 24. Patton, J. T., et al. 1997. 71:9618-26. [0250] 25. Phan, T. G., et al. 2004. Journal of Medical Virology 74:173-9. [0251] 26. Rahman, M., et al. 2005. Journal of Clinical Microbiology 43:4460-5. [0252] 27. Riepenhoff-Talty, M., K. et al. 1997. Journal of Clinical Microbiology 35:486-8. [0253] 28. Sanchez-Fauquier, A., E. et al. 2003. Archives of Virology 148:399-404. [0254] 29. Schnagl, R. D., et al. 2004. Journal of Clinical Microbiology 42:2127-33. [0255] 30. Souza, D. F., et al. 1998. An outbreak of group C rotavirus gastroenteritis among adults living in Valentim Gentil, Sao Paulo State, Brazil.[erratum appears in J Diarrhoeal Dis Res 1998 September; 16(3):following x]. Journal of Diarrhoeal Diseases Research 16:59-65. [0256] 31. Steele, A. D., and V. L. James. 1999. Journal of Clinical Microbiology 37:4142-4. [0257] 32. Steyer, A., M. et al. 2006. Journal of Medical Virology 78:1250-5. [0258] 33. Terrett, L. A., and L. J. Saif. 1987. Journal of Clinical Microbiology 25:1316-9. [0259] 34. Tsunemitsu, H., B. et al. 1992. Journal of Clinical Microbiology 30:2129-34. [0260] 35. Zeng, C. Q., et al. 1998. Journal of Virology 72:201-8. [0261] 36. Zeng, C. Q., et al. 1996. Journal of Virology 70:2736-42.
[0262] Any patents or publications mentioned in this specification are incorporated herein by reference to the same extent as if each individual publication is specifically and individually indicated to be incorporated by reference.
[0263] The compositions and methods described herein are presently representative of preferred embodiments, exemplary, and not intended as limitations on the scope of the invention. Changes therein and other uses will occur to those skilled in the art. Such changes and other uses can be made without departing from the scope of the invention as set forth in the claims. All numerical ranges described herein include all integers and decimal values within the range and are also inclusive of the endpoints.
Sequence CWU
1
471884PRTHuman rotavirus CMISC_FEATUREHuman rotavirus group C strain ASP88
VP2 protein 1Met Ile Ser Arg Asn Arg Arg Arg Asn Asn Gln Gln Lys Asp
Ile Gly1 5 10 15Lys Glu
Lys Gln Leu Glu Thr Ile Ile Asp Lys Glu Val Lys Glu Asn 20
25 30Lys Asp Ser Thr Lys Glu Asp Lys Leu
Val Val Thr Glu Glu Ser Asn 35 40
45Gly Asp Val Thr Ala Val Lys Glu Gln Ser Asn Asn Ile Asn Leu Gln 50
55 60Lys Asn Asp Leu Val Lys Glu Val Met
Asn Ile Gln Asn Gln Thr Leu65 70 75
80Asn Thr Val Val Ala Glu Asn Lys Val Glu Ile Glu Glu Ile
Val Lys 85 90 95Lys Tyr
Ile Pro Ser Tyr Asn Thr Asp Ser Leu Ile Val Lys Lys Leu 100
105 110Thr Glu Ile Gln Glu Ser Ser Ala Lys
Thr Tyr Asn Thr Leu Phe Arg 115 120
125Leu Phe Thr Pro Val Lys Ser Tyr Leu Tyr Asp Ile Asn Gly Glu Lys
130 135 140Lys Leu Ser Thr Arg Trp Tyr
Trp Lys Leu Leu Lys Asp Asp Leu Pro145 150
155 160Ala Gly Asp Tyr Ser Val Arg Gln Phe Phe Leu Ser
Leu Tyr Leu Asn 165 170
175Val Leu Glu Gly Met Pro Asp Tyr Ile Met Leu Arg Asp Met Ala Val
180 185 190Asp Asn Pro Tyr Ser Ala
Glu Ala Gly Lys Ile Val Asp Gly Lys Ser 195 200
205Lys Glu Ile Leu Val Glu Leu Tyr Gln Asp Gln Met Thr Glu
Gly Tyr 210 215 220Ile Arg Arg Tyr Met
Ser Glu Leu Arg His Lys Ile Ser Gly Glu Thr225 230
235 240Asn Thr Ala Lys Tyr Pro Ala Ile Leu His
Pro Val Asp Asn Glu Leu 245 250
255Asn Gln Tyr Phe Leu Glu His Gln Leu Ile Gln Pro Leu Thr Thr Arg
260 265 270Asn Ile Ala Glu Leu
Ile Pro Thr Gln Leu Tyr His Asp Pro Asn Tyr 275
280 285Val Phe Asn Ile Asp Ala Ala Phe Leu Thr Asn Ser
Arg Phe Val Pro 290 295 300Pro Tyr Leu
Thr Gln Asp Arg Ile Gly Leu His Asp Gly Phe Glu Ser305
310 315 320Ile Trp Asp Ser Lys Thr His
Ala Asp Tyr Val Ser Ala Arg Arg Phe 325
330 335Ile Pro Asp Leu Thr Glu Leu Val Asp Ala Glu Lys
Gln Ile Lys Glu 340 345 350Met
Ala Ala His Leu Gln Leu Glu Ala Ile Thr Val Gln Val Glu Ser 355
360 365Gln Phe Leu Ala Gly Ile Ser Ala Ala
Ala Ala Asn Glu Ala Phe Lys 370 375
380Phe Ile Ile Gly Ser Val Leu Ser Thr Arg Thr Ile Ala Val Glu Phe385
390 395 400Ile Thr Ser Asn
Tyr Met Ser Leu Ala Ser Cys Met Tyr Leu Met Thr 405
410 415Ile Met Pro Ser Glu Ile Phe Leu Arg Glu
Ser Leu Val Ala Met Arg 420 425
430Leu Ala Ile Ile Asn Thr Leu Ile Tyr Pro Ala Leu Gly Leu Ala Gln
435 440 445Met His Tyr Gln Ala Gly Glu
Val Arg Thr Pro Phe Glu Leu Ala Glu 450 455
460Met Arg Val Ala Asn Arg Ser Ile Arg Gln Trp Leu His His Cys
Asn465 470 475 480Thr Leu
Gln Phe Gly Arg Gln Ile Thr Glu Gly Ile Ile His Leu Arg
485 490 495Phe Thr Asn Asp Ile Met Thr
Gly Arg Ile Val Asn Leu Phe Ser Thr 500 505
510Met Leu Val Ala Leu Ser Ser Gln Pro Phe Ala Thr Tyr Pro
Leu Asp 515 520 525Tyr Lys Arg Ser
Val Gln Arg Ala Leu Gln Leu Leu Ser Asn Arg Thr 530
535 540Ala Gln Ile Ala Asp Leu Thr Arg Leu Ile Val Tyr
Asn Tyr Thr Thr545 550 555
560Leu Ser Ala Cys Ile Val Met Asn Met His Leu Val Gly Thr Leu Thr
565 570 575Val Glu Arg Ile Gln
Ala Thr Ser Leu Thr Ser Leu Met Met Leu Ile 580
585 590Ser Asn Lys Thr Val Ile Pro Glu Pro Ser Ser Leu
Phe Ser Tyr Phe 595 600 605Ser Ser
Asn Ile Asn Phe Leu Thr Asn Tyr Asn Glu Gln Ile Asp Asn 610
615 620Val Val Ala Glu Ile Met Ala Ala Tyr Arg Leu
Asn Leu Tyr Gln Gln625 630 635
640Lys Met Leu Met Leu Val Thr Arg Phe Val Ser Arg Leu Tyr Ile Phe
645 650 655Asp Ala Pro Lys
Ile Pro Pro Asp Gln Met Tyr Arg Leu Arg Asn Arg 660
665 670Leu Arg Asn Ile Pro Val Glu Arg Arg Arg Ala
Asp Val Phe Arg Ile 675 680 685Ile
Met Asn Asn Arg Asp Leu Ile Glu Lys Thr Ser Glu Arg Ile Cys 690
695 700Gln Gly Val Leu Leu Ser Tyr Thr Pro Met
Pro Leu Thr Tyr Val Glu705 710 715
720Asp Val Gly Leu Thr Asn Val Ile Asn Asp Thr Asn Asn Phe Gln
Ile 725 730 735Ile Asn Ile
Glu Glu Ile Glu Lys Thr Gly Asp Tyr Ser Ala Ile Thr 740
745 750Asn Ala Leu Leu Arg Asp Thr Pro Ile Ile
Leu Lys Gly Ala Ile Pro 755 760
765Tyr Val Thr Asn Ser Ser Val Ile Asp Val Leu Ser Lys Val Asp Thr 770
775 780Thr Val Phe Ala Ser Ile Val Lys
Asp Arg Asp Ile Ser Lys Leu Lys785 790
795 800Pro Ile Lys Phe Ile Ile Asn Ser Asp Ser Ser Glu
Tyr Tyr Leu Val 805 810
815His Asn Asn Lys Trp Thr Pro Thr Thr Thr Thr Ala Val Tyr Lys Ala
820 825 830Arg Ser Gln Gln Phe Asp
Ile Gln His Ser Val Ser Met Leu Glu Ser 835 840
845Asn Leu Phe Phe Val Val Tyr Asn Asp Leu Phe Lys Tyr Ile
Lys Thr 850 855 860Thr Thr Val Leu Pro
Ile Asn Ala Val Ser Tyr Asp Gly Ala Arg Ile865 870
875 880Met Gln Glu Thr233PRTArtificial
sequenceAntigenic epitope of human rotavirus group C VP2 protein
2Leu Glu Thr Ile Ile Asp Lys Glu Val Lys Glu Asn Lys Asp Ser Thr1
5 10 15Lys Asp Glu Lys Leu Val
Val Thr Glu Glu Ser Asn Gly Asp Val Thr 20 25
30Ala333PRTArtificial SequenceAntigenic epitope of human
rotavirus group C VP2 protein 3Leu Glu Thr Ile Ile Asn Lys Glu Val
Lys Glu Asn Lys Asp Ser Met1 5 10
15Lys Glu Asp Lys Leu Val Val Thr Glu Glu Ser Asn Gly Asp Val
Thr 20 25
30Thr433PRTArtificial SequenceAntigenic epitope of porcine rotavirus
group C VP2 protein 4Thr Glu Asn Val Glu Glu Lys Glu Ile Lys Glu Ala
Lys Glu Gln Val1 5 10
15Lys Asp Glu Lys Gln Val Ile Thr Glu Glu Asn Val Asp Ser Pro Lys
20 25 30Asp522PRTArtificial
SequenceAntigenic epitope of human rotavirus group C VP2 protein
5Lys Leu Thr Glu Ile Gln Glu Ser Ser Ala Lys Thr Tyr Asn Thr Leu1
5 10 15Phe Arg Leu Phe Thr Pro
20618PRTArtificial SequenceAntigenic epitope of porcine
rotavirus group C VP2 protein 6Asn Tyr Arg Asn Ser Arg Ile Lys Cys
Gln Thr Tyr Asn Lys Leu Phe1 5 10
15Arg Leu718PRTArtificial SequenceAntigenic epitope of human
rotavirus group C VP2 protein 7Leu Asn Val Leu Glu Gly Met Pro Asp
Tyr Ile Met Leu Arg Asp Met1 5 10
15Ala Val818PRTArtificial SequenceAntigenic epitope of human
rotavirus group C VP2 protein 8Leu Asn Val Leu Glu Glu Met Pro Asp
Tyr Ile Met Leu Arg Asp Met1 5 10
15Ala Val918PRTArtificial SequenceAntigenic epitope of porcine
rotavirus group C VP2 protein 9Leu Asn Val Leu Asp Glu Met Pro Asp
Tyr Val Met Leu Arg Asp Met1 5 10
15Ala Val1029PRTArtificial SequenceAntigenic epitope of human
rotavirus group C VP2 protein 10Ala Ala His Leu Gln Leu Glu Ala Ile
Thr Val Gln Val Glu Ser Gln1 5 10
15Phe Leu Ala Gly Ile Ser Ala Ala Ala Ala Asn Glu Ala
20 251119PRTArtificial SequenceAntigenic epitope of
porcine rotavirus group C VP2 protein 11Leu Gln Cys Lys Leu Asn His
Asn Ser Trp Gln Glu Leu Val His Gly1 5 10
15Arg Asn Glu1213PRTArtificial SequenceAntigenic epitope
of human rotavirus group C VP2 protein 12Leu Ser Ala Cys Ile Val Met
Asn Met His Leu Val Gly1 5
101317PRTArtificial SequenceAntigenic epitope of human rotavirus group C
VP2 protein 13Ile Pro Pro Asp Gln Met Tyr Arg Leu Arg Asn Arg Leu Arg
Asn Ile1 5 10
15Pro1420DNAArtificial Sequencesynthetic primer 14agccacatag ttcacatttc
201519DNAArtificial
Sequencesynthetic primer 15atctcattca caatggatg
1916884PRTHuman rotavirus CMISC_FEATUREHuman
rotavirus group C Bristol strain VP2 protein 16Met Ile Ser Arg Asn
Arg Arg Arg Asn Asn Gln Gln Lys Asn Ile Glu1 5
10 15Lys Glu Lys Gln Leu Glu Thr Ile Ile Asn Lys
Glu Val Lys Glu Asn 20 25
30Lys Asp Ser Met Lys Glu Asp Lys Leu Val Val Thr Glu Glu Ser Asn
35 40 45Gly Asp Val Thr Thr Ala Lys Glu
Gln Ser Asn Asn Ile Asn Leu Gln 50 55
60Lys Asn Asp Leu Val Lys Glu Val Met Asn Ile Gln Asn Gln Thr Leu65
70 75 80Asn Thr Val Val Thr
Glu Asn Lys Val Glu Ile Glu Glu Ile Val Lys 85
90 95Lys Tyr Ile Pro Ser Tyr Asn Thr Asp Ser Leu
Ile Val Lys Lys Leu 100 105
110Thr Glu Ile Gln Glu Ser Ser Ala Lys Thr Tyr Asn Thr Leu Phe Arg
115 120 125Leu Phe Thr Pro Val Lys Ser
Tyr Leu Tyr Asp Ile Asn Gly Glu Lys 130 135
140Lys Leu Ser Thr Arg Trp Tyr Trp Lys Leu Leu Lys Asp Asp Leu
Pro145 150 155 160Ala Gly
Asp Tyr Ser Val Arg Gln Phe Phe Leu Ser Leu Tyr Leu Asn
165 170 175Val Leu Glu Glu Met Pro Asp
Tyr Ile Met Leu Arg Asp Met Ala Val 180 185
190Asp Asn Pro Tyr Ser Ala Glu Ala Gly Lys Ile Val Asp Gly
Lys Ser 195 200 205Lys Glu Ile Leu
Ile Glu Leu Tyr Gln Asp Gln Met Thr Glu Gly Tyr 210
215 220Ile Arg Arg Tyr Met Ser Glu Leu Arg His Lys Ile
Ser Gly Glu Thr225 230 235
240Asn Thr Ala Lys Tyr Pro Ala Ile Leu His Pro Val Asp Asn Glu Leu
245 250 255Asn Gln Tyr Phe Leu
Glu His Gln Leu Ile Gln Pro Leu Thr Thr Arg 260
265 270Asn Ile Ala Glu Leu Ile Pro Thr Gln Leu Tyr His
Asp Pro Asn Tyr 275 280 285Val Phe
Asn Ile Asp Ala Ala Phe Leu Thr Asn Ser Arg Phe Val Pro 290
295 300Pro Tyr Leu Thr Gln Asp Arg Ile Gly Leu His
Asp Gly Phe Glu Ser305 310 315
320Ile Trp Asp Ser Lys Thr His Ala Asp Tyr Val Ser Ala Arg Arg Phe
325 330 335Ile Pro Asp Leu
Thr Glu Leu Val Asp Ala Glu Lys Gln Ile Lys Glu 340
345 350Met Ala Ala His Leu Gln Leu Glu Ala Ile Thr
Val Gln Val Glu Ser 355 360 365Gln
Phe Leu Ala Gly Ile Ser Ala Ala Ala Ala Asn Glu Ala Phe Lys 370
375 380Phe Ile Ile Gly Ser Val Leu Ser Thr Arg
Thr Ile Ala Val Glu Phe385 390 395
400Ile Thr Ser Asn Tyr Met Ser Leu Ala Ser Cys Met Tyr Leu Met
Thr 405 410 415Ile Met Pro
Ser Glu Ile Phe Leu Arg Glu Ser Leu Val Ala Met Gln 420
425 430Leu Ala Ile Ile Asn Thr Leu Ile Tyr Pro
Ala Leu Gly Leu Ala Gln 435 440
445Met His Tyr Gln Ala Gly Glu Val Arg Thr Pro Phe Glu Leu Ala Glu 450
455 460Met Gln Val Ala Asn Arg Ser Ile
Arg Gln Trp Leu His His Cys Asn465 470
475 480Thr Leu Gln Phe Gly Arg Gln Ile Thr Glu Gly Ile
Ile His Leu Arg 485 490
495Phe Thr Asn Asp Ile Met Thr Gly Arg Ile Val Asn Leu Phe Ser Thr
500 505 510Met Leu Val Ala Leu Ser
Ser Gln Pro Phe Ala Thr Tyr Pro Leu Asp 515 520
525Tyr Lys Arg Ser Val Gln Arg Ala Leu Gln Leu Leu Ser Asn
Arg Thr 530 535 540Ala Gln Ile Ala Asp
Leu Thr Arg Leu Ile Val Tyr Asn Tyr Thr Thr545 550
555 560Leu Ser Ala Cys Ile Val Met Asn Met His
Leu Val Gly Thr Leu Thr 565 570
575Val Glu Arg Ile Gln Ala Thr Ser Leu Thr Ser Leu Met Met Leu Ile
580 585 590Ser Asn Lys Thr Val
Ile Pro Glu Pro Ser Ser Leu Phe Ser Tyr Phe 595
600 605Ser Ser Asn Ile Asn Phe Leu Thr Asn Tyr Asn Glu
Gln Ile Asp Asn 610 615 620Val Val Ala
Glu Ile Met Ala Ala Tyr Arg Leu Asn Leu Tyr Gln Gln625
630 635 640Lys Met Leu Met Leu Val Thr
Arg Phe Val Ser Arg Leu Tyr Ile Phe 645
650 655Asp Ala Pro Lys Ile Pro Pro Asp Gln Met Tyr Arg
Leu Arg Asn Arg 660 665 670Leu
Arg Asn Ile Pro Val Glu Arg Arg Arg Ala Asp Val Phe Arg Ile 675
680 685Ile Met Asn Asn Arg Asp Leu Ile Glu
Lys Thr Ser Glu Arg Ile Cys 690 695
700Gln Gly Val Leu Leu Ser Tyr Thr Pro Met Pro Leu Thr Tyr Val Glu705
710 715 720Asp Val Gly Leu
Thr Asn Val Ile Asn Asp Thr Asn Asn Phe Gln Ile 725
730 735Ile Asn Ile Glu Glu Ile Glu Lys Thr Gly
Asp Tyr Ser Ala Ile Thr 740 745
750Asn Ala Leu Leu Arg Asp Thr Pro Ile Ile Leu Lys Gly Ala Ile Pro
755 760 765Tyr Val Thr Asn Ser Ser Val
Ile Asp Val Leu Ser Lys Val Asp Thr 770 775
780Thr Val Phe Ala Ser Ile Val Lys Asp Arg Asp Ile Ser Lys Leu
Lys785 790 795 800Pro Ile
Lys Phe Ile Ile Asn Ser Asp Ser Ser Glu Tyr Tyr Leu Val
805 810 815His Asn Asn Lys Trp Thr Pro
Thr Thr Thr Thr Ala Val Tyr Lys Ala 820 825
830Arg Ser Gln Gln Phe Asp Ile Gln His Ser Val Ser Met Leu
Glu Ser 835 840 845Asn Leu Phe Phe
Val Val Tyr Asn Asp Leu Phe Lys Tyr Ile Lys Thr 850
855 860Thr Thr Val Leu Pro Ile Asn Ala Val Ser Tyr Asp
Gly Ala Arg Ile865 870 875
880Met Gln Glu Thr17884PRTPorcine rotavirusMISC_FEATUREporcine rotavirus
group C Cowden strain VP2 protein 17Met Ile Ser Arg Asn Arg Arg Arg
Asn Thr Gln Gln Lys Asp Ala Glu1 5 10
15Lys Glu Lys Gln Thr Glu Asn Val Glu Glu Lys Glu Ile Lys
Glu Ala 20 25 30Lys Glu Gln
Val Lys Asp Glu Lys Gln Val Ile Thr Glu Glu Asn Val 35
40 45Asp Ser Pro Lys Asp Val Lys Glu Gln Ser Asn
Thr Val Asn Leu Gln 50 55 60Lys Asn
Asp Leu Val Lys Glu Val Ile Asn Ile Gln Asn Gln Thr Leu65
70 75 80Asn Thr Ile Val Ala Glu Asn
Lys Val Glu Ile Glu Glu Val Val Lys 85 90
95Lys Tyr Ile Pro Ser Tyr Ser Thr Asp Lys Leu Ile Val
Lys Lys Leu 100 105 110Thr Glu
Ile Gln Glu Ser Ser Ala Lys Thr Tyr Asn Lys Leu Phe Arg 115
120 125Leu Phe Thr Pro Val Lys Ser Tyr Leu Tyr
Asp Val Asn Gly Glu Lys 130 135 140Lys
Leu Ser Thr Arg Trp Tyr Trp Lys Leu Leu Lys Asp Asp Leu Pro145
150 155 160Ala Gly Asp Tyr Ser Val
Arg Gln Phe Phe Leu Ser Leu Tyr Leu Asn 165
170 175Val Leu Asp Glu Met Pro Asp Tyr Val Met Leu Arg
Asp Met Ala Val 180 185 190Asp
Asn Pro Tyr Ser Ala Glu Ala Gly Lys Ile Val Asp Glu Lys Ser 195
200 205Lys Glu Ile Leu Val Glu Ile Tyr Gln
Asp Gln Met Thr Glu Gly Tyr 210 215
220Ile Arg Arg Tyr Met Ser Asp Leu Arg His Arg Ile Ser Gly Glu Thr225
230 235 240Asn Thr Ala Lys
Tyr Pro Ala Ile Leu His Pro Val Asp Glu Glu Leu 245
250 255Asn Lys Tyr Phe Leu Glu His Gln Leu Ile
Gln Pro Leu Thr Thr Arg 260 265
270Asn Ile Ala Glu Leu Ile Pro Thr Gln Leu Tyr His Asp Pro Asn Tyr
275 280 285Val Phe Asn Ile Asp Ala Ala
Phe Leu Thr Asn Ser Arg Phe Val Pro 290 295
300Pro Tyr Leu Thr Gln Asp Arg Ile Gly Leu His Asp Gly Phe Glu
Ser305 310 315 320Ile Trp
Asp Ala Lys Thr His Ala Asp Tyr Val Ser Ala Arg Arg Phe
325 330 335Val Pro Asp Leu Thr Glu Leu
Val Asp Ala Glu Lys Gln Met Lys Glu 340 345
350Met Ala Ala His Leu Gln Leu Glu Ala Ile Thr Val Gln Val
Glu Ser 355 360 365Gln Phe Leu Ala
Gly Ile Ser Ala Ala Ala Ala Asn Glu Ala Phe Lys 370
375 380Phe Ile Ile Gly Thr Val Leu Ser Thr Arg Thr Ile
Ala Val Glu Phe385 390 395
400Ile Thr Ser Asn Tyr Met Ser Leu Ala Ser Cys Met Tyr Leu Met Thr
405 410 415Ile Met Pro Ser Glu
Ile Phe Leu Arg Glu Ser Leu Val Ala Met Gln 420
425 430Leu Ala Val Ile Asn Thr Leu Thr Tyr Pro Ala Leu
Gly Leu Ala Gln 435 440 445Met His
Tyr Gln Ala Gly Glu Ile Arg Thr Pro Phe Glu Leu Ala Glu 450
455 460Met Gln Val Ala Asn Arg Pro Ile Arg Gln Trp
Leu His His Cys Asn465 470 475
480Thr Leu Gln Phe Gly Arg Gln Val Thr Glu Gly Val Thr His Leu Arg
485 490 495Phe Thr Asn Asp
Ile Met Thr Gly Arg Ile Val Asn Leu Phe Ser Thr 500
505 510Met Leu Val Ala Leu Ser Ser Gln Pro Phe Ala
Thr Tyr Pro Leu Asp 515 520 525Tyr
Lys Arg Ser Val Gln Arg Ala Leu Gln Leu Leu Ser Asn Arg Thr 530
535 540Ala Gln Ile Ala Asp Leu Thr Arg Leu Ile
Val Tyr Asn Tyr Thr Thr545 550 555
560Leu Ser Ala Cys Ile Val Met Asn Met His Leu Val Gly Thr Leu
Thr 565 570 575Val Glu Arg
Ile Gln Ala Thr Ala Leu Thr Ser Leu Ile Met Leu Ile 580
585 590Ser Asn Lys Thr Val Ile Pro Glu Pro Ser
Ser Leu Phe Ser Tyr Phe 595 600
605Ser Ser Asn Ile Asn Phe Leu Thr Asn Tyr Asn Glu Gln Ile Asp Asn 610
615 620Val Val Ala Glu Ile Met Ala Ala
Tyr Arg Leu Asp Leu Tyr Gln Gln625 630
635 640Lys Met Leu Met Leu Val Thr Arg Phe Val Ser Arg
Leu Tyr Ile Phe 645 650
655Asp Ala Pro Lys Ile Pro Pro Asp Gln Met Tyr Arg Leu Arg Asn Arg
660 665 670Leu Arg Asn Ile Pro Val
Glu Arg Arg Arg Ala Asp Val Phe Arg Ile 675 680
685Ile Met Asn Asn Arg Asp Leu Ile Glu Lys Thr Ser Glu Arg
Ile Cys 690 695 700Gln Gly Val Leu Leu
Ser Tyr Ser Pro Met Pro Leu Thr Tyr Val Glu705 710
715 720Asp Val Gly Leu Thr Asn Val Val Asn Asp
Thr Asn Gly Phe Gln Ile 725 730
735Ile Asn Ile Glu Glu Ile Glu Lys Thr Gly Asp Tyr Ser Ala Ile Thr
740 745 750Asn Ala Leu Leu Arg
Asp Thr Pro Ile Ile Leu Lys Gly Ala Ile Pro 755
760 765Tyr Val Thr Asn Ser Ser Val Ile Asp Val Leu Ser
Lys Ile Asp Thr 770 775 780Thr Val Phe
Ala Ser Ile Val Lys Asp Arg Asp Ile Ser Lys Leu Lys785
790 795 800Pro Ile Lys Phe Thr Ile Asn
Ser Asp Ser Ser Glu Tyr Tyr Leu Val 805
810 815His Asn Asn Lys Trp Thr Pro Thr Thr Thr Thr Ala
Val Tyr Lys Ala 820 825 830Arg
Ser Gln Gln Phe Asn Ile Gln His Ser Val Ser Met Leu Glu Ser 835
840 845Asn Leu Phe Phe Val Val Tyr Asn Asp
Leu Phe Lys Tyr Ile Lys Thr 850 855
860Thr Thr Val Leu Pro Ile Asn Ala Val Ser Tyr Asp Gly Ala Arg Ile865
870 875 880Met Gln Glu
Thr182720DNAHuman rotavirus Cmisc_featureHuman rotavirus group C strain
ASP88 VP2 nucleotide sequence 18tcgaggacaa atcgtccaag atgataagca
gaaacaggcg cagaaataac caacaaaaag 60atataggaaa agagaaacaa ttagagacta
taattgacaa agaagtaaag gaaaacaaag 120attctacaaa agaagataag ctagtagtta
cggaagaaag taatggtgac gtcacagctg 180ttaaagaaca atcgaataat attaatttac
aaaagaatga tttggttaaa gaagtcatga 240atatacagaa tcaaacatta aatacagtag
ttgctgagaa taaagttgaa atagaagaaa 300tagttaaaaa atacattccc tcatataata
ctgacagcct tattgttaaa aagttaactg 360aaatccagga atcaagtgct aaaacatata
atacattatt cagattattt actccagtta 420aaagttattt atatgacata aatggtgaga
aaaaattatc gactagatgg tattggaaat 480tgctcaaaga tgatttacct gctggtgatt
actcagttag acaattcttc ctgtcactat 540atttaaatgt tttagaggga atgcccgatt
acataatgct tcgtgatatg gcagtggata 600acccatattc agcagaagca ggtaaaatcg
tagatggaaa gtctaaagaa attttagttg 660aactatatca agaccaaatg acagaagggt
atattagaag atatatgtct gaattaagac 720ataaaatatc tggagaaaca aatactgcaa
aatatccagc tattctacat cccgtggata 780atgagcttaa tcaatacttt cttgagcatc
agttaattca accattaact acaagaaata 840ttgcagaatt gattccaact caattatatc
atgatccaaa ttacgttttt aatattgatg 900cagccttttt aacaaattca agatttgttc
caccatactt aacacaggat aggattggat 960tacatgatgg attcgaatca atatgggatt
caaaaaccca tgctgattac gtttcagcta 1020gaagatttat acctgattta actgaactgg
tagatgctga aaagcaaata aaagaaatgg 1080ctgcacattt acaactagag gctattacag
tacaggttga atcacaattt ttagcgggaa 1140ttagtgctgc tgcagctaat gaagcgttca
aatttataat tggctcagtt ttatctacca 1200gaacaatagc tgtagaattc ataacctcaa
actatatgtc gttagcatca tgtatgtatt 1260taatgactat tatgccatca gagattttct
tgagagaatc attagttgct atgcgattag 1320caataataaa tacccttatt tatccagctc
taggtttagc gcaaatgcat tatcaagcag 1380gtgaagtgag gaccccattc gaattagctg
agatgcgagt agctaataga tctattagac 1440aatggttaca tcattgtaat acacttcaat
ttggtagaca gataacggaa gggataattc 1500atctacgatt tactaatgat atcatgacag
gtaggatagt gaacttattt tcaacaatgc 1560tagtagcttt atcatctcag ccattcgcta
catatccttt agactataaa agatctgtac 1620aaagagcatt acaactttta tcaaatagaa
cagcccaaat agcagattta accagattaa 1680tagtatacaa ttatactaca ttatctgcat
gtatagtcat gaatatgcat ttagtaggaa 1740ctcttactgt tgaacgtata caggccactt
ctctaacttc tttaatgatg ttaatttcta 1800ataagacagt tattccagaa ccatcgtctc
ttttttcata tttctctagt aacattaatt 1860ttcttacaaa ttataatgag caaattgata
atgtggtagc agaaataatg gccgcatata 1920gattgaattt atatcaacag aaaatgttga
tgctcgttac caggtttgtg tcaaggttgt 1980acatatttga tgctcctaaa ataccgccag
atcagatgta tagattaaga aaccgattaa 2040gaaatattcc agttgaaaga agaagagctg
atgtgttcag aattattatg aataatagag 2100atttaatcga aaaaacatca gaacgtatat
gtcagggtgt gttgttatct tatacaccaa 2160tgcctttaac ttacgttgaa gatgtcgggt
taacaaatgt aattaatgac actaataact 2220tccaaataat taatatagaa gaaattgaga
agaccggtga ctattcagcc ataacgaatg 2280cattacttcg ggatactcca attatattga
aaggtgcgat tccatatgtt actaactcat 2340cagtaattga tgttttatct aaagtggaca
ccacagtgtt cgcaagcatc gtaaaagata 2400gggatatttc aaagttaaaa ccaataaaat
tcataattaa ttcagattca tccgaatatt 2460atttagtaca caataataaa tggacaccaa
caacaactac agcagtatat aaagctagat 2520ctcagcaatt tgatatacaa cattcagtat
caatgctaga gtcaaactta ttttttgtgg 2580tatataatga tttatttaaa tacattaaaa
ccactacagt tctgccgata aatgctgtct 2640cttatgatgg tgcaagaatt atgcaagaaa
cataaatgat tgtatagtat catcttgtaa 2700cgacctcaaa ctctgtggct
2720192736DNAHuman rotavirus
Cmisc_featureHuman rotavirus group C Bristol strain VP2 nucleotide
sequence 19ggcttaaaaa gatcagttga ggacaaatcg ttcaagatga taagcagaaa
caggcgcaga 60aataatcaac aaaaaaacat agaaaaagag aaacaattag agactataat
taacaaagaa 120gttaaggaaa acaaagattc tatgaaagaa gataagctag tagttacaga
agaaagcaat 180ggagacgtca caactgctaa agaacaatcg aataatatta atttacaaaa
gaatgatttg 240gttaaagaag tcatgaatat acagaatcaa acattaaata cagtagttac
tgagaataaa 300gttgaaatag aagaaatagt taaaaaatac attccatcat ataatactga
tagcctcatt 360gttaaaaagt taactgaaat ccaggaatca agtgctaaaa catataatac
attgtttaga 420ttatttactc cagttaaaag ttatttatat gatataaatg gtgagaaaaa
attatcgact 480agatggtatt ggaaattgct caaagatgat ttacctgctg gtgattactc
agttagacaa 540ttcttcctgt cactatattt aaatgtttta gaggaaatgc ccgattacat
aatgcttcgt 600gatatggcag tggataaccc atattcagca gaagcaggta aaatcgtaga
tggaaagtct 660aaagaaattt tgattgaact atatcaagac cagatgacag aaggatatat
tagaagatat 720atgtctgaat taagacataa aatatctgga gagacaaata ctgcaaaata
cccagctatt 780ctacatcccg tggataatga acttaatcaa tactttcttg agcatcagtt
aattcaacca 840ttaactacaa ggaacattgc agaattgatt ccaactcaat tatatcatga
tccaaattac 900gtttttaata ttgatgcagc ctttttaaca aattcaagat ttgttccacc
atacttaaca 960caggatagga ttggattaca tgatggattt gaatcaatat gggattcaaa
aactcatgct 1020gattacgttt cagctagaag atttatacct gatttaactg aactggtgga
tgctgaaaag 1080caaataaaag aaatggctgc acatttacaa ctagaggcta ttacggtaca
ggttgaatca 1140caatttttag caggaattag tgctgctgca gctaatgaag cgtttaaatt
tataattggc 1200tcagttttat ctaccagaac aatagctgta gaattcataa cctcaaacta
tatgtcacta 1260gcatcatgta tgtatttaat gactattatg ccatcagaga ttttcttaag
agaatcatta 1320gttgctatgc aattagcaat aataaatacc cttatttatc cagctctagg
tttagcgcaa 1380atgcattatc aagcaggtga agtgaggact ccattcgaat tagctgaaat
gcaagtagct 1440aatagatcta ttagacaatg gttacatcat tgtaatacac ttcaatttgg
tagacagata 1500acggaaggga taattcatct acgatttact aatgatatca tgacaggcag
gatagtgaac 1560ttattttcaa caatgttagt ggctctatca tctcagcctt tcgctacata
tcctttagac 1620tataaaagat ctgtacaaag agcgttacaa cttttatcaa atagaacagc
tcaaatagca 1680gatttaacca gattaatagt atacaattat actacattat ctgcttgtat
agttatgaat 1740atgcatttag taggaactct tactgttgaa cgtatacaag ccacttctct
aacttcttta 1800atgatgttaa tctctaataa gacagttatt ccggaaccat cgtctctttt
ttcatatttc 1860tctagtaaca ttaattttct tacaaattat aatgagcaaa ttgataatgt
ggtagcagaa 1920ataatggccg catatagatt gaatttatat caacagaaaa tgttgatgct
cgttaccaga 1980tttgtgtcaa agttatacat atttgatgct cctaagatac caccagatca
gatgtataga 2040ttaagaaacc gattaagaaa tattccagtt gaaagaagaa gagctgacgt
attcagaatt 2100attatgaata atagagattt aatcgaaaaa acatcagaac gtatatgcca
gggtgtgctg 2160ttatcttata caccaatgcc tttaacttac gttgaagatg tcgggttaac
aaatgtaatt 2220aatgacacta atagctttca aataattaat attgaagaaa ttgagaagac
cggtgactat 2280tcagctataa cgaatgcatt acttcgggat actccaatca tattgaaagg
tgcgattcca 2340tatgttacta actcatcagt aattgatgtt ttatctaaag tggacaccac
agtgttcgca 2400agcattgtaa aagataggga catttcaaag ttaaaaccaa taaaattcat
aattaattca 2460gattcatccg aatattattt agtacataat aataaatgga caccaacaac
aactacagca 2520gtatataaag ctagatctca gcaatttgat atacaacatt cagtatcaat
gctagagtca 2580aacttatttt ttgtggtata taatgattta tttaaataca ttaaaaccac
tacagttctg 2640ccgataaatg ctgtctctta tgacggtgca agaattatgc aagaaacata
aatgattgta 2700tagtatcatc ttgtgacgac ctcaaacttt gtggct
27362018DNAArtificial Sequencesynthetic primer 20agccacatga
tcttgttt
182118DNAArtificial Sequencesynthetic primer 21ggcatttaaa aaagaaga
182220DNAArtificial
Sequencesynthetic primer 22tcgaggacaa atcgtccaag
202319DNAArtificial Sequencesynthetic primer
23agccacagag tttgaggtc
1924395PRTHuman rotavirus CMISC_FEATUREHuman rotavirus group C VP6
consensus sequence 24Met Asp Val Leu Phe Ser Ile Ala Lys Thr Val Ser Asp
Leu Lys Lys1 5 10 15Lys
Val Val Val Gly Thr Ile Tyr Thr Asn Val Glu Asp Val Val Gln 20
25 30Gln Thr Asn Glu Leu Ile Arg Thr
Leu Asn Gly Asn Ile Phe His Thr 35 40
45Gly Gly Ile Gly Thr Gln Pro Gln Lys Glu Trp Asn Phe Gln Leu Pro
50 55 60Gln Leu Gly Thr Thr Leu Leu Asn
Leu Asp Asp Asn Tyr Val Gln Ser65 70 75
80Thr Arg Gly Ile Ile Asp Phe Leu Ser Ser Phe Ile Glu
Ala Val Cys 85 90 95Asp
Asp Glu Ile Val Arg Glu Ala Ser Arg Asn Gly Met Gln Pro Gln
100 105 110Ser Pro Ala Leu Ile Leu Leu
Ser Ser Ser Lys Phe Lys Thr Ile Asn 115 120
125Phe Asn Asn Ser Ser Gln Ser Ile Lys Asn Trp Asn Ala Gln Ser
Arg 130 135 140Arg Glu Asn Pro Val Tyr
Glu Tyr Lys Asn Pro Met Leu Phe Glu Tyr145 150
155 160Lys Asn Ser Tyr Ile Leu Gln Arg Ala Asn Pro
Gln Phe Gly Ser Val 165 170
175Met Gly Leu Arg Tyr Tyr Thr Thr Ser Asn Thr Cys Gln Ile Ala Ala
180 185 190Phe Asp Ser Thr Leu Ala
Glu Asn Ala Pro Asn Asn Thr Gln Arg Phe 195 200
205Val Tyr Asn Gly Arg Leu Lys Arg Pro Ile Ser Asn Val Leu
Met Lys 210 215 220Ile Glu Ala Gly Ala
Pro Asn Ile Ser Asn Pro Thr Ile Leu Pro Asp225 230
235 240Pro Asn Asn Gln Thr Thr Trp Leu Phe Asn
Pro Val Gln Leu Met Asn 245 250
255Gly Thr Phe Thr Ile Glu Phe Tyr Asn Asn Gly Gln Leu Ile Asp Met
260 265 270Val Arg Asn Met Gly
Ile Val Thr Val Arg Thr Phe Asp Ser Tyr Arg 275
280 285Ile Thr Ile Asp Met Ile Arg Pro Ala Ala Met Thr
Gln Tyr Val Gln 290 295 300Arg Ile Phe
Pro Gln Gly Gly Pro Tyr His Phe Gln Ala Thr Tyr Met305
310 315 320Leu Thr Leu Ser Ile Leu Asp
Ala Thr Thr Glu Ser Val Leu Cys Asp 325
330 335Ser His Ser Val Glu Tyr Ser Ile Val Ala Asn Val
Arg Arg Asp Ser 340 345 350Ala
Met Pro Ala Gly Thr Val Phe Gln Pro Gly Phe Pro Trp Glu His 355
360 365Thr Leu Ser Asn Tyr Thr Val Ala Gln
Glu Asp Asn Leu Glu Arg Leu 370 375
380Leu Leu Ile Ala Ser Val Lys Arg Met Val Met385 390
395251185DNAHuman rotavirus Cmisc_featureHuman rotavirus
group C VP6 Bristol nucleotide sequence 25atggatgtac ttttttctat
agcgaaaact gtgtcagatc ttaaaaagaa agttgtggtt 60ggaacaattt atactaatgt
agaagatgtt gtacaacaga cgaatgaatt gattagaact 120ttaaatggaa atatttttca
tactggtggc attggaacac agcctcagaa agagtggaat 180tttcagctgc cacaattggg
tacaacttta ttaaatttag atgataatta tgttcaatca 240actagaggca taatcgattt
tttatcatct tttatagaag ctgtatgtga tgatgaaatt 300gttagagaag cttcaagaaa
tggtatgcaa cctcaatcac cagctcttat attattatct 360tcatcaaaat ttaaaacaat
taattttaat aatagttctc aatctattaa aaattggaat 420gctcaatcaa gacgtgagaa
tcctgtatat gagtataaaa atccaatgtt gtttgaatat 480aaaaattcgt atattttaca
acgcgcaaat ccacaatttg gaagcgtcat gggtttaaga 540tattatacaa caagtaatac
ttgtcaaatt gcagcatttg attccaccct agctgaaaat 600gcaccaaaca atacacaacg
cttcgtttat aatggcagac taaaaagacc catatcaaac 660gttttaatga aaatagaagc
tggtgctcca aatataagca acccaactat tttacctgat 720cctaataatc aaacaacttg
gctttttaat ccggtacaat taatgaatgg aacatttacc 780attgaattct ataataatgg
tcaactaatt gatatggttc gaaatatggg aatagttact 840gtaagaactt ttgattctta
tagaataaca attgacatga ttagaccagc tgctatgact 900caatacgttc aacgaatttt
tccacaaggt ggaccttatc attttcaggc tacatatatg 960ttaacattaa gtatattaga
tgctaccaca gagtccgttc tatgtgattc tcattcagtg 1020gaatattcaa tagtagcaaa
cgttagaaga gattcagcga tgccagctgg aactgttttt 1080caaccgggat ttccatggga
acacacacta tccaattaca ctgttgctca agaagataat 1140ttagaaagat tattgttaat
tgcatctgtg aagagaatgg taatg 1185261185DNAHuman
rotavirus Cmisc_featureHuman rotavirus group C VP6 Jajeri nucleotide
sequence 26atggatgtac ttttttctat agcgaaaact gtgtcagatc ttaaaaagaa
agttgtagtt 60ggaacaattt atactaatgt agaagatgtt gtacaacaga cgaatgaatt
gattagaact 120ttgaatggaa atatttttta tactggtggt attggaacac agcctcagaa
agagtggaat 180tttcagctac cacaattggg tacaacttta ttaaatttag atgacaatta
tgttcaatca 240accagaggca taattgattt tttatcatct tttatagaag ctgtatgtga
tgatgaaatt 300gttagagaag cttcaagaaa tggtatgcaa cctcaatcac cagctcttat
attattatct 360tcatcaaaat ttaaaacaat taattttaat aatagttctc aatctatcaa
aaattggaat 420gctcaatcaa gacgtgagaa tcctgtatat gagtacaaaa atccaatggt
gtttgaatat 480aaaaattctt atattttaca acgcgcaaat ccacaatttg gaagcgtcat
gggtttaaga 540tattatacaa caagtaatac ttgtcaaatt gcagcatttg attccaccct
agctgaaaat 600gcaccaaaca atacgcaacg cttcgtttat aatggcagac taaaaagacc
catatcaaat 660gttttaatga aaatagaagc tggtgcccca aatataagca acccaactat
tttacctgat 720cctaataatc aaacaacttg gctttttaat ccagtacagt taatgaatgg
aacattcact 780attgaattct ataataatgg tcaactaatt gatatggttc gaaatatggg
aatagttacc 840gtaagaactt ttgattctta tagaataaca attgacataa ttagaccagc
tgctatgact 900caatacgttc aacgaatttt tccacaaggt ggaccttatc attttcaggc
tacatatatg 960ttaacattaa gtatattaga tgctactaca gagtccgttc tatgtgattc
tcattcagta 1020gaatattcaa tagtagcaaa cgtcagaaga gattcagcga tgccagctgg
aactgttttt 1080caaccaggat ttccatggga acacacacta tccaattaca ctgttgctca
agaagataat 1140ttagaaagat tattgttaat cgcatctgtg aagagaatgg taatg
1185271185DNAHuman rotavirus Cmisc_featureHuman rotavirus
group C VP6 CMH004 nucleotide sequence 27atggatgtac ttttttctat
agcgaaaact gtatcagatc ttaaaaagaa agttgtagtt 60ggaacaattt atactaatgt
agaagatgtt gtacaacaga cgaatgaatt gattagaact 120ttgaatggaa atatttttca
tactggtggc attggaacac agcctcataa agagtggaat 180tttcagctac cacaattagg
tacaacttta ttaaatttag atgataatta tgttcaatca 240actagaggca taattgattt
tttatcatct tttatagaag ctgtatgtga tgatgaaatt 300gttagagaag cttcaagaaa
tggtatgcaa cctcaatcac cagctcttat attattatct 360tcatcaaaat ttaaaacaat
taattttaat aatagttctc aatctatcaa aaattggaat 420gctcaatcaa gacgtgagaa
tcctgtatat gagtacaaaa atccaatgtt gtttgaatat 480aaaaattctt atattttaca
acgcgcaaat ccacaatttg gaagtgttat gggtttaaga 540tattacacaa caagtaatac
ttgtcaaatt gcagcatttg attccaccct agctgaaaat 600gcaccgaaca atacgcaacg
cttcgtttat aatggcagac taaaaagacc catatcaaat 660gttttaatga aaatagaagc
tggtgctcca aatataagca acccaactat tttacctgat 720cctaataatc aaacaacttg
gctttttaat ccggtacaat taatgaatgg aacatttacc 780attgaattct ataataatgg
tcaactaatt gatatggttc gaaatatggg aatagttact 840gtaagaactt ttgattctta
tagaataaca attgacatga ttagaccagc tgctatgacc 900caatacgttc aacgaatttt
tccacaaggt ggaccttatc attttcaggc tacatatatg 960ttaacattaa gtatattaga
tgctaccaca gaatccgttc tatgtgattc tcattcagta 1020gaatattcaa tagtagcaaa
cgtcagaaga gattcagcga tgccagctgg aactgttttt 1080caaccgggat ttccatggga
acacacacta tccaattaca ctgttgctca agaagataat 1140ttagagagat tattgttaat
cgcatctgtg aagagaatgg taatg 1185281185DNAHuman
rotavirus Cmisc_featureHuman rotavirus group C VP6 V508 nucleotide
sequence 28atggatgtac ttttttctat agcgaaaact gtgtcagatc ttaaaaagaa
agttgtagtt 60ggaacaattt ataccaatgt agaagatgtt gtacaacaga cgaatgaatt
gattagaact 120ttgaatggaa atgtttttca tactggtggc attggaacac agcctcagaa
agagtggaat 180tttcaactac cacaattggg tacaacttta ttaaatttag atgataatta
tgttcaatca 240actagaggca taattgattt tttatcatct tttatagaag ctgtatgtga
tgatgaaatt 300gttagagaag cttcaagaaa tggtatgcaa ccccaatcac cagctcttat
attattatct 360tcatcaaaat ttaaaacaat taatttgaat aatagttctc aatctatcaa
aaattggaat 420gctcaatcaa gacgtgagaa tcctgtatat gagtacaaaa atccaatgtt
gtttgaatat 480aaaaattctt atattttaca acgcgcaaat ccacaatttg gaagcgtcat
gggtttaaga 540tattatacaa caagtaatac ttgtcaaatt gcagcatttg attccaccct
agctgaaaat 600gcaccaaaca atacgcaacg cttcgtttat aatggcagac taaaaagacc
catatcaaat 660gttttaatga aaatagaagc tggtgctcca aatataagca acccaactat
tttacctgat 720cctaataatc aaacaacttg gctttttaat ccagtacaat tgatgaatgg
aacattcacc 780attgagttct ataataatgg tcaactaatt gatatggttc gaaatatggg
aatagttact 840gtaagaactt ttgattctta tagaataaca attgacatga ttagaccagc
tgctatgact 900caatacgttc aacgaatttt tccacaaggc ggaccttatc attttcaggc
tacatatatg 960ttaacgttaa gtatattaga tgctaccaca gagtccgttt tatgtgattc
tcattcagta 1020gaatattcaa tagtagccaa cgtcagaaga gattcagcga tgccagctgg
aaccgttttt 1080caaccgggat ttccatggga acacacacta tccaattaca ctgttgctca
agaagataat 1140ttagaaagat tattgttaat cgcatctgtg aagagaatgg taatg
1185291185DNAHuman rotavirus Cmisc_featureHuman rotavirus
group C VP6 China nucleotide sequence 29atggatgtac ttttttctat
agcgaaaact gtgtcagatc ttaaaaagaa agttgtagtt 60ggaacaattt atactaatgt
agaagatgtt gtacaacaga cgaatgaact gattagaact 120ttgaatggaa atatttttca
tactggtggt attggaacac agccccagaa agagtggaat 180tttcagctac cacaattggg
tacaacttta ttaaatttag atgataatta tgttcaatca 240actagaggca taattgattt
tttatcatct tttatagaag ctgtatgtga tgatgaaatt 300gttagagaag cttcaagaaa
tggtatgcaa cctcaatcac cagctcttat attattatct 360tcatcaaaat ttaagacaat
taattttaat aatagttctc aatctatcaa aaattggaat 420gctcaatcaa gacgtgagaa
tcctgtatat gaatacaaaa atccaatgtt gttggaatat 480aaaaattctt atattttaca
acgcgcaatt ccacaatttg gaagcgtcat gggtttaaga 540tattatacaa caagtaatac
ttgtcaaatt gcagcatttg attccaccct agctgaaaat 600gcaccaaaca atacgcaacg
cttcgtttat aatggcagac taaaaagacc catatcaaat 660gttttaatga aaatagaagc
tggtgctcca aatataagca acccaactat tttacctgat 720cctaataatc aaacaacttg
gctttttaat ccggtacaat taatgaatgg aacattcacc 780attgaattct ataataatgg
tcaactaatt gatatggttc gaaatatggg aatagttact 840gtaaggactt ttgattctta
tagaataaca attgacatga ttagaccagc tgctaggact 900caatacgttc aacaaatttt
tccacaaggt ggaccttatc attttcaggc tacatatatg 960ttaacattaa gtatattaga
tgctaccaca gagtccgttc tatgtgattc tcattcagta 1020gaatattcaa tagtagcaaa
cgtcagaaga gattcagcga tgccagctgg aactgttttt 1080caaccgggtt ttccatggga
acacacacta tccaattaca ctgttgctca agaagataat 1140ttagaaagat tattgttaat
cgcatctgtg aagagaatgg taatg 1185301185DNAHuman
rotavirus Cmisc_featureHuman rotavirus group C VP6 BCN6 nucleotide
sequence 30atggatgtac ttttttctat agcgaaaact gtgtcagatc ttaaaaagaa
agttgtagtt 60ggaacaattt atactaatgt agaagatgtt gtacaacaga cgaatgaatt
gattagaact 120ttaaatggaa atgtttttca tactggtggc attggaacac agcctcagaa
agagtggaat 180tttcagctgc cacaattggg tacaacttta ttaaatttag atgataatta
tgttcaatca 240actagaggca taattgattt tttatcatct tttatagaag ctgtatgtga
tgatgaaatt 300gttagagaag cttcaagaaa tggtatgcaa cctcaatcac cagctcttat
attattatct 360tcatcaaagt ttaaaacaat taattttaat aatagttctc aatccattaa
aaattggaat 420gctcaatcaa gacgtgagaa tcctgtatat gagtataaaa atccaatgtt
gtttgaatat 480aaaaattcgt atattttaca acgcgcaaat ccacaatttg gaagcgtcat
gggtttaaga 540tactatacaa caagtaatac ttgtcaaatt gcagcatttg attccacctt
agctgaaaat 600gcaccaaaca atacacaacg tttcgtttat aatggcagac taaaaagacc
catatcaaac 660gttttaatga aaattgaagc tggtgctcca aatataagca acccaactat
tttacctgat 720cctaataatc aaacaacttg gctatttaat ccggtacaat taatgaatgg
aacatttacc 780attgaattct ataataatgg tcaactaatt gatatggttc gaaatatggg
aatagttact 840gtaagaactt ttgattctta tagaataaca attgacatga ttagaccagc
tgctatgact 900caatacgttc aacgaatctt tccacaaggt ggaccttatc attttcaggc
tacatatatg 960ttaacattaa gtatattaga tgctaccaca gagtccgttc tatgtgattc
tcattcagtg 1020gaatattcaa tagtagcaaa cgttagaaga gattcagcga tgccaactgg
aactgttttt 1080caaccgggat ttccatggga acacacacta tccaattaca ctgttgctca
agaagataat 1140ttagaaagat tattgttaat tgcatctgtg aagagaatgg taatg
1185311185DNAHuman rotavirus Cmisc_featureHuman rotavirus
group C VP6 S1 nucleotide sequence 31atggatgtac ttttttctat
agcgaaaacc gtgtcagatc ttaaagagaa agttgtagtt 60ggaacaattt atactaatgt
agaagatgtt gtacaacaga cgaatgaatt gattagaact 120ttgaatggaa atatttttca
tactggtggc attggaacac agcctcagaa agagtggaat 180tttcagctcc cacaattggg
taccacttta ttaaatttag atgataatta tgttcaatca 240actagaggca taattgattt
tttatcatct tttatagaag ctgtatgtga tgatgaaatt 300gttagagaag cttcaagaaa
tggtatgcaa cctcaatcac cagctcttat attattatct 360tcatcaaaat ttaaaacaat
taattttaat aatagttctc aatctatcaa aaattggaat 420gctcaatcaa gacgtgagaa
tcctgtatat gagtacaaaa atccaatgtt gtttgaatat 480aaaaattctt atattttaca
acgcgcaaat ccacaatttg gaagcgtcat gggtttaaga 540tattatacaa caagtaatat
ttgtcaaatt gcagcatttg attccaccct agctgaaaat 600gcaccaaata atacgcaacg
cttcgtttat aatggcagac taaaaagacc catatcaaat 660gttttaatga aaatagaagc
tggtgctcca aatataagca acccaactat tttacctgat 720cctaataatc aaacaacttg
gctttttaat ccggtacaat taatgaatgg aacatttacc 780attgaattct ataataatgg
tcaactaatt gatatggttc gaaatatggg aatagttact 840gtaagaactt ttgattctta
tagaataaca attgacatga ttagaccagc tgctatgact 900caatacgttc aacgaatttt
tccacaaggt ggaccttatc attttcaggc tacatatatg 960ttaacattaa gtatattaga
tgctaccaca gagtccgttc tatgtgattc tcattcagta 1020gaatattcaa tagtagcaaa
cgtcagaaga gattcagcaa tgccagctgg aactgttttt 1080caaccgggat ttccatggga
acacacacta tccaattaca ctgttgctca agaagataat 1140ttagaaagat tattgttaat
cgcatctgtg aagagaatgg taatg 118532395PRTHuman rotavirus
CMISC_FEATUREHuman rotavirus group C VP6 S1 protein sequence 32Met Asp
Val Leu Phe Ser Ile Ala Lys Thr Val Ser Asp Leu Lys Glu1 5
10 15Lys Val Val Val Gly Thr Ile Tyr
Thr Asn Val Glu Asp Val Val Gln 20 25
30Gln Thr Asn Glu Leu Ile Arg Thr Leu Asn Gly Asn Ile Phe His
Thr 35 40 45Gly Gly Ile Gly Thr
Gln Pro Gln Lys Glu Trp Asn Phe Gln Leu Pro 50 55
60Gln Leu Gly Thr Thr Leu Leu Asn Leu Asp Asp Asn Tyr Val
Gln Ser65 70 75 80Thr
Arg Gly Ile Ile Asp Phe Leu Ser Ser Phe Ile Glu Ala Val Cys
85 90 95Asp Asp Glu Ile Val Arg Glu
Ala Ser Arg Asn Gly Met Gln Pro Gln 100 105
110Ser Pro Ala Leu Ile Leu Leu Ser Ser Ser Lys Phe Lys Thr
Ile Asn 115 120 125Phe Asn Asn Ser
Ser Gln Ser Ile Lys Asn Trp Asn Ala Gln Ser Arg 130
135 140Arg Glu Asn Pro Val Tyr Glu Tyr Lys Asn Pro Met
Leu Phe Glu Tyr145 150 155
160Lys Asn Ser Tyr Ile Leu Gln Arg Ala Asn Pro Gln Phe Gly Ser Val
165 170 175Met Gly Leu Arg Tyr
Tyr Thr Thr Ser Asn Ile Cys Gln Ile Ala Ala 180
185 190Phe Asp Ser Thr Leu Ala Glu Asn Ala Pro Asn Asn
Thr Gln Arg Phe 195 200 205Val Tyr
Asn Gly Arg Leu Lys Arg Pro Ile Ser Asn Val Leu Met Lys 210
215 220Ile Glu Ala Gly Ala Pro Asn Ile Ser Asn Pro
Thr Ile Leu Pro Asp225 230 235
240Pro Asn Asn Gln Thr Thr Trp Leu Phe Asn Pro Val Gln Leu Met Asn
245 250 255Gly Thr Phe Thr
Ile Glu Phe Tyr Asn Asn Gly Gln Leu Ile Asp Met 260
265 270Val Arg Asn Met Gly Ile Val Thr Val Arg Thr
Phe Asp Ser Tyr Arg 275 280 285Ile
Thr Ile Asp Met Ile Arg Pro Ala Ala Met Thr Gln Tyr Val Gln 290
295 300Arg Ile Phe Pro Gln Gly Gly Pro Tyr His
Phe Gln Ala Thr Tyr Met305 310 315
320Leu Thr Leu Ser Ile Leu Asp Ala Thr Thr Glu Ser Val Leu Cys
Asp 325 330 335Ser His Ser
Val Glu Tyr Ser Ile Val Ala Asn Val Arg Arg Asp Ser 340
345 350Ala Met Pro Ala Gly Thr Val Phe Gln Pro
Gly Phe Pro Trp Glu His 355 360
365Thr Leu Ser Asn Tyr Thr Val Ala Gln Glu Asp Asn Leu Glu Arg Leu 370
375 380Leu Leu Ile Ala Ser Val Lys Arg
Met Val Met385 390 395331063DNAHuman
rotavirus Cmisc_featureHuman rotavirus group C VP7 S1 nucleotide
sequence 33ggcatttaaa aaagaagaag ctgtctgaca aactggtctt ctttttaaat
ggtttgtaca 60acattgtaca ctgtttgcgc cattctcttc attcttttca tttatatatt
attatttaga 120aaaatgttcc acctaataac tgatacttta atagtgatgc ttattttatc
taattgtgta 180gagtggtcac aaggtcagat gtttattgat gatatacatt ataatggtaa
cgttgagact 240atcataaatt ctactgatcc ttttaatgtt gaatctttat gtatttattt
tccaaatgca 300gttgtaggat cacagggacc aggtaaatcc gatggacatt tgaatgatgg
taattatgca 360cagactatcg ccactttgtt tgaaacaaaa ggattcccaa aaggttcaat
aataattaaa 420acatatacac agacatcaga ctttataaat tcagtagaaa tgacatgctc
ttataatata 480gttatcattc ctgatagccc aaatgattca gaatctattg aacagatagc
agaatggatt 540ttaaatgttt ggagatgtga tgacatgaat ttggaaattt atacttatga
acaaattgga 600ataaacaatt tatgggctgc atttggtagt gactgtgata tatctgtctg
tccattagat 660actacaagta atggaatcgg atgttcacca gctagtacag aaacttatga
agttgtatca 720aatgacaccc aattggcctt aattaatgtt gtggataatg ttagacatag
aatacagatg 780aacactgctc aatgtaaatt gaaaaattgt attaagggtg aagctcgact
gaatactgca 840ctaataagaa tttcaacatc atcaagtttt gataattcat tgtcaccatt
aaataacggc 900caaacaacaa gatcgtttaa aataaatgca aagaaatggt ggactatatt
ttatacaata 960attgattata ttaatacaat tgtacaatca atgactccca gacatcgggc
gatttatcca 1020gaagggtgga tgttgaggta tgcgtaaaca agatcatgtg gct
106334332PRTHuman rotavirus CMISC_FEATUREHuman rotavirus group
C VP7 S1 nucleotide sequence 34Met Val Cys Thr Thr Leu Tyr Thr Val
Cys Ala Ile Leu Phe Ile Leu1 5 10
15Phe Ile Tyr Ile Leu Leu Phe Arg Lys Met Phe His Leu Ile Thr
Asp 20 25 30Thr Leu Ile Val
Met Leu Ile Leu Ser Asn Cys Val Glu Trp Ser Gln 35
40 45Gly Gln Met Phe Ile Asp Asp Ile His Tyr Asn Gly
Asn Val Glu Thr 50 55 60Ile Ile Asn
Ser Thr Asp Pro Phe Asn Val Glu Ser Leu Cys Ile Tyr65 70
75 80Phe Pro Asn Ala Val Val Gly Ser
Gln Gly Pro Gly Lys Ser Asp Gly 85 90
95His Leu Asn Asp Gly Asn Tyr Ala Gln Thr Ile Ala Thr Leu
Phe Glu 100 105 110Thr Lys Gly
Phe Pro Lys Gly Ser Ile Ile Ile Lys Thr Tyr Thr Gln 115
120 125Thr Ser Asp Phe Ile Asn Ser Val Glu Met Thr
Cys Ser Tyr Asn Ile 130 135 140Val Ile
Ile Pro Asp Ser Pro Asn Asp Ser Glu Ser Ile Glu Gln Ile145
150 155 160Ala Glu Trp Ile Leu Asn Val
Trp Arg Cys Asp Asp Met Asn Leu Glu 165
170 175Ile Tyr Thr Tyr Glu Gln Ile Gly Ile Asn Asn Leu
Trp Ala Ala Phe 180 185 190Gly
Ser Asp Cys Asp Ile Ser Val Cys Pro Leu Asp Thr Thr Ser Asn 195
200 205Gly Ile Gly Cys Ser Pro Ala Ser Thr
Glu Thr Tyr Glu Val Val Ser 210 215
220Asn Asp Thr Gln Leu Ala Leu Ile Asn Val Val Asp Asn Val Arg His225
230 235 240Arg Ile Gln Met
Asn Thr Ala Gln Cys Lys Leu Lys Asn Cys Ile Lys 245
250 255Gly Glu Ala Arg Leu Asn Thr Ala Leu Ile
Arg Ile Ser Thr Ser Ser 260 265
270Ser Phe Asp Asn Ser Leu Ser Pro Leu Asn Asn Gly Gln Thr Thr Arg
275 280 285Ser Phe Lys Ile Asn Ala Lys
Lys Trp Trp Thr Ile Phe Tyr Thr Ile 290 295
300Ile Asp Tyr Ile Asn Thr Ile Val Gln Ser Met Thr Pro Arg His
Arg305 310 315 320Ala Ile
Tyr Pro Glu Gly Trp Met Leu Arg Tyr Ala 325
33035395PRTHuman rotavirus CMISC_FEATUREHuman rotavirus group C VP6
Bristol protein sequence 35Met Asp Val Leu Phe Ser Ile Ala Lys Thr
Val Ser Asp Leu Lys Lys1 5 10
15Lys Val Val Val Gly Thr Ile Tyr Thr Asn Val Glu Asp Val Val Gln
20 25 30Gln Thr Asn Glu Leu Ile
Arg Thr Leu Asn Gly Asn Ile Phe His Thr 35 40
45Gly Gly Ile Gly Thr Gln Pro Gln Lys Glu Trp Asn Phe Gln
Leu Pro 50 55 60Gln Leu Gly Thr Thr
Leu Leu Asn Leu Asp Asp Asn Tyr Val Gln Ser65 70
75 80Thr Arg Gly Ile Ile Asp Phe Leu Ser Ser
Phe Ile Glu Ala Val Cys 85 90
95Asp Asp Glu Ile Val Arg Glu Ala Ser Arg Asn Gly Met Gln Pro Gln
100 105 110Ser Pro Ala Leu Ile
Leu Leu Ser Ser Ser Lys Phe Lys Thr Ile Asn 115
120 125Phe Asn Asn Ser Ser Gln Ser Ile Lys Asn Trp Asn
Ala Gln Ser Arg 130 135 140Arg Glu Asn
Pro Val Tyr Glu Tyr Lys Asn Pro Met Leu Phe Glu Tyr145
150 155 160Lys Asn Ser Tyr Ile Leu Gln
Arg Ala Asn Pro Gln Phe Gly Ser Val 165
170 175Met Gly Leu Arg Tyr Tyr Thr Thr Ser Asn Thr Cys
Gln Ile Ala Ala 180 185 190Phe
Asp Ser Thr Leu Ala Glu Asn Ala Pro Asn Asn Thr Gln Arg Phe 195
200 205Val Tyr Asn Gly Arg Leu Lys Arg Pro
Ile Ser Asn Val Leu Met Lys 210 215
220Ile Glu Ala Gly Ala Pro Asn Ile Ser Asn Pro Thr Ile Leu Pro Asp225
230 235 240Pro Asn Asn Gln
Thr Thr Trp Leu Phe Asn Pro Val Gln Leu Met Asn 245
250 255Gly Thr Phe Thr Ile Glu Phe Tyr Asn Asn
Gly Gln Leu Ile Asp Met 260 265
270Val Arg Asn Met Gly Ile Val Thr Val Arg Thr Phe Asp Ser Tyr Arg
275 280 285Ile Thr Ile Asp Met Ile Arg
Pro Ala Ala Met Thr Gln Tyr Val Gln 290 295
300Arg Ile Phe Pro Gln Gly Gly Pro Tyr His Phe Gln Ala Thr Tyr
Met305 310 315 320Leu Thr
Leu Ser Ile Leu Asp Ala Thr Thr Glu Ser Val Leu Cys Asp
325 330 335Ser His Ser Val Glu Tyr Ser
Ile Val Ala Asn Val Arg Arg Asp Ser 340 345
350Ala Met Pro Ala Gly Thr Val Phe Gln Pro Gly Phe Pro Trp
Glu His 355 360 365Thr Leu Ser Asn
Tyr Thr Val Ala Gln Glu Asp Asn Leu Glu Arg Leu 370
375 380Leu Leu Ile Ala Ser Val Lys Arg Met Val Met385
390 39536395PRTHuman rotavirus
CMISC_FEATUREHuman rotavirus group C VP6 Jajeri protein sequence
36Met Asp Val Leu Phe Ser Ile Ala Lys Thr Val Ser Asp Leu Lys Lys1
5 10 15Lys Val Val Val Gly Thr
Ile Tyr Thr Asn Val Glu Asp Val Val Gln 20 25
30Gln Thr Asn Glu Leu Ile Arg Thr Leu Asn Gly Asn Ile
Phe Tyr Thr 35 40 45Gly Gly Ile
Gly Thr Gln Pro Gln Lys Glu Trp Asn Phe Gln Leu Pro 50
55 60Gln Leu Gly Thr Thr Leu Leu Asn Leu Asp Asp Asn
Tyr Val Gln Ser65 70 75
80Thr Arg Gly Ile Ile Asp Phe Leu Ser Ser Phe Ile Glu Ala Val Cys
85 90 95Asp Asp Glu Ile Val Arg
Glu Ala Ser Arg Asn Gly Met Gln Pro Gln 100
105 110Ser Pro Ala Leu Ile Leu Leu Ser Ser Ser Lys Phe
Lys Thr Ile Asn 115 120 125Phe Asn
Asn Ser Ser Gln Ser Ile Lys Asn Trp Asn Ala Gln Ser Arg 130
135 140Arg Glu Asn Pro Val Tyr Glu Tyr Lys Asn Pro
Met Val Phe Glu Tyr145 150 155
160Lys Asn Ser Tyr Ile Leu Gln Arg Ala Asn Pro Gln Phe Gly Ser Val
165 170 175Met Gly Leu Arg
Tyr Tyr Thr Thr Ser Asn Thr Cys Gln Ile Ala Ala 180
185 190Phe Asp Ser Thr Leu Ala Glu Asn Ala Pro Asn
Asn Thr Gln Arg Phe 195 200 205Val
Tyr Asn Gly Arg Leu Lys Arg Pro Ile Ser Asn Val Leu Met Lys 210
215 220Ile Glu Ala Gly Ala Pro Asn Ile Ser Asn
Pro Thr Ile Leu Pro Asp225 230 235
240Pro Asn Asn Gln Thr Thr Trp Leu Phe Asn Pro Val Gln Leu Met
Asn 245 250 255Gly Thr Phe
Thr Ile Glu Phe Tyr Asn Asn Gly Gln Leu Ile Asp Met 260
265 270Val Arg Asn Met Gly Ile Val Thr Val Arg
Thr Phe Asp Ser Tyr Arg 275 280
285Ile Thr Ile Asp Ile Ile Arg Pro Ala Ala Met Thr Gln Tyr Val Gln 290
295 300Arg Ile Phe Pro Gln Gly Gly Pro
Tyr His Phe Gln Ala Thr Tyr Met305 310
315 320Leu Thr Leu Ser Ile Leu Asp Ala Thr Thr Glu Ser
Val Leu Cys Asp 325 330
335Ser His Ser Val Glu Tyr Ser Ile Val Ala Asn Val Arg Arg Asp Ser
340 345 350Ala Met Pro Ala Gly Thr
Val Phe Gln Pro Gly Phe Pro Trp Glu His 355 360
365Thr Leu Ser Asn Tyr Thr Val Ala Gln Glu Asp Asn Leu Glu
Arg Leu 370 375 380Leu Leu Ile Ala Ser
Val Lys Arg Met Val Met385 390
39537395PRTHuman rotavirus CMISC_FEATUREHuman rotavirus group C VP6
CMH004 protein sequence 37Met Asp Val Leu Phe Ser Ile Ala Lys Thr
Val Ser Asp Leu Lys Lys1 5 10
15Lys Val Val Val Gly Thr Ile Tyr Thr Asn Val Glu Asp Val Val Gln
20 25 30Gln Thr Asn Glu Leu Ile
Arg Thr Leu Asn Gly Asn Ile Phe His Thr 35 40
45Gly Gly Ile Gly Thr Gln Pro His Lys Glu Trp Asn Phe Gln
Leu Pro 50 55 60Gln Leu Gly Thr Thr
Leu Leu Asn Leu Asp Asp Asn Tyr Val Gln Ser65 70
75 80Thr Arg Gly Ile Ile Asp Phe Leu Ser Ser
Phe Ile Glu Ala Val Cys 85 90
95Asp Asp Glu Ile Val Arg Glu Ala Ser Arg Asn Gly Met Gln Pro Gln
100 105 110Ser Pro Ala Leu Ile
Leu Leu Ser Ser Ser Lys Phe Lys Thr Ile Asn 115
120 125Phe Asn Asn Ser Ser Gln Ser Ile Lys Asn Trp Asn
Ala Gln Ser Arg 130 135 140Arg Glu Asn
Pro Val Tyr Glu Tyr Lys Asn Pro Met Leu Phe Glu Tyr145
150 155 160Lys Asn Ser Tyr Ile Leu Gln
Arg Ala Asn Pro Gln Phe Gly Ser Val 165
170 175Met Gly Leu Arg Tyr Tyr Thr Thr Ser Asn Thr Cys
Gln Ile Ala Ala 180 185 190Phe
Asp Ser Thr Leu Ala Glu Asn Ala Pro Asn Asn Thr Gln Arg Phe 195
200 205Val Tyr Asn Gly Arg Leu Lys Arg Pro
Ile Ser Asn Val Leu Met Lys 210 215
220Ile Glu Ala Gly Ala Pro Asn Ile Ser Asn Pro Thr Ile Leu Pro Asp225
230 235 240Pro Asn Asn Gln
Thr Thr Trp Leu Phe Asn Pro Val Gln Leu Met Asn 245
250 255Gly Thr Phe Thr Ile Glu Phe Tyr Asn Asn
Gly Gln Leu Ile Asp Met 260 265
270Val Arg Asn Met Gly Ile Val Thr Val Arg Thr Phe Asp Ser Tyr Arg
275 280 285Ile Thr Ile Asp Met Ile Arg
Pro Ala Ala Met Thr Gln Tyr Val Gln 290 295
300Arg Ile Phe Pro Gln Gly Gly Pro Tyr His Phe Gln Ala Thr Tyr
Met305 310 315 320Leu Thr
Leu Ser Ile Leu Asp Ala Thr Thr Glu Ser Val Leu Cys Asp
325 330 335Ser His Ser Val Glu Tyr Ser
Ile Val Ala Asn Val Arg Arg Asp Ser 340 345
350Ala Met Pro Ala Gly Thr Val Phe Gln Pro Gly Phe Pro Trp
Glu His 355 360 365Thr Leu Ser Asn
Tyr Thr Val Ala Gln Glu Asp Asn Leu Glu Arg Leu 370
375 380Leu Leu Ile Ala Ser Val Lys Arg Met Val Met385
390 39538395PRTHuman rotavirus
CMISC_FEATUREHuman rotavirus group C VP6 V508 protein sequence 38Met
Asp Val Leu Phe Ser Ile Ala Lys Thr Val Ser Asp Leu Lys Lys1
5 10 15Lys Val Val Val Gly Thr Ile
Tyr Thr Asn Val Glu Asp Val Val Gln 20 25
30Gln Thr Asn Glu Leu Ile Arg Thr Leu Asn Gly Asn Val Phe
His Thr 35 40 45Gly Gly Ile Gly
Thr Gln Pro Gln Lys Glu Trp Asn Phe Gln Leu Pro 50 55
60Gln Leu Gly Thr Thr Leu Leu Asn Leu Asp Asp Asn Tyr
Val Gln Ser65 70 75
80Thr Arg Gly Ile Ile Asp Phe Leu Ser Ser Phe Ile Glu Ala Val Cys
85 90 95Asp Asp Glu Ile Val Arg
Glu Ala Ser Arg Asn Gly Met Gln Pro Gln 100
105 110Ser Pro Ala Leu Ile Leu Leu Ser Ser Ser Lys Phe
Lys Thr Ile Asn 115 120 125Leu Asn
Asn Ser Ser Gln Ser Ile Lys Asn Trp Asn Ala Gln Ser Arg 130
135 140Arg Glu Asn Pro Val Tyr Glu Tyr Lys Asn Pro
Met Leu Phe Glu Tyr145 150 155
160Lys Asn Ser Tyr Ile Leu Gln Arg Ala Asn Pro Gln Phe Gly Ser Val
165 170 175Met Gly Leu Arg
Tyr Tyr Thr Thr Ser Asn Thr Cys Gln Ile Ala Ala 180
185 190Phe Asp Ser Thr Leu Ala Glu Asn Ala Pro Asn
Asn Thr Gln Arg Phe 195 200 205Val
Tyr Asn Gly Arg Leu Lys Arg Pro Ile Ser Asn Val Leu Met Lys 210
215 220Ile Glu Ala Gly Ala Pro Asn Ile Ser Asn
Pro Thr Ile Leu Pro Asp225 230 235
240Pro Asn Asn Gln Thr Thr Trp Leu Phe Asn Pro Val Gln Leu Met
Asn 245 250 255Gly Thr Phe
Thr Ile Glu Phe Tyr Asn Asn Gly Gln Leu Ile Asp Met 260
265 270Val Arg Asn Met Gly Ile Val Thr Val Arg
Thr Phe Asp Ser Tyr Arg 275 280
285Ile Thr Ile Asp Met Ile Arg Pro Ala Ala Met Thr Gln Tyr Val Gln 290
295 300Arg Ile Phe Pro Gln Gly Gly Pro
Tyr His Phe Gln Ala Thr Tyr Met305 310
315 320Leu Thr Leu Ser Ile Leu Asp Ala Thr Thr Glu Ser
Val Leu Cys Asp 325 330
335Ser His Ser Val Glu Tyr Ser Ile Val Ala Asn Val Arg Arg Asp Ser
340 345 350Ala Met Pro Ala Gly Thr
Val Phe Gln Pro Gly Phe Pro Trp Glu His 355 360
365Thr Leu Ser Asn Tyr Thr Val Ala Gln Glu Asp Asn Leu Glu
Arg Leu 370 375 380Leu Leu Ile Ala Ser
Val Lys Arg Met Val Met385 390
39539395PRTHuman rotavirus CMISC_FEATUREHuman rotavirus group C VP6 China
protein sequence 39Met Asp Val Leu Phe Ser Ile Ala Lys Thr Val Ser
Asp Leu Lys Lys1 5 10
15Lys Val Val Val Gly Thr Ile Tyr Thr Asn Val Glu Asp Val Val Gln
20 25 30Gln Thr Asn Glu Leu Ile Arg
Thr Leu Asn Gly Asn Ile Phe His Thr 35 40
45Gly Gly Ile Gly Thr Gln Pro Gln Lys Glu Trp Asn Phe Gln Leu
Pro 50 55 60Gln Leu Gly Thr Thr Leu
Leu Asn Leu Asp Asp Asn Tyr Val Gln Ser65 70
75 80Thr Arg Gly Ile Ile Asp Phe Leu Ser Ser Phe
Ile Glu Ala Val Cys 85 90
95Asp Asp Glu Ile Val Arg Glu Ala Ser Arg Asn Gly Met Gln Pro Gln
100 105 110Ser Pro Ala Leu Ile Leu
Leu Ser Ser Ser Lys Phe Lys Thr Ile Asn 115 120
125Phe Asn Asn Ser Ser Gln Ser Ile Lys Asn Trp Asn Ala Gln
Ser Arg 130 135 140Arg Glu Asn Pro Val
Tyr Glu Tyr Lys Asn Pro Met Leu Leu Glu Tyr145 150
155 160Lys Asn Ser Tyr Ile Leu Gln Arg Ala Ile
Pro Gln Phe Gly Ser Val 165 170
175Met Gly Leu Arg Tyr Tyr Thr Thr Ser Asn Thr Cys Gln Ile Ala Ala
180 185 190Phe Asp Ser Thr Leu
Ala Glu Asn Ala Pro Asn Asn Thr Gln Arg Phe 195
200 205Val Tyr Asn Gly Arg Leu Lys Arg Pro Ile Ser Asn
Val Leu Met Lys 210 215 220Ile Glu Ala
Gly Ala Pro Asn Ile Ser Asn Pro Thr Ile Leu Pro Asp225
230 235 240Pro Asn Asn Gln Thr Thr Trp
Leu Phe Asn Pro Val Gln Leu Met Asn 245
250 255Gly Thr Phe Thr Ile Glu Phe Tyr Asn Asn Gly Gln
Leu Ile Asp Met 260 265 270Val
Arg Asn Met Gly Ile Val Thr Val Arg Thr Phe Asp Ser Tyr Arg 275
280 285Ile Thr Ile Asp Met Ile Arg Pro Ala
Ala Arg Thr Gln Tyr Val Gln 290 295
300Gln Ile Phe Pro Gln Gly Gly Pro Tyr His Phe Gln Ala Thr Tyr Met305
310 315 320Leu Thr Leu Ser
Ile Leu Asp Ala Thr Thr Glu Ser Val Leu Cys Asp 325
330 335Ser His Ser Val Glu Tyr Ser Ile Val Ala
Asn Val Arg Arg Asp Ser 340 345
350Ala Met Pro Ala Gly Thr Val Phe Gln Pro Gly Phe Pro Trp Glu His
355 360 365Thr Leu Ser Asn Tyr Thr Val
Ala Gln Glu Asp Asn Leu Glu Arg Leu 370 375
380Leu Leu Ile Ala Ser Val Lys Arg Met Val Met385
390 39540395PRTHuman rotavirus CMISC_FEATUREHuman
rotavirus group C VP6 BCN6 protein sequence 40Met Asp Val Leu Phe
Ser Ile Ala Lys Thr Val Ser Asp Leu Lys Lys1 5
10 15Lys Val Val Val Gly Thr Ile Tyr Thr Asn Val
Glu Asp Val Val Gln 20 25
30Gln Thr Asn Glu Leu Ile Arg Thr Leu Asn Gly Asn Val Phe His Thr
35 40 45Gly Gly Ile Gly Thr Gln Pro Gln
Lys Glu Trp Asn Phe Gln Leu Pro 50 55
60Gln Leu Gly Thr Thr Leu Leu Asn Leu Asp Asp Asn Tyr Val Gln Ser65
70 75 80Thr Arg Gly Ile Ile
Asp Phe Leu Ser Ser Phe Ile Glu Ala Val Cys 85
90 95Asp Asp Glu Ile Val Arg Glu Ala Ser Arg Asn
Gly Met Gln Pro Gln 100 105
110Ser Pro Ala Leu Ile Leu Leu Ser Ser Ser Lys Phe Lys Thr Ile Asn
115 120 125Phe Asn Asn Ser Ser Gln Ser
Ile Lys Asn Trp Asn Ala Gln Ser Arg 130 135
140Arg Glu Asn Pro Val Tyr Glu Tyr Lys Asn Pro Met Leu Phe Glu
Tyr145 150 155 160Lys Asn
Ser Tyr Ile Leu Gln Arg Ala Asn Pro Gln Phe Gly Ser Val
165 170 175Met Gly Leu Arg Tyr Tyr Thr
Thr Ser Asn Thr Cys Gln Ile Ala Ala 180 185
190Phe Asp Ser Thr Leu Ala Glu Asn Ala Pro Asn Asn Thr Gln
Arg Phe 195 200 205Val Tyr Asn Gly
Arg Leu Lys Arg Pro Ile Ser Asn Val Leu Met Lys 210
215 220Ile Glu Ala Gly Ala Pro Asn Ile Ser Asn Pro Thr
Ile Leu Pro Asp225 230 235
240Pro Asn Asn Gln Thr Thr Trp Leu Phe Asn Pro Val Gln Leu Met Asn
245 250 255Gly Thr Phe Thr Ile
Glu Phe Tyr Asn Asn Gly Gln Leu Ile Asp Met 260
265 270Val Arg Asn Met Gly Ile Val Thr Val Arg Thr Phe
Asp Ser Tyr Arg 275 280 285Ile Thr
Ile Asp Met Ile Arg Pro Ala Ala Met Thr Gln Tyr Val Gln 290
295 300Arg Ile Phe Pro Gln Gly Gly Pro Tyr His Phe
Gln Ala Thr Tyr Met305 310 315
320Leu Thr Leu Ser Ile Leu Asp Ala Thr Thr Glu Ser Val Leu Cys Asp
325 330 335Ser His Ser Val
Glu Tyr Ser Ile Val Ala Asn Val Arg Arg Asp Ser 340
345 350Ala Met Pro Thr Gly Thr Val Phe Gln Pro Gly
Phe Pro Trp Glu His 355 360 365Thr
Leu Ser Asn Tyr Thr Val Ala Gln Glu Asp Asn Leu Glu Arg Leu 370
375 380Leu Leu Ile Ala Ser Val Lys Arg Met Val
Met385 390 395411185DNAHuman rotavirus
Cmisc_featureHuman rotavirus group C VP6 nucleotide consensus
sequence 41atggatgtac ttttttctat agcgaaaact gtgtcagatc ttaaaaagaa
agttgtagtt 60ggaacaattt atactaatgt agaagatgtt gtacaacaga cgaatgaatt
gattagaact 120ttgaatggaa atatttttca tactggtggc attggaacac agcctcagaa
agagtggaat 180tttcagctac cacaattggg tacaacttta ttaaatttag atgataatta
tgttcaatca 240actagaggca taattgattt tttatcatct tttatagaag ctgtatgtga
tgatgaaatt 300gttagagaag cttcaagaaa tggtatgcaa cctcaatcac cagctcttat
attattatct 360tcatcaaaat ttaaaacaat taattttaat aatagttctc aatctatcaa
aaattggaat 420gctcaatcaa gacgtgagaa tcctgtatat gagtacaaaa atccaatgtt
gtttgaatat 480aaaaattctt atattttaca acgcgcaaat ccacaatttg gaagcgtcat
gggtttaaga 540tattatacaa caagtaatac ttgtcaaatt gcagcatttg attccaccct
agctgaaaat 600gcaccaaaca atacgcaacg cttcgtttat aatggcagac taaaaagacc
catatcaaat 660gttttaatga aaatagaagc tggtgctcca aatataagca acccaactat
tttacctgat 720cctaataatc aaacaacttg gctttttaat ccggtacaat taatgaatgg
aacatttacc 780attgaattct ataataatgg tcaactaatt gatatggttc gaaatatggg
aatagttact 840gtaagaactt ttgattctta tagaataaca attgacatga ttagaccagc
tgctatgact 900caatacgttc aacgaatttt tccacaaggt ggaccttatc attttcaggc
tacatatatg 960ttaacattaa gtatattaga tgctaccaca gagtccgttc tatgtgattc
tcattcagta 1020gaatattcaa tagtagcaaa cgtcagaaga gattcagcga tgccagctgg
aactgttttt 1080caaccgggat ttccatggga acacacacta tccaattaca ctgttgctca
agaagataat 1140ttagaaagat tattgttaat cgcatctgtg aagagaatgg taatg
1185422655DNAHuman rotavirus Cmisc_featureHuman rotavirus
group C strain ASP88 VP2 S1 nucleotide sequence of open reading
frame 42atgataagca gaaacaggcg cagaaataac caacaaaaag atataggaaa agagaaacaa
60ttagagacta taattgacaa agaagtaaag gaaaacaaag attctacaaa agaagataag
120ctagtagtta cggaagaaag taatggtgac gtcacagctg ttaaagaaca atcgaataat
180attaatttac aaaagaatga tttggttaaa gaagtcatga atatacagaa tcaaacatta
240aatacagtag ttgctgagaa taaagttgaa atagaagaaa tagttaaaaa atacattccc
300tcatataata ctgacagcct tattgttaaa aagttaactg aaatccagga atcaagtgct
360aaaacatata atacattatt cagattattt actccagtta aaagttattt atatgacata
420aatggtgaga aaaaattatc gactagatgg tattggaaat tgctcaaaga tgatttacct
480gctggtgatt actcagttag acaattcttc ctgtcactat atttaaatgt tttagaggga
540atgcccgatt acataatgct tcgtgatatg gcagtggata acccatattc agcagaagca
600ggtaaaatcg tagatggaaa gtctaaagaa attttagttg aactatatca agaccaaatg
660acagaagggt atattagaag atatatgtct gaattaagac ataaaatatc tggagaaaca
720aatactgcaa aatatccagc tattctacat cccgtggata atgagcttaa tcaatacttt
780cttgagcatc agttaattca accattaact acaagaaata ttgcagaatt gattccaact
840caattatatc atgatccaaa ttacgttttt aatattgatg cagccttttt aacaaattca
900agatttgttc caccatactt aacacaggat aggattggat tacatgatgg attcgaatca
960atatgggatt caaaaaccca tgctgattac gtttcagcta gaagatttat acctgattta
1020actgaactgg tagatgctga aaagcaaata aaagaaatgg ctgcacattt acaactagag
1080gctattacag tacaggttga atcacaattt ttagcgggaa ttagtgctgc tgcagctaat
1140gaagcgttca aatttataat tggctcagtt ttatctacca gaacaatagc tgtagaattc
1200ataacctcaa actatatgtc gttagcatca tgtatgtatt taatgactat tatgccatca
1260gagattttct tgagagaatc attagttgct atgcgattag caataataaa tacccttatt
1320tatccagctc taggtttagc gcaaatgcat tatcaagcag gtgaagtgag gaccccattc
1380gaattagctg agatgcgagt agctaataga tctattagac aatggttaca tcattgtaat
1440acacttcaat ttggtagaca gataacggaa gggataattc atctacgatt tactaatgat
1500atcatgacag gtaggatagt gaacttattt tcaacaatgc tagtagcttt atcatctcag
1560ccattcgcta catatccttt agactataaa agatctgtac aaagagcatt acaactttta
1620tcaaatagaa cagcccaaat agcagattta accagattaa tagtatacaa ttatactaca
1680ttatctgcat gtatagtcat gaatatgcat ttagtaggaa ctcttactgt tgaacgtata
1740caggccactt ctctaacttc tttaatgatg ttaatttcta ataagacagt tattccagaa
1800ccatcgtctc ttttttcata tttctctagt aacattaatt ttcttacaaa ttataatgag
1860caaattgata atgtggtagc agaaataatg gccgcatata gattgaattt atatcaacag
1920aaaatgttga tgctcgttac caggtttgtg tcaaggttgt acatatttga tgctcctaaa
1980ataccgccag atcagatgta tagattaaga aaccgattaa gaaatattcc agttgaaaga
2040agaagagctg atgtgttcag aattattatg aataatagag atttaatcga aaaaacatca
2100gaacgtatat gtcagggtgt gttgttatct tatacaccaa tgcctttaac ttacgttgaa
2160gatgtcgggt taacaaatgt aattaatgac actaataact tccaaataat taatatagaa
2220gaaattgaga agaccggtga ctattcagcc ataacgaatg cattacttcg ggatactcca
2280attatattga aaggtgcgat tccatatgtt actaactcat cagtaattga tgttttatct
2340aaagtggaca ccacagtgtt cgcaagcatc gtaaaagata gggatatttc aaagttaaaa
2400ccaataaaat tcataattaa ttcagattca tccgaatatt atttagtaca caataataaa
2460tggacaccaa caacaactac agcagtatat aaagctagat ctcagcaatt tgatatacaa
2520cattcagtat caatgctaga gtcaaactta ttttttgtgg tatataatga tttatttaaa
2580tacattaaaa ccactacagt tctgccgata aatgctgtct cttatgatgg tgcaagaatt
2640atgcaagaaa cataa
2655432736DNAHuman rotavirus Cmisc_featureHuman rotavirus group C strain
ASP88 VP2 nucleotide sequence 43ggcttaaaaa gatcagtcga ggacaaatcg
tccaagatga taagcagaaa caggcgcaga 60aataaccaac aaaaagatat aggaaaagag
aaacaattag agactataat tgacaaagaa 120gtaaaggaaa acaaagattc tacaaaagaa
gataagctag tagttacgga agaaagtaat 180ggtgacgtca cagctgttaa agaacaatcg
aataatatta atttacaaaa gaatgatttg 240gttaaagaag tcatgaatat acagaatcaa
acattaaata cagtagttgc tgagaataaa 300gttgaaatag aagaaatagt taaaaaatac
attccctcat ataatactga cagccttatt 360gttaaaaagt taactgaaat ccaggaatca
agtgctaaaa catataatac attattcaga 420ttatttactc cagttaaaag ttatttatat
gacataaatg gtgagaaaaa attatcgact 480agatggtatt ggaaattgct caaagatgat
ttacctgctg gtgattactc agttagacaa 540ttcttcctgt cactatattt aaatgtttta
gagggaatgc ccgattacat aatgcttcgt 600gatatggcag tggataaccc atattcagca
gaagcaggta aaatcgtaga tggaaagtct 660aaagaaattt tagttgaact atatcaagac
caaatgacag aagggtatat tagaagatat 720atgtctgaat taagacataa aatatctgga
gaaacaaata ctgcaaaata tccagctatt 780ctacatcccg tggataatga gcttaatcaa
tactttcttg agcatcagtt aattcaacca 840ttaactacaa gaaatattgc agaattgatt
ccaactcaat tatatcatga tccaaattac 900gtttttaata ttgatgcagc ctttttaaca
aattcaagat ttgttccacc atacttaaca 960caggatagga ttggattaca tgatggattc
gaatcaatat gggattcaaa aacccatgct 1020gattacgttt cagctagaag atttatacct
gatttaactg aactggtaga tgctgaaaag 1080caaataaaag aaatggctgc acatttacaa
ctagaggcta ttacagtaca ggttgaatca 1140caatttttag cgggaattag tgctgctgca
gctaatgaag cgttcaaatt tataattggc 1200tcagttttat ctaccagaac aatagctgta
gaattcataa cctcaaacta tatgtcgtta 1260gcatcatgta tgtatttaat gactattatg
ccatcagaga ttttcttgag agaatcatta 1320gttgctatgc gattagcaat aataaatacc
cttatttatc cagctctagg tttagcgcaa 1380atgcattatc aagcaggtga agtgaggacc
ccattcgaat tagctgagat gcgagtagct 1440aatagatcta ttagacaatg gttacatcat
tgtaatacac ttcaatttgg tagacagata 1500acggaaggga taattcatct acgatttact
aatgatatca tgacaggtag gatagtgaac 1560ttattttcaa caatgctagt agctttatca
tctcagccat tcgctacata tcctttagac 1620tataaaagat ctgtacaaag agcattacaa
cttttatcaa atagaacagc ccaaatagca 1680gatttaacca gattaatagt atacaattat
actacattat ctgcatgtat agtcatgaat 1740atgcatttag taggaactct tactgttgaa
cgtatacagg ccacttctct aacttcttta 1800atgatgttaa tttctaataa gacagttatt
ccagaaccat cgtctctttt ttcatatttc 1860tctagtaaca ttaattttct tacaaattat
aatgagcaaa ttgataatgt ggtagcagaa 1920ataatggccg catatagatt gaatttatat
caacagaaaa tgttgatgct cgttaccagg 1980tttgtgtcaa ggttgtacat atttgatgct
cctaaaatac cgccagatca gatgtataga 2040ttaagaaacc gattaagaaa tattccagtt
gaaagaagaa gagctgatgt gttcagaatt 2100attatgaata atagagattt aatcgaaaaa
acatcagaac gtatatgtca gggtgtgttg 2160ttatcttata caccaatgcc tttaacttac
gttgaagatg tcgggttaac aaatgtaatt 2220aatgacacta ataacttcca aataattaat
atagaagaaa ttgagaagac cggtgactat 2280tcagccataa cgaatgcatt acttcgggat
actccaatta tattgaaagg tgcgattcca 2340tatgttacta actcatcagt aattgatgtt
ttatctaaag tggacaccac agtgttcgca 2400agcatcgtaa aagataggga tatttcaaag
ttaaaaccaa taaaattcat aattaattca 2460gattcatccg aatattattt agtacacaat
aataaatgga caccaacaac aactacagca 2520gtatataaag ctagatctca gcaatttgat
atacaacatt cagtatcaat gctagagtca 2580aacttatttt ttgtggtata taatgattta
tttaaataca ttaaaaccac tacagttctg 2640ccgataaatg ctgtctctta tgatggtgca
agaattatgc aagaaacata aatgattgta 2700tagtatcatc ttgtaacgac ctcaaactct
gtggct 2736442736DNAPorcine
rotavirusmisc_featurePorcine rotavirus group C strain Cowden VP2
nucleotide sequence 44ggtttaaaaa gatcaatcga ggacaaatcg tccaagatga
taagcagaaa tagacgtaga 60aacactcaac agaaagatgc tgaaaaggaa aagcagacag
aaaatgtgga ggagaaagag 120ataaaggaag ctaaagaaca agttaaagat gaaaagcaag
tgattacaga agaaaacgtc 180gatagtccta aggatgttaa agaacaatca aacaccgtaa
atctacaaaa aaatgactta 240gttaaagaag ttataaatat ccaaaatcaa acattgaata
caatagttgc tgagaataaa 300gtggaaattg aagaagtggt taaaaagtat attccatcat
actcaactga caagctaata 360gttaaaaaat taactgaaat tcaagaatca agtgctaaaa
catacaataa attgtttaga 420ttatttacac cggttaagag ttatctatat gatgtaaatg
gagagaaaaa actatccact 480agatggtatt ggaaactact taaagatgat ctacctgctg
gtgattactc agttagacaa 540ttctttctat ctttatactt gaatgtatta gatgaaatgc
ctgattatgt tatgcttcgt 600gatatggctg tggataatcc atattcagca gaggcaggaa
aaatagtaga tgaaaaatca 660aaagaaatcc tagtagaaat atatcaagat caaatgactg
aagggtatat acggagatat 720atgtctgatt tgagacatag aatatctggt gaaacgaata
ctgctaaata tccagctatt 780ttacatcctg tagatgaaga actaaataaa tactttcttg
agcaccaact gattcaacca 840ttgactacaa gaaatatagc agaattaatt ccaactcaat
tgtatcatga tccaaattat 900gtgtttaaca ttgatgctgc atttttaaca aactcaagat
ttgttccacc gtatctaaca 960caagatagaa ttggattaca tgatggattt gagtcaattt
gggatgcaaa aacacatgct 1020gattacgttt cagctagaag atttgtacct gatttaactg
agttagttga tgctgaaaaa 1080cagatgaaag aaatggcagc acatttacag cttgaagcta
ttacagtgca agttgaatca 1140caattcttgg caggaattag tgcagcggca gctaatgaag
catttaagtt tataattggt 1200actgtgctgt caactagaac aatagcagta gaattcatca
catcaaatta tatgtcatta 1260gcgtcatgta tgtatttaat gacgattatg ccatcagaaa
tctttttgag agaatcgcta 1320gttgcaatgc agttagcagt aataaatact cttacctatc
cagctttagg attagcacaa 1380atgcattatc aggcaggtga aataagaacg ccctttgaac
tagcagaaat gcaagtagca 1440aataggccca ttaggcaatg gttgcatcat tgtaatacac
ttcaatttgg cagacaagta 1500actgaaggag taacacatct acggtttaca aatgacatca
tgacaggtag aatagttaat 1560ctcttttcaa ctatgttggt agctttatca tctcagcctt
ttgctacata tccattagat 1620tacaaaagat ctgtccagag agcattacag cttctttcaa
acaggactgc tcaaatagct 1680gatttgacta gattaatagt gtacaactat acaacattgt
cagcatgcat agtcatgaac 1740atgcatttgg ttggaacctt aactgtagaa cgtatacaag
ctacagcttt aacttcactg 1800ataatgttga tatccaataa aacggttatt ccagaaccat
catccctctt ttcatacttt 1860tctagtaata ttaatttctt gacaaactac aatgaacaga
ttgataacgt agtggctgaa 1920ataatggcag catacagact agatctatat caacagaaaa
tgctaatgct tgttactcga 1980tttgtttcac gactgtacat atttgatgct cctaagatac
caccagacca gatgtataga 2040ttaagaaata gactgaggaa cattccagtt gaaagaagaa
gagcagatgt gttcagaatc 2100attatgaata acagagatct tatagagaaa acatcagaac
gcatttgtca aggagtgtta 2160ctatcatatt caccaatgcc attaacatat gttgaggatg
ttggtttgac aaatgtggtt 2220aatgacacta atggttttca gataataaac attgaagaaa
tcgagaagac aggtgattat 2280tcagcaatta caaacgcatt actccgtgat actccaatca
tactgaaggg cgctattccg 2340tacgttacca attcatcagt aatagatgtt ctatctaaaa
tagatacaac agtgtttgcg 2400agtatcgtta aagacagaga tatttcaaaa ttaaaaccga
taaaattcac aattaattca 2460gactcatcag aatactattt agtacacaat aataaatgga
caccaacaac aacaactgct 2520gtgtacaaag ccagatctca gcaatttaat atacaacatt
cagtgtcaat gttagagtca 2580aacttgttct ttgttgtata taatgatctg tttaagtaca
tcaaaacaac tacagtatta 2640ccaatcaatg ccgtgtctta tgatggtgcg agaattatgc
aggaaacatg aactgattaa 2700ttatatcatc ttgtgatgac ctcaaactct gtggct
273645999DNAHuman rotavirus Cmisc_featureHuman
rotavirus group C VP7 S1 nucleotide sequence of open reading frame
45atggtttgta caacattgta cactgtttgc gccattctct tcattctttt catttatata
60ttattattta gaaaaatgtt ccacctaata actgatactt taatagtgat gcttatttta
120tctaattgtg tagagtggtc acaaggtcag atgtttattg atgatataca ttataatggt
180aacgttgaga ctatcataaa ttctactgat ccttttaatg ttgaatcttt atgtatttat
240tttccaaatg cagttgtagg atcacaggga ccaggtaaat ccgatggaca tttgaatgat
300ggtaattatg cacagactat cgccactttg tttgaaacaa aaggattccc aaaaggttca
360ataataatta aaacatatac acagacatca gactttataa attcagtaga aatgacatgc
420tcttataata tagttatcat tcctgatagc ccaaatgatt cagaatctat tgaacagata
480gcagaatgga ttttaaatgt ttggagatgt gatgacatga atttggaaat ttatacttat
540gaacaaattg gaataaacaa tttatgggct gcatttggta gtgactgtga tatatctgtc
600tgtccattag atactacaag taatggaatc ggatgttcac cagctagtac agaaacttat
660gaagttgtat caaatgacac ccaattggcc ttaattaatg ttgtggataa tgttagacat
720agaatacaga tgaacactgc tcaatgtaaa ttgaaaaatt gtattaaggg tgaagctcga
780ctgaatactg cactaataag aatttcaaca tcatcaagtt ttgataattc attgtcacca
840ttaaataacg gccaaacaac aagatcgttt aaaataaatg caaagaaatg gtggactata
900ttttatacaa taattgatta tattaataca attgtacaat caatgactcc cagacatcgg
960gcgatttatc cagaagggtg gatgttgagg tatgcgtaa
999461343DNAHuman rotavirus Cmisc_featureHuman rotavirus group C VP6 S1
nucleotide sequence 46atctcattca caatggatgt acttttttct atagcgaaaa
ccgtgtcaga tcttaaagag 60aaagttgtag ttggaacaat ttatactaat gtagaagatg
ttgtacaaca gacgaatgaa 120ttgattagaa ctttgaatgg aaatattttt catactggtg
gcattggaac acagcctcag 180aaagagtgga attttcagct cccacaattg ggtaccactt
tattaaattt agatgataat 240tatgttcaat caactagagg cataattgat tttttatcat
cttttataga agctgtatgt 300gatgatgaaa ttgttagaga agcttcaaga aatggtatgc
aacctcaatc accagctctt 360atattattat cttcatcaaa atttaaaaca attaatttta
ataatagttc tcaatctatc 420aaaaattgga atgctcaatc aagacgtgag aatcctgtat
atgagtacaa aaatccaatg 480ttgtttgaat ataaaaattc ttatatttta caacgcgcaa
atccacaatt tggaagcgtc 540atgggtttaa gatattatac aacaagtaat atttgtcaaa
ttgcagcatt tgattccacc 600ctagctgaaa atgcaccaaa taatacgcaa cgcttcgttt
ataatggcag actaaaaaga 660cccatatcaa atgttttaat gaaaatagaa gctggtgctc
caaatataag caacccaact 720attttacctg atcctaataa tcaaacaact tggcttttta
atccggtaca attaatgaat 780ggaacattta ccattgaatt ctataataat ggtcaactaa
ttgatatggt tcgaaatatg 840ggaatagtta ctgtaagaac ttttgattct tatagaataa
caattgacat gattagacca 900gctgctatga ctcaatacgt tcaacgaatt tttccacaag
gtggacctta tcattttcag 960gctacatata tgttaacatt aagtatatta gatgctacca
cagagtccgt tctatgtgat 1020tctcattcag tagaatattc aatagtagca aacgtcagaa
gagattcagc aatgccagct 1080ggaactgttt ttcaaccggg atttccatgg gaacacacac
tatccaatta cactgttgct 1140caagaagata atttagaaag attattgtta atcgcatctg
tgaagagaat ggtaatgtag 1200ataagctaga agactaaaca tcttctatgc ggcctacata
ccatgtagca tgaatcacga 1260ctgggtttag tccatgctcg cataggggca aatatgcatg
atatggatga tccccagaag 1320gatgaaatgt gaactatgtg gct
1343471353DNAHuman rotavirus Cmisc_featureHuman
rotavirus group C VP6 Bristol nucleotide sequence 47ggcatttaaa
atctcattca caatggatgt acttttttct atagcgaaaa ctgtgtcaga 60tcttaaaaag
aaagttgtgg ttggaacaat ttatactaat gtagaagatg ttgtacaaca 120gacgaatgaa
ttgattagaa ctttaaatgg aaatattttt catactggtg gcattggaac 180acagcctcag
aaagagtgga attttcagct gccacaattg ggtacaactt tattaaattt 240agatgataat
tatgttcaat caactagagg cataatcgat tttttatcat cttttataga 300agctgtatgt
gatgatgaaa ttgttagaga agcttcaaga aatggtatgc aacctcaatc 360accagctctt
atattattat cttcatcaaa atttaaaaca attaatttta ataatagttc 420tcaatctatt
aaaaattgga atgctcaatc aagacgtgag aatcctgtat atgagtataa 480aaatccaatg
ttgtttgaat ataaaaattc gtatatttta caacgcgcaa atccacaatt 540tggaagcgtc
atgggtttaa gatattatac aacaagtaat acttgtcaaa ttgcagcatt 600tgattccacc
ctagctgaaa atgcaccaaa caatacacaa cgcttcgttt ataatggcag 660actaaaaaga
cccatatcaa acgttttaat gaaaatagaa gctggtgctc caaatataag 720caacccaact
attttacctg atcctaataa tcaaacaact tggcttttta atccggtaca 780attaatgaat
ggaacattta ccattgaatt ctataataat ggtcaactaa ttgatatggt 840tcgaaatatg
ggaatagtta ctgtaagaac ttttgattct tatagaataa caattgacat 900gattagacca
gctgctatga ctcaatacgt tcaacgaatt tttccacaag gtggacctta 960tcattttcag
gctacatata tgttaacatt aagtatatta gatgctacca cagagtccgt 1020tctatgtgat
tctcattcag tggaatattc aatagtagca aacgttagaa gagattcagc 1080gatgccagct
ggaactgttt ttcaaccggg atttccatgg gaacacacac tatccaatta 1140cactgttgct
caagaagata atttagaaag attattgtta attgcatctg tgaagagaat 1200ggtaatgtag
ataagctaga gggctaaaca tcttctatgc ggcctacata ccatgtagca 1260tgaatcacga
ctgggtttag tccatgcttg cataggggca aatatgcatg atatggatga 1320tccccagaag
gatgaaatgt gaactatgtg gct 1353
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