Patent application title: INFECTIOUS cDNA CLONE OF NORTH AMERICAN PORCINE REPRODUCTIVE AND RESPIRATORY SYNDROME (PRRS) VIRUS AND USES THEREOF
Inventors:
Jay Gregory Calvert (Otsego, MI, US)
Michael G. Sheppard (Victoria, AU)
Siao-Kun W. Welch (Kalamazoo, MI, US)
Siao-Kun W. Welch (Kalamazoo, MI, US)
Raymond R. R. Rowland (Manhattan, KS, US)
Dal-Young Kim (Birmingham, AL, US)
IPC8 Class: AC12N700FI
USPC Class:
4241991
Class name: Drug, bio-affecting and body treating compositions antigen, epitope, or other immunospecific immunoeffector (e.g., immunospecific vaccine, immunospecific stimulator of cell-mediated immunity, immunospecific tolerogen, immunospecific immunosuppressor, etc.) recombinant virus encoding one or more heterologous proteins or fragments thereof
Publication date: 2015-02-12
Patent application number: 20150044255
Abstract:
The invention provides isolated polynucleotide molecules that comprise a
DNA sequence encoding an infectious RNA sequence encoding a
genetically-modified North American PRRS virus, wherein the
polynucleotide molecule lacks at least one detectable antigenic epitope
of North American PRRS virus. The invention also provides vaccines
comprising genetically modified North American PRRS virus, RNA molecules,
plasmids and viral vectors comprising the isolated polynucleotide
molecules. Also provided are isolated polynucleotide molecules further
comprising at least one nucleotide sequence that encodes a detectable
heterologous antigenic epitope, and vaccines comprising North American
PRRS virus, RNA molecules, plasmids and viral vectors comprising such
isolated polynucleotide molecules.Claims:
1. An isolated polynucleotide molecule comprising a DNA sequence encoding
an infectious RNA molecule encoding a North American PRRS virus that is
genetically modified such that when it infects a porcine animal it is
unable to produce PRRS in the animal yet able to elicit an effective
immunoprotective response against a PRRS virus in the porcine animal,
wherein said DNA sequence is SEQ ID NO:1 or a sequence homologous
thereto, except that it lacks at least one DNA sequence encoding a
detectable antigenic epitope of North American PRRS virus.
2. The isolated polynucleotide molecule of claim 1, wherein said molecule lacks at least one DNA sequence encoding a detectable antigenic epitope of North American PRRS virus in OAF1a or OAF1b within said DNA sequence.
3. The isolated polynucleotide molecule of claim 1, wherein said molecule lacks at least one DNA sequence encoding a detectable antigenic epitope of North American PRRS virus in the nonstructural protein 2 coding region of ORF1a within said DNA sequence.
4. The isolated polynucleotide molecule of claim 1, wherein said molecule lacks at least one DNA sequence encoding a detectable antigenic epitope of North American PRRS virus in the hypervariable region in the nonstructural protein 2 coding region of ORF1a within said DNA sequence.
5. The isolated polynucleotide molecule of claim 1, wherein said molecule lacks at least one DNA sequence encoding a detectable antigenic epitope of North American PRRS virus between amino acids from 616 to 752 in the hypervariable region in the nonstructural protein 2 coding region of ORF1a within said DNA sequence.
6. The isolated polynucleotide molecule of claim 1, wherein said molecule lacks the DNA sequence encoding amino acids 628 and 759 in the hypervariable region in the nonstructural protein 2 coding region of ORF1a within said DNA sequence.
7. A vaccine for protecting a porcine animal from infection by a PRRS virus, wherein said vaccine comprises: a) A genetically modified North American PRRS virus encoded by an infectious RNA molecule encoded by the isolated polynucleotide molecule according to claim 1; b) An infectious RNA molecule encoded by the isolated polynucleotide molecule according to claim 1; c) The isolated polynucleotide molecule according to claim 1 in the form of a plasmid, or d) A viral vector comprising the isolated polynucleotide molecule according to claim 1; and a carrier acceptable for veterinary use.
8. The isolated polynucleotide molecule of claim 1, wherein said isolated polynucleotide molecule further comprises at least one nucleotide sequence that encodes a detectable heterologous antigenic epitope.
9. The isolated polynucleotide molecule of claim 8, wherein translation of the encoded infectious RNA molecule results in formation of at least one protein consisting of a fusion between a PRRS virus protein and said heterologous antigenic epitope.
10. The isolated polynucleotide molecule of claim 6, wherein said nucleotide sequence that encodes a heterologous antigenic epitope is inserted in ORF1a or ORF1b within said DNA sequence.
11. The isolated polynucleotide molecule of claim 8, wherein said nucleotide sequence that encodes a heterologous antigenic epitope is inserted in the nonstructural protein 2 coding region of ORF1a within said DNA sequence.
12. The isolated polynucleotide molecule of claim 8, wherein said nucleotide sequence that encodes a heterologous antigenic epitope is inserted in the hypervariable region in the nonstructural protein 2 coding region of ORF1a within said DNA sequence.
13. The isolated polynucleotide molecule of claim 8, wherein said nucleotide sequence that encodes a heterologous antigenic epitope is inserted between amino acids from 628 to 759 in the hypervariable region in the nonstructural protein 2 coding region of ORF1a within said DNA sequence.
14. The isolated polynucleotide molecule of claim 8, wherein said nucleotide sequence that encodes a heterologous antigenic epitope is inserted between amino acids from 818 to 752 in the hypervariable region in the nonstructural protein 2 coding region of ORF1a within said DNA sequence.
15. A vaccine for protecting a porcine animal from infection by a PRRS virus, wherein said vaccine comprises: a) A genetically modified North American PRRS virus encoded by an infectious RNA molecule encoded by the isolated polynucleotide molecule according to claim 8; b) An infectious RNA molecule encoded by the isolated polynucleotide molecule according to claim 8; c) The isolated polynucleotide molecule according to claim 8 in the form of a plasmid, or d) A viral vector comprising the isolated polynucleotide molecule according to claim 8; and a carrier acceptable for veterinary use.
16. A diagnostic kit comprising reagents which are useful for differentiating between porcine animals naturally infected with a field strain of a PRRS virus and porcine animals vaccinated with the vaccine of claim 15.
17. The diagnostic it of claim 15 having as one of its components a peptide comprising the amino acid sequences selected from SEQ ID NO. 64 to 115.
18. The diagnostic kit of claim 16, wherein one of its components is a peptide comprising the amino acid sequence of SEQ ID NO. 64, or a peptide fragment thereof 10 amino acid residues in length or longer.
19. The diagnostic kit of claim 17, where said peptide is optionally a fusion protein.
Description:
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] The present application is a continuation-in-part of U.S. patent application Ser. No. 11/366,851, filed on Mar. 1, 2006, which is a continuation-in-part of U.S. patent application Ser. No. 10/241,332, filed Sep. 11, 2002, which is a continuation-in-part of U.S. patent application Ser. No. 10/127,391, filed on Apr. 22, 2002, which is a continuation of U.S. patent application Ser. No. 09/470,661, filed Dec. 22, 1999, U.S. Pat. No. 6,500,662, which claims the benefit of priority of U.S. Provisional Application Ser. No. 60/113,345, filed Dec. 22, 1998, the disclosures of which are incorporated herein by reference in their entirety.
FIELD OF THE INVENTION
[0002] The present invention is in the field of animal health and is directed to infectious cDNA clones of positive polarity RNA viruses and the construction of vaccines, in particular, swine vaccines, using such cDNA clones.
BACKGROUND OF THE INVENTION
[0003] Porcine reproductive and respiratory syndrome (PRRS) is a new disease of swine, first described in 1987 in North America and in 1990 in Europe. The disease has since spread to Asia and affects most of the major swine producing countries of the world. Primary symptoms are reproductive problems in sows and gilts, including late term abortions, stillbirths and mummies, and litters of small weak pigs which are born viremic and often fail to survive. In addition, the syndrome manifests itself as a respiratory disease in young pigs which spreads horizontally and causes fever, lethargy, labored breathing, loss of appetite, slow growth, and occasionally death, often in association with other respiratory pathogens. The disease furthermore can be transmitted to sows and gilts via the semen of infected boars, either naturally or by artificial insemination. For these, and other reasons, PRRS has proven to be a difficult disease to control and therefore one of the most economically damaging diseases to the swine industry.
[0004] The causative agent of PRRS is the PRRS virus, which exists as two genetically and serologically distinct types (Murtaugh, M. P. et al., 1995, Arch-Virol. 140, 1451-1460; Suarez, P. et al., 1996, Virus Research 42:159-165). The two types are believed to have first entered swine populations independently, one in North America and the other in Europe, in the1980's, from unknown biological reservoirs, possibly of rodent or avian origin. The European type, represented by the prototype "Lelystad Virus", was isolated and sequenced in the Netherlands in 1991 (Terpstra, C. et al., 1991, Vet. Quart. 13:131-136; Wensvoort, G. et al., 1991, Vet. Quart. 13:121-130; Wensvoort, G. et al., WO 92/213751992 (PCT/NL92/00096), 1992; Meulenberg, J. J. M. et al., 1993, Virol. 192:62-72).
[0005] Both the North American PRRS virus and the European PRRS virus are classified within the family Arteriviridae, which also includes equine arteritis virus, lactate dehydrogenase-elevating virus, and simian haemorrhagic fever virus. The arteriviruses are in turn placed within the order Nidovirales, which also includes the coronaviruses and toroviruses. The nidoviruses are enveloped viruses having genomes consisting of a single strand of positive polarity RNA. The genomic RNA of a positive-stranded RNA virus fulfills the dual role in both storage and expression of genetic information. No DNA is involved in replication or transcription in nidoviruses. The reproduction of nidoviral genomic RNA is thus a combined process of genome replication and mRNA transcription. Moreover, some proteins are translated directly from the genomic RNA of nidoviruses. The molecular biology of the family Arteriviridae has recently been reviewed by Snijder and Meulenberg (Snijder, E. J. and Meulenberg, J. J. M., 1998, Journal of General Virology 79:961-979).
[0006] Currently available commercial vaccines against PRRS are either conventional modified live virus (cell culture, attenuated) or conventional killed (inactivated cell culture preparations of virulent virus). Several of these vaccines have been criticized based on safety and/or efficacy concerns. The development of a second generation of PRRS vaccines, based upon specific additions, deletions, and other modifications to the PRRS genome, is therefore highly desirable. However, since the PRRS viruses do not include any DNA intermediates during their replication, such vaccines have thus far awaited the construction of full-length cDNA clones of PRRS viruses for manipulation by molecular biology techniques at the DNA level. Very recently, a full-length infectious cDNA clone of the European PRRS virus has been reported (Meulenberg, J. J. M. et al., 1998, supra; Meulenberg, J. J. M. et at, 1988, J. Virol. 72, 380-387.).
[0007] The preceding publications, as well as all other references discussed below in this application, are hereby incorporated by reference in their entireties.
SUMMARY OF THE INVENTION
[0008] The subject invention provides an isolated polynucleotide molecule comprising a DNA sequence encoding an infectious RNA molecule encoding a North American PRRS virus that is genetically modified such that when it infects a porcine animal it is unable to produce PRRS in the animal yet able to elicit an effective immunoprotective response against a PRRS virus in the porcine animal, wherein said DNA sequence is SEQ ID NO:1 or a sequence homologous thereto, except that it lacks at least one DNA sequence encoding a detectable antigenic epitope of North American PRRS virus.
[0009] The subject invention further provides an isolated polynucleotide molecule where at least one DNA sequence encoding a detectable antigenic epitope of North American PRRS virus in ORF1a or ORF1b within said DNA sequence is deleted.
[0010] The subject invention further provides an isolated polynucleotide molecule where at least one DNA sequence encoding a detectable antigenic epitope of North American PRRS virus in the nonstructural protein 2 coding region of ORF1a within said DNA sequence is deleted.
[0011] The subject invention further provides an isolated polynucleotide molecule where at least one DNA sequence encoding a detectable antigenic epitope of North American PRRS virus in the hypervariable region in the nonstructural protein 2 coding region of ORF1a within said DNA sequence is deleted.
[0012] The subject invention further provides an isolated polynucleotide molecule where the DNA sequence encoding amino acids 628 and 759 in the hypervariable region in the nonstructural protein 2 coding region of ORF1a within said DNA sequence is deleted.
[0013] The subject invention further provides a vaccine for protecting a porcine animal from infection by a PRRS virus, wherein said vaccine comprises a genetically modified North American PRRS virus encoded by an infectious RNA molecule encoded by the isolated polynucleotide molecule as described above; an infectious RNA molecule encoded by the isolated polynucleotide molecule as described above; an isolated polynucleotide molecule as described above in the form of a plasmid, or a viral vector comprising the isolated polynucleotide molecule as described above, and a carrier acceptable for veterinary use.
[0014] The subject invention also provides an isolated polynucleotide molecule comprising a DNA sequence encoding an infectious RNA molecule encoding a North American PRRS virus that is genetically modified such that when it infects a porcine animal it is unable to produce PRRS in the animal yet able to elicit an effective immunoprotective response against a PRRS virus in the porcine animal, wherein the DNA sequence is SEQ ID NO:1 or a sequence homologous thereto, except that it lacks at least one DNA sequence encoding a detectable antigenic epitope of North American PRRS virus, wherein it further comprises at least one nucleotide sequence that encodes a detectable heterologous antigenic epitope.
[0015] The present invention further provides an isolated polynucleotide molecule comprising at least one nucleotide sequence encoding a heterologous antigenic epitope, wherein said nucleotide sequence encoding a heterologous antigenic epitope is inserted in OAF1a or ORF1b within said DNA sequence encoding an infectious RNA molecule encoding a North American PRRS virus.
[0016] The present invention further provides an isolated polynucleotide molecule comprising at least one nucleotide sequence encoding a heterologous antigenic epitope, wherein said nucleotide sequence encoding a heterologous antigenic epitope is inserted in the nonstructural protein 2 coding region of OAF 1a within said DNA sequence encoding an infectious RNA molecule encoding a North American PRRS virus.
[0017] The present invention further provides an isolated polynucleotide molecule comprising at least one nucleotide sequence encoding a heterologous antigenic epitope, wherein said nucleotide sequence encoding a heterologous antigenic epitope is inserted in the hypervariable region in the nonstructural protein 2 coding region of OAF 1a within said DNA sequence encoding an infectious RNA molecule encoding a North American PRRS virus.
[0018] The present invention further provides an isolated polynucleotide molecule comprising at least one nucleotide sequence encoding a heterologous antigenic epitope, wherein said nucleotide sequence encoding a heterologous antigenic epitope is inserted in the hypervariable region in the nonstructural protein 2 coding region of ORF1a between the DNA sequence encoding amino acids 628 and 759 in the hypervariable region in the nonstructural protein 2 coding region of ORF1a within said DNA sequence.
[0019] The present invention further provides a vaccine for protecting a porcine animal from infection by a PRRS virus, wherein said vaccine comprises a genetically modified North American PRRS virus encoded by an infectious RNA molecule encoded by an isolated polynucleotide molecule as described above, an infectious RNA molecule encoded by an isolated polynucleotide molecule as described above, an isolated polynucleotide molecule as described above, or a viral vector comprising an isolated polynucleotide molecule as described above, and a carrier acceptable for veterinary use.
[0020] The present invention also provides a diagnostic kit for differentiating between porcine animals vaccinated with the above described vaccines and porcine animals infected with field strains of PRRS virus. Said kit contains as one of its components at least one peptide having a sequence comprising the 132 amino acid region deleted within the PRRS virus strains presently described. Alternative, the kit can have as one of its components the corresponding peptide from any PRRS virus strain. The kit can otherwise have as one of its components a smaller peptide corresponding to a portion of the 132 amino acid sequence comprising the nsp2 deletion, from 10 amino acid residues or upward in length. The peptide can additionally be in the form of a fusion protein.
BRIEF DESCRIPTION OF THE FIGURES
[0021] FIG. 1: Cloning strategy for construction of full-length infectious cDNA clone of North American PRRS virus, pT7P129A. Arrowheads represent T7 promoter sequences.
[0022] FIG. 2: Serum viremia following infection with P129A or recombinant PRRS virus rP129A-1. Determined by plaque assay on MARC-145 cells. The lower limit of detection is 5 pfu/ml (or 0.7 on the log scale).
[0023] FIG. 3: Anti-PRRS virus serum antibody following infection with P129A or recombinant PRRS virus rP129A-1. Determined by HerdChek PRRS ELISA assay (IDEXX (Westbrook, Me., USA)).
[0024] FIG. 4: Genome organization of PRRSV virus. Location or range of various deletions and insertion sites, including: OAF 1a and 1b, Nsp2, the hypervariable region of Nsp2, the deleted region within Nsp2 encoding amino acids 628-759, and the MluI and SgrA1sites at genome positions 3219 and 3814, between which GFP or the 9 amino acid HA tag were inserted. The active site residues of the cysteine protease domain (Cys437 and His507) and the hypervariable region are indicated. The hypervariable region of nsp2 corresponds approximately to genome coordinates 2720-3980 in P129.
DETAILED DESCRIPTION OF THE INVENTION
[0025] Production and manipulation of the isolated polynucleotide molecules described herein are within the skill in the art and can be carried out according to recombinant techniques described, among other pieces, in Maniatis, et al., 1989, Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.; Ausubel, et al. 1989, Current Protocols In Molecular Biology, Greene Publishing Associates & Wiley Interscience, NY; Sambrook, et al., 1989, Molecular Cloning: A Laboratory Manual, 2d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.; Innis et al. (eds), 1995, PCR Strategies, Academic Press, Inc., San Diego; and Erlich (ed), 1992, PCR Technology, Oxford University Press, New York, all of which are incorporated herein by reference.
[0026] A. Isolated Polynucleotide Molecules and RNA Molecules Encoding Genetically Modified North American PRRS Viruses.
[0027] The present invention provides isolated polynucleotide molecules comprising DNA sequences that encode infectious RNA molecules that encode genetically modified North American PRRS viruses.
[0028] The subject invention provides an isolated polynucleotide molecule comprising a DNA sequence encoding an infectious RNA molecule that encodes a North American PRRS virus, wherein said DNA sequence is SEQ ID NO:1 or a sequence homologous thereto. The present invention provides an isolated polynucleotide molecule comprising a DNA sequence encoding an infectious RNA molecule that encodes a North American PRRS virus, wherein said DNA sequence is the sequence beginning with and including nucleotide 1 through and including nucleotide 15,416 of SEQ ID NO:1, except that the nucleotide corresponding to nucleotide 12,622 of SEQ ID NO:1 is a guanine instead of an adenine and the nucleotide corresponding to nucleotide 1,559 of SEQ ID NO:1 is a thymine instead of a cytosine. Said DNA sequence encodes an infectious RNA molecule that is the RNA genome of the North American PRRS isolate P129.
[0029] It is understood that terms herein referring to nucleic acid molecules such as "isolated polynucleotide molecule", "nucleotide sequence", "open reading frame (OAF)", and the like, unless otherwise specified, include both DNA and RNA molecules and include both single-stranded and double-stranded molecules. Also, when reference to a particular sequence from the "Sequence Listing" section of the subject application is made, it is intended, unless otherwise specified, to refer to both the DNA of the "Sequence Listing", as well as RNA corresponding to the DNA sequence, and includes sequences complementary to the DNA and RNA sequences. In such contexts in this application, "corresponding to" refers to sequences of DNA and RNA that are identical to one another but for the fact that the RNA sequence contains uracil in place of thymine and the backbone of the RNA molecule contains ribose instead of deoxyribose.
[0030] For example, SEQ ID NO:1 is a DNA sequence corresponding to the RNA genome of a North American PRRS virus. Thus, a DNA sequence complementary to the DNA sequence set forth in SEQ ID NO:1 is a template for, i.e. is complementary to or "encodes", the RNA genome of the North American PRRS virus (i.e., RNA that encodes the North American PRRS virus). Nonetheless, a reference herein to SEQ ID NO:1 includes both the RNA sequence corresponding to SEQ ID NO:1 and a DNA sequence complementary to SEQ ID NO:1.
[0031] Furthermore, when reference is made herein to sequences homologous to a sequence in the Sequence Listing, it is to be understood that sequences homologous to a sequence corresponding to the sequence in the Sequence Listing and sequences homologous to a sequence complementary to the sequence in the Sequence Listing are also included.
[0032] An "infectious RNA molecule", for purposes of the present invention, is an RNA molecule that encodes the necessary elements for viral replication, transcription, and translation into a functional virion in a suitable host cell, provided, if necessary, with a peptide or peptides that compensate for any genetic modifications, e.g. sequence deletions, in the RNA molecule.
[0033] An "isolated infectious RNA molecule" refers to a composition of matter comprising the aforementioned infectious RNA molecule purified to any detectable degree from its naturally occurring state, if such RNA molecule does indeed occur in nature. Likewise, an "isolated polynucleotide molecule" refers to a composition of matter comprising a polynucleotide molecule of the present invention purified to any detectable degree from its naturally occurring state, if any.
[0034] For purposes of the present invention, the nucleotide sequence of a second polynucleotide molecule (either RNA or DNA) is "homologous" to the nucleotide sequence of a first polynucleotide molecule where the nucleotide sequence of the second polynucleotide molecule encodes the same polyaminoacid as the nucleotide sequence of the first polynucleotide molecule as based on the degeneracy of the genetic code, or when it encodes a polyaminoacid that is sufficiently similar to the polyaminoacid encoded by the nucleotide sequence of the first polynucleotide molecule so as to be useful in practicing the present invention. For purposes of the present invention, a polynucleotide molecule is useful in practicing the present invention where it can be used as a diagnostic probe to detect the presence of the North American PRRS virus in a fluid or tissue sample of an infected pig, e.g. by standard hybridization or amplification techniques. It is to be understood that the polyaminoacid encoded by the nucleotide sequence of the polynucleotide molecule can comprise a group of two or more polyaminoacids. Generally, the nucleotide sequence of a second polynucleotide molecule is homologous to the nucleotide sequence of a first polynucleotide molecule if it has at least about 70% nucleotide sequence identity to the nucleotide sequence of the first polynucleotide molecule as based on the BLASTN algorithm (National Center for Biotechnology Information, otherwise known as NCBI, (Bethesda, Md., USA) of the United States National Institute of Health). Preferably, a homologous nucleotide sequence has at least about 75% nucleotide sequence identity, even more preferably at least about 85% nucleotide sequence identity. Since the genetic code is degenerate, a homologous nucleotide sequence can include any number of "silent" base changes, i.e. nucleotide substitutions that nonetheless encode the same amino acid. A homologous nucleotide sequence can further contain non-dent mutations, i.e. base substitutions, deletions, or additions resulting in amino acid differences in the encoded polyaminoacid, so long as the sequence remains at least about 70% identical to the polyaminoacid encoded by the first nucleotide sequence or otherwise is useful for practicing the present invention. Homologous nucleotide sequences can be determined by comparison of nucleotide sequences, for example by using BLASTN, above. Alternatively, homologous nucleotide sequences can be determined by hybridization under selected conditions. For example, the nucleotide sequence of a second polynucleotide molecule is homologous to SEQ ID NO:1 if it hybridizes to the complement of SEQ ID NO:1 under moderately stringent conditions, e.g., hybridization to filter-bound DNA in 0.5 M NaHPO4, 7% sodium dodecyl sulfate (SDS), 1 mM EDTA at 65° C., and washing in 0.2×SSC/0.1% SDS at 42° C. (see Ausubel et al. above), or conditions which will otherwise result in hybridization of sequences that encode a North American PRRS virus as defined below. In another embodiment, a second nucleotide sequence is homologous to SEQ ID NO:1 if it hybridizes to the complement of SEQ ID NO:1 under highly stringent conditions, e.g. hybridization to fitter-bound DNA in 0.5 M NaHPO4, 7% SDS, 1 mM EDTA at 65° C., and washing in 0.1×SSC/0.1% SOS at 68° C. (Ausubel et al., above).
[0035] It is furthermore to be understood that the isolated polynucleotide molecules and the isolated RNA molecules of the present invention include both synthetic molecules and molecules obtained through recombinant techniques, such as by in vitro cloning and transcription.
[0036] As used herein, the term "PRRS" encompasses disease symptoms in swine caused by a PRRS virus infection. Examples of such symptoms include, but are not limited to, abortion in pregnant females, and slow growth, respiratory difficulties, loss of appetite, and mortality in young pigs. As used herein, a PRRS virus that is "unable to produce PRRS" refers to a virus that can infect a pig, but which does not produce any disease symptoms normally associated with a PRRS infection in the pig, or produces such symptoms, but to a lesser degree, or produces a fewer number of such symptoms, or both.
[0037] The terms "porcine" and "swine" are used interchangeably herein and refer to any animal that is a member of the family Suidae such as, for example, a pig. "Mammals" include any warm-blooded vertebrates of the Mammalia class, including humans.
[0038] The term "PRRS virus", as used herein, unless otherwise indicated, means any strain of either the North American or European PRRS viruses.
[0039] The term "field strain" or "field strains", as used herein, unless otherwise indicated, means any strain of either North American or European PRRS virus which occurs in nature, and is infectious to a pig.
[0040] The term "North American PRRS virus" means any PRRS virus having genetic characteristics associated with a North American PRRS virus isolate, such as, but not limited to the PRRS virus that was first isolated in the United States around the early 1990's (see, e.g., Collins, J. E., et al., 1992, J. Vet. Diego. Invest. 4:117-126); North American PRRS virus isolate MN-1b (Kwang, J. et al., 1994, J. Vet. Diagn. Invest. 6:293-296); the Quebec IAF-exp91 strain of PRRS (Mardassi, H. et at., 1995, Arch. Virol. 140:1405-1418); and North American PRRS virus isolate VR 2385 (Meng, X.-J. et al., 1994, J. Gen. Virol. 75:1795-1801). Genetic characteristics refers to genomic nucleotide sequence similarity and aminoacid sequence similarity shared by North American PRRS virus strains. For purposes of the present invention, a North American PRRS virus is a virus that is encoded by an RNA sequence the same as or homologous to SEQ ID NO:1, wherein the term "homologous" is as defined previously. Thus, strains of North American PRRS viruses have, preferably, at least about 70% genomic nucleotide sequence identity with SEQ ID NO:1, and more preferably at least about 75% genomic nucleotide sequence identity with SEQ ID NO:1, at least about 85% genomic nucleotide sequence identity with SEQ ID NO:1 being even more preferred.
[0041] The term "European PRRS virus" refers to any strain of PRRS virus having the genetic characteristics associated with the PRRS Virus that was first isolated in Europe around 1991 (see, e.g., Wensvoort, G., et al., 1991, Vet Q. 13:121-130), "European PRRS virus" is also sometimes referred to in the art as "Lelystad virus".
[0042] Unless otherwise indicated, a North American PRRS virus is "useful in practicing the present invention" if its characteristics are within the definition of a North American PRRS virus set forth herein. For example, a virus encoded by one of the isolated polynucleotide molecules of the present invention is a "North American PRRS virus useful in practicing the present invention" if it, e.g., has genetic characteristics associated with a North American PRRS virus.
[0043] Other polyaminoacids are "useful in practicing the present invention", e.g., peptides encoded by polynucleotide sequences homologous to North American PRRS virus OAFs, if they can compensate for an RNA molecule encoding a genetically modified PRRS virus, deficient in a gene essential for expressing functional PRRS virions, in a transfected host cell so that functional PRRS virions can be generated by the cell.
[0044] The term "open reading frame", or "OAF", as used herein, means the minimal nucleotide sequence required to encode a particular PRRS virus protein without an intervening stop codon.
[0045] Terms such as "suitable host cell" and "appropriate host cell", unless otherwise indicated, refer to cells into which RNA molecules (or isolated polynucleotide molecules or viral vectors comprising DNA sequences encoding such RNA molecules) of the present invention can be transformed or transfected. "Suitable host cells" for transfection with such RNA molecules, isolated polynucleotide molecules, or viral vectors, include mammalian, particularly porcine, and avian cells, and are described in further detail below.
[0046] A "functional virion" is a virus particle that is able to enter a cell capable of hosting a PRRS virus, and express genes of its particular RNA genome (either an unmodified genome or a genetically modified genome as described herein) within the cell. Cells capable of hosting a PRRS virus include porcine alveolar macrophage cells and MARC 145 monkey kidney cells. Other mammalian or avian cells, especially other porcine cells, may also serve as suitable host cells for PRRS virions.
[0047] The isolated polynucleotide molecules of the present invention encode North American PRRS viruses that can be used to prepare live, killed, or attenuated vaccines using art-recognized methods for protecting swine from infection by a PRRS virus, as described in further detail below. These isolated polynucleotide molecules are also useful as vectors for delivering heterologous genes into mammals, including swine, or birds, as is also described in detail below. Furthermore, these isolated polynucleotide molecules are useful because they can be mutated using molecular biology techniques to encode genetically-modified North American PRRS viruses useful, inter alia, as vaccines for protecting swine from PRRS infection. Such genetically-modified North American PRRS viruses, as well as vaccines comprising them, are also described in further detail below.
[0048] The term "genetically modified", as used herein and unless otherwise indicated, means genetically mutated, i.e. having one or more nucleotides replaced, deleted and/or added. Polynucleotide molecules can be genetically mutated using recombinant techniques known to those of ordinary skill in the art, including by site-directed mutagenesis, or by random mutagenesis such as by exposure to chemical mutagens or to radiation, as known in the art. In one embodiment, genetic modification of the North American PRRS virus of the present invention renders the virus unable to replicate effectively, or reduces its ability to replicate effectively, in a bird or mammal in which the wild-type virus otherwise can effectively replicate. In another embodiment, the genetically modified North American PRRS virus of the present invention remains able to replicate effectively in birds or mammals infected therewith. "Effective replication" means the ability to multiply and produce progeny viruses (virions) in an infected animal, i.e. the ability to "productively infect" an animal.
[0049] The subject invention further provides an isolated polynucleotide molecule comprising a DNA sequence encoding an infectious RNA molecule which encodes a genetically modified North American PRRS virus that is unable to produce PRRS in a porcine animal, wherein the DNA sequence encoding the infectious RNA molecule encoding said North American PRRS virus is SEQ ID NO:1 or a sequence homologous thereto, except that it contains one or more mutations that genetically disable the encoded PRRS virus in its ability to produce PRRS. "Genetically disabled" means that the PRRS virus is unable to produce PRRS in a swine animal infected therewith.
[0050] The subject invention also provides an isolated polynucleotide molecule comprising a DNA sequence encoding an infectious RNA molecule which encodes a North American PRRS virus that is genetically modified such that when it infects a porcine animal it: a) is unable to produce PRRS in the animal, and b) is able to elicit an effective immunoprotective response against infection by a PRRS virus in the animal, wherein the DNA sequence encoding said North American PRRS virus is SEQ ID NO:1 or a sequence homologous thereto, except that it contains one or more mutations that genetically disable the encoded PRRS virus in its ability to produce PRRS.
[0051] The term "immune response" for purposes of this invention means the production of antibodies and/or cells (such as T lymphocytes) that are directed against, or assist in the decomposition or inhibition of, a particular antigenic epitope or particular antigenic epitopes. The phrases "an effective immunoprotective response", "immunoprotection", and like terms, for purposes of the present invention, mean an immune response that is directed against one or more antigenic epitopes of a pathogen so as to protect against infection by the pathogen in a vaccinated animal. For purposes of the present invention, protection against infection by a pathogen includes not only the absolute prevention of infection, but also any detectable reduction in the degree or rate of infection by a pathogen, or any detectable reduction in the severity of the disease or any symptom or condition resulting from infection by the pathogen in the vaccinated animal as compared to an unvaccinated infected animal. An effective immunoprotective response can be induced in animals that have not previously been infected with the pathogen and/or are not infected with the pathogen at the time of vaccination. An effective immunoprotective response can also be induced in an animal already infected with the pathogen at the time of vaccination.
[0052] An "antigenic epitope" is, unless otherwise indicated, a molecule that is able to elicit an immune response in a particular animal or species. Antigenic epitopes are proteinaceous molecules, i.e. polyaminoacid sequences, optionally comprising non-protein groups such as carbohydrate moieties and/or lipid moieties.
[0053] The term "pathogenically infecting" used herein refers to the ability of a pathogen to infect an animal and cause a disease in the animal. As an example, a PRRS virus is capable of pathogenically infecting a porcine animal since it can cause PRRS in swine. However, although PRRS virus may be able to infect, either productively or non-productively, a bird or another mammal, such as a human, it does not pathogenically infect any animal other than a porcine animal since it does not cause any disease in animals other than porcine animals.
[0054] The genetically modified North American PRRS viruses encoded by the above-described isolated polynucleotide molecules are, in one embodiment, able to elicit an effective immunoprotective response against infection by a PRRS virus. Such genetically modified North American PRRS viruses are preferably able to elicit an effective immunoprotective response against any strain of PRRS viruses, including both European and North American strains.
[0055] The present invention provides a mutation or mutations in the isolated polynucleotide molecule encoding the genetically disabled North American PRRS virus that are non-silent and occur in one or more open reading frames of the nucleotide sequence encoding the North American PRRS virus; i.e., the mutation or mutations occur in one or more of the sequences within the nucleotide sequence encoding the North American PRRS virus that are the same as or homologous to ORFs 1a, 1b, 2, 3, 4, 5, 6, or 7 of SEQ ID NO:l. The mutation or mutations may occur in one or more noncoding regions of the North American PRRS virus genome, such as, for example, in the leader sequence of the North American PRRS virus genome; i.e., the mutation or mutations occur within the sequence that is the same as or homologous to the sequence of nucleotides 1-191 of SEQ ID NO:1 in the same isolated polynucleotide molecule, mutations can occur in both coding and noncoding regions.
[0056] As used herein, unless otherwise indicated, "noncoding regions" of the nucleotide sequence encoding the North American PRRS virus refer to those sequences of RNA that are not translated into a protein and those sequences of cDNA that encode such RNA sequences. Coding regions refer to those sequences of RNA from which North American PRRS virus proteins are expressed, and also refer to cDNA that encodes such RNA sequences. Likewise, "ORFs" refer both to RNA sequences that encode North American PRRS virus proteins and to cDNA sequence encoding such RNA sequences.
[0057] Determining suitable locations for a mutation or mutations that will encode a North American PRRS virus that is genetically disabled so that it is unable to produce PRRS yet remains able to elicit an effective immunoprotective response against infection by a PRRS virus can be made based on the SEQ ID NO:1 provided herein. One of ordinary skill can refer to the sequence of the infectious cDNA clone of North American PRRS virus provided by this invention, make sequence changes which will result in a mutation, and test the viruses encoded thereby both for their ability to produce PRRS in swine, and to elicit an effective immunoprotective response against infection by a PRRS virus. In so doing, one of ordinary skill can refer to techniques known in the art and also those described and/or exemplified herein.
[0058] For example, an ORF of the sequence encoding tee infectious RNA molecule encoding the North American PRRS virus can be mutated and the resulting genetically modified North American PRRS virus tested for its ability to cause PRRS. The ORF of a North American PRRS virus encodes proteins as follows: OAF1a encodes a polyprotein comprising protease function; ORF1b encodes a polyprotein comprising replicase (RNA polymerase) and helicase functions; ORFs 2, 3, and 4 encode small membrane glycoproteins; ORF 5 encodes a major envelope glycoprotein; ORF 6 encodes a nonglycosylated integral membrane protein; and OAF 7 encodes a nucleocapsid protein. Genetic mutations of one or more of these ORFs can be used in preparing the genetically modified North American PRRS viruses described infra.
[0059] The subject invention also provides an isolated polynucleotide molecule comprising a DNA sequence encoding an infectious RNA molecule encoding a North American PRRS virus that is genetically modified such that it lacks at least one DNA sequence encoding a detectable antigenic epitope of North American PRRS virus, wherein the DNA sequence encoding the RNA molecule encoding the North American PRRS virus is SEQ ID NO:1 or a sequence homologous thereto.
[0060] A "detectable antigenic epitope" is, unless otherwise indicated, a molecule that is able to elicit an immune response in a particular animal or species, which immune response is measureable via an assay or method.
[0061] In a non-limiting embodiment, the detectable antigenic epitope which is deleted is within OAF1a or ORF1b. In another embodiment, the detectable antigenic epitope which is deleted is within the sequences encoding for the nonstructural protein 2 (nsp2), which is part of OFT 1a. In a separate embodiment, the detectable antigenic epitope which is deleted is within the hypervariable C-terminal portion of nsp2. In a further embodiment, the detectable antigenic epitope which is deleted, or lacking, is the DNA sequence encoding any 10 aa polypeptides or longer, up to 400 aa length polypeptides, where the DNA sequence that may be deleted is the DNA that encodes any amino acids from 616 to 752 of the hypervariable region of nsp2. This region, from 616 to 752 of nsp2, is the conserved sub-region within the hypervariable region of nsp2.
[0062] One example of such a deletion is Example XI. In Example XI a deletion is described which results in the lack of a DNA sequence encoding amino acids 628 through 759 of nsp2. This deletion is within the region encoding the hypervariable C-terminal portion of nsp2. This Example XI deletion, which lacks the DNA sequence encoding amino acids from 628 to 759 in the hypervariable region is considered as being taken from the region of the DNA sequence encoding amino acids 616 to 762 in the hypervariable region because the region begins in the region of the DNA sequence encoding amino acids 616 to 752 in the hypervariable region. The deleted region can extend beyond the conserved sub-region in either direction, but the entire deleted region should be no greater than the amount of DNA that encodes for a polypeptide of about 400 aa. Table 1 shows the relative positions of some of the nucleotides and amino acids in relation to certain regions of the genome for SEQ. ID. NO. 1.
TABLE-US-00001 TABLE 1 Genome coordinates of nsp2 regions based on SEQ ID NO. 1. Amino acid Genome position Amino acid range range Landmark (nucleotides) (w/in ORF1a) (w/in nsp2) Entire 1-15,450 NA NA genome (1-15,395 w/o polyA tail) ORF1a 192-7685 1-2497 NA (7491 nt) (2497 aa) ATGTCT . . . TGCTAG MSGIL . . . QCLNC Nsp2 1341-4262 384-1357 1-974 (2922 nt) (974 aa) (974 aa) GCTGGA . . . CTGGGC AGKRA . . . GRLLG AGKRA . . . GRLLG Hypervariable 2721-3980 844-1263 461-880 region of nsp2 (1260 nt) (420 aa) (420 aa) AGATCT . . . CTCTTT RSDYG . . . LFCLF RSDYG . . . LFCLF Conserved 3136-3596 999-1135 616-752 subregion w/in (411 nt) (137 aa) (137 aa) hypervariable region TCATCA . . . CGCATC SSSSS . . . DIPRI SSSSS . . . DIPRI Deletion in 3222-3617 1011-1142 628-759 Example XI (396 nt) (132 aa) (132 aa) CGCCCA . . . AATACC RPKYS . . . KIENT RPKYS . . . KIENT
[0063] Such deletions may be anywhere within the hypervariable region, including the region of the DNA encoding the conserved sub-region of amino acids from 616 to 752 in nsp2, and such deletions may extend into the flanking regions as well, provided the DNA encodes at least 10 amino acids from the conserved sub-region or from other locations in the hypervariable region.
[0064] In a further embodiment, the detectable antigenic epitope which is removed is taken from the DNA sequence encoding amino acids 628 through 759 of nsp2, which is both within the conserved sub-region and within the region encoding the hypervariable C-terminal portion of nsp2.
[0065] In a further embodiment, the detectable antigenic epitope which is deleted is taken from the DNA sequence encoding polypeptides as short as ten amino acids, or as long as the entire hypervariable region of nsp2 which can extend up to 400 amino acids. Specifically the region may be from 10 to 100 aa in length, from 100 to 150, from 150 to 200, from 200 to 300 or from 300 to 400 amino acids in length. For example, the deleted DNA can encode the peptide that comprises amino acids 1-10 of the hypervariable region of the nsp2 peptide. The peptide can be amino acids 2-11 of the hypervariable region. The peptide can be amino acids 3-12 of the hypervariable region. Peptides are selected in this fashion by progressively "walking" down the amino acid sequence of the hypervariable region of nsp2, all of the way through the hypervariable region, more particularly through the region of the DNA encoding the conserved subregion of amino acids from 616 to 752 of nsp2 within the hypervariable region, and it may extend into the flanking regions. A specific example of this is Example XI where the deleted region is the DNA sequence encoding amino acids 628 through 759 in the hypervariable region.
[0066] In addition to 10-mer peptides, the deleted fragment can be a DNA encoding 11 amino acid residues in length, beginning with a fragment comprising amino acid 1-11 of the hypervariable region, and progressing to the C-terminal end of the hypervariable region. Similarly, the fragment can be DNA encoding 12, 13, 14, 15, 16, 17, 18, 19, 20 or more residues in length, up to 400 residues in length, and such peptides are readily identifiable by beginning at residue 1 and "walking" down the amino acid sequence of the corresponding hypervariable region of nsp2. Such a strategy can be applied to the corresponding hypervariable region of the nsp2 sequence from any strain of PRRS virus.
[0067] The subject invention also provides an isolated polynucleotide molecule comprising a DNA sequence encoding an infectious RNA molecule encoding a North American PRRS virus that is genetically modified such that it lacks at least one DNA sequence encoding a detectable antigenic epitope of North American PRRS virus, wherein it further comprises one or more heterologous antigenic epitopes, wherein the DNA sequence encoding the RNA molecule encoding the North American PRRS virus is SEQ ID NO:1 or a sequence homologous thereto, and further comprising one or more additional nucleotide sequences that each encode a heterologous antigenic epitope, and wherein each heterologous antigenic epitope is capable of inducing an effective immunoprotective response against a particular pathogen in a mammal or a bird.
[0068] A pathogen against which an effective immunoprotective response can be induced by means of the above recited aspect of the present invention is any pathogen, such as a virus, bacteria, fungus, or protozoan, capable of causing a disease in a mammal or bird, which pathogen comprises or has associated therewith one or more antigenic epitopes which can be used to induce an effective immunoprotective response against the pathogen in the mammal or bird.
[0069] The term "heterologous antigenic epitope" for purposes of the present invention means an antigenic epitope, as defined above, not normally found in a wild-type North American PRRS virus. A nucleotide sequence encoding a heterologous antigenic epitope can be inserted into a North American PRRS viral genome using known recombinant techniques. Antigenic epitopes useful as heterologous antigenic epitopes for the present invention include additional North American PRRS virus antigenic epitopes, antigenic epitopes from European PRRS viruses, antigenic epitopes from swine pathogens other than PRRS viruses, or antigenic epitopes from pathogens that pathogenically infect birds or mammals other than swine, including humans. Sequences encoding such antigenic epitopes are known in the art or are provided herein. For example, a second North American PRRS virus envelope protein, encoded by North American PRRS ORF 5 described herein, can be inserted into a DNA sequence encoding an RNA molecule encoding a North American PRRS virus of the present invention to generate a genetically modified North American PRRS virus comprising an additional envelope protein as a heterologous antigenic epitope. Such a genetically modified North American PRRS virus can be used to induce a more effective immunoprotective response against PRRS viruses in a porcine animal vaccinated therewith.
[0070] Examples of an antigenic epitope from a swine pathogen other than a North American PRRS virus include, but are not limited to, an antigenic epitope from a swine pathogen selected from the group consisting of European PRRS, porcine parvovirus, porcine circovirus, a porcine rotavirus, swine influenza, pseudorabies virus, transmissible gastroenteritis virus, porcine respiratory coronavirus, classical swine fever virus, African swine fever virus, encephalornyocatditis virus, porcine paramyxovirus, Actinobacillus pleuropneumoni, Bacillus anthraci, Bordetella bronchiseptica, Clostridium haemolyticum, Clostridium pertringens, Clostridium tetani, Escherichia coil, Etysipelothrix rhuslopathiae, Haemophilus parasuis, Leptospira spp., Mycoplasma hyopneumoniae, Mycoplasma hyorhinis, Pasteurella haemolytica, Pasteurella multocida, Salmonella choleraesuis, Salmonella typhimurium, Streptococcus equismilis, and Streptococcus suis. Nucleotide sequences encoding antigenic epitopes from the aforementioned swine pathogens are known in the art and can be obtained from public gene databases such as GenBank (http://www.ncbi.nlm.nih.gov/Web/Genbank/index.html) provided by NCBI.
[0071] If the heterologous antigenic epitopes are antigenic epitopes from one or more other swine pathogens, then the isolated polynucleotide molecule can further contain one or more mutations that genetically disable the encoded PRRS virus in its ability to produce PRRS. Such isolated polynucleotide molecules and the viruses they encode are useful for preparing vaccines for protecting swine against the swine pathogen or pathogens from which the heterologous antigenic epitopes are derived.
[0072] The present invention provides a genetically modified North American PRRS virus able to elicit an effective immunoprotective response against infection by a PRRS virus in a porcine animal. Such isolated polynucleotide molecules and the viruses they encode are useful for preparing dual-function vaccines for protecting swine against infection by both a North American PRRS virus and the swine pathogen or pathogens from which the heterologous antigenic epitopes are derived.
[0073] The isolated polynucleotide molecules of the present invention comprising nucleotide sequences encoding heterologous antigenic epitopes can be prepared as described above based on the sequence encoding a North American PRRS virus described herein using known techniques in molecular biology.
[0074] In one embodiment, the additional nucleotide sequences that each encode a heterologous antigenic epitope are inserted into the DNA sequence encoding an infectious RNA molecule encoding a North American PRRS virus within an open reading frame encoding for a PRRS virus protein. In a non-limiting embodiment, the additional nucleotide sequences are inserted in ORF1a or ORF1b, in another embodiment, the additional nucleotide sequences are inserted within sequences encoding for the nonstructural protein 2 (nsp2), which is part of ORF1a. In a separate embodiment, the additional nucleotide sequences are inserted within the region encoding the hypervariable C-terminal portion of nsp2. In a further embodiment, the additional nucleotide sequences are inserted between the DNA sequence encoding amino acids 628 and 759 in the hypervariable region in the nonstructural protein 2 coding region of ORE1a within said DNA sequence, which is within the region encoding the hypervariable C-terminal portion of nsp2. See FIG. 4.
[0075] The hypervariable region of nsp2 corresponds approximately to genome coordinates 2720-3980 in P129. See FIG. 4.
[0076] In a separate embodiment, the additional nucleotide sequences that each encode a heterologous antigenic epitope are inserted into the DNA sequence encoding an infectious RNA molecule encoding a North American PRRS virus within the sequences encoding any of ORFs 1a, 1b, 2, 3, 4, 5, 6, or 7. See FIG. 4.
[0077] In another embodiment, a heterologous antigenic epitope of the genetically modified North American PRRS virus of the present invention is a detectable antigenic epitope. Such isolated polynucleotide molecules and the North American PRRS viruses they encode are useful, inter alia, for studying PRRS infections in swine, determining successfully vaccinated swine, and/or for distinguishing vaccinated swine from swine infected by a wild-type PRRS virus. Preferably, such isolated polynucleotide molecules further contain one or more mutations that genetically disable the encoded PRRS virus in its ability to produce PRRS, and more preferably are able to elicit an effective immunoprotective response in a porcine animal against infection by a PRRS virus.
[0078] Heterologous antigenic epitopes that are detectable, and the sequences that encode them, are known in the art. Techniques for detecting such antigenic epitopes are also known in the art and include serological detection of antibody specific to the heterologous antigenic epitope by means of, for example. Western blot, ELISA, or fluorescently labeled antibodies capable of binding to the antibodies specific to the heterologous antigenic epitope. Techniques for serological detection useful in practicing the present invention can be found in texts recognized in the art, such as Coligan, J. E., et al. (eds), 1998, Current Protocols in Immunology, John Willey & Sons, Inc., which is hereby incorporated by reference in its entirety. Alternatively, the heterologous antigenic epitope itself can be detected by, for example, contacting samples that potentially comprise the antigenic epitope with fluorescently-labeled antibodies or radioactively-labeled antibodies that specifically bind to the antigenic epitopes.
[0079] The present invention further provides an isolated polynucleotide molecule comprising a DNA sequence encoding an infectious RNA molecule which encodes a genetically modified North American PRRS virus that detectably lacks a North American PRRS virus antigenic epitope, wherein the DNA sequence encoding the RNA molecule encoding the North American PRRS virus is SEQ ID NO:1 or a sequence homologous thereto, except that it lacks one or more nucleotide sequences encoding a detectable North American PRRS virus antigenic epitope. Such isolated polynucleotide molecules are useful for distinguishing between swine infected with a recombinant North American PRRS virus of the present invention and swine infected with a wild-type PRRS virus. For example, animals vaccinated with killed, live or attenuated North American PRRS virus encoded by such an isolated polynucleotide molecule can be distinguished from animals infected with wild-type PRRS based on the absence of antibodies specific to the missing antigenic epitope, or based on the absence of the antigenic epitope itself. If antibodies specific to the missing antigenic epitope, or if the antigenic epitope itself, are detected in the animal, then the animal was exposed to and infected by a wild-type PRRS virus. Means for detecting antigenic apitopes and antibodies specific thereto are known in the art, as discussed above. Preferably, such an isolated polynucleotide molecule further contains one or more mutations that genetically disable the encoded PRRS virus in its ability to produce PRRS. More preferably, the encoded virus remains able to elicit an effective immunoprotective response against infection by a PRRS virus.
[0080] B. Vaccines and Uses Thereof.
[0081] The present invention also provides vaccines comprising North American PRRS viruses, including genetically modified North American PRRS viruses disabled in their ability to produce PRRS in a swine animal as described herein; infectious RNA molecules and plasmids encoding such North American PRRS viruses as described herein; and viral vectors encoding such North American PRRS viruses and isolated RNA molecules as described herein.
[0082] In on embodiment, the subject invention provides a vaccine comprising a genetically modified North American PRRS virus which lacks at least one detectable antigenic epitope as described herein, an infectious RNA molecule encoding such a genetically modified North American PRRS virus, a plasmid as described herein encoding such a genetically modified North American PRRS virus, or a viral vector encoding such a genetically modified North American PRRS virus, and a carrier acceptable for pharmaceutical or veterinary use, in an amount effective to elicit an immunoprotective response against PRRS virus infection in the porcine animal.
[0083] In another embodiment, the subject invention provides a vaccine comprising a genetically modified North American PRRS virus comprising one or more detectable heterologous antigenic epitopes as described herein, an infectious RNA molecule encoding such a genetically modified North American PRRS virus, a plasmid as described herein encoding such a genetically modified North American PRRS virus, or a viral vector encoding such a genetically modified North American PRRS virus, and a carrier acceptable for pharmaceutical or veterinary use.
[0084] Such vaccines can be used to protect from infection a mammal or a bird capable of being pathogenically infected by the pathogen or pathogens from which the detectable heterologous antigenic epitope(s) are derived. If such a vaccine comprises the genetically modified North American PRRS virus, the genetic modification of the North American PRRS virus preferably preferably renders the virus unable to cause PRRS in swine. Thus, such would provide a dual-vaccine for swine, protecting swine from infection by the swine pathogen or pathogens from which the heterologous antigenic epitope(s) are derived as well as from infection by a PRRS virus. If the vaccine comprises an infectious RNA molecule or a plasmid encoding a genetically-modified North American PRRS virus comprising one or more heterologous antigenic epitopes from another swine pathogen, then the sequence encoding the infectious RNA molecule encoding the genetically modified PRRS virus preferably comprises one or more further mutations that genetically disable the encoded North American PRRS virus so that it is unable to cause PRRS. In another preferred embodiment, the encoded genetically modified, disabled North American PRRS virus is able to elicit an immunoprotective response against a PRRS infection in a swine animal, thus providing a dual-vaccine for swine, able to protect swine from infection by the swine pathogen or pathogens from which the heterologous antigenic epitope(s) are derived as well as from infection by a PRRS virus. All of these vaccines also further comprise a carrier acceptable for veterinary use.
[0085] Vaccines of the present invention can be formulated following accepted convention to include acceptable carriers for animals, including humans (if applicable), such as standard buffers, stabilizers, diluents, preservatives, and/or solubilizers, and can also be formulated to facilitate sustained release. Diluents include water, saline, dextrose, ethanol, glycerol, and the like. Additives for isotonicity include sodium chloride, dextrose, mannitol, sorbitol, and lactose, among others. Stabilizers include albumin, among others. Other suitable vaccine vehicles and additives, including those that are particularly useful in formulating modified live vaccines, are known or will be apparent to those skilled in the art, See, e.g., Remington's Pharmaceutical Science, 18th ed., 1990, Mack Publishing, Mack is incorporated herein by reference.
[0086] Vaccines of the present invention may further comprise one or more additional immunomodulatory components such as, e.g., an adjuvant or cylokine, among others. Non-limiting examples of adjuvants that can be used in the vaccine of the present invention include the RIBI adjuvant system (Ribi Inc., Hamilton, Mont.), alum, mineral gels such as aluminum hydroxide gel, oil-in-water emulsions, water-in-oil emulsions such as, e.g., Freund's complete and incomplete adjuvants, Block copolymer (CytRx, Atlanta Ga.), QS-21 (Cambridge Biotech Inc., Cambridge Mass.), SAF-M (Chiron, Emeryville Calif.). AMPHIGEN® adjuvant, saponin, Quil A or other saponin fraction, monophosphoryl lipid A, and Avridine lipid-amine adjuvant. Non-limiting examples of oil-in-water emulsions useful in the vaccine of the invention include modified SEAM62 and SEAM 1/2 formulations. Modified SEAM62 is an oil-in-water emulsion containing 5% (v/v) squalene (Sigma), 1% (v/v) SPAN® 85 detergent (ICI Surfactants), 0.7% (v/v) TWEEN® 80 detergent (ICI Surfactants), 2.5% (v/v) ethanol, 200 μg/ml Quil A, 100 μg/ml cholesterol, and 0.5% (v/v) lecithin. Modified SEAM 1/2 is an oil-in-water emulsion comprising 5% (v/v) squalene, 1% (v/v) SPA 85 detergent, 0.7% (v/v) Tween 80 detergent, 2.5% (v/v) ethanol, 100 μg/ml Quil A, and 50 μg/ml cholesterol. Other immunomodulatory agents that can be included in the vaccine include, e.g., one or more interleukins, interferons, or other known cytokines.
[0087] Vaccines of the present invention can optionally be formulated for sustained release of the virus, infectious RNA molecule, plasmid, or viral vector of the present invention. Examples of such sustained release formulations include virus, infectious RNA molecule, plasmid, or viral vector in combination with composites of biocompatible polymers, such as, e.g., poly(lactic acid), poly(lactic-co-glycolic acid), methylcellulose, hyaluronic acid, collagen and the like. The structure, selection and use of degradable polymers in drug delivery vehicles have been reviewed in several publications, including A. Domb et al., 1992, Polymers for Advanced Technologies 3: 279-292, which is incorporated herein by reference. Additional guidance in selecting and using polymers in pharmaceutical formulations can be found in texts known in the art, for example M. Chasin and R. Langer (eds), 1990, "Biodegradable Polymers as Drug Delivery Systems"in: Drugs and the Pharmaceutical Sciences, Vol. 45, M. Dekker, N.Y., which is also incorporated herein by reference. Alternatively, or additionally, the virus, plasmid, or viral vector can be microencapsulated to improve administration and efficacy. Methods for microencapsulating antigens are well-known in the art, and include techniques described, e.g., in U.S. Pat. No. 3,137,631; U.S. Pat. No. 3,959,457; U.S. Pat. No. 4,205,050; U.S. Pat. No. 4,606,940: U.S. Pat. No. 4,744,933; U.S. Pat. No. 5,132,117; and International Patent Publication WO 95/28227, all of which are incorporated herein by reference.
[0088] Liposomes can also be used to provide for the sustained release of virus, plasmid, or viral vector. Details concerning how to make and use liposomal formulations can be found in, among other places, U.S. Pat. No. 4,016,100; U.S. Pat. No. 4,452,747; U.S. Pat. No. 4,921,706; U.S. Pat. No. 4,927,637; U.S. Pat. No. 4,944,948; U.S. Pat. No. 5,008,050; and U.S. Pat. No. 5,009,956, all of which are incorporated herein by reference.
[0089] An effective amount of any of the above-described vaccines can be determined by conventional means, starting with a low dose of virus, plasmid or viral vector and then increasing the dosage while monitoring the effects. An effective amount may be obtained after a single administration of a vaccine or after multiple administrations of a vaccine. Known factors can be taken into consideration when determining an optimal dose per animal. These include the species, size, age and general condition of the animal, the presence of other drugs in the animal, and the like. The actual dosage is preferably chosen after consideration of the results from other animal studies.
[0090] One method of detecting whether an adequate immune response has been achieved is to determine seroconversion and antibody titer in the animal after vaccination. The timing of vaccination and the number of boosters, if any, will preferably be determined by a doctor or veterinarian based on analysis of all relevant factors, some of which are described above.
[0091] The effective dose amount of virus, infectious RNA molecule, plasmid, or viral vector, of the present invention can be determined using known techniques, taking into account factors that can be determined by one of ordinary skill in the art such as the weight of the animal to be vaccinated. The dose amount of virus of the present invention in a vaccine of the present invention preferably ranges from about 101 to about 109 pfu (plaque forming units), more preferably from about 102 to about 108 pfu, and most preferably from about 103 to about 107 pfu. The dose amount of a plasmid of the present invention in a vaccine of the present invention preferably ranges from about 0.1 μg to about 100 mg, more preferably from about 1 μg to about 10 mg, even more preferably from about 10 μg to about 1 mg. The dose amount of an infectious RNA molecule of the present invention in a vaccine of the present invention preferably ranges from about 0.1 μg to about 100 mg, more preferably from about 1 μg to about 10 mg, even more preferably from about 10 μg to about 1 mg. The dose amount of a viral vector of the present invention in a vaccine of the present invention preferably ranges from about 101 pfu to about 109 btu, more preferably from about 102 pfu to about 108 pfu, and even more preferably from about 103 to about 107 pfu. A suitable dosage size ranges from about 0.5 ml to about 10 ml, and more preferably from about 1 ml to about 5 ml.
[0092] It is to be understood that the term "North American PRRS viruses of the present invention" and like terms, unless otherwise indicated, include any of the genetically modified North American PRRS viruses described herein as well as the unmodified North American PRRS virus described herein encoded by SEQ ID NO:1 or a sequence homologous thereto.
[0093] C. Diagnostic Kits
[0094] The present invention also provides diagnostic kits. The kit can be valuable for differentiating between porcine animals naturally infected with a field strain of a PRRS virus and porcine animals vaccinated with any of the vaccines described herein. The kits can also be of value because animals potentially infected with field strains of PRRS virus can be detected prior to the existence in the absence of clinical symptoms and removed from the herd, or kept in isolation away from naive or vaccinated animals. The kits include reagents for analyzing a sample from a porcine animal for the presence of antibodies to a PRRS virus. Diagnostic kits of the present invention can include as a component a peptide or peptides which comprise all or a portion of a protein sequence which is no longer present in a genetically modified North American PRRS virus which may optionally comprise one or more detectable heterologous antigenic epitopes. Kits of the present invention can alternatively include as a component a peptide which is a fusion protein. The term "fusion peptide" or "fusion protein" for purposes of the present invention means a single polypeptide chain consisting of at least a portion of a PRRS virus protein and a heterologous peptide or protein.
[0095] D. Plasmids Encoding a North American PRRS Virus or a Genetically Modified North American PRRS Virus.
[0096] The present invention also provides any of the above-described isolated polynucleotide molecules in the form of a plasmid capable of expressing the North A PRRS virus encoded thereby.
[0097] Plasmids of the present invention can express the encoded North American PRRS virus outside of a living organism, to produce North American PRRS viruses of the invention useful, inter alia, for preparing vaccines. In one embodiment, a plasmid of the present invention capable of expressing a North American PRRS virus outside of a living organism is a plasmid wherein transcription of viral RNA therefrom occurs in vitro (i.e. extracellularly); the resulting viral RNA molecule is transfected into a suitable host cell using known mechanisms of transfection, such as electroporation, lipofection (in some cases using a commercially available reagent, such as Lipofectin® (Life Technologies Inc., Rockville, Md., USA)), or DEAE dextran mediated transfection. Other methods of transfection are known in the art and can be employed in the present invention. An example of such a plasmid for in vitro transcription of North American PRRS viral RNA is the plasmid pT7P129A (ATCC Accession No. 203488). Any promoter useful for in vitro transcription can be used in such plasmids of this invention. T7 is one such promoter, but other promoters can be used, such as SP6 promoter or a T3 promoter. The sequences of such promoters can be artificially synthesized or cloned from commercially available plasmids. Suitable plasmids for preparing such plasmids capable of expressing North American PRRS virus include, but are not limited to, general purpose cloning vector plasmids such as pCR2.1 (Invitrogen, Carlsbad, Calif., USA), pBR322, and pUC18. A nucleotide sequence of the present invention encoding the North American PRRS virus can be inserted into any of these plasmids using known recombinant techniques. Other plasmids into which the polynucleotide molecules of the present invention can be inserted will be recognized by those of ordinary skill in the art.
[0098] Suitable conditions for in vitro transcription of viral RNA from any of the above-described recombinant plasmids comprising a DNA sequence encoding an infectious RNA molecule encoding a North American PRRS virus depends on the type of plasmid, for example, its particular promoter, and can be ascertained by one of ordinary skill in the art. For example, if a plasmid of the present invention is based on a pCR2.1 plasmid comprising a T7 promoter, then an example of suitable conditions for in vitro transcription includes reacting the plasmid with T7 RNA polymerase and ribonucleotides in a standard buffer and incubating the reaction at 37° C. for about 30 minutes. In some cases, commercial kits are available for transcribing RNA from a particular plasmid, and such kits can be used in the present invention. The reaction mixture following transcription can be directly transfected into a suitable host cell without purification, or the transcribed North American PRRS virus RNA can be purified by known RNA purification techniques, for example by organic (e.g. phenol) extraction and alcohol (e.g. ethanol or isopropanol) precipitation, prior to transfection.
[0099] Transcription of the DNA sequence encoding an infectious RNA molecule encoding a North American PRRS virus, and further comprising one or more additional nucleotide sequences that each encode a heterologous antigenic epitope, results in the formation of a genomic and subgenomic RNA molecules. In one embodiment, at least one heterologous antigenic epitope is translated directly from The genomic RNA molecule. In another embodiment, at least one heterologous antigenic epitope is translated from a subgenomic RNA molecule that is synthesized from the genomic RNA.
[0100] The term "translation" for purposes of the present invention means the synthesis of protein from nucleotides such as an RNA molecule. In one embodiment, translation of the genomic or a subgenomic RNA molecule encoding a heterologous antigenic epitope results in synthesis of a peptide or protein consisting of the heterologous antigenic epitope itself. In another embodiment, translation of the genomic or a subgenomic RNA molecule results in synthesis of a fusion peptide or protein. The term "fusion peptide or protein" for purposes of the present invention means a single polypeptide chain consisting of at least a portion of a PRRS virus protein and at least one heterologous antigenic epitope.
[0101] Practically any mammalian or avian cell culture can be transfected with the North American PRRS virus RNA obtained as described above in order to generate a first round of North American PRRS virions. An example of cells which one might find particularly useful because of their ready availability and ease of use are BHK (baby hamster kidney) cells. However, if one wishes to generate a cell culture capable of sustained production of North American PRRS virions, then porcine alveolar macrophage cells or MARC-145 cells (Kim, H. S., et al., supra) are preferred since these cells excrete high levels of new generation PRRS virions subsequent to PRRS virus infection. Other cell lines derived from the MA-104 cell line may also be used for sustained generation of North American PRRS virions of the present invention. Primary porcine alveolar macrophage cells can be obtained by lung lavages from pigs, and the MARC-145 monkey kidney cell line can be obtained from the National Veterinary Services Laboratories otherwise known as NVSL (Ames, Iowa, USA).
[0102] In another embodiment, a plasmid capable of expressing a North American PRRS virus of the present invention outside of a living organism is a plasmid which is transfected into a suitable host cell, for example by electroporation or lipofection, transcription of the infectious RNA molecule and expression of the North American PRRS virus therefrom occurring within the transfected host cell. The transfected host cell therefore generates North American PRRS virions. Such a completely cellular method has heretofore never been disclosed or suggested for any virus within the order of Nidovirales. Because of possible cryptic splicing and termination sequences present in the RNA genome of viruses of the Nidovirales order, a completely cellular method of expressing a Nidovirales virus was believed unlikely. Cryptic sequences include RNA splice donor and splice acceptor sequences, which could cause inappropriate splicing of the RNA transcript, as well as polyadenylation sequences, which could cause premature termination by the cellular RNA polymerase II. The present invention demonstrates, however, that the presence of such sequences in a plasmid comprising a cDNA clone of a Nidovirus does not prevent the plasmid's ability to express the Nidovirus when the plasmid is directly transfected into a suitable host cell.
[0103] Accordingly, the subject invention also provides plasmids and a completely cellular method for expressing a Nidovirales virus, wherein the plasmid comprises: a) a DNA sequence encoding an infectious RNA molecule encoding the Nidovirales virus; and b) a promoter capable of transcribing said encoding sequence in a cell, wherein said promoter is in operative association with the DNA sequence encoding the infectious RNA molecule. The method comprises transfecting a suitable host cell with such a plasmid, subjecting the transfected host cell to conditions suitable for expression of gene sequences transfected therein, and collecting the expressed Nidovirales virus therefrom. An example of a plasmid suitable for completely cellular expression of North American PRRS virus outside of a living organism is the plasmid pCMV-S-P129 (ATCC Accession No. 203489). In a preferred embodiment, the promoter of such a plasmid is a CMV promoter. In a preferred embodiment, a plasmid of the invention suitable for a completely cellular method of expressing a Nidovirales virus comprises a eukaryotic promoter, such as a CMV promoter, immediately upstream and adjacent to the nucleotide sequence encoding the Nidovirales virus, in a preferred embodiment, the nucleotide sequence encoding the Nidovirales virus encodes a PRRS virus, either European or North American. Other examples of Nidovirales viruses that can be expressed by means of the above-described completely cellular method include other Arteriviruses such as, equine arteritis virus, lactate dehydrogenase-elevating virus, and simian haemorrhagic fever virus; viruses that are members of the genus Coronavitidae, such as, but not limited to, feline infectious peritinitis virus, feline enteric coronavirus, canine coronavirus, bovine coronavirus, porcine respiratory coronavirus, turkey coronavirus, porcine transmissible gastroenteritis virus, human coronavirus, murine hepatitis virus, and avian infectious bronchitis virus; and members of the genus Toroviridae, such as, but not limited to, Berne virus, Breda virus, and human torovirus. Thus, plasmids suitable for completely cellular expression comprising a nucleotide sequence encoding one of these viruses are also encompassed by the present invention.
[0104] Suitable plasmids that can be used to prepare recombinant plasmids of the present invention for completely cellular expression outside of a living organism of a Nidovirales virus, such as a PRRS virus, include virtually any plasmid useful for transfection and expression in eukaryotic An examples of a plasmid suitable for preparing recombinant plasmids of the present invention for completely cellular expression of a Nidovirales virus is the plasmid pCMVbeta (Clontech, Palo Alto, Calif., USA). Other plasmids which are able to transfect and express genes in eukaryotic cells which can be used to prepare plasmids of the present invention include, but are not limited to, pcDNA3.1, pRc/RSV, and pZeoSV2 (all from Invitrogen); and pCMV-Sport3 and pSV-Sport1 (both from Life Technologies Inc.). However, almost any eukaryotic expression vector will work for the present invention. Constructs based on cosmids can also be used for completely cellular ex vivo expression of a Nidovirales virus.
[0105] Suitable host cells for the completely cellular method of the present invention for expressing PRRS virus include porcine alveolar macrophage cells and the MARC-145 cells, described above. Methods of transfecting these cells with a plasmid are basically the same as those methods for transfecting cells with viral RNA described above. Such methods include, but are not limited to, electroporation, lipofection, DEAE dextran mediated transfection, and calcium phosphate coprecipitation.
[0106] Once host cells, such as porcine alveolar macrophage cells or a MARC-145 cells, have been transfected according to the subject invention, either with viral RNA or with a plasmid comprising a nucleotide sequence encoding a virus, then the cells can be frozen at about -80° C. or below for storage for up to several years. For longer periods of time, i.e. decades, storage in liquid nitrogen is preferred. If relatively frequent use of the encoded virus is envisioned, then cells hosting the virus can also be maintained (unfrozen) in culture using known techniques, for shorter periods of time. Moreover, viral particles excreted by such cells can be stored frozen at about -80° C. or below as a source of virus. Transfection of such cell lines with the polynucleotide molecule encoding the virus can be confirmed if desired, for example, by testing exhausted medium excreted by the cell line for a PRRS virus antigen using an immunofluorescent antibody test. Antibodies which are specific for PRRS virus antigens are known in the art (see, e.g., Collins, E. J., et al., WO 93/03780 Mar. 4, 1993).
[0107] In another embodiment, a plasmid of the present invention comprising a nucleotide sequence encoding a North American PRRS virus is suitable for in vivo expression of the North American PRRS virus, i.e. expression in a living organism. Plasmids which can be used for preparing recombinant plasmids for in vivo expression of a North American PRRS virus include, but are not limited to the plasmids capable of transfecting eukaryotic cells described above, such as pCMVbeta.
[0108] Animals that can be transfected with plasmids of the present invention include mammals and birds. If the animal is other than a porcine animal, for example, a mallard duck, then the plasmid can comprise a nucleotide sequence encoding a North American PRRS virus comprising further antigenic epitopes from pathogens which are capable of pathogenically infecting the animal; in such a case, the plasmid encode a North American PRRS virus serving as a vector for transporting epitopes into the animal. If the animal is a porcine animal, then the plasmid can usefully encode any of the North American PRRS viruses described herein, including the genetically-modified North American PRRS viruses described herein.
[0109] E. Viral Vectors Encoding a North American PRRS Virus, including Viral Vectors Encoding Genetically Modified North American PRRS Viruses:
[0110] The present invention also provides viral vectors comprising a DNA sequence encoding an infectious RNA molecule encoding any of the North American PRRS viruses described herein, including the genetically-modified North American PRRS viruses described herein. Such viral vectors are useful for transfecting eukaryotic cells for production of PRRS viruses of the present invention outside of a living organism, or for transfecting swine, or other mammals, or avians, with the sequence encoding the North American PRRS virus, for in vivo expression of the North American PRRS virus therein.
[0111] Some examples of viruses that can be used as vectors for preparing the viral vectors of the present invention include, but are not limited to, swine viruses such as, but not limited to, swine pox virus, pseudorabies virus, or African swine fever virus. Such swine viruses can be obtained from The National Veterinary Services Laboratories (Ames, Iowa, USA) of the United States Department of Agriculture; the American Type Culture Collection, otherwise known as the ATCC (Manassas, Va., USA); and other known sources Recombinant viral vectors based on suitable swine viruses such as the aforementioned swine viruses are useful for transfecting swine animals with a nucleotide sequence encoding a North American PRRS virus of the present invention.
[0112] Viral vectors comprising a DNA sequence encoding an infectious RNA molecule encoding a North American PRRS virus of the present invention based on these and other viruses can be prepared using known recombinant techniques described in texts such as those cited previously in this application.
[0113] F. Transfected Host Cells Encoding or a Genetically Modified North American PRRS Viruses.
[0114] The present invention also provides transfected host cells that comprise a DNA sequence encoding an infectious RNA molecule encoding any of the North American PRRS viruses described herein, including the genetically-modified North American PRRS viruses described herein, which transfected host cells are capable of expressing the North American PRRS virus. Such transfected host cells are useful for producing North American PRRS viruses of the present invention. Examples of transfected host cells of the present invention include the transfected porcine alveolar macrophage cells and the transfected MARC-145 cells described above.
[0115] Other transfected host cells of the invention include, but are not limited to, transfected MA-104 cells and other derivatives of MA-104 cells that are transfected; transfected Baby Hamster Kidney (BHK) cells; transfected Chinese Hamster Ovary (CHO) cells, and African Green Monkey kidney cells other than MA-104 cells or MARC-145 cells, such as VERO cells; that are transfected,
[0116] G. North American PRRS Viruses, including Generically Modified North American PRRS Viruses:
[0117] The present invention also provides North American PRRS viruses as described herein, including genetically-modified North American PRRS viruses as described herein, expressed and/or encoded by any of the above-described isolated polynucleotide molecules, RNA molecules, plasmids, viral vectors, or transfected host cells.
[0118] In certain situations, for example where the North American PRRS virus is to be used in a vaccine for swine and the North American PRRS virus has not been genetically modified as described above so as to be unable to cause PRRS, it is desirable to treat the North American PRRS virus, for example by inactivating or attenuating it, so that it is unable to cause PRRS in swine to which it is administered. Known methods can be used to inactivate a North American PRRS virus of the present invention so that it is unable to cause PRRS in an animal. Examples of such methods include, but are not limited to, treatment with formaldehyde, BEI (binary ethyleneimine), or BPL (beta-propiolactone). Methods of attenuation are also known in the art, and such methods can be used to attenuate a North American PRRS virus of the present invention. A North American PRRS virus of the present invention can, for example, be attenuated by serial passage in cell culture.
[0119] If a North American PRRS virus of the present invention is for use in an animal other than a porcine animal, or if it has been genetically modified as described herein so that it is unable to produce PRRS in a porcine animal, then it is not necessary to treat the virus as described in the preceding paragraph prior to using it in a vaccine.
[0120] The following examples are provided to merely illustrate aspects of the subject invention. They are not intended, and should not be construed, to limit the invention set forth in the claims and more fully described herein.
EXAMPLES
Example I
Preparation of an Infectious cDNA Clone of a North American PRRS Virus Isolate
[0121] Source of PRRS virus and MARC-145 cells: A North American PRRS virus isolate designated P129 was obtained from Drs. Gregory W. Stevenson, William G. Van Alstine, and Charles L. Kanitz of Purdue University's Animal Disease Diagnostic Laboratory in West Lafayette, Ind. The P129 virus was originally isolated in the autumn of 1995 from a swine herd in southern Indiana experiencing a severe PRRS outbreak. This farm had no previous history of PRRS problems or PRRS vaccination. The P129 isolate was more virulent than several other field isolates from the same time period and geographic area, in that it produced more severe and more consistent respiratory disease in young pigs. The virus was initially isolated on primary porcine alveolar macrophage (the natural host cell), and subsequently passaged on MARC-145 cells (Kim at al., 1993). Genes encoding structural proteins of P129 were found to be homologous to corresponding known North American PRRS gene sequences.
[0122] The MARC-145 cell line that was used to propagate PRRS viruses is a clone of the MA-104 Rhesus Macaque Monkey Kidney cell line. The MARC-145 cells were obtained from the National Veterinary Services Laboratories (NVSL, Ames, Iowa) of the USDA. These cells have been tested and found negative for mycoplasmas and for common porcine extraneous agents. MARC-145 cells are routinely grown at 37 C in OptiMEM (Life Technologies Inc.) with 2% fetal bovine serum and antibiotics.
[0123] Five biological clones were plaque purified from the P129 virus stock, and these were designated P129A through P129E. Plaque purification was carded out by infecting monolayers of MARC-145 cells with P129 virus, adding an overlay of OptiMEM containing 1.25% SeaPlaque agarose (FMC BioProducts), 2% fetal bovine serum, and antbiotics. Plaques were clearly visible following incubation for 7 days, when 5 well-isolated plaques were picked and passaged onto fresh MARC-145 monolayers. When cytopathic effect (virus induced cell death) became apparent the progeny virus from each of these cultures was subjected to another round of plaque purification. One well-isolated plaque from each of the five clones was picked and expanded to produce large stocks. The 5 clones were tested for virulence in young pigs, either individually (clones A and E) or in combination (clones B-D, or clones A-E). In all cases, the plaque purified virus replicated well in pigs and caused clinical disease. The severity of clinical symptoms was less than that caused by the uncloned P129 virus, even when all five clones were used together. P129A was chosen for sequencing, and was used in subsequent molecular manipulations.
[0124] Determination of the genome sequence of P129A: Plaque purified virus P129A was used for sequence determination after 10 serial passages from the pig (including two plaque purifications and one subsequent passage). SEQ ID NO:1 shows the cDNA sequence corresponding to the P129A RNA genome. The genome is 15,395 nucleotides in length (excluding the polyadenosine tail), begins with ATGACGTA, and ends with CCGCAATT. A typical polyadenosine tail of 55 residues is also provided in SEQ ID NO:1. For the structural genes of P129A (ORFs 2 through 7), which comprise the 3' 20% of the genome, various PCR primers were chosen based on several partial cDNA sequences of other North American PRRS virus isolates available in the public DNA sequence database GenBank (for example PRU00153). Purified viral RNA was reverse transcribed into cDNA using reverse transcriptase and random hexamer primers. This cDNA was then used in PCR with gene-specific primers. PCR products were excised from gels and T/A cloned into plasmid pCR2.1 (Invitrogen). For each primer pair, multiple plasmids (from independent PCR reactions) were DNA sequenced. Sequences were assembled using the Seqman program from the Lasergene package (DNASTAR, Inc). This permitted completing the sequence of positions 11,992 through 15,347 of the P129A genome.
[0125] Also in the GenBank database are a series of short sequences (approximately 218 nucleotides total) which comprise a portion of the ORF1b gene of several isolates of PRRS virus. One of these (PPSSEQB) was used to design PCR primers (forward 5'-ACAGTTTGGTGATCTATG-3' (SEQ ID NO:10), corresponding to positions 9063-9080; reverse 5'-CAGATTCAGATGTTCAA-3' (SEQ ID NO:11), corresponding to positions 9252-9268). These amplified a 206 nucleotide fragments, which includes 171 nucleotides of new sequence from the P129A ORF1b gene, corresponds to positions 9081 to 9251. A new forward primer was designed within this region (5'-ACCTCGTGCTGTATGCCGAATCTC-3' (SEQ ID NO:12), positions 9201-9224), and a matching primer was designed within ORF1b immediately upstream of ORF2 (5'-TCAGGCCTAAAGTTGGTTCAATGA-3' (SEQ ID NO:13), positions 12,027-12,050). These primers were used in RT-PCR to amplify a 2850 nucleotide fragment of ORF1b, corresponding to positions 9201-12,050 of the P129A genome.
[0126] During RT-PCR amplification of ORF5 of another North American field isolate of PRRS virus, a minor band was seen which was smaller than the expected size. This was sequenced and found to have limited homology with ORF1a of Lelystad virus (resulting from false priming). New primers within this region wore chosen to amplify P129A (forward 5'-GATGACTGGGCTACTGACGAGGAT-3' (SEQ ID NO:14), corresponding to positions 1587-1610; reverse 5'-AGAGCGGCTGGGATGACACTG-3' (SEQ ID NO:15), corresponding to positions 1877-1897). In addition to the product of 311 nucleotides (266 nucleotides of new P129A sequence between the primers corresponding to positions 1611-1876), a larger minor PCR product of 701 nucleotides was cloned and sequenced (656 nucleotides of new P129A sequence between the primers corresponding to positions 1611-2264). The larger band results from false priming of the reverse primer at positions 2265-2269.
[0127] The extreme 5' end of the genome of P129A was determined by 5' RACE (rapid amplification of cDNA ends) using a commercially available kit (Life Technologies Inc). Two nested reverse primers were chosen from within the known ORF1a sequence ("RACE2" 5'-CCGGGGAAGCCAGACGATTGAA-3' (SEQ ID NO:16), positions 1917-1938; and "RACE3" 5'-AGGGGGAGCAAAGAAGGGGTCATC-3' (SEQ ID NO:17), positions 1733-1756). RACE2 was used to prime cDNA synthesis, while RACE3 was used in PCR. The resulting PCR products were cloned and sequenced. The two longest products ended at precisely the same base (position 1 in SEQ ID NO:1).
[0128] The large gap between known sequence in ORF1a and ORF1b was bridged using long RT-PCR. Two new primers were used (forward 5'-AGCACGCTCTGGTGCAACTG-3' (SEQ ID NO:18), positions 1361-1380; reverse 5'-GCCGCGGCGTAGTATTCAG-3' (SEQ ID NO:19), positions 9420-9438). The resulting 8078 nucleotide RT-PCR product was cloned and sequenced.
[0129] The extreme 3' end of the genome of P129A was determined by ligating the 3' and 5' ends of the viral RNA together and using RT-PCR to amplify the junction fragment. The resulting junction fragments were cloned and sequenced. Briefly, RNA extracted from pelleted virions was treated with tobacco acid pyrophosphatase (to remove 5' cap structures), then self-ligated with T4 RNA ligase (both from Epicentre Technologies). The primers used were 5-CGCGTCACAGCATCACCCTCAG-3' (SEQ ID NO:20) (forward, positions 15,218-15,239) and either 5'-CGGTAGGTTGGTTAACACATGAGTT-3' (SEQ ID NO:21) (reverse, positions 656-660) or 5'-TGGCTCTTCGGGCCTATAAAATA-3' (SEQ ID NO:22) (reverse, positions 337-359). All of the resulting clones were truncated at the 5' end of the genome (the most complete came to within 57 nucleotides of the actual 5' end, as revealed by 5' RACE), however two of these clones contained the complete 3' end of the genome including the polyadenosine tail (42 and 55 adenosine residues in length). This completed the sequencing of the cDNA 15,450 base genome of PRRS isolate P129A, including polyA tail, as shown in SEQ ID NO:1.
[0130] Creation of an infectious full-length cDNA clone of P129A: A full-length infectious cDNA clone of P129A, designated pT7P129A, was assembled from four overlapping cloned RT-PCR products. The four RT-PCR products were first T/A cloned into plasmid pCR2.1 (Invitrogen) and transfected into Escherichia coli strain DH5-alpha. Bacterial colonies were screened, and those which contained inserts of the expected sizes in the "T7 to M13" orientation were chosen for sequencing and further manipulation. All four cloned RT-PCR products contained one or more non-silent mutations (deviations from the consensus nucleotide sequence for P129A of SEQ ID NO:1 which would result in a change in amino acid sequence of the encoded OAFs). These non-silent mutations (save one at position 12,622 in ORF2) were repaired by subcloning fragments from other cloned RT-PCR products. The four repaired subgenomic clones were assembled into a full-length clone in a stepwise manner, using available restriction sites (see FIG. 1). The 5' and 3' ends of the cDNA corresponding to the P129A genome in PT7P129A were modified by the addition of a T7 promoter and appropriate restriction endonuclease sites. The construction of pT7P129A is described in further detail in the following paragraphs:
[0131] The 5' end of the genome (positions 1-1756), generated by 5'-RACE and cloned into pCR2.1 as described above, was modified to include a T7 promoter immediately upstream of the cDNA corresponding to the P129A genome and a Pac site for future cloning. A 3-way ligation was performed using the 1216 bp DsaI-BseRI fragment of this plasmid (containing bases 27-1242 of P129A), the 4407 bp BseRI-XbaI fragment of the same plasmid (containing bases 1243-1756 of P129A and the entire plasmid vector up to the XbaI site), and the following synthetic double-stranded adapter (SEQ ID NO: 23, first below and SEQ ID NO: 24, second below):
TABLE-US-00002 5'-CTAGATTAATTAATACGACTCACTATAGGGATGACGTATAGGTGTTGGCTCTATGC-3' 3'-TAATTAATTATGCTGAGTGATATCCCTACTGCATATCCACAACCGAGATACGGTGC-5' XbaI T7 promoter DsaI PacI P129A genome
[0132] The predicted transcript from the T7 promoter includes a single "G" residue from the promoter immediately upstream of the first "A" of the viral genome. A non-silent mutation at position 1230 (A to G) was repaired by replacing the 906 bp AaflI-SacII fragment (bases 740-1645) with the same fragment from another clone. This plasmid was designated "pT7R3A-2".
[0133] The 8078 nucleotide PGA product described above was used to cover bases 1361-9438 of the P129A genome. A 58 bp deletion (positions 2535-2592) and 7 non-silent point mutations were corrected by subcloning fragments from other cloned RT-PCR products, yielding plasmid "pGRP10B-4". The 7837 bp Bsu36I-SpeI fragment from this plasmid was ligated to the 5482 bp Bsu36I-SpeI fragment from pT7R3A-2. The resulting plasmid "pT7RG" contains the first 9438 bases of the P129A genome behind the T7 promoter.
[0134] The 2860 nucleotide fragment of ORF1b described above (genome positions 9201-12,050) was corrected to repair non-silent mutations and designated "p1B3A-2". The 2682 bp NdeI-SpeI fragment of this plasmid was ligated to the 13,249 bp NdeI-SpeI fragment of pT7RG to yield plasmid "pT71A1B", which contains the first 12,050 bases of the P129A genome.
[0135] The fourth and final fragment of the P129A genome was derived by RT-PCR of ORFs 2 through 7, including the 3' non-translated region and a portion of the polyA tail. The forward primer was 5'-ACTCAGTCTAAGTGCTGGAAAGTTATG-3' (SEQ ID NO:25) (positions 11,504-11,530) and the reverse primer was 5'-GGGATTTAAATATGCATTTTTTTTTTTTTTT TTTTTTAATTGCGGCCGCATGGTTCTCG-3' (SEQ ID NO:26). The reverse primer contains the last 22 bases of the P129A genome (positions 15,374-15,395), a polyA tail of 21 bases, an NsiI site (ATGCAT) and a SwaI site (ATTTAAAT). Non-silent point mutations and a single base deletion were repaired by subcloning fragments from other clones. An additional non-silient point mutation at position 12,622 (A to G) was inadvertently introduced at this stage. This results in a change from glutamine to arginine near the C-terminus of the ORF2 protein (amino acid residue 189 of the 256 amino acids in ORF2, which does not affect the overlapping OAF 3). This mutation had no apparent influence on viral growth, in cell culture or in pigs, and was not repaired. This mutation served as a genetic marker to distinguish virus derived from the cDNA clone from possible contamination with parental P129A or other PRRS viruses. The plasmid was designated "p2--7D-4". The structural genes of P129A were added to the rest of the genome by ligating the 3678 bp Eco47III-SpeI fragment of p2--7D-4 to the 15,635 bp ECo47III-SpeI fragment of pT71A1B.
[0136] This yields the final construct "pT7P129A", which comprises cDNA corresponding almost identically to the entire genome of P129A (however, with only a 21 base polyA tail, as opposed to 55 base polyA tail) behind a T7 promoter, cloned into the pCA2.1 vector between unique restriction enzyme sites (PacI and SwaI). The total length of pTP7129A is 19,313 bp, and it is stable in E. coli strain DH5-alpha. pT7P129A contains an A to G non-silent point mutation at position 12,622 that results in an arginine at position 189 of ORF2 rather than a glutamine (as is encoded by SEQ ID NO:1) and a silent C to T mutation at position1559. Neither of these mutations affected viral growth under the conditions examined, both in cell culture and in pigs. For example, pT7P129A was used for in vitro transcription and the resulting RNA transcripts produced live North American PRRS virus when transfected into MARC-145 cells, thus demonstrating that this full-length clone is infectious.
[0137] In vitro transcription and transfection of RNA transcripts: In plasmid pT7P129A there are two T7 promoters in tandem upstream of the viral genome. One of these is positioned immediately upstream of the viral genome and was built into the PCR primer as described above. The other is present in the pCR2.1 cloning vector and is located outside of the multiple cloning site (initiating transcription 44 bases upstream of the viral genome). PacI was used to cut between these T7 promoters prior to in vitro transcription to generate a transcript that is closer to authentic viral RNA (a single extra G immediately upstream the viral genome, as opposed to 44 extra bases from the distal T7 promoter). In addition, pT7P129A was cut with SwaI prior to in vitro transcription. The resulting run-off transcripts include a 21 base long polyA tail and nine non-PRRS nucleotides, including an NsiI site (which was not used to linearize the plasmid, since the site also occurs once in the viral genome). The digested plasmid was purified by phenol extraction and ethanol precipitation prior to use.
[0138] A commercial kit (T7 Cap-Scribe, Boehringer Mannheim) was used for in vitro transcription. The DNA pellet from above, containing about 0.6 μg of PacI/SwaI digested pT7P129A, was resuspended in 20 μl of T7 Cap-Scribe buffer/T7 polymerase and incubated at 37° C. for 30 minutes. A portion of the reaction was analyzed by agarose gel electrophoresis and shown to contain full-length RNA transcripts in addition to the expected DNA bands of 15,445 bp and 3868 bp. The in vitro transcription reaction was used fresh, immediately following incubation, without purification. Freshly confluent monolayers of MARC-145 cells were washed once in OptiMEM (without serum), and covered with 1 ml per 35 mm well of OptiMEM (without serum) containing 500 μg/ml DEAE dextran (molecular weight approx. 500,000, Pharmacia Biotech). In vitro transcription reaction (15 μl) was added immediately. After 1 hour at 37° C., the transfection mixture was removed, monolayers were washed once with PBS and overlaid with 1.25% SeaPlaque agarose (FMC corporation) in OptiMEM with 2% fetal bovine serum and antibiotics. After 5 days at 37° C., a single plaque was visible. This virus was designated "rP129A-1" and was expanded on MARC-145 cells and characterized in cell culture and in pigs. Subsequent transfections of in vitro transcribed RNA from pT7P129A, using both DEAE dextran and electroporation, have yielded many additional plaques.
[0139] Characterization of recombinant virus rP129A-1: There are no apparent differences in growth kinetics, yield, or plaque morphology between cDNA-derived recombinant virus rP129A-1 and its non-recombinant parent P129A. As discussed above, there are two differences in nucleotide sequence between the coding sequence of pT7P129A and the consensus sequence of P129A (shown in SEQ ID NO:1). Firstly, at position 1559 pT7P129A contains a T, whereas P129A contains a C (this is a silent mutation). Secondly, at position 12,622 pT7P129A contains a G, whereas P129A contains an A (this is the glutamine to arginine change in ORF2 described above). In order to rule out the possibility that rP129A-1 actually a non-recombinant PRRS virus contaminant, RT-PCR and sequencing were performed on the regions surrounding these two differences. In the case of both genetic markers, rP129A-1 was identical to plasmid pT7P129A and different from parental virus P129A, thus confirming that rP129A-1 is derived from the infectious cDNA clone.
[0140] Characterization of recombinant virus rP129A-1 in pigs: The cDNA-derived virus rP129A-1 was compared to its non-recombinant parent P129A for its ability to infect and cause clinical disease in young pigs. Three groups of 10 pigs each from a PRRS-negative herd were infected at 4 weeks of age with either P129A, rP129A-1, or mock-infected with cell culture medium. Clinical signs, rectal temperatures, and body weights were monitored. Blood was collected on days 0, 2, 6, 10, and 13 post-infection for determination of serum viremia (by plaque assay on MARC-145 cells, FIG. 2) and serum antibody (by ELISA using HerdChek PRRS from IDEXX, FIG. 3). Gross and microscopic lesions of the lung were observed upon necropsy. There were no significant differences between the two virus-infected groups, indicating that rP129A-1 replicates in pigs and causes clinical disease which is quantitatively and qualitatively similar to its non-recombinant parent virus.
Example II
Deletion of ORF7 (Nucleocapsid Gene) from the North American PRRS Virus; Preparation of a Negatively-Marked, Replication-Defective Vaccine Thereby
[0141] The viral nucleocapsid gene (ORF7) was partially deleted from an infectious cDNA clone of the PRRS virus of the present invention. The resulting recombinant modified PRRS virus would be expected to be replication-defective in pigs. This recombinant modified PRRS virus can be used as a vaccine to induce an immune response to the other PRRS virus proteins without the risks of clinical disease, spread to non-vaccinated animals, or reversion to virulence associated with attenuated live vaccines. In addition to being very safe, such a vaccine virus would also be "negatively marked", in the sense that it would allow exposure to field isolates of PRRS virus to be determined serologically, even in the presence of antibody to the vaccine virus. Antibodies to the ORF7 protein are commonly found in the sera of PRRS virus-infected pigs, whereas pigs vaccinated with an ORF7-deleted PRRSV would lack antibodies to the ORF7 protein.
[0142] Deletion of ORF7 from an infectious clone was accomplished as follows: Plasmid p2--7D-4 (see FIG. 1) was used as template in PCR to amplify the 5' and 3' flanking regions upstream and downstream of ORF7. The upstream flank forward primer 5'-ATTAGATCTTGC CACCATGGTGGGGAAATGCTTGAC-3' (SEQ ID NO:27) (which binds to genome positions 13,776-13,791 near the beginning of OAF5 and contains additional restriction sites which are irrelevant to the current cloning) and the upstream flank reverse primer 5'-CTTTACGCGTTTG CTTAAGTTATTTGGCGTATTTGACAAGGTTTAC-3' (SEQ ID NO:28) (which binds to genome positions 14,857-14,902 at the junction of ORFs 6 and 7) amplified a fragment of 1147 bp. The reverse primer introduced MluI and AflII sites and a single base change at position 14,874, destroying the ATG start codon for ORF7 without altering the tyrosine encoded in the overlapping ORF6. For the downstream flank, the forward primer 5'-CAACAC GCGTCAGCAAAAGAAAAAGAAGGGG-3' (SEQ ID NO:29) (positions 14,884-14,914 near the 5' end of ORF7, introduced an MluI site) and reverse primer 5'-GCGCGTTGGCCGATTC ATTA-3' (SEQ ID NO:30) (downstream of the viral genome in the pCR2.1 plasmid) amplified a 462 bp fragment. A 3-way ligation was performed, using the 611 bp BsfEII-MluI fragment of the upstream flank PCR product, the 575 by MluI-SpeI fragment of the downstream flank PCR product, and the 6653 by BstEII-SpeI fragment from plasmid p2--7D-4 (all fragments were gel purified following digestion). The resulting plasmid p2--7Ddelta7+7023 was deleted in the first seven amino acids of ORF7, and lacks a functional ATG start codon. Two new restriction sites which are absent in both the viral genome and the plasmid backbone, AflII and MluI, have been inserted to facilitate directional cloning of foreign genes into the space previously occupied by the 5' end of ORF7.
[0143] The changes made in p2--7Ddelta7+7023 were incorporated into a full-length genomic clone by ligating the 3683 bp Eco47III-SpeI fragment of p2--7Ddelta7+7023 with the 15,214 bp Eco47III-SpeI fragment of pCMV-S-p129. The resulting plasmid pCMV-S-p129delta7+7023 was used to transfect cells.
[0144] Since nucleocapsid is essential for viral growth, it is necessary to provide this protein in order to allow generation and replication of an ORF7-deficient PRRS virus. This can be accomplished using a helper virus or a complementing cell line, for example. ORF7-expressing MARC-145 cell lines can be created by stably transfecting cells with a plasmid containing both the ORF7 gene from P129A and the neomycin resistance gene. After selecting for neomycin resistance using the antibiotic G418, single-cell colonies can then be expanded and characterized. Clonal MARC-145-derived cell lines that are positive for ORF7 expression by both immunofluorescence and RT-PCR can be transfected with RNA from pT7P129delta7 in order to generate ORF7-deficient P129 virus.
[0145] Similar strategies can be used to generate PRRS viruses deficient in other structural genes (ORFs 2, 3, 4, 5, or 6), or deficient in all or portions of non-structural genes 1a and 1b. In addition, multiple deletions can be engineered into a single PRRS virus, and these can be grown in complementing cells which provide all necessary functions. Such gene-deficient PRRS viruses are likely to be either partially or completely attenuated in pigs, making them useful as vaccines against PRRS. They can also be used to distinguish vaccinated animals from animals infected with a wild-type PRRS virus as discussed above and/or as vectors for vaccinating animals with epitopes of other porcine pathogens (see Example III, below).
Example III
Insertion of Heterologous Genes into the North American PRRS Virus Genome; use of PRRS Virus as a Vector, and a Positively-Marked North American PRRS Virus
[0146] In Example II, above, AflII and MluI restriction enzyme sites were inserted into the region formerly occupied by the 5' end of ORF7. These sites are absent in the P129A genome and in the pCR2.1 and pCMV plasmids, and can be used in the directional cloning of foreign (heterologous) genes into the viral genome for expression. Potential leader-junction sites for transcription of the ORF7 subgenomic RNA at positions 14,744-14,749 (ATAACC) and 14358-14,863 (TAAACC) are not affected by deletion of the ORF7 coding sequence, and can function in transcription of a foreign gene. Foreign (heterologous) genes can include genes from other PRRS virus isolates or genotypes, and/or genes from other non-PRRS pathogens, either pathogens that infect swine or pathogens that infect mammals other than swine, or avians.
[0147] In addition, these foreign genes (or portions thereof) can provide antigenic epitopes which are not normally found in swine. Such epitopes can be used to "positively mark" a vaccine, so that successful vaccination can be monitored serologically, even in the presence of antibody to field or conventional vaccine strains of PRRS virus. A positive marker needs not be a separate expression cassette. An antigenic epitope can be fused to a structural gene of the PRRS virus. For example, the upstream flank reverse primer described in Example I, above, can be extended in such a way as to add a carboxyl-terminal fusion of a non-PRRS virus antigenic epitope to the ORF6 membrane protein. The presence of antibody to this epitope in swine indicates successful vaccination.
Example IV
Cellular Expression of a PRRS Virus by Direct Transfection of cDNA into Cells
[0148] The eukaryotic expression vector pCMV-MC1 (SEQ ID NO:31) was derived from the commercially available plasmid pCMVbeta (Clontech) by replacing the LacZ coding sequence between two Not I sites with a linker containing Not I, EroR V, Avr II, Bgl II, Cla I, Kpn I, Pac I, Nhe I, Swa I, Sma I, Spe I and Not I sites. Modification of the human CMV immediate early promoter was accomplished by substituting the sequence between the Sac I and the second Not I sites of pCMV-MC1 with a synthetic linker (shown below). The linker contains a half site for Sac I following by Pac I, Spe I and a half site for Not I. After annealing the two single stranded oligonucletides, the linker was cloned into pCMV-MC1 between the Sac I and Not I sites, and a selected clone was designated pCMV-S1. The Spe I site of pCMV-S1 could not be cut, possibly due to a mistake in the oligo sequence. Therefore, the fragment between Pac I and Hind III in pCMV-S1 was replaced with Pac I (at position 877)--Hind III (at position 1162) fragment from pCMV-MC1. Thus, a Spa I site was regained. This final construct (pCMV-S) was used to clone the full length P129 genome.
[0149] Linker sequence (SEQ ID NO 32, first below and SEQ ID NO: 33, second below):
TABLE-US-00003 5' CGTTATTAAACCGACTAGTGC 3' 3' TCGAGCAATTAATTTGGCTGATCACGCCGG 5' Pac I Spe I Sac I Not I
[0150] The sequence immediately upstream of the 5' end of the P129 genome was modified to contain proper spacing and a convenient restriction enzyme site (Pac I). This was done by designing appropriate PCR primers (SEQ ID NO:34 and SEQ ID NO:35) for amplification from pT7P129. After digestion with Pac I and Aat II, this PCR fragment was subcloned into the Pac I and Aat II sites of pT7RG (FIG. 1). The resulting plasmid was designated pT7AG-deltaT7.
[0151] The final construction was completed by subcloning the viral sequences from pT7RG-deltaT7 at the Pac I and Nde I sites into pT7P129, creating pT7P129-deltaT7. The full length P129 genome was digested from pT7P129-deltaT7 at Pac I and Spe I and transferred into pCMV-S at the Pac I and Spe I sites. This constructed was named pCMV-S-P129.
[0152] The sequence of the region of modification between the CMV promoter TATA box and the 5' end of the P129 sequence in pCMV-S-P129 is shown in SEQ ID NO:36 and schematically presented below:
TABLE-US-00004 TATATAAGCAGAGCTCGTTAATTAAACCGTCATGACGTATAGGTGTTGGC 5' TATA box Sac I Pac I Start of P129 3'
[0153] To test the use of the CMV promoter to initiate PRRS virus infection in cells, pCMV-S-P129 plasmid DNA (0.5 or 1.0 μg) was transfected into MARC-145 cells by lipofection using Lipofectamine® (Life Technologies Inc.). PRRS virus specific cytopathic effect was observed after transfection and the presence of PRRS virus antigen was determined by the immunofluorescent antibody test.
[0154] PRRS virus was generated efficiently from pCMV-S-P129, and the progeny virus can be passaged on MARC-145 cells. This demonstrates that a PRRS virus infection can be initiated directly from a plasmid cDNA encoding a PRRS virus, without an in vitro transcription step. Furthermore, pCMV-S-P129 generated a greater amount of progeny virus compared to plasmids wherein the 3' end of the pCMV promoter was not immediately in front of the start of the sequence encoding the North American PRRS virus.
Example V
Deletion of ORF4 from the North American PRRS Virus; Preparation of a Replication-Defective Vaccine Thereby
[0155] A portion of the gene for ORF4, which encodes a membrane glycoprotein, was deleted from an infectious cDNA clone of the PRRS virus of the present invention. The resulting recombinant modified PRRS virus is expected to be replication-defective in pigs and to induce an immune response to the other PRRS virus proteins without the risks of clinical disease, spread to non-vaccinated animals, or reversion to virulence associated with attenuated live vaccines.
[0156] Deletion of ORF4 from an infectious clone was accomplished as follows. Plasmid p2--7D-4 (see FIG. 1) was used as template in PCR to amplify the 5' and 3' flanking regions upstream and downstream of ORF4. The upstream flank forward primer was V-AGGTCGAC GGCGGCAATTGGTTTCACCTAGAGTGGCTGCGTCCCTTCT-3' (SEQ ID NO:37). This primer binds to genome positions 13194-13241, near the beginning of ORF4, and introduces a mutation at position 13225 which destroys the ATG start codon of ORF4 without altering the overlapping amino acid sequence of ORF3. The upstream flank reverse primer was 5'-TCTTAAGCATTGGCTGTGATGGTGATATAC-3' (SEQ ID NO:38). This primer binds to genome positions 13455-13477 within the ORF4 coding region, downstream of ORF3, and introduces an AflII site. For the downstream flanking region, the forward primer was 5'-CTTC TTAAGTCCACGCGTTTTCTTCTTGCCTTTTCTATGCTTCT-3' (SEQ ID NO:39). This primer binds to genome positions 13520-13545 in the middle of ORF4, and introduces AflII and MluI sites for directional cloning of foreign genes. The reverse primer was 5'-TGCCCGGTCCCTT GCCTCT3' (SE0 ID NO:0). This primer binds to genome positions 14981-14999 in the ORF7 coding sequence. A three-way ligation was performed using the SalI-AflII fragment of the upstream flank PCR product, the AflII-BstEII fragment of the downstream flank PCR product, and the SalI-BstEII fragment from plasmid p2--7D-4. All fragments were gel-purified following digestion. The resulting plasmid p2--7D-4delta4N has 42 bases of the central portion of ORF4 deleted and replaced with a 15 base artificial cloning site. The cloning site contains two restriction sites (AflII and MluI) that are absent from both the viral genome and the plasmid backbone. These can be used to facilitate directional cloning of foreign genes into the space previously occupied by ORF4. The cloning site also contains a stop codon (TAA) that is in frame with ORF4 and further assures that functional ORF4 protein is not produced.
[0157] It was found that a more extensive deletion of the ORF4 coding sequence can be made without interfering with expression of the downstream ORF5 envelope gene. In this case a shorter downstream flanking region was amplified by PCR using the same template and reverse primer, and using forward primer 5'-GTTTACGCGTCGCTCCTTGGTGGTCG-3' (SEQ ID NO:41). This primer binds to genome positions 13654-13669 near the 3' end of ORF4, and contains an AflII site. Two-way ligation between the AflII-BstEII fragment of the downstream flank PCR product and the AflII-BstEII fragment from plasmid p2--7D-4delta4N yielded the new plasmid p2--7D-4delta4NS. This plasmid has 176 bases of the ORF4 coding sequence deleted and replaced with the 15 base cloning site.
[0158] The changes made in p2--7D-4delta4N and p2--7Ddelta4NS were Incorporated into the full-length genomic clone by replacing the BsrGI-SpeI fragment from pCMV-S-P129 with the modified BsrGI-SpeI fragments from p2--7D-4delta4N and p2--7D-4delta4NS. The resulting plasmids pCMV-S-P129delta4N and pCMV-S-P129delta4NS were used to transfect cells.
[0159] In contrast to pCMV-S-P129, transfection of MARC-145 cells with plasmids pCMV-S-P129delta4N or pCMV-S-P129delta4NS did not result in viral plaques or fluorescent foci. Individual transfected cells can be seen to be producing the ORF7 nucleocapsid protein, suggesting that the ORF4 gene product is not required for RNA replication or expression of viral genes, but is essential for release of infectious progeny virus. Since deletion of ORF4 is lethal to virus replication, it is necessary to provide this protein. This can be accomplished by using a complementing cell line. We created ORF4-expressing MARC-145 cell lines by stable transfecting cells with a plasmid containing both ORF4 and the neomycin resistance gene. After selection for neomycin resistance using the antibiotic G418, single-cell colonies were expanded and characterized. After transfection with pCMV-S-P129delta4NS, three ORF4-expressing cell clones yielded live virus that can be propagated in these cells but not in MARC-145 cells. One of these, MARC400E9, was further characterized. Immunofluorescent staining for viral nucleocapsid in MARC400E9 cells transfected with plasmid pCMV-S-P129delta4NS was positive.
[0160] Virus derived by transfecting MARC400E9 cells with the pCMV-S-P129delta4NS plasmid (designated P129delta4NS) was amplified by passaging several times on MARC400E9 cells and was used to vaccinate pigs against a virulent PRRSV challenge. Live virus was formulated with one of three different types of adjuvant or used without adjuvant, and was delivered in two doses by one of two vaccination routes (intranasal followed by intramuscular, or intramuscular followed by a second intramuscular dose).
[0161] At approximately three weeks (Day 0) and five weeks (Day 14) of age, seronegative conventional pigs were vaccinated according to the Table 2, below. The pigs in Group J were considered sentinel pigs and were necropsied approximately one week prior to challenge to evaluate the health status of all pigs.
TABLE-US-00005 TABLE 2 Test groups selected for vaccination derived from P129delta4NS Group Vaccine N Route* Adjuvant A P129delta4NS 10 IN/IM None B P129delta4NS 10 IM/IM None C P129delta4NS 10 IN/IM rmLT** D P129delta4NS 10 IM/IM rmLT E P129delta4NS 10 IN/IM QuilA/Cholesterol F P129delta4NS 10 IM/IM QuilA/Cholesterol G P129delta4NS 10 IN/IM Amphigen H P129delta4NS 10 IM/IM Amphigen I PBS only 10 IM/IM None J None 10 NA NA *IN--intranasal; IM--intramuscular. **recombinant mutant heat-labile enterotoxin from E. coli.
[0162] The first and second intramuscular vaccinations were administered in the left and right sides of the neck, respectively. On Days 0, 13, 21 and 28, blood samples were collected for serology (IDEXX HerdChek PRRS ELISA). At approximately three weeks following the second vaccination (Day 35), all pigs were challenged with 1 mL of parental P129 virus instilled drop-wise in each nostril (a total of 2 mL/pig or 1.2×105 pfu). On Days 35, 38, 42 and 45, blood samples were collected from all pigs for serology and for PRRSV titraton (by TCID50 assay). On Days 45 and 46, pigs were euthanized and necropsied. Lungs were removed from the chest cavity, examined for PRRS lesions, and photographed. Necropsies were performed at the Animal Disease Research and Diagnostic Laboratory (ADRDL) at South Dakota State University, Brookings, S. Dak.
[0163] The serological results showed induction of anti-PRRSV antibody in several of the vaccinated groups prior to challenge, and priming of the humoral immune system for a rapid post-challenge antibody response in all vaccinated groups. Immediately before challenge (Day 35), the average S/P ratio (Sample/Positive control ratio) value in the unvaccinated controls (group I) was 0.003. All eight of the vaccinated groups had higher values, ranging from 0.005 (group B) up to 0.512 (group H). By one week after challenge (Day 42), the control group was beginning to mount an antibody response to the challenge virus (S/P value of 0.171). All eight vaccinated groups had much higher antibody levels, ranging from 0.858 (group 8) to 1.191 (group 6). This represents an increase in anti-PRRSV antibodies of approximately 5- to 10-fold as a result of priming of the humoral immune system.
TABLE-US-00006 TABLE 3 Anti-PRRSV antibody levels (S/P ratios) Day-1 Day-13 Day-21 Day-28 Day-35 Day-38 Day-42 Day-45 Group A None/IN 0.003 0.008 0.004 0.002 0.015 0.102 0.862 2.270 Group B None/IM 0.005 0.015 0.024 0.022 0.005 0.105 0.858 2.053 Group C rmLT/IN 0.002 0.030 0.015 0.021 0.016 0.108 1.219 2.653 Group D rmLT/IM 0.002 0.042 0.143 0.149 0.138 0.229 1.271 2.679 Group E QuilA/IN 0.015 0.039 0.084 0.048 0.013 0.097 1.217 2.299 Group F QuilA/IM 0.001 0.020 0.104 0.115 0.085 0.109 1.240 2.593 Group G Amph/IN 0.002 0.001 0.047 0.051 0.061 0.080 1.919 3.484 Group H Amph/IM 0.022 0.006 0.460 0.531 0.512 0.492 1.601 3.371 Group I PBS only 0.008 0.020 0.001 0.001 0.003 0.053 0.171 0.881
[0164] Following challenge with virulent PRRSV, serum viremia was determined. At three days post challenge (Day 38) the titer of virus in the unvaccinated control group had reached 2.58 logs. All eight of the vaccinated groups showed reduced viremia, ranging from 2.52 (group E) down to 1.07 logs (group H). This represents more than a 30-fold reduction in virus levels relative to the control group, see Table 4.
TABLE-US-00007 TABLE 4 Serum viremia (log 10 TCID50 titers) on day 38 (three days post challenge) Group A None/IN 2.18 Group B None/IM 1.98 Group C rmLT/IN 2.15 Group D rmLT/IM 2.48 Group E QuilA/IN 2.52 Group F QuilA/IM 1.65 Group G Amph/IN 1.74 Group H Amph/IM 1.07 Group I PBS only 2.58
[0165] At necropsy, all pigs were examined for the presence of macroscopic lung lesions resulting from the PRRS challenge virus. In the unvaccinated control group, 90% of the animals showed lung lesions. In contrast, all eight of the vaccinated groups showed reduced levels of lung lesions (ranging from 80% in group D down to 22% in group A). Vaccination reduces damage to the lungs that result from PRRSV infection. See Table 5.
TABLE-US-00008 TABLE 5 Number of pigs with macroscopic lung lesions/total pigs (percent positive) Group A None/IN 2/9 (22%) Group B None/IM 7/10 (70%) Group C rmLT/IN 6/10 (60%) Group D rmLT/IM 8/10 (80%) Group E QuilA/IN 5/10 (50%) Group F QuilA/IM 4/10 (40%) Group G Amph/IN 7/10 (70%) Group H Amph/IM 5/9 (56%) Group I PBS only 9/10 (90%)
[0166] The above swine data demonstrates the process of vaccinating swine with the recombinant North American PRRS virion containing the ORF4 deletion. The results of the above data illustrate that the P129delta4NS virus, which is deleted in ORF4, is useful as a vaccine to protect pigs from disease caused by the PRRS virus. Efficacy of the vaccine may be increased by formulation with various adjuvants and by changing the route of administration.
Example VI
Use of Gene-Deleted PRRS Virus as a Vector for Expression of Foreign Genes
[0167] In order to determine whether heterologous genes can be expressed from a gene-deleted PRRS virus, we inserted a copy of green fluorescent protein (GFP) into the site of ORF4 deletion in plasmid pCMV-S-P129delta4N. The GFP gene was amplified from a commercially available plasmid vector using PCR primers that introduced an AflI site at the 5' end of the gene and an MluI site at the 3' end of the gene. The resulting plasmid pCMV-S-P129delta4N-GFP was used to transfect MARC-145 and MARC400E9 cells. As anticipated, MARC-145 cells did not support replication of the ORF4-deleted virus. Single green cells resulting from primary tranfection were seen under the UV scope, indication that GFP was being expressed, but the virus did not spread to neighboring cells and no CPE was observed. In contrast, large green foci were observed in transfected MARC400E9 cells. Visible plaques formed and merged, destroying the monolayer. These results indicate that foreign genes can be expressed from ORF4-deleted PRRS virus, and that increasing the size of the viral genome by 692 bases (4.5%) does not interfere with packaging of the viral RNA into infectious particles.
Example VII
Use of Replication-Competent PRRS Virus as a Vector for Expression of Foreign Genes
[0168] In order to determine whether heterologous genes can be expressed from a replication-competent PRRS virus, we inserted a copy of GFP into the region between ORF1b and ORF2. Since the leader/junction (L/J) sequence for ORF2 lies within ORF1b, this L/J sequence was used to drive expression of the GFP gene and copy of the ORF6 L/J sequence was inserted downstream from GFP to drive expression of ORF2.
[0169] Plasmid p2--7D-4 (see FIG. 1) was used as template in PCR to amplify the 5' and 3' flanking regions upstream and downstream of the insertion site. The upstream flank forward primer was 5'-AACAGAAGAGTTGTCGGGTCCAC-3' (SEQ ID NO:42). This primer binds to genome positions 11699-11721 in OAF1b. The upstream flank reverse primer was 5'-GCTTT GACGCGTCCCCACTTAAGTTCAATTCAGGCCTAAAGTTGGTTCA-3' (SEQ ID NO:43). This primer binds to genome positions 12031-12055 in ORF1b and adds AflII and MluI sites for directional cloning of foreign genes between ORF1b and ORF2. The downstream flank forward primer was 5'-GCGACGCGTGTTCCGTGGCAACCCCTTTAACCAGAGTTTCAGCGG AACAATGAAATGGGGTCTATACAAAGCCTCTTCGACA-3' (SEQ ID NO:44). This primer binds to genome positions 12056-12089 in ORF2 and contains an MluI site followed by the 40 bases that precede the start of ORF6 (containing the ORF6 L/J sequence). The downstream flank reverse primer was 5'-AACAGAACGGCACGATACACCACAAA-3' (SEQ ID NO:45). This primer binds to genome positions 13819-13844 in OAF5. A three-way ligation was performed using the Eco47III-MluI fragment of the upstream flank PCR product, the MluI BsrGI fragment from the downstream flank PCR product, and the Eco47III-BsrGI fragment from pCMV-S-P129. The resulting plasmid pCMV-S-P129-1bMCS2 contains the entire P129 genome with a cloning site and an additional L/J site between ORF1b and ORF2. The plasmid produces functionally normal virus when transfected into MARC-145 cells.
[0170] The GFP gene from a commercially available plasmid was PCR amplified using primers that add an AflII site to the 5' end and an MluI site to the 3 end of the gene. After digestion of the PCR fragment and PCMV-5P129-1bMCS2 with AflII and MluI, the insert was ligated into the vector to yield plasmid pCMV-S-P129-1bGFP2. This plasmid produced green plaques when transfected into MARC-145 cells. The resulting virus can be passaged onto MARC-145 cells and continued to produce green plaques when observed under the UV scope. Thus, foreign genes may be expressed from replication-comptetent PRRS virus vectors. The P129-1bGFP2 virus contains a genome which is 774 bases (5%) longer than that of its P129 parent, yet it is packaged normally.
Example VIII
Deletion of OAF2 (Minor Structural Protein) from the North American PRRS Virus; Preparation of a Replication-Defective Vaccines
[0171] The following example illustrates the preparation of the North American PRRS virions containing the OAF2 deletion.
[0172] The viral minor structural protein encoded by ORF2 is deleted from an infectious cDNA clone of the PRRS virus of the present invention. The resulting recombinant modified PRRS virus is replication-defective in pigs and can induce an immune response to the other PRRS virus proteins without the risks of clinical disease, spread to non-vaccinated animals, or reversion to virulence associated with attenuated live vaccines.
[0173] Deletion of ORF2 from an infectious clone was accomplished as follows. Plasmid p2--7D-4 (see FIG. 1) was used as template in PCR to amplify the 5' and 3' flanking regions upstream and downstream of ORF2. The upstream flank forward primer was 5'-CAAAGGGC GAAAAACCGTCTATCA-3' (SEQ ID NO:46), which binds within the cloning vector. The upstream flank reverse primer was 5'-CCCCACTTAAGTTCAATTCAGGC-3' (SEQ ID NO:47), which binds to genome positions 12045-12067 near the beginning of ORF2. The downstream flank forward primer was 5'-GCTCCTTAAGAACAACGCGTCGCCATTGAAGGCGAGA-3' (SKI ID NO:48), which binds to genome positions 12492-12528. The downstream flank reverse primer was 5'-GCAAGCCTAATAACGAAGCAAATC (SEQ ID NO:49), which binds to genome positions 14131-14154. A 3-way ligation was performed, using the XhoI-AflII fragment of the 5' flanking PCR product, the AflII-BsrG1 fragment of the 3' flanking PCR product, and the large XhoI-BsrGI fragment from plasmid p2--7D-4. All fragments were gel purified following restriction enzyme digestion. The resulting plasmid p2--7D4delta2 was deleted in the region of ORF2 that does not overlap ORF3 or it's 5' transcriptional regulatory sequences. Two restriction sites that are absent in both the viral genome and the plasmid backbone, AflII and MluI, have been introduced to facilitate directional cloning of foreign genes into the space previously occupied by ORF2.
[0174] The changes made in p2--7D4delta2 were incorporated into a full-length genomic clone by replacing the AfeI-BsrGI fragment from pCMV-S-P129 with the modified sequence from p2--7D4delta2. The resulting plasmid pCMV-S-P129delta2 was used for transfections.
[0175] Since the deletion of ORF2 is lethal to virus replication, it is necessary to provide this protein in trans to allow generation of ORF2-deficient PRRS virus. This can be accomplished by using a complementing cell line. We created ORF2-expressing MARC-145 cell lines were created by stably transfecting cells with a plasmid containing both the ORF2 gene from P129A and the neomycin resistance gene. After selecting for neomycin resistance using the antibiotic G418, single-cell colonies were expanded and characterized. After transfecting with pCMV-S-P129delta2, three cell clones yielded live viruses that can be propagated and passed only in these ORF2-expressing cells. This virus was designated P129delta2.
[0176] Recombinant virus P129delta2 was used to vaccinate groups of 20 sero-negative conventional pigs. At three weeks of age (Day 0) and six weeks of age (Day 20), pigs were vaccinated according to Table 6, below. Controls were mock vaccinated with phosphate buffered saline (PBS). Amphigen adjuvant was not viricidal to the live vaccine virus.
TABLE-US-00009 TABLE 6 Various groups of 20 sero-negative conventional pigs vaccinated with P129delta2. Treatment Group Route* Adjuvant Titer/Dose (log10 TCID50) P129delta2 IM IM/IM 5% Amphigen 5.8 P129delta2 IT IT/IT None 5.7 Mock vaccinated IT/IT None NA (PBS) Controls *IM--intramuscular; IT--intratracheal.
[0177] At three weeks following second vaccination (Day 42, 9 weeks of age), pigs were challenged with approximately 2.8 mL of virulent P129 challenge virus (3.41×105 TCID50/mL) in each nostril, to achieve a total challenge volume of 5.6 mL (1.91×106 TCID50). Pigs were bled periodically for determination of anti-PRRSV antibody (IDEXX HerdChek PRRS ELISA) and serum viremia (TCID50 assay).
TABLE-US-00010 TABLE 7 Anti-PRRSV antibody levels (S/P ratios) Day 2 Day 19 Day 28 Day 34 Day 42 Day 52 Day 55 Day 63 P129delta2 IM 0.026 0.132 1.265 1.319 1.389 1.995 2.322 2.02 P129delta2 IT 0.011 0.004 0.026 0.115 0.036 1.306 1.584 1.501 Mock vaccinated 0.001 0.002 0.071 0.003 0.004 0.763 1.362 1.395 (PBS) Controls
[0178] Two vaccinations with live Amphigen-adjuvanted P129delta2 intramuscularly resulted in a marked increase in anti-PRRSV antibody in the serum prior to challenge, as well as an anamnestic response following challenge. Two intratracheal vaccinations with unadjuvanted P129delta2 were less effective, but nevertheless resulted in a slight increase in antibody level prior to challenge and a more rapid response following challenge, relative to the mock-vaccinated control group.
TABLE-US-00011 TABLE 8 Results from two vaccinations with live Amphigen-adjuvanted P129delta2 and control. Serum titer (log 10 TCID50) and number of positive pigs following PRRSV challenge Days post-challenge Treatment 0 5 8 10 14 21 P129delta2 IM Titer 0.0 1.5 0.3 0.0 0.0 0.0 Positive/Total 0/19 13/18 4/18 1/18 0/8 0/8 (%) (0%) (72%) (22%) (6%) (0%) (0%) P129delta2 IT Titer 0.0 2.3 2.1 1.5 0.5 0.2 Positive/Total 0/20 19/20 19/20 17/20 4/10 1/10 (%) (0%) (95%) (95%) (85%) (40%) (10%) Mock vaccinated Titer 0.0 2.4 2.0 1.4 0.3 0.0 (PBS) Controls Positive/Total 0/20 19/19 19/19 16/19 3/9 0/9 (%) (0%) (100%) (100%) (84%) (33%) (0%)
[0179] Intramuscular vaccination with two doses of live Amphigen-adjuvanted P129delta2 virus resulted in a reduction of the number of viremic pigs (from 100% to 72%), reduction in the duration of viremia (from about 21 days to about 14 days), and reduction in the average serum virus load of nearly one log at 5 days post-infection (the peak of viremia) and more than one log at later times. Intratracheal vaccination with unadjuvanted P129delta2 showed little or no reduction in viremia relative to the mock-vaccinated control group.
Example IX
Insertion of Unique MluI and SgrAI Restriction Sites into the PRRS Virus Genome within nsp2
[0180] The QuikChange® II XL Site-Directed Mutagenesis kit (Stratagene; La Jolla, Calif.) was used to introduce a unique MluI restriction enzyme site into the PRRS virus genome at nucleotide position 3,219 using plasmid pCMV-S-P129 as template. Genome position 3,219 lies within the nonstructural protein 2 (nsp2) coding region, which is part of ORF1a (FIG. 5). This site is located within the hypervariable C-terminal portion of nsp2. The sequence of the parental virus beginning at genome position 3,219 (ACACGC) is such that changing only two nucleotides (ACGCGT) allows the introduction of an MluI site without altering the encoded amino acid sequence. The mutagenic primers used to insert the MluI site (MluI F and MluI R) are shown in Table 1. QuikChange II XL reactions were as recommended by the manufacturer and contained 150 picomoles of each primer and 10 ng of plasmid DNA as template. Reaction conditions were as follows: initial denaturation at 95° C. for 60 seconds, 18 cycles of 95° C. denaturation for 50 seconds, 60° C. annealing for 50 seconds, and 68° C. extension for 20 minutes, concluding with a 7 minute incubation at 68° C. Successful introduction of the new site was determined based on the ability of the resulting plasmid to be cut with MluI, and confirmed by DNA sequencing. The new plasmid was named pCMV-S-P129-Nsp2-Mlu, and was used as a backbone for the insertion foreign DNA fragments.
[0181] A unique SgrAI restriction enzyme site was inserted into plasmid pCMV-S-P129-Nsp2-Mlu as follows. A 4500 bp PCR product (genome positions 3201-7700) was amplified using template pCMV-S-P129-Nsp2-Mlu, primers Mlu-F and 7700-R (Table 1), and ExTaq® polymerase (TakeRa Minis Bio; Madison, Wis.) according to the manufacturer's directions. The forward primer Mlu-F contains the unique MluI site added in the previous step, and the reverse primer 7700-R contains a unique PmeI site to be used in a subsequent cloning step. Reactions conditions were: initial denaturation at 95° C. for 120 seconds, 30 cycles of 94° C. denaturation for 35 seconds, 58° C. annealing for 35 seconds, and 72° C. extension for 4.5 minutes. The resulting PCR product was cloned the TOPO PCS XL cloning kit (invitrogen Corp., Carlsbad, Calif.). The QuikChange II XL Site-Directed Mutagenesis Kit was used to introduce a unique SgrAI site beginning at genome position 3614 according to the manufacturer's directions. The original sequence TACCGGTG was changed to CACCGGTG without altering the encoded amino acid sequence, using primers SgrAI-F and SgrAI-R (Table 1). Reactions conditions were: initial denaturation at 95° C. for 60 seconds, 18 cycles of 95° C. denaturation for 50 seconds. 60° C. annealing for 50 seconds, and 68° C. extension for 9 minutes, followed by a 68° C. polishing reaction for 7 minutes. After transformation, plasmids containing the new SgrAI site were selected by restriction digestion and sequence analysis. The 4,456 bp MluI-PmeI fragment from this plasmid was ligated to the 14,436 bp MluI-PmeI fragment of PCMV-S-P129-Nsp2-Mlu to yield pCMV-S-P129-Nsp2-Mlu/SgrA. This plasmid was transfected into MARC-145 cells using Lipofectamine 2000 (Invitrogen Corp.; Carlsbad, Calif.) according to the manufacturers instructions. Three days post-transfection lysates were titrated by limiting dilution. Infectious virus was recovered from plasmid pCMV-S-P129-Nsp2-Mlu/SgrA at levels that were similar to plasmids pCMV-S-P129 and pCMV-S-P129-Nsp2-Mlu.
Example X
Insertion of Green Fluorescent Protein (GFP) into nsp2
[0182] MluI sites were placed on the 5' and 3' ends of GFP, amplified by PCR from plasmid pEGFP-C3 (Clontech, Mountain View, Calif.), using gene-specific PCR primers GFP-Mlu-F and GFP-Mlu-R (Table 1). ExTaq® polymerase (TakeRa Mirus Bio; Madison, Wis.) was used according to the manufacturer's recommendation. Reactions conditions were: initial denaturation at 95° C. for 120 seconds, 30 cycles of 94° C. denaturation for 35 seconds, 56° C. annealing for 35 seconds, and 72° C. extension for 60 seconds. The PCR product was digested with MluI and ligated into MluI-digested pCMV-S-P129-Nsp2-Mlu, DNA sequencing confirmed that GFP had been inserted in-frame, in the forward orientation, into nsp2. The new plasmid was designated pCMV-S-P129-Nsp2-Mlu-GFP.
[0183] Plasmids pCMV-S-P129, pCMV-S-P129-Nsp2-Mlu, and pCMV-S-P129-Nsp2-Mlu-GFP were transfected Into the MARC-145 monkey kidney cell line in order to generate infectious virus. Cell lines were maintained at 37° C., 5% CO2 in modified Eagle's medium (MEM) supplemented with 8% fetal bovine serum, 0.008% Fungizone and 0.01% penicilln/streptomycin. Approximately one day prior to transfection, 2×105 cells/well were seeded into a 12-weil plate. Transfection of infectious cDNA clones was performed using Lipofectamine 2000 (Invitrogen, Carlsbad, Calif.) according to the manufacturer's instructions. At 4 days post-transfection, virus was harvested by subjecting the infected cells to three freeze-thaw cycles. Cell debris was removed by centrifugation at 1000×g for 10 minutes, and virus-containing culture supernatants collected. Recombinant viruses rescued from plasmids pCMV-S-P129, pCMV-S-P129-Nsp2-Mlu, and pCMV-S-P129-Nsp24-Mlu-GFP were designated P129, P129-Nsp2-Mlu, and P129-Nsp2-Mlu-GFP, respectively.
[0184] To determine virus titer, end-point dilutions were performed on MARC-145 cells in 96 well plates. Titrations were performed in triplicate and results presented as TCID50/ml. Cells were fixed at 3 days post-transfection and immunostained either directly with the nucleocapsid-specific monoclonal antibody SDOW-17 conjugated to FITC, or indirectly with the nucleocapsid-specific primary monoclonal antibodies SR30 or JP24 followed by Alexaflour 594-conjugated goat anti-mouse IgG secondary antibody (Molecular Probes/invitrogen Corp,: Carlsbad, Calif.). Titers of P129 and P129-Nsp2-Mlu were similar, while the titer of P129-Nsp2-Mlu-GFP was slightly lower. The P129-Nsp2-Mlu-GFP virus expressed GFP and formed fluorescent green foci. Within the focus of infection, cells with perinuclear green fluorescence are seen, due to expression of the Nsp2-GFP fusion peptide.
[0185] For western blot analysis, cells were lysed with 100 ul of 2× Laemmli sample buffer and boiled for 10 min. Proteins were fractionated on SDS-polyacrylamide gels (Bio-Rad; Hercules, Calif.) under reducing conditions. Proteins were transferred onto nitrocellulose membranes and processed at room temperature. Membranes were blocked for 1 hr with 5% nonfat dry milk in washing solution (0.2% Tween 20 in PBS). Affinity purified goat anti-GFP antibody (Rockland immunochemicals Inc., Gilbertsville, Pa.) was diluted 1:1000 in blocking buffer and incubated with membranes for 1 hr. Membranes were washed three times for 15 minutes with TBS-Tween. Rabbit anti-goat horseradish peroxidase-conjugated antibody (Rockland) was diluted 1:1000 in blocking buffer and incubated with membranes for 1 hr. Membranes were washed three times for 16 minutes with TBS-Tween, Peroxidase activity was detected using the Supersignal West Pico Chemiluminescent substrate (Pierce Chemical Company; Rockford, Ill.). Western blots identified a 70 kDa peptide from P129-Nsp2-Mlu-GFP infected cells that reacted with anti-GFP antibody.
[0186] Using a similar procedure, the GFP gene was also inserted into nsp2 at the SgrAI site in the plasmid pCMV-S-P129-Nsp2-Mlu/SgrA. Primers GFP-SgrA-F and GFP-SgrA-R (Table 1) were used to add SgrAI sites to the ends of the GFP gene, using plasmid pEGFP-C3 as template. ExTac® polymerase (TakeRa Minus Bio; Madison, Wis.) was used according to the manufacturer's recommendation. Reactions conditions were: initial denaturation at 95° C. for 120 seconds, 30 cycles of 94° C. denaturation for 35 seconds, 55° C. annealing for 35 seconds, and 72° C. extension for 35 seconds. The PCR product was digested with SgrAI and ligated into SgrAI-digested pCMV-S-P129-Nsp2-Mlu/SgrA. DNA sequencing confirmed that GFP had been inserted in-frame, in the forward orientation, into nsp2. The new plasmid was designated pCMV-S-P129-Nsp2-SgrA-GFP. This plasmid produced infectious virus and fluorescent green foci when transfected into MARC-145 cells.
TABLE-US-00012 TABLE 9 PCR primers used to introduce the heteroogous genes. Primer (genome SEQ position) Sequence Orientation ID NO MluI-F (3201- CTGTCAAGTGTTAGGATCACGCGTCCAAAATACTCAG Forward 50 3242) GTCAA MluI-R (3201- TTGAGCTGAGTATTTTGGACGCGTGATCCTAACACTT Reverse 51 3242) GACAG GFP-Mlu-F GCCACGCGTGCCACCATGGTGAGCAAGGGCGAG Forward 52 GFP-Mlu-R GCCACGCGTGTTATCTAGATCCGGTGGATCC Reverse 53 7700-R (7661- TCAAGCCGCTGGCGGCTAGCAGTTTAAACACTGCTC Reverse 54 7700) CTTA SgrAI-F (3597- CTCGGGAAAATAGAAAACACCGGTGAGATGATCAACC Forward 55 3637) AGGG SgrAI-R (3597- CCCTGGTTGATCATCTCACCGGTGTTTTCTATTTTCC Reverse 56 3637) CGAG GFP-SgrA-F GCCGCCCACCGGTGAGGTGAGCAAGGGCGAGGAGC Forward 57 TGTTC GFP-SgrA-R GCCGCCCACCGGTGTTGTTATCTAGATCCGGTGGAT Reverse 58 C All sequences are 5' to 3'. Restriction sites are underlined. Nucleotide positions are based on the sequence of PRRSV strain P129.
Example XI
Construction of PRRS Viruses having a Deleted Portion of nsp2
[0187] The previously made plasmid pCMV-S-P129-Mlu/SgrA (see Example IX) was used as the backbone plasmid for construction of recombinant PRRS viruses lacking a 132 amino acid region of nsp2, and with either green fluorescent protein (GFP) or the influenza hemagglutinin (HA) tag inserted into the deleted region.
[0188] The enhanced GFP gene from a commercially available plasmid (pEGFP-C3, Clontech) was PCR amplified using primers that add MluI and SgrAI restriction enzyme recognition sites to the 5' and 3' ends, respectively. The forward primer was GFP-MLU-F2 (5'-GCCACGCGTGTGAGCAAGGGCGAGGAGCTG-3') SEQ ID NO. 59 and the reverse primer was GFP-SgrA-R (5'-GCCCACCGGTGTTGTTATCTAGATCCGGTGGATC-3') SEQ ID NO. 58. PCR reactions used ExTag polymerase (TakeRa Mirus Bio, Madison, Wis.) according to the manufacturer's instructions. Reaction conditions included an initial 120 second denaturation at 95 C, followed by 30 cycles of denaturation at 94 C for 35 seconds, annealing at 55 C for 35 seconds, and extension at 72 C for 35 seconds. After digestion of the PCR fragment and of pCMV-S-P129-Mlu/SgrA with MluI and SgrAI, the insert was ligated into the vector to yield plasmid pCMV-S-P129-Mlu-GFP-SgrA. Deletion of the 132 amino acid region and insertion of GFP (257 amino acids) was confirmed by DNA sequencing. This plasmid produced viable virus, which in turn produced green fluorescent foci of infection when transfected into MARC-145 cells and observed under a fluorescence microscope. This virus was designated P129-Mlu-GFP-SgrA.
[0189] The HA tag is a small peptide epitope (YPYDVPDYA), derived from the influenza hemagglutinin gene, that reacts with commercially available monoclonal antibodies. A synthetic adapter was created by heating and slowly annealing two phosphorylated oligonucleotides, designated "HA tag F" (5'-CGCGTATACCCATACGACGTCCCAGACTACGCA-3') SEQ ID NO. 60 and "HA tag R" (5'-CCGGTGCGTAGTCTGGGACGTCGTATGGGTATA-3') SEQ ID NO. 61. This adapter, with MluI and SgrAI half-sites, was then ligating into pCMV-S-P129-Mlu/SgrA which was previously digested with MluI and SgrAI. The structure of the resulting plasmid, pCMV-S-P129-Mlu-HA-SgrA, was confirmed by DNA sequencing. An in-frame fusion of the HA tag peptide 9-mer replaces the 132 amino acid deletion in nsp2. The plasmid produced viable virus when transfected into MARC-145 cells. This virus was designated P129-Mlu-HA-SgrA.
[0190] In a separate experiment, a simple deletion of the same region (without a simultaneous insertion) was engineered into a different infectious cDNA clone, using a different methodology. The plasmid pCMV-S-P129-PK contains a full-length infectious COMA clone of the P129 isolate of PRRS virus that was isolated from the serum of an infected pig on primary porcine alveolar macrophage cells, and subsequently passaged 16 times on the porcine kidney cell line PK.9. The PK.9 cell line expresses the CD163 PRRS virus receptor, and is fully permissive to PRRSV infection. A precise deletion of 396 nucleotides, which encodes the 132 amino acid region described above, was generated in pCMV-S-P129-PK using the QuikChange II XL Site-Directed Mutagenesis kit (Stratagene; La Jolla, Calif.) and mutagenic primers P129-PK-3199F (5'-CCCTGTCAAGTGTTAAGATCACAGGTGAGATGATCAACC-3') SEQ ID NO. 62 and P129-PK-3637R (5'-CCCTGGTTGATCATCTCACCTGTGATCTTAACACTTGACAG-3') SEQ ID NO. 63. The resulting plasmid pCMV-S-P129-PK-dnsp2 contained a deletion of nsp2 amino acids 628-759 (confirmed by sequencing), and produced viable progeny virus when transfected into PK.9 cells. This virus was designated P129-PK-dnsp2.
Example XII
Development of an ELISA for the Detection of Serum Antibodies to a 132-Amino Acid Portion of nsp2
[0191] The 132 amino acid portion of nsp2 that is deleted in viruses P129-Mlu-GFP-SgrA and P129-Mlu-HA-SgrA is designated P129-nsp2 (628-759). This peptide, RPKYSAQAIIDLGGPCSGHLQREKEACLRIMREACDAAKLSDPATQEWLSRMWDRVDMLTW RNTSAYQAFRTLDGRFGFLPKMILETPPPYPCGFVMLPHTPAPSVSAESDLTIGSVATEDIPRI LGKIENT) (SEQ ID NO. 64) was expressed as a recombinant peptide in Escherichia coli, using the pHUE system (Catanzariti A M, Soboleva T A. Jens D A, Board P G, Baker R T, 2004. An efficient system for high-level expression and easy purification of authentic recombinant proteins. Protein Sci.13:1331-1339). In this expression system, the peptide of interest is cloned and expressed as a histidine-tagged/ubiquitin/PRRS peptide fusion (25 kDa apparent molecular weight on SDS-PAGE). After an initial round of purification by nickel ion affinity chromatography, the histidine-tagged/ubiquitin/PRRS peptide is optionally incubated with histidine-tagged/deubiquinating enzyme to cleave histidine-tagged/ubiquitin from the PRRS peptide. In a second round of nickel ion affinity chromatography, the histidine-tagged/ubiquitin and the histidine-tagged/deubiquiriating enzyme are retained on the nickel ion affinity column and the free PRRS peptide is eluted.
[0192] The ubiquitin-conjugated P129-nsp2 (628-759) peptide from the first nickel affinity chromatography step was coated on ELISA plates (0.1 ug/ml). Plates were blocked with 10% goal serum. Dilutions of sera from infected and non-infected pigs were made in PBS with 10% goat serum, and added to the blocked plates. After incubation and washing, bound porcine antibodies were detected using biotin-labeled goat anti-swine antibody, followed by avidin-peroxidase. Peroxidase activity was detected using a chromagenic substrate.
[0193] Serum from an uninfected control pig was serially diluted from 1:10 to 1:20,480 and applied to the ELISA plates for analysis. Optical density (OD) was below 0.4 in all cases. In contrast, serum from a pig that had previously been infected with the heterologous PRRS virus isolate VR2332 about 200 days earlier reacted in a dilution-dependent manner, giving OD values in excess of 1.2 at 1:10 and 1:20 dilutions, and dropping below an OD of 0.4 only after reaching a 1:640 dilution. The VR2332 virus is quite divergent from the P129 isolate, and the 132 amino acid nsp2 (628-759) peptide from VR2332 (RPKYSAQAIIDSGGPCSGHLQEVKETCLSVMREACDATKLDDPATQEWLSRMWDRVDMLT WRNTSVYQAICTLDGRLKFLPKMILETPPPYPCEFVMMPHTPAPSVGAESDLTIGSVATEDVP RILEKIENV) (SEQ. ID NO. 65) shares only 85.6% identity with its homolog that was used in the ELISA assay. This suggests that antigenic epitopes are shared, and Mat an ELISA of this sort is likely to be robust enough to detect infection with most, if not all, type II ("North American" genotype) PRRS viruses. In addition, the ELISA is sensitive enough to show a strong positive reaction with serum from a pig whose antibody levels have been waning for a long period of time (200 days post infection).
[0194] Pigs vaccinated with a virus that lacks the nsp2 (628-759) peptide, due to genetic deletion, are not expected to induce antibodies to this region. It is this ability to differentiate vaccinated and naive (negative) pigs from pigs that have been exposed to field strains of the PRRS virus (positive) that form the basis of the utility of this invention. Pigs that test positive can be removed from the herd or placed in isolation, thus aiding in the management and possible eradication of the PRRS virus.
[0195] Alternatively to the peptides disclosed above, peptides representing the corresponding region from other PRRS viruses can be employed in a diagnostic assay. Numerous examples of such peptides are provided for by the sequences disclosed in SEQ ID. NOs 66-115. That is 50 examples of similarly suitable peptides are provided by the polypeptides disclosed in SEQ ID. NOs 66-115 In addition to those examples, fragments of any of the peptides described can be used as a reagent. Fragments can be as short as ten amino acids, capable of forming and being recognized as an epitope by an antibody molecule, or as long as the entire region. For example, the peptide can comprise amino acids 1-10 of the respective 132 amino acid nsp2 peptide. The peptide can comprise amino acids 2-11 of the peptide. The peptide can comprise amino acids 3-12 of the peptide. Peptides are selected in this fashion by progressively "walking" down the amino acid sequence of the nsp2 fragment, ail of the way through to amino acid 132, generating 123 possible 10-mer peptides.
[0196] In addition to 10-mer peptides, the fragment can be 11 amino acid residues in length. Beginning with a fragment comprising amino acid 1-11, and progressing to the C-terminal residue, 122 different peptides are possible for the corresponding 132 amino acid region in nsp2. Similarly, the fragment can be 12, 13, 14, 15, 16, 17, 16, 19, 20 or more residues in length, up to 131 residues in length, and such peptides are readily identifiable by beginning at residue 1 and "walking" down the amino acid sequence of the corresponding peptide. There are 7626 such fragments in a peptide of 132 animo acid residues (including the full length peptide). Such a strategy can be applied to the corresponding nsp2 sequence from any strain of PRRS virus, making it possible to have a diagnostic kit specific for that and possibly other related strains of PRRS virus.
Example XIII
Infection of Pigs with Recombinant Viruses
[0197] To establish the level of attenuation achieved by the deletion of the nsp2 (628-759) peptide, and to evaluate the ability of the deletion to serve as a serological marker, young pigs are infected with the undeleted parental virus P129-Mlu/SgrA, P129-Mlu-GFP-SgrA or P129-Mlu-HA-SgrA. Three groups of 10 conventional weaned pigs, 3 weeks of age, are housed separately. A mock-infected group (6 pigs) is included in the study, and nave pigs (contact controls, 2 per vaccine group) are commingled with infected pigs in order to evaluate the ability of the viruses to spread. After a week of acclamation, at about 4 weeks of age, pigs are infected with the three viruses, or mock infected, by delivering 2 ml of a diluted virus stock (at 1×104 TCID50/ml) intramuscularly and 1 ml intranasally to each nostril (total dose is 4×104 TCID50/ml). Pigs are monitored daily for clinical signs of disease from day -3 to day 10 (general condition, depression, loss of appetite, sneezing, coughing, and respiratory distress), and rectal temperatures are taken on day -1, 3, 7, and 10, On day 10, half of the pigs in each group (selected by prior allotment) are necropsied in order to examine lung lesions and to sample tissues for virus isolation and histopathology. The remaining pigs are necropsied on day 28. All pigs are weighed on arrival, on day -1 or 0, and at necropsy (day 10 or 28).
[0198] Blood (10 ml) is collected from all pigs on days -1 or 0, 3, 7, 10, 14, 21, and 28, into serum separator tubes. Sera are frozen at -80 C until used for the determination of anti-PRRSV antibodies (by IDEXX HerdChek ELISA), neutralizing antibodies (by serum neutralization assay), and virus isolation (titration of virus infectivity by TCID50 assay). In addition, sera will be assayed for antibodies against the nsp2 (628-759) peptide using the ELISA described in Example XII above. Also, the induction of serum antibodies to GFP or to the HA tag can be determined using commercial kits or reagents.
[0199] The viruses with nsp2 (628-759) deleted are attenuated (less virulent) relative to the parental virus P129-Mlu/SgrA, as evidenced by a combination of one or more of the following characteristics in infected pigs: (1) reduced clinical signs, (2) reduced fever, (3) increased weight gain, (4) reduced number or severity of lung lesions, (5) increased neutralizing antibody levels, (6) decreased level or duration of viremia in serum, lung, and/or tonsil tissue, or (7) reduced spread to naive contact control pigs. Attenuated viruses with these characteristics are useful as live vaccines.
[0200] All infected pigs test positive for anti-PRRSV antibodies using a commercial ELISA (IDEXX HerdChek), with seroconversion occurring no later than 21 days post-infection. Only those pigs infected with undeleted parental virus P129-Mlu/SgrA are expected to have antibodies specific to the nsp2 (628-759) peptide, as measured using the ELISA described in Example XII. Such a vaccine virus is useful for differentiating infected from vaccinated animals (DIVA), otherwise known as a negatively marked vaccine.
[0201] Sera from pigs infected with P129-Mlu-GFP-SgrA or P129-Mlu-HA-SgrA are assayed for antibodies to GFP and the HA tag, respectively, by detecting these antibodies using commercially available reagents and kits. Such positive serological markers or "compliance markers", when incorporated into a vaccine, are useful for determining which pigs in a herd have been successfully immunized with the vaccine. In addition, the waning of antibodies against the marker can be used as surrogate for the waning of antibodies against the PRRS virus, giving an indication of when to revaccinate.
[0202] In a separate pig study, the ability of an nsp2 (828-759)-deleted PRRS virus to function as a vaccine is tested directly. The P129-PK-dnsp2 virus (Example XI) is used to immunize 4 week old pigs by delivering 2 ml of virus at 2×104 TCID50/ml intramuscularly. After 28 days, these pigs are challenged by infection with a virulent PRRS isolate (NADC20). The severity of the disease induced by the NADC20 virus is compared to disease induced in non-vaccinated (naive) pigs. A useful vaccine will result in at least partial reduction of disease caused by NADC20, including clinical signs, fever, reduced weight gain, lung lesions, persistent viremia, and spread to contact control pigs.
Deposit of Biological Materials
[0203] The following biological material was deposited with the American Type Culture Collection (ATCC) at 10801 University Blvd., Manassas, Va., 20110-2209, USA, on Nov. 19, 1998 and were assigned the following accession numbers:
TABLE-US-00013 Plasmid Accession No. pT7P129A 203488 pCMV-S-P129 203489
[0204] All patents, patent applications, and publications cited above are incorporated herein by reference in their entirety.
[0205] The present invention is not to be limited in scope by the specific embodiments described herein, which are intended as single illustrations of individual aspects of the invention, and functionally equivalent methods and components are within the scope of the invention. Indeed, various modifications of the invention, in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description and accompanying drawings. Such modifications are intended to fall within the scope of the appended claims.
Sequence CWU
1
1
115115450DNAPorcine reproductive and respiratory syndrome virus
1atgacgtata ggtgttggct ctatgccacg gcatttgtat tgtcaggagc tgtgaccatt
60ggcacagccc aaaacttgct gcacggaaaa cgcccttctg tgacagcctt cttcagggga
120gcttaggggt ctgtccctag caccttgctt ctggagttgc actgctttac ggtctctcca
180cccctttaac catgtctggg atacttgatc ggtgcacgtg cacccccaat gccagggtgt
240ttatggcgga gggccaagtc tactgcacac gatgtctcag tgcacggtct ctccttcctc
300tgaatctcca agttcctgag cttggggtgc tgggcctatt ttataggccc gaagagccac
360tccggtggac gttgccacgt gcattcccca ctgtcgagtg ctcccccgcc ggggcctgct
420ggctttctgc gatctttcca attgcacgaa tgaccagtgg aaacctgaac tttcaacaaa
480gaatggtgcg ggttgcagct gagatttaca gagccggcca actcacccct gcagttttga
540aggctctaca agtttatgaa cggggttgtc gctggtaccc cattgtcgga cctgtccctg
600gagtggccgt tcacgccaac tccctacatg tgagtgacaa acctttcccg ggagcaactc
660atgtgttaac caacctaccg ctcccgcaga ggcccaagcc tgaagacttt tgcccttttg
720agtgtgctat ggctgacgtc tatgacatta gccatgacgc cgtcatgtat gtggccagag
780ggaaagtctc ctgggcccct cgtggcgggg atgaagtgaa atttgaaacc gtccccgaag
840agttgaagtt gattgcgaac cgactccaca tctccttccc gccccaccac gcagtggaca
900tgtctgagtt tgccttcata gcccctggga gtggtgtctc cttgcgggtc gagcaccaac
960acggttgcct tcccgctgat actgtccctg aagggaactg ctggtggtgc ttgtttgact
1020tgctcccacc ggaagttcag aataaagaaa ttcgccgtgc taaccaattt ggctatcaaa
1080ccaagcatgg tgtccctggc aagtacctac agcggaggct gcaagttaat ggtctccgag
1140cagtgactga tacagatgga cctattgtcg tacagtactt ctctgttagg gagagttgga
1200tccgccactt cagactggcg gaagaaccta gcctccctgg gtttgaagac ctcctcagaa
1260taagggtaga gcctaatacg tcgccattgg gtggcaaggg tgaaaaaatc ttccggtttg
1320gcagtcacaa gtggtacggt gctggaaaga gagcaaggag agcacgctct ggtgcaactg
1380ccacggtcgc tcactgcgct ttgcccgctc gcgaagccca gcaggccaag aagctcgagg
1440ttgccagcgc caacagggct gagcatctca agtactattc cccgcctgcc gacgggaact
1500gtggttggca ctgcatttcc gccattacca accggatggt gaattccaaa tttgaaacca
1560ctcttcccga gagagtgaga ccttcagatg actgggctac tgacgaggat cttgtgaata
1620ccatccaaat cctcaggctc cccgcggcct tggacaggaa cggtgcttgt gctggcgcca
1680agtacgtgct caagctggaa ggtgagcact ggaccgtctc tgtgacccct gggatgaccc
1740cttctttgct cccccttgaa tgtgttcagg gttgttgtga gcataagagc ggtcttggtt
1800tcccagacgt ggtcgaagtt tccggatttg accctgcctg tcttgaccga cttgctgaga
1860taatgcactt acctagcagt gtcatcccag ctgctctggc cgagatgtcc gacgacttca
1920atcgtctggc ttccccggcc gccactgtgt ggactgtttc gcaattcttt gcccgccaca
1980gaggaggaga gcatcctgac caggtgtgct tagggaaaat tatcaacctt tgtcaggtga
2040ttgaggaatg ctgctgttcc cggaacaaag ccaaccgggc taccccggaa gaggttgcgg
2100caaaagttga ccagtacctc cgtggtgcag caagccttgg agaatgcttg gccaagcttg
2160agagggctcg cccgccgagc gcgatggaca cctcctttga ttggaatgtt gtgcttcctg
2220gggttgagac ggcggatcag acaaccaaac agctccatgt caaccagtgc cgcgctctgg
2280ttcctgtcgt gactcaagag cctttggaca gagactcggt ccctctgacc gccttctcgc
2340tgtccaattg ctactaccct gcacaaggtg acgaggtccg tcaccgtgag aggctaaact
2400ccgtgctctc taagttggag ggggttgttc gtgaggaata tgggctcacg ccaactggac
2460ctggcccgcg acccgcactg ccgaacgggc tcgacgagct taaagaccag atggaggagg
2520atctgctgaa attagtcaac gcccaggcaa cttcagaaat gatggcctgg gcagccgagc
2580aggttgatct aaaagcttgg gtcaaaaatt acccacggtg gacaccgcca ccccctccac
2640caagagttca gcctcgaaaa acgaagtctg tcaagagctt gctagagaac aagcctgtcc
2700ctgctccgcg caggaaggtc agatctgatt atggcagccc gattttgatg ggcgacaatg
2760ttcctaacgg ttgggaagat tcgactgttg gtggtcccct tgacctttcg gcaccatccg
2820agccgatgac acctctgagt gagcctgtac ttatttccag gccagtgaca tctttgagtg
2880tgccggcccc agttcctgca ccgcgtagag ctgtgtctcg accgatgacg ccctcgagtg
2940agccaatttt tgtgtctgca ctgcgacaca aatttcagca ggtggaaaaa gcaaatctgg
3000cggcagcagc gccgatgtac caggacgaac ccttagattt gtctgcatcc tcacagactg
3060aatatggggc ttctccccta acaccaccgc agaacgtggg cattctggag gtaagggggc
3120aagaagctga ggaagttctg agtgaaatct cggatattct gaatgatacc aaccctgcac
3180ctgtgtcatc aagcagctcc ctgtcaagtg ttaggatcac acgcccaaaa tactcagctc
3240aagccattat cgacttgggc gggccctgca gtgggcacct ccaaagggaa aaagaagcat
3300gcctccgcat catgcgtgag gcttgtgatg cggccaagct tagtgaccct gccacgcagg
3360aatggctttc tcgcatgtgg gatagggtgg acatgctgac ttggcgcaac acgtctgctt
3420accaggcgtt tcgcacctta gatggcaggt ttgggtttct cccaaagatg atactcgaga
3480cgccgccgcc ctacccgtgt gggtttgtga tgttgcctca cacccctgca ccttccgtga
3540gtgcagagag cgaccttacc atcggttcag tcgccactga agatattcca cgcatcctcg
3600ggaaaataga aaataccggt gagatgatca accagggacc cttggcatcc tctgaggaag
3660aaccggtata caaccaacct gccaaagact cccggatatc gtcgcggggg tctgacgaga
3720gcacagcagc tccgtccgca ggtacaggtg gcgccggctt atttactgat ttgccacctt
3780cagacggcgt agatgcggac ggtggggggc cgttgcagac ggtaagaaag aaagctgaaa
3840ggctcttcga ccaattgagc cgtcaggttt ttaacctcgt ctcccatctc cctgttttct
3900tctcacacct cttcaaatct gacagtggtt attctccggg tgattggggt tttgcagctt
3960ttactctatt ttgcctcttt ttgtgttaca gctacccatt cttcggtttc gttcccctct
4020tgggtgtatt ttctgggtct tctcggcgtg tgcgcatggg ggtttttggc tgctggctgg
4080cttttgctgt tggcctgttc aagcctgtgt ccgacccagt cggcactgct tgtgagtttg
4140actcgccaga gtgtaggaac gtccttcatt cttttgagct tctcaaacct tgggaccctg
4200ttcgcagcct tgttgtgggc cccgtcggtc tcggtcttgc cattcttggc aggttactgg
4260gcggggcacg ctacatctgg cattttttgc ttaggcttgg cattgttgca gattgtatct
4320tggctggagc ttatgtgctt tctcaaggta ggtgtaaaaa gtgctgggga tcttgtataa
4380gaactgctcc taatgaaatc gccttcaacg tgttcccttt tacacgtgcg accaggtcgt
4440cactcatcga cctgtgcgat cggttttgtg cgccaaaagg catggacccc attttcctcg
4500ccactgggtg gcgtgggtgc tggaccggcc gaagtcccat tgagcaaccc tctgaaaaac
4560ccatcgcgtt cgcccagttg gatgaaaaga ggattacggc tagaactgtg gtcgctcagc
4620cttatgatcc taatcaagcc gtaaagtgct tgcgggtgtt acaggcgggt ggggcgatgg
4680tggccgaggc agtcccaaaa gtggtcaaag tttctgctat tccattccga gccccctttt
4740ttcccaccgg agtgaaagtt gatcccgagt gcaggatcgt ggtcgacccc gatactttta
4800ctacagccct ccggtctggt tactctacca caaacctcgt ccttggtgtg ggggactttg
4860cccagctgaa tggactaaag atcaggcaaa tttccaagcc ttcgggagga ggcccacacc
4920tcattgctgc cctgcatgtt gcctgctcga tggcgttgca catgcttgct ggggtttatg
4980taacttcagt ggggtcttgc ggtgccggca ccaacgatcc atggtgcact aatccgtttg
5040ccgttcctgg ctacggacca ggctctctct gcacgtccag attgtgcatc tcccaacatg
5100gccttaccct gcccttgaca gcacttgtgg cgggattcgg tcttcaggaa atcgccttgg
5160tcgttttgat tttcgtttcc atcggaggca tggctcatag gttgagttgt aaggctgata
5220tgctgtgcat cttacttgca atcgccagct atgtttgggt accccttacc tggttgcttt
5280gtgtgtttcc ttgttggttg cgctggttct ctttgcaccc cctcaccatc ctatggttgg
5340tgtttttctt gatttctgta aatatgcctt cgggaatctt ggccgtggtg ttattggttt
5400ctctttggct tttgggacgt tatactaaca ttgctggtct tgtcaccccc tatgatattc
5460atcattacac cagtggcccc cgcggtgttg ccgccttagc taccgcacca gatggaacct
5520acttggctgc cgtccgccgc gctgcgttga ctggtcgcac catgctgttc accccgtctc
5580agcttgggtc ccttcttgag ggcgctttca gaactcgaaa gccctcactg aacaccgtca
5640atgtggttgg gtcctccatg ggctctggtg gagtgttcac catcgacggg aaaattaggt
5700gcgtgactgc cgcacatgtc cttacgggta attcggctag ggtttccgga gtcggcttca
5760atcaaatgct tgactttgat gtgaaagggg acttcgccat agctgattgc ccgaattggc
5820aaggagctgc tcccaagacc caattctgcg aggatggatg ggctggccgt gcctattggc
5880tgacatcctc tggcgtcgaa cccggtgtta ttgggaatgg attcgccttc tgcttcaccg
5940cgtgcggcga ttccgggtcc ccagtgatca ccgaagctgg tgagcttgtc ggcgttcaca
6000caggatcaaa taaacaagga ggtggcatcg tcacgcgccc ttcaggccag ttttgtaacg
6060tggcacccat caagctgagc gaattaagtg aattctttgc tggacccaag gtcccgctcg
6120gtgatgtgaa ggttggcagc cacataatta aagatacgtg cgaagtacct tcagatcttt
6180gcgccttgct tgctgccaaa cctgaactgg agggaggcct ctccaccgtc caacttctgt
6240gtgtgttttt cctactgtgg agaatgatgg gacatgcctg gacgcccttg gttgctgtgg
6300ggtttttcat tctgaatgag gttctcccag ctgtcctggt tcggagtgtt ttctcctttg
6360ggatgtttgt gctatcttgg ctcacaccat ggtctgcgca agttctgatg atcaggcttc
6420taacagcagc tcttaacagg aacagatggt cacttgcctt ttacagcctt ggtgcggtga
6480ccggttttgt cgcagatctt gcggcaactc aagggcaccc gttgcaggca gtaatgaatt
6540tgagcaccta tgccttcctg cctcggatga tggttgtgac ctcaccagtc ccagtgattg
6600cgtgtggtgt tgtgcaccta cttgccatca ttttgtactt gttcaagtac cgcggcctgc
6660acaatgttct tgttggtgat ggagcgtttt ctgcagcttt cttcttgcga tactttgccg
6720agggaaagtt gagggaaggg gtgtcgcaat cctgcggaat gaatcatgag tcattaactg
6780gtgccctcgc tatgagactc aatgacgagg acttggactt ccttacgaaa tggactgatt
6840ttaagtgctt tgtttctgcg tccaacatga ggaatgcagc aggccaattc atcgaggctg
6900cctatgcaaa agcacttaga attgaacttg cccagttggt gcaggttgat aaggttcgag
6960gtactttggc caagcttgag gcttttgctg ataccgtggc accccaactc tcgcccggtg
7020acattgttgt tgctcttggc catacgcctg ttggcagcat cttcgaccta aaggttggtg
7080gtaccaagca tactctccaa gtcattgaga ccagagtcct tgccgggtcc aaaatgaccg
7140tggcgcgcgt cgttgaccca acccccacgc ccccacccgc acccgtgccc atccccctcc
7200caccgaaagt tctagagaat ggtcccaacg cctgggggga tggggaccgt ttgaataaga
7260agaagaggcg taggatggaa accgtcggca tctttgtcat gggtgggaag aagtaccaga
7320aattttggga caagaattcc ggtgatgtgt tttacgagga ggtccatgac aacacagatg
7380cgtgggagtg cctcagagtt ggtgaccctg ccgactttga ccctgagaag ggaactctgt
7440gtgggcatac tactattgaa gataaggatt acaaagtcta cgcctcccca tctggcaaga
7500agttcctggt ccccgtcaac tcagagagcg gaagagccca atgggaagct gcaaagcttt
7560ccgtggagca ggcccttggc atgatgaatg tcgacggtga actgacggcc aaagaagtgg
7620agaaactgaa aagaataatt gacaaacttc agggcctgac taaggagcag tgtttaaact
7680gctagccgcc agcggcttga cccgctgtgg tcgcggcggc ttggttgtta ctgagacagc
7740ggtaaaaata gtcaaatttc acaaccggac tttcacccta gggcctgtga atttaaaagt
7800ggccagtgag gttgagctga aagacgcggt cgagcacaac caacacccgg ttgcaagacc
7860ggttgacggt ggtgttgtgc tcctgcgttc cgcagttcct tcgcttatag atgtcctgat
7920ctccggtgct gacgcatctc ctaagttact cgctcgtcac gggccgggga acactgggat
7980cgatggcacg ctttgggact ttgaggccga ggccaccaaa gaggaaattg cactcagtgc
8040gcaaataata caggcttgtg acattaggcg cggcgacgca cctgaaattg gtctccctta
8100caagctgtac cctgttaggg gcaaccctga gcgggtaaaa ggagttttac agaatacaag
8160gtttggagac ataccttaca aaacccccag tgacactgga agcccagtgc acgcggctgc
8220ctgcctcacg cccaatgcca ctccggtgac tgatgggcgc tctgtcttgg ctactaccat
8280gccctccggt tttgaattgt atgtaccgac cattccagcg tctgtccttg attatcttga
8340ctctaggcct gactgcccca aacagttgac agagcacggc tgtgaggatg ccgcattgag
8400agacctctcc aagtatgact tgtccaccca aggctttgtt ttacctgggg ttcttcgcct
8460tgtgcgtaag tacctgtttg cccatgtggg taagtgcccg cccgttcatc ggccttccac
8520ttaccctgcc aagaattcta tggctggaat aaatgggaac aggtttccaa ccaaggacat
8580tcagagcgtc cctgaaatcg acgttctgtg cgcacaggcc gtgcgagaaa actggcaaac
8640tgttacccct tgtaccctca agaaacagta ttgtgggaag aagaagacta ggacaatact
8700cggcaccaat aatttcattg cgttggccca ccgggcagcg ttgagtggtg tcacccaggg
8760cttcatgaaa aaggcgttta actcgcccat cgccctcggg aaaaacaaat ttaaggagct
8820acagactccg gtcttaggca ggtgccttga agctgatctt gcatcctgtg atcgatccac
8880acctgcaatt gtccgctggt ttgccgccaa tcttctttat gaacttgcct gtgctgaaga
8940gcacctaccg tcgtacgtgc tgaactgctg ccatgaccta ttggtcacgc agtccggcgc
9000agtgactaag aggggtggcc tatcgtctgg cgacccgatc acttctgtgt ctaacaccat
9060ttacagcttg gtgatatatg cacagcacat ggtgcttagt tactttaaaa gtggtcaccc
9120tcatggcctt ctgttcctac aagaccagct gaagttcgag gacatgctca aagtccaacc
9180cctgatcgtc tattcggacg acctcgtgct gtatgccgaa tctcccacca tgccgaacta
9240ccactggtgg gtcgaacatc tgaatttgat gctgggtttt cagacggacc caaagaagac
9300agccataacg gactcgccat catttctagg ctgtaggata ataaatggac gccagctagt
9360ccccaaccgt gacaggatcc tcgcggccct cgcttaccat atgaaggcaa gcaatgtttc
9420tgaatactac gccgcggcgg ctgcaatact catggacagc tgtgcttgtt tagagtatga
9480tcctgaatgg tttgaagagc ttgtggttgg gatagcgcag tgcgcccgca aggacggcta
9540cagctttccc ggcccgccgt tcttcttgtc catgtgggaa aaactcagat ccaatcatga
9600ggggaagaag tccagaatgt gcgggtattg cggggccccg gctccgtacg ccactgcctg
9660tggcctcgac gtctgtattt accacaccca cttccaccag cattgtccag tcataatctg
9720gtgtggccac ccggctggtt ctggttcttg tagtgagtgc aaaccccccc tagggaaagg
9780cacaagccct ctagatgagg tgttagaaca agtcccgtat aagcctccac ggactgtaat
9840catgcatgtg gagcagggtc tcacccctct tgacccaggc agataccaga ctcgccgcgg
9900attagtctcc gttaggcgtg gcatcagagg aaacgaagtt gacctaccag acggtgatta
9960tgctagcacc gccctactcc ccacttgtaa agagatcaac atggtcgctg tcgcctctaa
10020tgtgttgcgc agcaggttca tcatcggtcc gcccggtgct gggaaaacat actggctcct
10080tcagcaggtc caggatggtg atgtcattta cacaccgact caccagacca tgctcgacat
10140gattagggct ttggggacgt gccggttcaa cgtcccagca ggtacaacgc tgcaattccc
10200tgccccctcc cgtaccggcc cgtgggttcg catcctggcc ggcggttggt gtcctggtaa
10260gaattccttc ctggatgaag cagcgtattg taatcacctt gatgtcttga ggctccttag
10320caaaaccacc cttacctgtc tgggagactt caaacaactc cacccagtgg gttttgattc
10380tcattgctat gtttttgaca tcatgcctca gacccagttg aagaccatct ggagattcgg
10440acagaacatc tgtgatgcca tccaaccaga ttacagggac aaacttgtgt ccatggtcaa
10500cacaacccgt gtaacctaca tggaaaaacc tgtcaagtat gggcaagtcc tcacccctta
10560ccacagggac cgagaggacg gcgccatcac aattgactcc agtcaaggcg ccacatttga
10620tgtggttaca ctgcatttgc ccactaaaga ttcactcaac aggcaaagag cccttgttgc
10680tatcaccagg gcaagacatg ctatctttgt gtatgaccca cacaggcaat tgcagagcat
10740gtttgatctt cctgcgaagg gcacacccgt caacctcgca gtgcaccgtg atgagcagct
10800gatcgtactg gatagaaata ataaagaatg cacagttgct caggctatag gcaacggaga
10860taaattcagg gccaccgaca agcgcgttgt agattctctc cgcgccattt gtgctgatct
10920ggaagggtcg agctccccgc tccccaaggt cgcacacaac ttgggatttt atttctcacc
10980tgatttgaca cagtttgcta aactcccggt agaccttgca ccccactggc ccgtggtgac
11040aacccagaac aatgaaaagt ggccggatcg gctggttgcc agccttcgcc ctgtccataa
11100gtatagccgt gcgtgcattg gtgccggcta tatggtgggc ccctcggtgt ttctaggcac
11160ccctggggtc gtgtcatact acctcacaaa atttgtcaag ggcgaggctc aagtgcttcc
11220ggagacagtc ttcagcaccg gccgaattga ggtggattgc cgggagtatc ttgatgacag
11280ggagcgagaa gttgctgagt ccctcccaca tgccttcatt ggcgacgtca aaggcaccac
11340cgttggggga tgtcatcatg tcacctccaa ataccttccg cgcttccttc ccaaggaatc
11400agtcgcggta gtcggggttt cgagccccgg gaaagccgca aaagcagtgt gcacattgac
11460ggatgtgtac ctcccagacc ttgaggccta cctccaccca gagactcagt ctaagtgctg
11520gaaagttatg ttggacttca aggaagttcg actgatggtc tggaaagaca agacggccta
11580tttccaactt gaaggccgct atttcacctg gtatcagctt gcaagctacg cctcgtacat
11640ccgtgttcct gtcaactcca cggtgtatct ggacccctgc atgggccctg ccctttgcaa
11700cagaagagtt gtcgggtcca cccattgggg agctgacctc gcagtcaccc cttatgatta
11760cggtgctaaa atcatcttgt ctagcgctta ccatggtgaa atgcctcctg gatacaagat
11820tctggcgtgc gcggagttct cgctcgacga cccagtcaag tacaaacaca cctggggttt
11880tgaatcggat acagcgtatc tgtatgagtt caccggaaac ggtgaggact gggaggatta
11940caatgatgcg tttcgtgcgc gccagaaagg gaaaatttat aaggccactg ctaccagcat
12000gaagttttat tttcccccgg gccccgtcat tgaaccaact ttaggcctga attgaaatga
12060aatggggtct atacaaagcc tcttcgacaa aattggccag ctttttgtgg atgctttcac
12120ggaatttttg gtgtccattg ttgatatcat catatttttg gccattttgt ttggcttcac
12180catcgccggt tggctggtgg tcttttgcat cagattggtt tgctccgcgg tattccgtgc
12240gcgccctgcc attcaccctg agcaattaca gaagatccta tgaggccttt ctttctcagt
12300gccgggtgga cattcccacc tggggggtaa aacacccttt ggggatgttt tggcaccata
12360aggtgtcaac cctgattgat gaaatggtgt cgcgtcgaat gtaccgcatc atggaaaaag
12420cagggcaagc tgcctggaaa caggtggtga gcgaggctac gctgtctcgc attagtagtt
12480tggatgtggt ggctcatttt caacatcttg ccgccattga agccgagacc tgtaaatatt
12540tggcttctcg actgcccatg ctacacaacc tgcgcatgac agggtcaaat gtaaccatag
12600tgtataatag cactttaaat caggtgtttg ctatttttcc aacccctggt tcccggccaa
12660agcttcatga ttttcagcaa tggctaatag ctgtacattc ctccatattt tcctctgttg
12720cagcttcttg tactcttttt gttgtgctgt ggttgcgggt tccaatgcta cgtactgttt
12780ttggtttccg ctggttaggg gcaatttttc tttcgaactc atggtgaatt acacggtgtg
12840tccaccttgc ctcacccgac aagcagccgc tgaggtcctt gaacccggta ggtctctttg
12900gtgcaggata gggcatgacc gatgtgggga ggacgatcac gacgaactag ggttcatggt
12960tccgcctggc ctctccagcg aaagccactt gaccagtgtt tacgcctggt tggcgttcct
13020gtccttcagc tacacggccc agttccatcc cgagatattt gggataggga acgtgagtga
13080agtttatgtt gacatcaagc accaattcat ctgcgccgtt catgacgggc agaacaccac
13140cttgcctcgc catgacaata tttcagccgt atttcagacc tactatcaac atcaggtcga
13200cggcggcaat tggtttcacc tagaatggct gcgtcccttc ttttcctctt ggttggtttt
13260aaatgtttcg tggtttctca ggcgttcgcc tgcaagccat gtttcagttc gagtctttca
13320gacatcaaaa ccaacactac cgcagcatca ggctttgttg tcctccagga catcagctgc
13380cttaggcatg gcgactcgtc ctttccgacg attcgcaaaa gctctcaatg ccgcacggcg
13440atagggacac ccgtgtatat caccatcaca gccaatgtga cagatgagaa ttacttacat
13500tcttctgatc tcctcatgct ttcttcttgc cttttctatg cttctgagat gagtgaaaag
13560ggattcaagg tggtgtttgg caatgtgtca ggcatcgtgg ctgtgtgtgt caactttacc
13620agctacgtcc aacatgtcaa agagtttacc caacgctcct tggtggtcga tcatgtgcgg
13680ctgcttcatt tcatgacacc tgagaccatg aggtgggcaa ccgttttagc ctgtcttttt
13740gccatcctac tggcaatttg aatgttcaag tatgttgggg aaatgcttga ccgcgggctg
13800ttgctcgcga ttgctttctt tgtggtgtat cgtgccgttc tgttttgctg tgctcggcag
13860cgccaacagc agcagcagct ctcattttca gttgatttat aacttgacgc tatgtgagct
13920gaatggcaca gattggctgg cagaaaaatt tgattgggca gtggagactt ttgtcatctt
13980tcccgtgttg actcacattg tttcctatgg tgcactcacc accagccatt tccttgacac
14040agttggtctg gttactgtgt ccaccgccgg gttttatcac gggcggtatg tcttgagtag
14100catctacgcg gtctgtgctc tggctgcgtt gatttgcttc gttattaggc ttgcgaagaa
14160ctgcatgtcc tggcgctact cttgtaccag atataccaac ttccttctgg acactaaggg
14220cagactctat cgttggcggt cgcccgttat catagaaaaa gggggtaagg ttgaggtcga
14280aggtcacctg atcgacctca aaagagttgt gcttgatggt tccgtggcaa cccctttaac
14340cagagtttca gcggaacaat ggggtcgtct ctagacgact tttgccatga tagcacggct
14400ccacaaaagg tgcttttggc gttttccatt acctacacgc cagtaatgat atatgctcta
14460aaggtaagtc gcggccgact actagggctt ctgcaccttt tgatctttct gaattgtgct
14520tttaccttcg ggtacatgac attcgagcac tttcagagca caaatagggt cgcgctcact
14580atgggagcag tagttgcact tctttggggg gtgtactcag ccatagaaac ctggaaattc
14640atcacctcca gatgccgttt gtgcttgcta ggccgcaagt acattctggc ccctgcccac
14700cacgtcgaaa gtgccgcggg ctttcatccg attgcggcaa atgataacca cgcatttgtc
14760gtccggcgtc ccggctccac tacggttaac ggcacattgg tgcccgggtt gaaaagcctc
14820gtgttgggtg gcagaaaagc tgttaaacag ggagtggtaa accttgtcaa atatgccaaa
14880taacaacggc aagcagcaaa agaaaaagaa ggggaatggc cagccagtca atcagctgtg
14940ccagatgctg ggtaaaatca tcgcccagca aaaccagtcc agaggcaagg gaccgggcaa
15000gaaaagtaag aagaaaaacc cggagaagcc ccattttcct ctagcgaccg aagatgacgt
15060caggcatcac ttcacccctg gtgagcggca attgtgtctg tcgtcgatcc agactgcctt
15120taaccagggc gctggaactt gtaccctgtc agattcaggg aggataagtt acactgtgga
15180gtttagtttg ccgacgcatc atactgtgcg cctgatccgc gtcacagcat caccctcagc
15240atgatgggct ggcattcttt aggcacctca gtgtcagaat tggaagaatg tgtggtggat
15300ggcactgatt gacattgtgc ctctaagtca cctattcaat tagggcgacc gtgtgggggt
15360aaaatttaat tggcgagaac catgcggccg caattaaaaa aaaaaaaaaa aaaaaaaaaa
15420aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa
1545027494DNAPorcine reproductive and respiratory syndrome virus
2atgtctggga tacttgatcg gtgcacgtgc acccccaatg ccagggtgtt tatggcggag
60ggccaagtct actgcacacg atgtctcagt gcacggtctc tccttcctct gaatctccaa
120gttcctgagc ttggggtgct gggcctattt tataggcccg aagagccact ccggtggacg
180ttgccacgtg cattccccac tgtcgagtgc tcccccgccg gggcctgctg gctttctgcg
240atctttccaa ttgcacgaat gaccagtgga aacctgaact ttcaacaaag aatggtgcgg
300gttgcagctg agatttacag agccggccaa ctcacccctg cagttttgaa ggctctacaa
360gtttatgaac ggggttgtcg ctggtacccc attgtcggac ctgtccctgg agtggccgtt
420cacgccaact ccctacatgt gagtgacaaa cctttcccgg gagcaactca tgtgttaacc
480aacctaccgc tcccgcagag gcccaagcct gaagactttt gcccttttga gtgtgctatg
540gctgacgtct atgacattag ccatgacgcc gtcatgtatg tggccagagg gaaagtctcc
600tgggcccctc gtggcgggga tgaagtgaaa tttgaaaccg tccccgaaga gttgaagttg
660attgcgaacc gactccacat ctccttcccg ccccaccacg cagtggacat gtctgagttt
720gccttcatag cccctgggag tggtgtctcc ttgcgggtcg agcaccaaca cggttgcctt
780cccgctgata ctgtccctga agggaactgc tggtggtgct tgtttgactt gctcccaccg
840gaagttcaga ataaagaaat tcgccgtgct aaccaatttg gctatcaaac caagcatggt
900gtccctggca agtacctaca gcggaggctg caagttaatg gtctccgagc agtgactgat
960acagatggac ctattgtcgt acagtacttc tctgttaggg agagttggat ccgccacttc
1020agactggcgg aagaacctag cctccctggg tttgaagacc tcctcagaat aagggtagag
1080cctaatacgt cgccattggg tggcaagggt gaaaaaatct tccggtttgg cagtcacaag
1140tggtacggtg ctggaaagag agcaaggaga gcacgctctg gtgcaactgc cacggtcgct
1200cactgcgctt tgcccgctcg cgaagcccag caggccaaga agctcgaggt tgccagcgcc
1260aacagggctg agcatctcaa gtactattcc ccgcctgccg acgggaactg tggttggcac
1320tgcatttccg ccattaccaa ccggatggtg aattccaaat ttgaaaccac tcttcccgag
1380agagtgagac cttcagatga ctgggctact gacgaggatc ttgtgaatac catccaaatc
1440ctcaggctcc ccgcggcctt ggacaggaac ggtgcttgtg ctggcgccaa gtacgtgctc
1500aagctggaag gtgagcactg gaccgtctct gtgacccctg ggatgacccc ttctttgctc
1560ccccttgaat gtgttcaggg ttgttgtgag cataagagcg gtcttggttt cccagacgtg
1620gtcgaagttt ccggatttga ccctgcctgt cttgaccgac ttgctgagat aatgcactta
1680cctagcagtg tcatcccagc tgctctggcc gagatgtccg acgacttcaa tcgtctggct
1740tccccggccg ccactgtgtg gactgtttcg caattctttg cccgccacag aggaggagag
1800catcctgacc aggtgtgctt agggaaaatt atcaaccttt gtcaggtgat tgaggaatgc
1860tgctgttccc ggaacaaagc caaccgggct accccggaag aggttgcggc aaaagttgac
1920cagtacctcc gtggtgcagc aagccttgga gaatgcttgg ccaagcttga gagggctcgc
1980ccgccgagcg cgatggacac ctcctttgat tggaatgttg tgcttcctgg ggttgagacg
2040gcggatcaga caaccaaaca gctccatgtc aaccagtgcc gcgctctggt tcctgtcgtg
2100actcaagagc ctttggacag agactcggtc cctctgaccg ccttctcgct gtccaattgc
2160tactaccctg cacaaggtga cgaggtccgt caccgtgaga ggctaaactc cgtgctctct
2220aagttggagg gggttgttcg tgaggaatat gggctcacgc caactggacc tggcccgcga
2280cccgcactgc cgaacgggct cgacgagctt aaagaccaga tggaggagga tctgctgaaa
2340ttagtcaacg cccaggcaac ttcagaaatg atggcctggg cagccgagca ggttgatcta
2400aaagcttggg tcaaaaatta cccacggtgg acaccgccac cccctccacc aagagttcag
2460cctcgaaaaa cgaagtctgt caagagcttg ctagagaaca agcctgtccc tgctccgcgc
2520aggaaggtca gatctgatta tggcagcccg attttgatgg gcgacaatgt tcctaacggt
2580tgggaagatt cgactgttgg tggtcccctt gacctttcgg caccatccga gccgatgaca
2640cctctgagtg agcctgtact tatttccagg ccagtgacat ctttgagtgt gccggcccca
2700gttcctgcac cgcgtagagc tgtgtctcga ccgatgacgc cctcgagtga gccaattttt
2760gtgtctgcac tgcgacacaa atttcagcag gtggaaaaag caaatctggc ggcagcagcg
2820ccgatgtacc aggacgaacc cttagatttg tctgcatcct cacagactga atatggggct
2880tctcccctaa caccaccgca gaacgtgggc attctggagg taagggggca agaagctgag
2940gaagttctga gtgaaatctc ggatattctg aatgatacca accctgcacc tgtgtcatca
3000agcagctccc tgtcaagtgt taggatcaca cgcccaaaat actcagctca agccattatc
3060gacttgggcg ggccctgcag tgggcacctc caaagggaaa aagaagcatg cctccgcatc
3120atgcgtgagg cttgtgatgc ggccaagctt agtgaccctg ccacgcagga atggctttct
3180cgcatgtggg atagggtgga catgctgact tggcgcaaca cgtctgctta ccaggcgttt
3240cgcaccttag atggcaggtt tgggtttctc ccaaagatga tactcgagac gccgccgccc
3300tacccgtgtg ggtttgtgat gttgcctcac acccctgcac cttccgtgag tgcagagagc
3360gaccttacca tcggttcagt cgccactgaa gatattccac gcatcctcgg gaaaatagaa
3420aataccggtg agatgatcaa ccagggaccc ttggcatcct ctgaggaaga accggtatac
3480aaccaacctg ccaaagactc ccggatatcg tcgcgggggt ctgacgagag cacagcagct
3540ccgtccgcag gtacaggtgg cgccggctta tttactgatt tgccaccttc agacggcgta
3600gatgcggacg gtggggggcc gttgcagacg gtaagaaaga aagctgaaag gctcttcgac
3660caattgagcc gtcaggtttt taacctcgtc tcccatctcc ctgttttctt ctcacacctc
3720ttcaaatctg acagtggtta ttctccgggt gattggggtt ttgcagcttt tactctattt
3780tgcctctttt tgtgttacag ctacccattc ttcggtttcg ttcccctctt gggtgtattt
3840tctgggtctt ctcggcgtgt gcgcatgggg gtttttggct gctggctggc ttttgctgtt
3900ggcctgttca agcctgtgtc cgacccagtc ggcactgctt gtgagtttga ctcgccagag
3960tgtaggaacg tccttcattc ttttgagctt ctcaaacctt gggaccctgt tcgcagcctt
4020gttgtgggcc ccgtcggtct cggtcttgcc attcttggca ggttactggg cggggcacgc
4080tacatctggc attttttgct taggcttggc attgttgcag attgtatctt ggctggagct
4140tatgtgcttt ctcaaggtag gtgtaaaaag tgctggggat cttgtataag aactgctcct
4200aatgaaatcg ccttcaacgt gttccctttt acacgtgcga ccaggtcgtc actcatcgac
4260ctgtgcgatc ggttttgtgc gccaaaaggc atggacccca ttttcctcgc cactgggtgg
4320cgtgggtgct ggaccggccg aagtcccatt gagcaaccct ctgaaaaacc catcgcgttc
4380gcccagttgg atgaaaagag gattacggct agaactgtgg tcgctcagcc ttatgatcct
4440aatcaagccg taaagtgctt gcgggtgtta caggcgggtg gggcgatggt ggccgaggca
4500gtcccaaaag tggtcaaagt ttctgctatt ccattccgag cccccttttt tcccaccgga
4560gtgaaagttg atcccgagtg caggatcgtg gtcgaccccg atacttttac tacagccctc
4620cggtctggtt actctaccac aaacctcgtc cttggtgtgg gggactttgc ccagctgaat
4680ggactaaaga tcaggcaaat ttccaagcct tcgggaggag gcccacacct cattgctgcc
4740ctgcatgttg cctgctcgat ggcgttgcac atgcttgctg gggtttatgt aacttcagtg
4800gggtcttgcg gtgccggcac caacgatcca tggtgcacta atccgtttgc cgttcctggc
4860tacggaccag gctctctctg cacgtccaga ttgtgcatct cccaacatgg ccttaccctg
4920cccttgacag cacttgtggc gggattcggt cttcaggaaa tcgccttggt cgttttgatt
4980ttcgtttcca tcggaggcat ggctcatagg ttgagttgta aggctgatat gctgtgcatc
5040ttacttgcaa tcgccagcta tgtttgggta ccccttacct ggttgctttg tgtgtttcct
5100tgttggttgc gctggttctc tttgcacccc ctcaccatcc tatggttggt gtttttcttg
5160atttctgtaa atatgccttc gggaatcttg gccgtggtgt tattggtttc tctttggctt
5220ttgggacgtt atactaacat tgctggtctt gtcaccccct atgatattca tcattacacc
5280agtggccccc gcggtgttgc cgccttagct accgcaccag atggaaccta cttggctgcc
5340gtccgccgcg ctgcgttgac tggtcgcacc atgctgttca ccccgtctca gcttgggtcc
5400cttcttgagg gcgctttcag aactcgaaag ccctcactga acaccgtcaa tgtggttggg
5460tcctccatgg gctctggtgg agtgttcacc atcgacggga aaattaggtg cgtgactgcc
5520gcacatgtcc ttacgggtaa ttcggctagg gtttccggag tcggcttcaa tcaaatgctt
5580gactttgatg tgaaagggga cttcgccata gctgattgcc cgaattggca aggagctgct
5640cccaagaccc aattctgcga ggatggatgg gctggccgtg cctattggct gacatcctct
5700ggcgtcgaac ccggtgttat tgggaatgga ttcgccttct gcttcaccgc gtgcggcgat
5760tccgggtccc cagtgatcac cgaagctggt gagcttgtcg gcgttcacac aggatcaaat
5820aaacaaggag gtggcatcgt cacgcgccct tcaggccagt tttgtaacgt ggcacccatc
5880aagctgagcg aattaagtga attctttgct ggacccaagg tcccgctcgg tgatgtgaag
5940gttggcagcc acataattaa agatacgtgc gaagtacctt cagatctttg cgccttgctt
6000gctgccaaac ctgaactgga gggaggcctc tccaccgtcc aacttctgtg tgtgtttttc
6060ctactgtgga gaatgatggg acatgcctgg acgcccttgg ttgctgtggg gtttttcatt
6120ctgaatgagg ttctcccagc tgtcctggtt cggagtgttt tctcctttgg gatgtttgtg
6180ctatcttggc tcacaccatg gtctgcgcaa gttctgatga tcaggcttct aacagcagct
6240cttaacagga acagatggtc acttgccttt tacagccttg gtgcggtgac cggttttgtc
6300gcagatcttg cggcaactca agggcacccg ttgcaggcag taatgaattt gagcacctat
6360gccttcctgc ctcggatgat ggttgtgacc tcaccagtcc cagtgattgc gtgtggtgtt
6420gtgcacctac ttgccatcat tttgtacttg ttcaagtacc gcggcctgca caatgttctt
6480gttggtgatg gagcgttttc tgcagctttc ttcttgcgat actttgccga gggaaagttg
6540agggaagggg tgtcgcaatc ctgcggaatg aatcatgagt cattaactgg tgccctcgct
6600atgagactca atgacgagga cttggacttc cttacgaaat ggactgattt taagtgcttt
6660gtttctgcgt ccaacatgag gaatgcagca ggccaattca tcgaggctgc ctatgcaaaa
6720gcacttagaa ttgaacttgc ccagttggtg caggttgata aggttcgagg tactttggcc
6780aagcttgagg cttttgctga taccgtggca ccccaactct cgcccggtga cattgttgtt
6840gctcttggcc atacgcctgt tggcagcatc ttcgacctaa aggttggtgg taccaagcat
6900actctccaag tcattgagac cagagtcctt gccgggtcca aaatgaccgt ggcgcgcgtc
6960gttgacccaa cccccacgcc cccacccgca cccgtgccca tccccctccc accgaaagtt
7020ctagagaatg gtcccaacgc ctggggggat ggggaccgtt tgaataagaa gaagaggcgt
7080aggatggaaa ccgtcggcat ctttgtcatg ggtgggaaga agtaccagaa attttgggac
7140aagaattccg gtgatgtgtt ttacgaggag gtccatgaca acacagatgc gtgggagtgc
7200ctcagagttg gtgaccctgc cgactttgac cctgagaagg gaactctgtg tgggcatact
7260actattgaag ataaggatta caaagtctac gcctccccat ctggcaagaa gttcctggtc
7320cccgtcaact cagagagcgg aagagcccaa tgggaagctg caaagctttc cgtggagcag
7380gcccttggca tgatgaatgt cgacggtgaa ctgacggcca aagaagtgga gaaactgaaa
7440agaataattg acaaacttca gggcctgact aaggagcagt gtttaaactg ctag
749434392DNAPorcine reproductive and respiratory syndrome virus
3ggagcagtgt ttaaactgct agccgccagc ggcttgaccc gctgtggtcg cggcggcttg
60gttgttactg agacagcggt aaaaatagtc aaatttcaca accggacttt caccctaggg
120cctgtgaatt taaaagtggc cagtgaggtt gagctgaaag acgcggtcga gcacaaccaa
180cacccggttg caagaccggt tgacggtggt gttgtgctcc tgcgttccgc agttccttcg
240cttatagatg tcctgatctc cggtgctgac gcatctccta agttactcgc tcgtcacggg
300ccggggaaca ctgggatcga tggcacgctt tgggactttg aggccgaggc caccaaagag
360gaaattgcac tcagtgcgca aataatacag gcttgtgaca ttaggcgcgg cgacgcacct
420gaaattggtc tcccttacaa gctgtaccct gttaggggca accctgagcg ggtaaaagga
480gttttacaga atacaaggtt tggagacata ccttacaaaa cccccagtga cactggaagc
540ccagtgcacg cggctgcctg cctcacgccc aatgccactc cggtgactga tgggcgctct
600gtcttggcta ctaccatgcc ctccggtttt gaattgtatg taccgaccat tccagcgtct
660gtccttgatt atcttgactc taggcctgac tgccccaaac agttgacaga gcacggctgt
720gaggatgccg cattgagaga cctctccaag tatgacttgt ccacccaagg ctttgtttta
780cctggggttc ttcgccttgt gcgtaagtac ctgtttgccc atgtgggtaa gtgcccgccc
840gttcatcggc cttccactta ccctgccaag aattctatgg ctggaataaa tgggaacagg
900tttccaacca aggacattca gagcgtccct gaaatcgacg ttctgtgcgc acaggccgtg
960cgagaaaact ggcaaactgt taccccttgt accctcaaga aacagtattg tgggaagaag
1020aagactagga caatactcgg caccaataat ttcattgcgt tggcccaccg ggcagcgttg
1080agtggtgtca cccagggctt catgaaaaag gcgtttaact cgcccatcgc cctcgggaaa
1140aacaaattta aggagctaca gactccggtc ttaggcaggt gccttgaagc tgatcttgca
1200tcctgtgatc gatccacacc tgcaattgtc cgctggtttg ccgccaatct tctttatgaa
1260cttgcctgtg ctgaagagca cctaccgtcg tacgtgctga actgctgcca tgacctattg
1320gtcacgcagt ccggcgcagt gactaagagg ggtggcctat cgtctggcga cccgatcact
1380tctgtgtcta acaccattta cagcttggtg atatatgcac agcacatggt gcttagttac
1440tttaaaagtg gtcaccctca tggccttctg ttcctacaag accagctgaa gttcgaggac
1500atgctcaaag tccaacccct gatcgtctat tcggacgacc tcgtgctgta tgccgaatct
1560cccaccatgc cgaactacca ctggtgggtc gaacatctga atttgatgct gggttttcag
1620acggacccaa agaagacagc cataacggac tcgccatcat ttctaggctg taggataata
1680aatggacgcc agctagtccc caaccgtgac aggatcctcg cggccctcgc ttaccatatg
1740aaggcaagca atgtttctga atactacgcc gcggcggctg caatactcat ggacagctgt
1800gcttgtttag agtatgatcc tgaatggttt gaagagcttg tggttgggat agcgcagtgc
1860gcccgcaagg acggctacag ctttcccggc ccgccgttct tcttgtccat gtgggaaaaa
1920ctcagatcca atcatgaggg gaagaagtcc agaatgtgcg ggtattgcgg ggccccggct
1980ccgtacgcca ctgcctgtgg cctcgacgtc tgtatttacc acacccactt ccaccagcat
2040tgtccagtca taatctggtg tggccacccg gctggttctg gttcttgtag tgagtgcaaa
2100ccccccctag ggaaaggcac aagccctcta gatgaggtgt tagaacaagt cccgtataag
2160cctccacgga ctgtaatcat gcatgtggag cagggtctca cccctcttga cccaggcaga
2220taccagactc gccgcggatt agtctccgtt aggcgtggca tcagaggaaa cgaagttgac
2280ctaccagacg gtgattatgc tagcaccgcc ctactcccca cttgtaaaga gatcaacatg
2340gtcgctgtcg cctctaatgt gttgcgcagc aggttcatca tcggtccgcc cggtgctggg
2400aaaacatact ggctccttca gcaggtccag gatggtgatg tcatttacac accgactcac
2460cagaccatgc tcgacatgat tagggctttg gggacgtgcc ggttcaacgt cccagcaggt
2520acaacgctgc aattccctgc cccctcccgt accggcccgt gggttcgcat cctggccggc
2580ggttggtgtc ctggtaagaa ttccttcctg gatgaagcag cgtattgtaa tcaccttgat
2640gtcttgaggc tccttagcaa aaccaccctt acctgtctgg gagacttcaa acaactccac
2700ccagtgggtt ttgattctca ttgctatgtt tttgacatca tgcctcagac ccagttgaag
2760accatctgga gattcggaca gaacatctgt gatgccatcc aaccagatta cagggacaaa
2820cttgtgtcca tggtcaacac aacccgtgta acctacatgg aaaaacctgt caagtatggg
2880caagtcctca ccccttacca cagggaccga gaggacggcg ccatcacaat tgactccagt
2940caaggcgcca catttgatgt ggttacactg catttgccca ctaaagattc actcaacagg
3000caaagagccc ttgttgctat caccagggca agacatgcta tctttgtgta tgacccacac
3060aggcaattgc agagcatgtt tgatcttcct gcgaagggca cacccgtcaa cctcgcagtg
3120caccgtgatg agcagctgat cgtactggat agaaataata aagaatgcac agttgctcag
3180gctataggca acggagataa attcagggcc accgacaagc gcgttgtaga ttctctccgc
3240gccatttgtg ctgatctgga agggtcgagc tccccgctcc ccaaggtcgc acacaacttg
3300ggattttatt tctcacctga tttgacacag tttgctaaac tcccggtaga ccttgcaccc
3360cactggcccg tggtgacaac ccagaacaat gaaaagtggc cggatcggct ggttgccagc
3420cttcgccctg tccataagta tagccgtgcg tgcattggtg ccggctatat ggtgggcccc
3480tcggtgtttc taggcacccc tggggtcgtg tcatactacc tcacaaaatt tgtcaagggc
3540gaggctcaag tgcttccgga gacagtcttc agcaccggcc gaattgaggt ggattgccgg
3600gagtatcttg atgacaggga gcgagaagtt gctgagtccc tcccacatgc cttcattggc
3660gacgtcaaag gcaccaccgt tgggggatgt catcatgtca cctccaaata ccttccgcgc
3720ttccttccca aggaatcagt cgcggtagtc ggggtttcga gccccgggaa agccgcaaaa
3780gcagtgtgca cattgacgga tgtgtacctc ccagaccttg aggcctacct ccacccagag
3840actcagtcta agtgctggaa agttatgttg gacttcaagg aagttcgact gatggtctgg
3900aaagacaaga cggcctattt ccaacttgaa ggccgctatt tcacctggta tcagcttgca
3960agctacgcct cgtacatccg tgttcctgtc aactccacgg tgtatctgga cccctgcatg
4020ggccctgccc tttgcaacag aagagttgtc gggtccaccc attggggagc tgacctcgca
4080gtcacccctt atgattacgg tgctaaaatc atcttgtcta gcgcttacca tggtgaaatg
4140cctcctggat acaagattct ggcgtgcgcg gagttctcgc tcgacgaccc agtcaagtac
4200aaacacacct ggggttttga atcggataca gcgtatctgt atgagttcac cggaaacggt
4260gaggactggg aggattacaa tgatgcgttt cgtgcgcgcc agaaagggaa aatttataag
4320gccactgcta ccagcatgaa gttttatttt cccccgggcc ccgtcattga accaacttta
4380ggcctgaatt ga
43924771DNAPorcine reproductive and respiratory syndrome virus
4atgaaatggg gtctatacaa agcctcttcg acaaaattgg ccagcttttt gtggatgctt
60tcacggaatt tttggtgtcc attgttgata tcatcatatt tttggccatt ttgtttggct
120tcaccatcgc cggttggctg gtggtctttt gcatcagatt ggtttgctcc gcggtattcc
180gtgcgcgccc tgccattcac cctgagcaat tacagaagat cctatgaggc ctttctttct
240cagtgccggg tggacattcc cacctggggg gtaaaacacc ctttggggat gttttggcac
300cataaggtgt caaccctgat tgatgaaatg gtgtcgcgtc gaatgtaccg catcatggaa
360aaagcagggc aagctgcctg gaaacaggtg gtgagcgagg ctacgctgtc tcgcattagt
420agtttggatg tggtggctca ttttcaacat cttgccgcca ttgaagccga gacctgtaaa
480tatttggctt ctcgactgcc catgctacac aacctgcgca tgacagggtc aaatgtaacc
540atagtgtata atagcacttt aaatcaggtg tttgctattt ttccaacccc tggttcccgg
600ccaaagcttc atgattttca gcaatggcta atagctgtac attcctccat attttcctct
660gttgcagctt cttgtactct ttttgttgtg ctgtggttgc gggttccaat gctacgtact
720gtttttggtt tccgctggtt aggggcaatt tttctttcga actcatggtg a
7715765DNAPorcine reproductive and respiratory syndrome virus 5atggctaata
gctgtacatt cctccatatt ttcctctgtt gcagcttctt gtactctttt 60tgttgtgctg
tggttgcggg ttccaatgct acgtactgtt tttggtttcc gctggttagg 120ggcaattttt
ctttcgaact catggtgaat tacacggtgt gtccaccttg cctcacccga 180caagcagccg
ctgaggtcct tgaacccggt aggtctcttt ggtgcaggat agggcatgac 240cgatgtgggg
aggacgatca cgacgaacta gggttcatgg ttccgcctgg cctctccagc 300gaaagccact
tgaccagtgt ttacgcctgg ttggcgttcc tgtccttcag ctacacggcc 360cagttccatc
ccgagatatt tgggataggg aacgtgagtg aagtttatgt tgacatcaag 420caccaattca
tctgcgccgt tcatgacggg cagaacacca ccttgcctcg ccatgacaat 480atttcagccg
tatttcagac ctactatcaa catcaggtcg acggcggcaa ttggtttcac 540ctagaatggc
tgcgtccctt cttttcctct tggttggttt taaatgtttc gtggtttctc 600aggcgttcgc
ctgcaagcca tgtttcagtt cgagtctttc agacatcaaa accaacacta 660ccgcagcatc
aggctttgtt gtcctccagg acatcagctg ccttaggcat ggcgactcgt 720cctttccgac
gattcgcaaa agctctcaat gccgcacggc gatag
7656537DNAPorcine reproductive and respiratory syndrome virus 6atggctgcgt
cccttctttt cctcttggtt ggttttaaat gtttcgtggt ttctcaggcg 60ttcgcctgca
agccatgttt cagttcgagt ctttcagaca tcaaaaccaa cactaccgca 120gcatcaggct
ttgttgtcct ccaggacatc agctgcctta ggcatggcga ctcgtccttt 180ccgacgattc
gcaaaagctc tcaatgccgc acggcgatag ggacacccgt gtatatcacc 240atcacagcca
atgtgacaga tgagaattac ttacattctt ctgatctcct catgctttct 300tcttgccttt
tctatgcttc tgagatgagt gaaaagggat tcaaggtggt gtttggcaat 360gtgtcaggca
tcgtggctgt gtgtgtcaac tttaccagct acgtccaaca tgtcaaagag 420tttacccaac
gctccttggt ggtcgatcat gtgcggctgc ttcatttcat gacacctgag 480accatgaggt
gggcaaccgt tttagcctgt ctttttgcca tcctactggc aatttga
5377603DNAPorcine reproductive and respiratory syndrome virus 7atgttgggga
aatgcttgac cgcgggctgt tgctcgcgat tgctttcttt gtggtgtatc 60gtgccgttct
gttttgctgt gctcggcagc gccaacagca gcagcagctc tcattttcag 120ttgatttata
acttgacgct atgtgagctg aatggcacag attggctggc agaaaaattt 180gattgggcag
tggagacttt tgtcatcttt cccgtgttga ctcacattgt ttcctatggt 240gcactcacca
ccagccattt ccttgacaca gttggtctgg ttactgtgtc caccgccggg 300ttttatcacg
ggcggtatgt cttgagtagc atctacgcgg tctgtgctct ggctgcgttg 360atttgcttcg
ttattaggct tgcgaagaac tgcatgtcct ggcgctactc ttgtaccaga 420tataccaact
tccttctgga cactaagggc agactctatc gttggcggtc gcccgttatc 480atagaaaaag
ggggtaaggt tgaggtcgaa ggtcacctga tcgacctcaa aagagttgtg 540cttgatggtt
ccgtggcaac ccctttaacc agagtttcag cggaacaatg gggtcgtctc 600tag
6038525DNAPorcine
reproductive and respiratory syndrome virus 8atggggtcgt ctctagacga
cttttgccat gatagcacgg ctccacaaaa ggtgcttttg 60gcgttttcca ttacctacac
gccagtaatg atatatgctc taaaggtaag tcgcggccga 120ctactagggc ttctgcacct
tttgatcttt ctgaattgtg cttttacctt cgggtacatg 180acattcgagc actttcagag
cacaaatagg gtcgcgctca ctatgggagc agtagttgca 240cttctttggg gggtgtactc
agccatagaa acctggaaat tcatcacctc cagatgccgt 300ttgtgcttgc taggccgcaa
gtacattctg gcccctgccc accacgtcga aagtgccgcg 360ggctttcatc cgattgcggc
aaatgataac cacgcatttg tcgtccggcg tcccggctcc 420actacggtta acggcacatt
ggtgcccggg ttgaaaagcc tcgtgttggg tggcagaaaa 480gctgttaaac agggagtggt
aaaccttgtc aaatatgcca aataa 5259372DNAPorcine
reproductive and respiratory syndrome virus 9atgccaaata acaacggcaa
gcagcaaaag aaaaagaagg ggaatggcca gccagtcaat 60cagctgtgcc agatgctggg
taaaatcatc gcccagcaaa accagtccag aggcaaggga 120ccgggcaaga aaagtaagaa
gaaaaacccg gagaagcccc attttcctct agcgaccgaa 180gatgacgtca ggcatcactt
cacccctggt gagcggcaat tgtgtctgtc gtcgatccag 240actgccttta accagggcgc
tggaacttgt accctgtcag attcagggag gataagttac 300actgtggagt ttagtttgcc
gacgcatcat actgtgcgcc tgatccgcgt cacagcatca 360ccctcagcat ga
3721018DNAPorcine
reproductive and respiratory syndrome virus 10acagtttggt gatctatg
181117DNAPorcine reproductive
and respiratory syndrome virus 11cagattcaga tgttcaa
171224DNAPorcine reproductive and
respiratory syndrome virus 12acctcgtgct gtatgccgaa tctc
241324DNAPorcine reproductive and respiratory
syndrome virus 13tcaggcctaa agttggttca atga
241424DNAPorcine reproductive and respiratory syndrome virus
14gatgactggg ctactgacga ggat
241521DNAPorcine reproductive and respiratory syndrome virus 15agagcggctg
ggatgacact g
211622DNAPorcine reproductive and respiratory syndrome virus 16ccggggaagc
cagacgattg aa
221724DNAPorcine reproductive and respiratory syndrome virus 17agggggagca
aagaaggggt catc
241820DNAPorcine reproductive and respiratory syndrome virus 18agcacgctct
ggtgcaactg
201919DNAPorcine reproductive and respiratory syndrome virus 19gccgcggcgt
agtattcag
192022DNAPorcine reproductive and respiratory syndrome virus 20cgcgtcacag
catcaccctc ag
222125DNAPorcine reproductive and respiratory syndrome virus 21cggtaggttg
gttaacacat gagtt
252223DNAPorcine reproductive and respiratory syndrome virus 22tggctcttcg
ggcctataaa ata
232356DNAPorcine reproductive and respiratory syndrome virus 23ctagattaat
taatacgact cactataggg atgacgtata ggtgttggct ctatgc
562456DNAPorcine reproductive and respiratory syndrome virus 24taattaatta
tgctgagtga tatccctact gcatatccac aaccgagata cggtgc
562527DNAPorcine reproductive and respiratory syndrome virus 25actcagtcta
agtgctggaa agttatg
272659DNAPorcine reproductive and respiratory syndrome virus 26gggatttaaa
tatgcatttt tttttttttt tttttttaat tgcggccgca tggttctcg
592736DNAPorcine reproductive and respiratory syndrome virus 27attagatctt
gccaccatgg tggggaaatg cttgac
362846DNAPorcine reproductive and respiratory syndrome virus 28ctttacgcgt
ttgcttaagt tatttggcgt atttgacaag gtttac
462931DNAPorcine reproductive and respiratory syndrome virus 29caacacgcgt
cagcaaaaga aaaagaaggg g
313020DNAPorcine reproductive and respiratory syndrome virus 30gcgcgttggc
cgattcatta
203137DNAPorcine reproductive and respiratory syndrome virus 31ctcgttaatt
aaaccgtcat gacgtatagg tgttggc
37323796DNAPorcine reproductive and respiratory syndrome virus
32gaattcgagc ttgcatgcct gcaggtcgtt acataactta cggtaaatgg cccgcctggc
60tgaccgccca acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg
120ccaataggga ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg
180gcagtacatc aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa
240tggcccgcct ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac
300atctacgtat tagtcatcgc tattaccatg gtgatgcggt tttggcagta catcaatggg
360cgtggatagc ggtttgactc acggggattt ccaagtctcc accccattga cgtcaatggg
420agtttgtttt ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca
480ttgacgcaaa tgggcggtag gcgtgtacgg tgggaggtct atataagcag agctcgttta
540gtgaaccgtc agatcgcctg gagacgccat ccacgctgtt ttgacctcca tagaagacac
600cgggaccgat ccagcctccg gactctagag gatccggtac tcgaggaact gaaaaaccag
660aaagttaact ggtaagttta gtctttttgt cttttatttc aggtcccgga tccggtggtg
720gtgcaaatca aagaactgct cctcagtgga tgttgccttt acttctaggc ctgtacggaa
780gtgttacttc tgctctaaaa gctgcggaat tgtacccgcg gccgcaagat atcgccctag
840gaagatctcg atcgattggt accaatccgc gaccccttaa ttaacagcta gcggatttaa
900atcagggccc gggatactag tgagcggccg cggggatcca gacatgataa gatacattga
960tgagtttgga caaaccacaa ctagaatgca gtgaaaaaaa tgctttattt gtgaaatttg
1020tgatgctatt gctttatttg taaccattat aagctgcaat aaacaagtta acaacaacaa
1080ttgcattcat tttatgtttc aggttcaggg ggaggtgtgg gaggtttttt cggatcctct
1140agagtcgacc tgcaggcatg caagcttggc gtaatcatgg tcatagctgt ttcctgtgtg
1200aaattgttat ccgctcacaa ttccacacaa catacgagcc ggaagcataa agtgtaaagc
1260ctggggtgcc taatgagtga gctaactcac attaattgcg ttgcgctcac tgcccgcttt
1320ccagtcggga aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg cggggagagg
1380cggtttgcgt attgggcgct cttccgcttc ctcgctcact gactcgctgc gctcggtcgt
1440tcggctgcgg cgagcggtat cagctcactc aaaggcggta atacggttat ccacagaatc
1500aggggataac gcaggaaaga acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa
1560aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa
1620tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc
1680ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc
1740cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta ggtatctcag
1800ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga
1860ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc
1920gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac
1980agagttcttg aagtggtggc ctaactacgg ctacactaga aggacagtat ttggtatctg
2040cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca
2100aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa
2160aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa
2220ctcacgttaa gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt
2280aaattaaaaa tgaagtttta aatcaatcta aagtatatat gagtaaactt ggtctgacag
2340ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat
2400agttgcctga ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc
2460cagtgctgca atgataccgc gagacccacg ctcaccggct ccagatttat cagcaataaa
2520ccagccagcc ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca
2580gtctattaat tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa
2640cgttgttgcc attgctacag gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt
2700cagctccggt tcccaacgat caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc
2760ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact
2820catggttatg gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc
2880tgtgactggt gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg
2940ctcttgcccg gcgtcaatac gggataatac cgcgccacat agcagaactt taaaagtgct
3000catcattgga aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc
3060cagttcgatg taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag
3120cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac
3180acggaaatgt tgaatactca tactcttcct ttttcaatat tattgaagca tttatcaggg
3240ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt
3300tccgcgcaca tttccccgaa aagtgccacc tgacgtctaa gaaaccatta ttatcatgac
3360attaacctat aaaaataggc gtatcacgag gccctttcgt ctcgcgcgtt tcggtgatga
3420cggtgaaaac ctctgacaca tgcagctccc ggagacggtc acagcttgtc tgtaagcgga
3480tgccgggagc agacaagccc gtcagggcgc gtcagcgggt gttggcgggt gtcggggctg
3540gcttaactat gcggcatcag agcagattgt actgagagtg caccatatgc ggtgtgaaat
3600accgcacaga tgcgtaagga gaaaataccg catcaggcgc cattcgccat tcaggctgcg
3660caactgttgg gaagggcgat cggtgcgggc ctcttcgcta ttacgccagc tggcgaaagg
3720gggatgtgct gcaaggcgat taagttgggt aacgccaggg ttttcccagt cacgacgttg
3780taaaacgacg gccagt
37963322DNAPorcine reproductive and respiratory syndrome virus
33cgttaattaa accgactagt gc
223430DNAPorcine reproductive and respiratory syndrome virus 34tcgagcaatt
aatttggctg atcacgccgg
303524DNAPorcine reproductive and respiratory syndrome virus 35cggggacggt
ttcaaatttc actt
243650DNAPorcine reproductive and respiratory syndrome virus 36tatataagca
gagctcgtta attaaaccgt catgacgtat aggtgttggc
503748DNAPorcine reproductive and respiratory syndrome virus 37aggtcgacgg
cggcaattgg tttcacctag agtggctgcg tcccttct
483830DNAPorcine reproductive and respiratory syndrome virus 38tcttaagcat
tggctgtgat ggtgatatac
303944DNAPorcine reproductive and respiratory syndrome virus 39cttcttaagt
ccacgcgttt tcttcttgcc ttttctatgc ttct
444019DNAPorcine reproductive and respiratory syndrome virus 40tgcccggtcc
cttgcctct
194126DNAPorcine reproductive and respiratory syndrome virus 41gtttacgcgt
cgctccttgg tggtcg
264223DNAPorcine reproductive and respiratory syndrome virus 42aacagaagag
ttgtcgggtc cac
234349DNAPorcine reproductive and respiratory syndrome virus 43gctttgacgc
gtccccactt aagttcaatt caggcctaaa gttggttca
494482DNAPorcine reproductive and respiratory syndrome virus 44gcgacgcgtg
ttccgtggca acccctttaa ccagagtttc agcggaacaa tgaaatgggg 60tctatacaaa
gcctcttcga ca
824526DNAPorcine reproductive and respiratory syndrome virus 45aacagaacgg
cacgatacac cacaaa
264624DNAPorcine reproductive and respiratory syndrome virus 46caaagggcga
aaaaccgtct atca
244723DNAPorcine reproductive and respiratory syndrome virus 47ccccacttaa
gttcaattca ggc
234837DNAPorcine reproductive and respiratory syndrome virus 48gctccttaag
aacaacgcgt cgccattgaa gccgaga
374924DNAPorcine reproductive and respiratory syndrome virus 49gcaagcctaa
taacgaagca aatc
245042DNAPorcine reproductive and respiratory syndrome virus 50ctgtcaagtg
ttaggatcac gcgtccaaaa tactcagctc aa
425142DNAPorcine reproductive and respiratory syndrome virus 51ttgagctgag
tattttggac gcgtgatcct aacacttgac ag
425233DNAPorcine reproductive and respiratory syndrome virus 52gccacgcgtg
ccaccatggt gagcaagggc gag
335331DNAPorcine reproductive and respiratory syndrome virus 53gccacgcgtg
ttatctagat ccggtggatc c
315440DNAPorcine reproductive and respiratory syndrome virus 54tcaagccgct
ggcggctagc agtttaaaca ctgctcctta
405541DNAPorcine reproductive and respiratory syndrome virus 55ctcgggaaaa
tagaaaacac cggtgagatg atcaaccagg g
415641DNAPorcine reproductive and respiratory syndrome virus 56ccctggttga
tcatctcacc ggtgttttct attttcccga g
415740DNAPorcine reproductive and respiratory syndrome virus 57gccgcccacc
ggtgaggtga gcaagggcga ggagctgttc
405837DNAPorcine reproductive and respiratory syndrome virus 58gccgcccacc
ggtgttgtta tctagatccg gtggatc
375930DNAPorcine reproductive and respiratory syndrome virus 59gccacgcgtg
tgagcaaggg cgaggagctg
306033DNAPorcine reproductive and respiratory syndrome virus 60cgcgtatacc
catacgacgt cccagactac gca
336133DNAPorcine reproductive and respiratory syndrome virus 61ccggtgcgta
gtctgggacg tcgtatgggt ata
336239DNAPorcine reproductive and respiratory syndrome virus 62ccctgtcaag
tgttaagatc acaggtgaga tgatcaacc
396341DNAPorcine reproductive and respiratory syndrome virus 63ccctggttga
tcatctcacc tgtgatctta acacttgaca g
4164132PRTPorcine reproductive and respiratory syndrome virus 64Arg Pro
Lys Tyr Ser Ala Gln Ala Ile Ile Asp Leu Gly Gly Pro Cys1 5
10 15Ser Gly His Leu Gln Arg Glu Lys
Glu Ala Cys Leu Arg Ile Met Arg 20 25
30Glu Ala Cys Asp Ala Ala Lys Leu Ser Asp Pro Ala Thr Gln Glu
Trp 35 40 45Leu Ser Arg Met Trp
Asp Arg Val Asp Met Leu Thr Trp Arg Asn Thr 50 55
60Ser Ala Tyr Gln Ala Phe Arg Thr Leu Asp Gly Arg Phe Gly
Phe Leu65 70 75 80Pro
Lys Met Ile Leu Glu Thr Pro Pro Pro Tyr Pro Cys Gly Phe Val
85 90 95Met Leu Pro His Thr Pro Ala
Pro Ser Val Ser Ala Glu Ser Asp Leu 100 105
110Thr Ile Gly Ser Val Ala Thr Glu Asp Ile Pro Arg Ile Leu
Gly Lys 115 120 125Ile Glu Asn Thr
13065132PRTPorcine reproductive and respiratory syndrome virus 65Arg
Pro Lys Tyr Ser Ala Gln Ala Ile Ile Asp Ser Gly Gly Pro Cys1
5 10 15Ser Gly His Leu Gln Glu Val
Lys Glu Thr Cys Leu Ser Val Met Arg 20 25
30Glu Ala Cys Asp Ala Thr Lys Leu Asp Asp Pro Ala Thr Gln
Glu Trp 35 40 45Leu Ser Arg Met
Trp Asp Arg Val Asp Met Leu Thr Trp Arg Asn Thr 50 55
60Ser Val Tyr Gln Ala Ile Cys Thr Leu Asp Gly Arg Leu
Lys Phe Leu65 70 75
80Pro Lys Met Ile Leu Glu Thr Pro Pro Pro Tyr Pro Cys Glu Phe Val
85 90 95Met Met Pro His Thr Pro
Ala Pro Ser Val Gly Ala Glu Ser Asp Leu 100
105 110Thr Ile Gly Ser Val Ala Thr Glu Asp Val Pro Arg
Ile Leu Glu Lys 115 120 125Ile Glu
Asn Val 13066132PRTPorcine reproductive and respiratory syndrome virus
66Arg Pro Lys Tyr Ser Ala Gln Ala Ile Ile Asp Ser Gly Gly Pro Cys1
5 10 15Ser Gly His Leu Gln Lys
Glu Lys Glu Ala Cys Leu Ser Ile Met Arg 20 25
30Glu Ala Cys Asp Ala Ser Lys Leu Ser Asp Pro Ala Thr
Gln Glu Trp 35 40 45Leu Ser Arg
Met Trp Asp Arg Val Asp Met Leu Thr Trp Arg Asn Thr 50
55 60Ser Ala Tyr Gln Ala Phe Arg Thr Leu Asn Gly Arg
Phe Glu Phe Leu65 70 75
80Pro Lys Met Ile Leu Glu Thr Pro Pro Pro His Pro Cys Gly Phe Val
85 90 95Met Leu Pro His Thr Pro
Ala Pro Ser Val Ser Ala Glu Ser Asp Leu 100
105 110Thr Ile Gly Ser Val Ala Thr Glu Asp Val Pro Arg
Ile Leu Gly Lys 115 120 125Ile Gly
Asp Thr 13067132PRTPorcine reproductive and respiratory syndrome virus
67Arg Pro Lys Tyr Ser Ala Gln Ala Ile Ile Asp Ser Gly Gly Pro Cys1
5 10 15Ser Gly His Leu Gln Lys
Glu Lys Glu Ala Cys Leu Ser Ile Met Arg 20 25
30Glu Ala Cys Asp Ala Ser Lys Leu Ser Asp Pro Ala Thr
Gln Glu Trp 35 40 45Leu Ser Arg
Met Trp Asp Arg Val Asp Met Leu Thr Trp Arg Asn Thr 50
55 60Ser Ala Tyr Gln Ala Phe Arg Thr Leu Asn Gly Arg
Phe Glu Phe Leu65 70 75
80Pro Lys Met Ile Leu Glu Thr Pro Pro Pro His Pro Cys Gly Phe Val
85 90 95Met Leu Pro His Thr Pro
Ala Pro Ser Val Ser Ala Glu Ser Asp Leu 100
105 110Thr Ile Gly Ser Val Ala Thr Glu Asp Val Pro Arg
Ile Leu Gly Lys 115 120 125Ile Gly
Asp Thr 13068132PRTPorcine reproductive and respiratory syndrome virus
68Arg Pro Lys Tyr Ser Ala Gln Ala Ile Ile Asp Ser Gly Gly Pro Cys1
5 10 15Ser Gly His Leu Gln Glu
Val Lys Glu Ala Cys Leu Ser Val Met Arg 20 25
30Glu Ala Cys Asp Ala Thr Lys Leu Asp Asp Pro Ala Thr
Gln Glu Trp 35 40 45Leu Ser Arg
Met Trp Asp Arg Val Asp Met Leu Thr Trp Arg Asn Thr 50
55 60Ser Val Tyr Gln Ala Ile Cys Thr Leu Asp Gly Arg
Leu Lys Phe Leu65 70 75
80Pro Lys Met Ile Leu Glu Thr Pro Pro Pro Tyr Pro Cys Glu Phe Val
85 90 95Met Met Pro His Thr Pro
Ala Pro Ser Val Gly Ala Glu Ser Asp Leu 100
105 110Thr Ile Gly Ser Val Ala Thr Glu Asp Val Pro Arg
Ile Leu Glu Lys 115 120 125Ile Glu
Asn Val 13069132PRTPorcine reproductive and respiratory syndrome virus
69Arg Pro Lys Tyr Ser Ala Gln Ala Ile Ile Asp Ser Gly Gly Pro Cys1
5 10 15Ser Gly His Leu Gln Glu
Val Lys Glu Thr Cys Leu Ser Val Met Arg 20 25
30Glu Ala Cys Asp Ala Thr Lys Leu Asp Asp Pro Ala Thr
Gln Glu Trp 35 40 45Leu Ser Arg
Met Trp Asp Arg Val Asp Met Leu Thr Trp Arg Asn Thr 50
55 60Ser Ala Tyr Gln Ala Ile Cys Thr Leu Asp Gly Arg
Leu Lys Phe Leu65 70 75
80Pro Lys Met Ile Leu Glu Thr Pro Pro Pro Tyr Pro Cys Glu Phe Val
85 90 95Met Met Pro His Thr Pro
Ala Pro Ser Val Gly Ala Glu Ser Asp Leu 100
105 110Thr Ile Gly Ser Val Ala Thr Glu Asp Val Pro Arg
Ile Leu Glu Lys 115 120 125Met Glu
Asn Val 13070132PRTPorcine reproductive and respiratory syndrome virus
70Arg Pro Lys Tyr Ser Ala Gln Ala Ile Ile Asp Ser Gly Gly Pro Cys1
5 10 15Ser Gly His Leu Gln Glu
Val Lys Glu Thr Cys Leu Ser Val Met Arg 20 25
30Glu Ala Cys Asp Ala Thr Lys Leu Asp Asp Pro Ala Thr
Gln Glu Trp 35 40 45Leu Ser Arg
Met Trp Asp Arg Val Asp Met Leu Thr Trp Arg Asn Thr 50
55 60Ser Val Tyr Gln Ala Ile Cys Thr Leu Asp Gly Arg
Leu Lys Phe Leu65 70 75
80Pro Lys Met Ile Leu Glu Thr Pro Pro Pro Tyr Pro Cys Glu Phe Val
85 90 95Met Met Pro His Thr Pro
Ala Pro Ser Val Gly Ala Glu Ser Asp Leu 100
105 110Thr Ile Gly Ser Val Ala Thr Glu Asp Val Pro Arg
Ile Leu Glu Lys 115 120 125Ile Glu
Asn Val 13071132PRTPorcine reproductive and respiratory syndrome virus
71Arg Pro Lys Tyr Ser Ala Gln Ala Ile Ile Asp Ser Gly Gly Pro Cys1
5 10 15Ser Gly His Leu Gln Glu
Val Lys Glu Thr Cys Leu Ser Val Met Arg 20 25
30Glu Ala Cys Asp Ala Thr Lys Leu Asp Asp Pro Ala Thr
Gln Glu Trp 35 40 45Leu Ser Arg
Met Trp Asp Arg Val Asp Met Leu Thr Trp Arg Asn Thr 50
55 60Ser Val Tyr Gln Ala Ile Cys Thr Leu Asp Gly Arg
Leu Lys Phe Leu65 70 75
80Pro Lys Met Ile Leu Glu Thr Pro Pro Pro Tyr Pro Cys Glu Phe Val
85 90 95Met Met Pro His Thr Pro
Ala Pro Ser Val Gly Ala Glu Ser Asp Leu 100
105 110Thr Ile Gly Ser Val Ala Thr Glu Asp Val Pro Arg
Ile Leu Glu Lys 115 120 125Ile Glu
Asn Val 13072132PRTPorcine reproductive and respiratory syndrome virus
72Arg Pro Lys Tyr Ser Ala Gln Ala Ile Ile Asp Ser Gly Gly Pro Cys1
5 10 15Ser Gly His Leu Gln Glu
Val Lys Glu Thr Cys Leu Ser Val Met Arg 20 25
30Glu Ala Cys Asp Ala Thr Lys Leu Asp Asp Pro Ala Thr
Gln Glu Trp 35 40 45Leu Ser Arg
Met Trp Asp Arg Val Asp Met Leu Thr Trp Arg Asn Thr 50
55 60Ser Val Tyr Gln Ala Ile Cys Thr Leu Asp Gly Arg
Leu Lys Phe Leu65 70 75
80Pro Lys Met Ile Leu Glu Thr Pro Pro Pro Tyr Pro Cys Glu Phe Val
85 90 95Met Met Pro His Thr Pro
Ala Pro Ser Val Gly Ala Glu Ser Asp Leu 100
105 110Thr Ile Gly Ser Val Ala Thr Glu Asp Val Pro Arg
Ile Leu Glu Lys 115 120 125Ile Glu
Asn Val 13073132PRTPorcine reproductive and respiratory syndrome virus
73Arg Pro Lys Tyr Ser Ala Gln Ala Ile Ile Asp Ser Gly Gly Pro Cys1
5 10 15Ser Gly His Leu Gln Glu
Val Lys Glu Thr Cys Leu Ser Val Met Arg 20 25
30Glu Ala Cys Asp Ala Thr Lys Leu Asp Asp Pro Ala Thr
Gln Glu Trp 35 40 45Leu Ser Arg
Met Trp Asp Arg Val Asp Met Leu Thr Trp Arg Asn Thr 50
55 60Ser Val Tyr Gln Ala Ile Cys Thr Leu Asp Gly Arg
Leu Lys Phe Leu65 70 75
80Pro Lys Met Ile Leu Glu Thr Pro Pro Pro Tyr Pro Cys Glu Phe Val
85 90 95Met Met Pro His Thr Pro
Ala Pro Ser Val Gly Ala Glu Ser Asp Leu 100
105 110Thr Ile Gly Ser Val Ala Thr Glu Asp Val Pro Arg
Ile Leu Glu Lys 115 120 125Ile Glu
Asn Val 13074132PRTPorcine reproductive and respiratory syndrome virus
74Arg Pro Lys Tyr Ser Ala Gln Ala Ile Ile Asp Ser Gly Gly Pro Cys1
5 10 15Ser Gly His Leu Gln Glu
Val Lys Glu Thr Cys Leu Ser Val Met Arg 20 25
30Glu Ala Cys Asp Ala Thr Lys Leu Asp Asp Pro Ala Thr
Gln Glu Trp 35 40 45Leu Ser Arg
Met Trp Asp Arg Val Asp Met Leu Thr Trp Arg Asn Thr 50
55 60Ser Val Tyr Gln Ala Ile Cys Thr Leu Asp Gly Arg
Leu Lys Phe Leu65 70 75
80Pro Lys Met Ile Leu Glu Thr Pro Pro Pro Tyr Pro Cys Glu Phe Val
85 90 95Met Met Pro His Thr Pro
Ala Pro Ser Val Gly Ala Glu Ser Asp Leu 100
105 110Thr Ile Gly Ser Val Ala Thr Glu Asp Val Pro Arg
Ile Leu Glu Lys 115 120 125Ile Glu
Asn Val 13075132PRTPorcine reproductive and respiratory syndrome virus
75Arg Pro Lys Tyr Ser Ala Gln Ala Ile Ile Asp Ser Gly Gly Pro Cys1
5 10 15Ser Gly His Leu Gln Glu
Val Lys Glu Thr Cys Leu Ser Val Met Arg 20 25
30Glu Ala Cys Asp Ala Thr Lys Leu Asp Asp Pro Ala Thr
Gln Glu Trp 35 40 45Leu Ser Arg
Met Trp Asp Arg Val Asp Met Leu Thr Trp Arg Asn Thr 50
55 60Ser Val Tyr Gln Ala Ile Cys Thr Leu Asp Gly Arg
Leu Lys Phe Leu65 70 75
80Pro Lys Met Ile Leu Glu Thr Pro Pro Pro Tyr Pro Cys Glu Phe Val
85 90 95Met Met Pro His Thr Pro
Ala Pro Ser Val Gly Ala Glu Ser Asp Leu 100
105 110Thr Ile Gly Ser Val Ala Thr Glu Asp Val Pro Arg
Ile Leu Glu Lys 115 120 125Ile Glu
Asn Val 13076132PRTPorcine reproductive and respiratory syndrome virus
76Arg Pro Lys Tyr Ser Ala Gln Ala Ile Ile Asp Ser Gly Gly Pro Cys1
5 10 15Ser Gly His Leu Gln Glu
Val Lys Glu Thr Cys Leu Ser Val Met Arg 20 25
30Glu Ala Cys Asp Ala Thr Lys Leu Asp Asp Pro Ala Thr
Gln Glu Trp 35 40 45Leu Ser Arg
Met Trp Asp Arg Val Asp Met Leu Thr Trp Arg Asn Thr 50
55 60Ser Val Tyr Gln Ala Ile Cys Thr Leu Asp Gly Arg
Leu Lys Phe Leu65 70 75
80Pro Lys Met Ile Leu Glu Thr Pro Pro Pro Tyr Pro Cys Glu Phe Val
85 90 95Met Met Pro His Thr Pro
Ala Pro Ser Val Gly Ala Glu Ser Asp Leu 100
105 110Thr Ile Gly Ser Val Ala Thr Glu Asp Val Pro Arg
Ile Leu Glu Lys 115 120 125Ile Glu
Asn Val 13077132PRTPorcine reproductive and respiratory syndrome virus
77Arg Pro Lys Tyr Ser Ala Gln Ala Ile Ile Asp Ser Gly Gly Pro Cys1
5 10 15Ser Gly His Leu Gln Glu
Val Lys Glu Thr Cys Leu Ser Val Met Arg 20 25
30Glu Ala Cys Asp Ala Thr Lys Leu Asp Asp Pro Ala Thr
Gln Glu Trp 35 40 45Leu Ser Arg
Met Trp Asp Arg Val Asp Met Leu Thr Trp Arg Asn Thr 50
55 60Ser Val Tyr Gln Ala Ile Cys Thr Leu Asp Gly Arg
Leu Lys Phe Leu65 70 75
80Pro Lys Met Ile Leu Glu Thr Pro Pro Pro Tyr Pro Cys Glu Phe Val
85 90 95Met Met Pro His Thr Pro
Ala Pro Ser Val Gly Ala Glu Ser Asp Leu 100
105 110Thr Ile Gly Ser Val Ala Thr Glu Asp Val Pro Arg
Ile Leu Glu Lys 115 120 125Ile Glu
Asn Val 13078132PRTPorcine reproductive and respiratory syndrome virus
78Arg Pro Lys Tyr Ser Ala Gln Ala Ile Ile Asp Ser Gly Gly Pro Cys1
5 10 15Ser Gly His Leu Gln Glu
Val Lys Glu Thr Cys Leu Ser Val Met Arg 20 25
30Glu Ala Cys Asp Ala Thr Lys Leu Asp Asp Pro Ala Thr
Gln Glu Trp 35 40 45Leu Ser Arg
Met Trp Asp Arg Val Asp Met Leu Thr Trp Arg Asn Thr 50
55 60Ser Val Tyr Gln Ala Ile Cys Thr Leu Asp Gly Arg
Leu Lys Phe Leu65 70 75
80Pro Lys Met Ile Leu Glu Thr Pro Pro Pro Tyr Pro Cys Glu Phe Val
85 90 95Met Met Pro His Thr Pro
Ala Pro Ser Val Gly Ala Glu Ser Asp Leu 100
105 110Thr Ile Gly Ser Val Ala Thr Glu Asp Val Pro Arg
Ile Leu Glu Lys 115 120 125Ile Glu
Asn Val 13079132PRTPorcine reproductive and respiratory syndrome virus
79Arg Pro Lys Tyr Ser Ala Gln Ala Ile Ile Asp Ser Gly Gly Pro Cys1
5 10 15Ser Gly His Leu Gln Glu
Val Lys Glu Thr Cys Leu Ser Val Met Arg 20 25
30Glu Ala Cys Asp Ala Thr Lys Leu Asp Asp Pro Ala Thr
Gln Glu Trp 35 40 45Leu Ser Arg
Met Trp Asp Arg Val Asp Met Leu Thr Trp Arg Asn Thr 50
55 60Ser Val Tyr Gln Ala Ile Cys Thr Leu Asp Gly Arg
Leu Lys Phe Leu65 70 75
80Pro Lys Met Ile Leu Glu Thr Pro Pro Pro Tyr Pro Cys Glu Phe Val
85 90 95Met Met Pro His Thr Pro
Ala Pro Ser Val Gly Ala Glu Ser Asp Leu 100
105 110Thr Ile Gly Ser Val Ala Thr Glu Asp Val Pro Arg
Ile Leu Glu Lys 115 120 125Ile Glu
Asn Val 13080132PRTPorcine reproductive and respiratory syndrome virus
80Arg Pro Lys Tyr Ser Ala Gln Ala Ile Ile Asp Ser Gly Gly Pro Cys1
5 10 15Ser Gly His Leu Gln Gly
Val Lys Glu Thr Cys Leu Ser Val Met Arg 20 25
30Glu Ala Cys Asp Ala Thr Lys Leu Asp Asp Pro Ala Thr
Gln Glu Trp 35 40 45Leu Ser Arg
Met Trp Asp Arg Val Asp Met Leu Thr Trp Arg Asn Thr 50
55 60Ser Val Cys Gln Ala Ile Arg Thr Leu Asp Gly Arg
Leu Lys Phe Leu65 70 75
80Pro Lys Met Ile Leu Glu Thr Pro Pro Pro Tyr Pro Cys Glu Phe Val
85 90 95Met Met Pro His Thr Pro
Ala Pro Ser Val Gly Ala Glu Ser Asp Leu 100
105 110Thr Ile Gly Ser Val Ala Thr Glu Asp Val Pro Arg
Ile Leu Glu Lys 115 120 125Ile Glu
Asn Val 13081132PRTPorcine reproductive and respiratory syndrome virus
81Arg Pro Lys Tyr Ser Ala Gln Ala Ile Ile Asp Ser Gly Gly Pro Cys1
5 10 15Ser Gly His Leu Gln Gly
Val Lys Glu Thr Cys Leu Ser Val Met Arg 20 25
30Glu Ala Cys Asp Ala Thr Lys Leu Asp Asp Pro Ala Thr
Gln Glu Trp 35 40 45Leu Ser Arg
Met Trp Asp Arg Val Asp Met Leu Thr Trp Arg Asn Thr 50
55 60Ser Val Cys Gln Ala Ile Arg Thr Leu Asp Gly Arg
Leu Lys Phe Leu65 70 75
80Pro Lys Met Ile Leu Glu Thr Pro Pro Pro Tyr Pro Cys Glu Phe Val
85 90 95Met Met Pro His Thr Pro
Ala Pro Ser Val Gly Ala Glu Ser Asp Leu 100
105 110Thr Ile Gly Ser Val Ala Thr Glu Asp Val Pro Arg
Ile Leu Glu Lys 115 120 125Ile Glu
Asn Val 13082132PRTPorcine reproductive and respiratory syndrome virus
82Arg Pro Lys Tyr Ser Ala Gln Ala Ile Ile Asp Ser Gly Gly Pro Cys1
5 10 15Ser Gly His Leu Gln Glu
Val Lys Glu Thr Cys Leu Ser Val Met Arg 20 25
30Glu Ala Cys Asp Ala Thr Lys Leu Asp Asp Pro Ala Thr
Gln Glu Trp 35 40 45Leu Ser Arg
Met Trp Asp Arg Val Asp Met Leu Thr Trp Arg Asn Thr 50
55 60Ser Val Tyr Gln Ala Ile Cys Thr Leu Asn Gly Arg
Leu Lys Phe Leu65 70 75
80Pro Lys Met Ile Leu Glu Thr Pro Pro Pro Tyr Pro Cys Glu Phe Val
85 90 95Met Met Pro His Thr Pro
Ala Pro Ser Val Gly Ala Glu Ser Asp Leu 100
105 110Thr Ile Gly Ser Val Ala Thr Glu Asp Val Pro Arg
Ile Leu Glu Lys 115 120 125Ile Glu
Asn Val 13083132PRTPorcine reproductive and respiratory syndrome virus
83Arg Pro Thr Tyr Ser Ala Gln Ala Ile Ile Asp Ser Gly Gly Pro Cys1
5 10 15Ser Gly His Leu Gln Glu
Val Lys Glu Thr Cys Leu Ser Val Met Arg 20 25
30Glu Ala Cys Asp Ala Thr Lys Leu Asp Asp Pro Ala Thr
Gln Glu Trp 35 40 45Leu Ser Arg
Met Trp Asp Arg Val Asp Met Leu Thr Trp Arg Asn Thr 50
55 60Ser Val Tyr Gln Ala Ile Cys Thr Leu Asp Gly Arg
Leu Lys Phe Leu65 70 75
80Pro Lys Met Ile Leu Glu Thr Pro Pro Pro Tyr Pro Cys Glu Phe Val
85 90 95Met Met Pro His Thr Pro
Ala Pro Ser Val Gly Ala Glu Ser Asp Leu 100
105 110Thr Ile Gly Ser Val Ala Thr Glu Asp Val Pro Arg
Ile Leu Glu Lys 115 120 125Ile Glu
Asn Val 13084132PRTPorcine reproductive and respiratory syndrome virus
84Arg Pro Lys Tyr Ser Ala Gln Ala Ile Ile Asp Ser Gly Gly Pro Cys1
5 10 15Ser Gly His Leu Gln Glu
Val Lys Glu Thr Cys Leu Ser Val Met Arg 20 25
30Glu Ala Cys Asp Ala Thr Lys Leu Asp Asp Pro Ala Thr
Gln Glu Trp 35 40 45Leu Ser Arg
Met Trp Asp Arg Val Asp Met Leu Thr Trp Arg Asn Thr 50
55 60Ser Val Tyr Gln Val Ile Cys Thr Leu Asp Gly Met
Leu Lys Phe Leu65 70 75
80Pro Lys Met Ile Leu Glu Thr Pro Pro Pro Tyr Pro Cys Glu Phe Val
85 90 95Met Met Pro His Thr Pro
Ala Pro Ser Val Gly Ala Glu Ser Asp Leu 100
105 110Thr Ile Gly Ser Val Thr Thr Glu Asp Val Pro Arg
Ile Leu Glu Lys 115 120 125Ile Gly
Asn Val 13085132PRTPorcine reproductive and respiratory syndrome virus
85Arg Pro Lys Tyr Ser Ala Gln Ala Ile Ile Asp Ser Gly Gly Pro Tyr1
5 10 15Ser Gly His Leu Gln Glu
Val Lys Glu Thr Cys Leu Ser Ile Met Ser 20 25
30Glu Ala Cys Asp Val Thr Lys Leu Asp Asp Pro Ala Thr
Gln Glu Trp 35 40 45Leu Ser Arg
Met Trp Asp Arg Val Asp Met Leu Thr Trp Arg Asn Thr 50
55 60Ser Val His Gln Ala Ser Arg Thr Leu Asp Asp Arg
Phe Lys Phe Leu65 70 75
80Pro Lys Met Ile Leu Glu Thr Pro Pro Pro Tyr Pro Cys Gly Phe Val
85 90 95Met Met Pro Arg Thr Pro
Ala Pro Ser Val Gly Ala Glu Ser Asp Leu 100
105 110Thr Ile Gly Ser Val Ala Thr Glu Asp Val Pro Arg
Ile Phe Gly Lys 115 120 125Val Asn
Asp Val 13086132PRTPorcine reproductive and respiratory syndrome virus
86Arg Pro Lys Tyr Ser Ala Gln Ala Ile Ile Asp Ser Gly Gly Pro Tyr1
5 10 15Ser Gly His Leu Gln Glu
Val Lys Glu Thr Cys Leu Ser Ile Met Ser 20 25
30Glu Ala Cys Asp Val Thr Lys Leu Asp Asp Pro Ala Thr
Gln Glu Trp 35 40 45Leu Ser Arg
Met Trp Asp Arg Val Asp Met Leu Thr Trp Arg Asn Thr 50
55 60Ser Val His Gln Ala Ser Arg Thr Leu Asp Asp Arg
Phe Lys Phe Leu65 70 75
80Pro Lys Met Ile Leu Glu Thr Pro Pro Pro Tyr Pro Cys Gly Phe Val
85 90 95Met Met Pro Arg Thr Pro
Ala Pro Ser Val Gly Ala Glu Ser Asp Leu 100
105 110Thr Ile Gly Ser Val Ala Thr Glu Asp Val Pro Arg
Ile Phe Gly Lys 115 120 125Val Asn
Asp Val 13087132PRTPorcine reproductive and respiratory syndrome virus
87Arg Pro Lys His Ser Ala Gln Ala Ile Ile Asp Leu Val Gly Pro Cys1
5 10 15Ser Gly His Leu Gln Glu
Val Lys Glu Ile Cys Leu Ser Val Met Arg 20 25
30Glu Ala Cys Asp Ala Thr Lys Leu Asn Asp Pro Ala Thr
Gln Glu Trp 35 40 45Leu Ser Arg
Met Trp Asp Arg Val Asp Met Leu Thr Trp Arg Asn Thr 50
55 60Ser Ala Tyr Gln Thr Leu Ser Thr Leu Asp Gly Trp
Ser Arg Phe Leu65 70 75
80Pro Lys Leu Ile Leu Glu Thr Pro Pro Pro Tyr Pro Cys Glu Phe Val
85 90 95Met Met Pro Tyr Thr Pro
Ala Pro Ser Val Gly Ala Glu Ser Asp Leu 100
105 110Thr Ile Gly Ser Val Ala Thr Glu Asp Val Pro Arg
Ile Leu Glu Asn 115 120 125Met Glu
Asn Val 13088131PRTPorcine reproductive and respiratory syndrome virus
88Arg Pro Lys Tyr Ser Ala Gln Ala Ile Ile Asp Ser Gly Gly Pro Cys1
5 10 15Ser Gly His Leu Gln Glu
Val Lys Glu Thr Cys Leu Ser Val Met Arg 20 25
30Glu Ala Cys Asp Ala Thr Lys Leu Asp Asp Pro Ala Thr
Gln Glu Trp 35 40 45Leu Ser Arg
Met Trp Asp Arg Val Asp Met Leu Thr Cys Asn Thr Ser 50
55 60Val Tyr Gln Ala Ile Cys Thr Leu Asp Gly Arg Leu
Lys Phe Leu Pro65 70 75
80Lys Leu Ile Leu Glu Thr Pro Pro Pro Tyr Pro Cys Glu Phe Val Met
85 90 95Met Pro His Thr Pro Ala
Pro Ser Val Gly Ala Glu Ser Asp Leu Thr 100
105 110Ile Gly Ser Val Ala Thr Glu Asp Val Pro Arg Ile
Leu Glu Lys Thr 115 120 125Glu Asn
Val 13089132PRTPorcine reproductive and respiratory syndrome virus
89Arg Pro Lys Tyr Ser Ala Gln Ala Ile Ile Asn Ser Gly Gly Pro Cys1
5 10 15Cys Gly His Leu Gln Glu
Val Lys Glu Lys Tyr Leu Asn Val Met Arg 20 25
30Glu Ala Cys Asp Ala Thr Lys Leu Asp Asp Pro Ala Thr
Gln Glu Trp 35 40 45Leu Ser Arg
Met Trp Asp Arg Val Asp Met Leu Thr Trp Arg Asn Thr 50
55 60Ser Ile Phe Gln Ala Pro Phe Thr Leu Ala Asp Lys
Phe Lys Ser Leu65 70 75
80Pro Lys Met Ile Leu Glu Thr Pro Pro Pro Tyr Pro Cys Gly Phe Val
85 90 95Met Met Pro Arg Thr Pro
Ala Pro Ser Val Gly Ala Glu Ser Asp Leu 100
105 110Thr Val Gly Ser Val Ala Thr Glu Asp Val Pro Arg
Ile Leu Gly Lys 115 120 125Val Gln
Gly Val 13090132PRTPorcine reproductive and respiratory syndrome virus
90Arg Pro Lys Tyr Ser Ala Gln Ala Ile Ile Asn Ser Gly Gly Pro Cys1
5 10 15Cys Gly His Leu Gln Glu
Val Lys Glu Lys Tyr Leu Asn Val Met Arg 20 25
30Glu Ala Cys Asp Ala Thr Lys Leu Asp Asp Pro Ala Thr
Gln Glu Trp 35 40 45Leu Ser Arg
Met Trp Asp Arg Val Asp Met Leu Thr Trp Arg Asn Thr 50
55 60Ser Ile Phe Gln Ala Pro Phe Thr Leu Ala Asp Lys
Phe Lys Thr Leu65 70 75
80Pro Lys Met Ile Leu Glu Thr Pro Pro Pro Tyr Pro Cys Gly Phe Val
85 90 95Met Met Pro Arg Thr Pro
Ala Pro Ser Val Gly Ala Glu Ser Asp Leu 100
105 110Thr Val Gly Ser Val Ala Thr Glu Asp Val Pro Arg
Ile Leu Gly Asn 115 120 125Val Gln
Gly Val 13091132PRTPorcine reproductive and respiratory syndrome virus
91Arg Pro Arg Tyr Ser Ala Gln Ala Leu Ile Asp Arg Gly Gly Pro Leu1
5 10 15Ala Ala Val His Ala Lys
Ile Lys Asn Arg Val Tyr Glu Gln Cys Leu 20 25
30Gln Ala Cys Glu Pro Gly Ser Arg Ala Thr Pro Ala Thr
Lys Glu Trp 35 40 45Leu Asp Lys
Met Trp Asp Arg Val Asp Met Lys Thr Trp Arg Cys Thr 50
55 60Ser Gln Phe Gln Ala Gly Arg Ile Leu Ala Ser Leu
Lys Phe Leu Pro65 70 75
80Asp Met Ile Gln Asp Thr Pro Pro Pro Val Pro Lys Lys Asn Arg Ala
85 90 95Ser Asp Asn Ala Gly Leu
Lys Gln Leu Val Ala Arg Trp Asp Lys Lys 100
105 110Leu Ser Val Thr Pro Pro Leu Lys Ser Ala Gly Leu
Ala Leu Asp Gln 115 120 125Thr Val
Pro Pro 13092132PRTPorcine reproductive and respiratory syndrome virus
92Arg Pro Arg Tyr Ser Ala Gln Ala Leu Ile Asp Arg Gly Gly Pro Leu1
5 10 15Ala Asp Val His Ala Lys
Ile Lys Asn Arg Val Tyr Glu Gln Cys Leu 20 25
30Gln Ala Cys Glu Pro Gly Ser Arg Ala Thr Pro Ala Thr
Lys Glu Trp 35 40 45Leu Asp Lys
Met Trp Asp Arg Val Asp Met Lys Thr Trp Arg Cys Thr 50
55 60Ser Gln Phe Gln Ala Gly Arg Ile Leu Ala Ser Leu
Lys Phe Leu Pro65 70 75
80Asp Met Ile Gln Asp Thr Pro Pro Pro Val Pro Arg Lys Asn Arg Ala
85 90 95Ser Asp Asn Ala Gly Leu
Arg Gln Leu Val Ala Arg Trp Asp Lys Lys 100
105 110Leu Ser Val Thr Pro Pro Pro Lys Ser Thr Gly Leu
Val Leu Asp Gln 115 120 125Thr Val
Pro Leu 13093132PRTPorcine reproductive and respiratory syndrome virus
93Arg Pro Arg Tyr Ser Ala Gln Ala Leu Ile Asp Arg Gly Gly Pro Leu1
5 10 15Ala Asp Val His Ala Lys
Ile Lys Asn Arg Val Tyr Glu Gln Cys Leu 20 25
30Gln Ala Cys Glu Pro Gly Ser Arg Ala Thr Pro Ala Thr
Lys Glu Trp 35 40 45Leu Asp Lys
Met Trp Asp Arg Val Asp Met Lys Thr Trp Arg Cys Thr 50
55 60Ser Gln Phe Gln Ala Gly Arg Ile Leu Ala Ser Leu
Lys Phe Leu Pro65 70 75
80Asp Met Ile Gln Asp Thr Pro Pro Pro Val Pro Lys Lys Asn Arg Ala
85 90 95Ser Asp Asn Ala Gly Leu
Lys Gln Leu Val Ala Arg Trp Asp Lys Lys 100
105 110Leu Ser Val Thr Pro Pro Pro Lys Ser Ala Gly Leu
Val Leu Asn Gln 115 120 125Thr Val
Pro Pro 13094132PRTPorcine reproductive and respiratory syndrome virus
94Arg Pro Arg Tyr Ser Ala Gln Ala Leu Ile Asp Arg Gly Gly Pro Leu1
5 10 15Ala Asp Val His Ala Lys
Ile Lys Asn Arg Val Tyr Glu Gln Cys Leu 20 25
30Gln Ala Cys Glu Pro Gly Ser Arg Ala Thr Pro Ala Thr
Lys Glu Trp 35 40 45Leu Asp Lys
Met Trp Asp Arg Val Asp Met Lys Thr Trp Arg Cys Thr 50
55 60Ser Gln Phe Gln Ala Gly Arg Ile Leu Ala Ser Leu
Lys Phe Leu Pro65 70 75
80Asp Met Ile Gln Asp Thr Pro Pro Pro Val Pro Arg Lys Asn Arg Ala
85 90 95Ser Asp Asn Ala Gly Leu
Arg Gln Leu Val Ala Arg Trp Asp Lys Lys 100
105 110Leu Ser Val Thr Pro Pro Pro Lys Ser Ala Gly Leu
Val Leu Asp Gln 115 120 125Thr Val
Pro Pro 13095132PRTPorcine reproductive and respiratory syndrome virus
95Arg Pro Arg Phe Ser Ala Gln Ala Leu Ile Asp Arg Gly Gly Pro Leu1
5 10 15Ala Asp Val His Ala Lys
Ile Lys Asn Arg Val Tyr Glu Gln Cys Leu 20 25
30Gln Ala Cys Glu Pro Gly Ser Arg Ala Thr Pro Ala Thr
Arg Glu Trp 35 40 45Leu Asp Lys
Met Trp Asp Arg Val Asp Met Lys Thr Trp Arg Cys Thr 50
55 60Ser Gln Phe Gln Ala Gly Arg Ile Leu Ala Ser Leu
Lys Phe Leu Pro65 70 75
80Asp Met Ile Gln Asp Thr Pro Pro Pro Val Pro Arg Lys Asn Arg Ala
85 90 95Ser Asp Asn Ala Gly Leu
Lys Gln Leu Val Ala Gln Trp Asp Arg Lys 100
105 110Leu Ser Val Thr Pro Pro Pro Lys Pro Val Gly Pro
Val Leu Asp Gln 115 120 125Ile Val
Pro Pro 13096132PRTPorcine reproductive and respiratory syndrome virus
96Arg Pro Arg Phe Ser Ala Gln Ala Leu Ile Asp Arg Gly Gly Pro Leu1
5 10 15Ala Asp Val His Ala Lys
Ile Lys Asn Arg Val Tyr Glu Gln Cys Leu 20 25
30Gln Ala Cys Glu Pro Gly Ser Arg Ala Thr Pro Ala Thr
Arg Glu Trp 35 40 45Leu Asp Lys
Met Trp Asp Arg Val Asp Met Lys Thr Trp Arg Cys Thr 50
55 60Ser Gln Phe Gln Ala Gly Arg Ile Leu Ala Ser Leu
Lys Phe Leu Pro65 70 75
80Asp Met Ile Gln Asp Thr Pro Pro Pro Val Pro Arg Lys Asn Arg Ala
85 90 95Ser Asp Asn Ala Gly Leu
Lys Gln Leu Val Ala Gln Trp Asp Arg Lys 100
105 110Leu Ser Val Thr Pro Pro Pro Lys Pro Val Gly Pro
Val Leu Asp Gln 115 120 125Ile Val
Pro Pro 13097132PRTPorcine reproductive and respiratory syndrome virus
97Arg Pro Arg Phe Ser Ala Gln Ala Leu Ile Asp Arg Gly Gly Pro Leu1
5 10 15Ala Asp Val His Ala Lys
Ile Lys Asn Arg Val Tyr Glu Gln Cys Leu 20 25
30Gln Ala Cys Glu Pro Gly Ser Arg Ala Thr Pro Ala Thr
Arg Glu Trp 35 40 45Leu Asp Lys
Met Trp Asp Arg Val Asp Met Lys Thr Trp Arg Cys Thr 50
55 60Ser Gln Phe Gln Ala Gly Arg Ile Leu Ala Ser Leu
Lys Phe Leu Pro65 70 75
80Asp Met Ile Gln Asp Thr Pro Pro Pro Val Pro Arg Lys Asn Arg Ala
85 90 95Ser Asp Ser Thr Gly Leu
Lys Gln Leu Val Ala Gln Trp Asp Arg Lys 100
105 110Leu Ser Val Ala Pro Pro Gln Lys Pro Val Gly Pro
Val Arg Asn Gln 115 120 125Ala Val
Pro Pro 13098132PRTPorcine reproductive and respiratory syndrome virus
98Arg Pro Arg Phe Ser Ala Gln Ala Leu Ile Asp Arg Gly Gly Pro Leu1
5 10 15Ala Asp Val His Ala Lys
Ile Lys Asn Arg Val Tyr Glu Gln Cys Leu 20 25
30Gln Ala Cys Glu Pro Gly Ser Arg Ala Thr Pro Ala Thr
Arg Glu Trp 35 40 45Leu Asp Lys
Met Trp Asp Arg Val Asp Met Lys Thr Trp Arg Cys Thr 50
55 60Ser Gln Phe Gln Ala Gly Arg Ile Leu Ala Ser Leu
Lys Phe Leu Pro65 70 75
80Asp Met Ile Gln Asp Thr Pro Pro Pro Val Pro Arg Lys Asn Arg Ala
85 90 95Ser Asp Asn Ala Gly Leu
Lys Gln Leu Val Ala Gln Trp Asp Arg Lys 100
105 110Leu Ser Val Thr Pro Pro Pro Lys Pro Val Gly Pro
Val Leu Asp Gln 115 120 125Ile Val
Pro Pro 13099132PRTPorcine reproductive and respiratory syndrome virus
99Arg Pro Arg Phe Ser Ala Gln Ala Leu Ile Asp Arg Gly Gly Pro Leu1
5 10 15Ala Asp Val His Ala Lys
Ile Lys Asn Arg Val Tyr Glu Gln Cys Leu 20 25
30Gln Ala Cys Glu Pro Gly Ser Arg Ala Thr Pro Ala Thr
Arg Glu Trp 35 40 45Leu Asp Lys
Met Trp Asp Arg Val Asp Met Lys Thr Trp Arg Cys Thr 50
55 60Ser Gln Phe Gln Ala Gly Arg Ile Leu Ala Ser Leu
Lys Phe Leu Pro65 70 75
80Asp Met Ile Gln Asp Thr Pro Pro Pro Val Pro Arg Lys Asn Arg Ala
85 90 95Ser Asp Asn Ala Gly Leu
Lys Gln Leu Val Ala Gln Trp Asp Arg Lys 100
105 110Leu Ser Val Thr Pro Pro Pro Lys Pro Val Gly Pro
Val Leu Asp Gln 115 120 125Thr Val
Pro Pro 130100132PRTPorcine reproductive and respiratory syndrome
virus 100Arg Pro Arg Phe Ser Ala Gln Ala Leu Ile Asp Arg Gly Gly Pro Leu1
5 10 15Ala Asp Val His
Ala Lys Ile Lys Asn Arg Val Tyr Glu Gln Cys Leu 20
25 30Lys Ala Cys Glu Pro Gly Ser Arg Ala Thr Pro
Ala Thr Lys Glu Trp 35 40 45Leu
Asp Lys Met Trp Asp Arg Val Asp Met Lys Thr Trp Arg Cys Thr 50
55 60Ser Gln Phe Gln Ala Gly Arg Ile Leu Ala
Ser Leu Lys Phe Leu Pro65 70 75
80Asp Met Ile Gln Asp Thr Pro Pro Pro Val Pro Arg Lys Asn Arg
Ala 85 90 95Ser Asp Asn
Ala Gly Leu Lys Gln Leu Val Ala Gln Trp Asp Arg Lys 100
105 110Phe Ser Val Thr Pro Pro Pro Lys Leu Ala
Gly Pro Val Leu Asp Gln 115 120
125Thr Val Leu Pro 130101132PRTPorcine reproductive and respiratory
syndrome virus 101Arg Pro Arg His Ser Ala Gln Ala Leu Ile Asp Arg Gly Gly
Pro Leu1 5 10 15Ala Asp
Val His Ala Lys Ile Lys Asn Arg Val Tyr Glu Gln Cys Leu 20
25 30Gln Ala Cys Glu Pro Gly Ser Arg Ala
Thr Pro Ala Thr Arg Glu Trp 35 40
45Leu Asp Lys Met Trp Asp Arg Val Asp Met Lys Thr Trp Arg Cys Thr 50
55 60Ser Gln Phe Gln Ala Gly Arg Ile Leu
Ala Ser Leu Lys Phe Leu Pro65 70 75
80Asp Met Ile Gln Asp Thr Pro Pro Pro Val Pro Arg Lys Asn
Arg Ala 85 90 95Ser Asp
Asn Ala Gly Leu Lys Gln Leu Val Ala Arg Trp Asp Lys Lys 100
105 110Leu Ser Val Thr Pro Pro Pro Lys Ser
Ala Gly Leu Val Leu Asp Gln 115 120
125Thr Val Pro Pro 130102132PRTPorcine reproductive and respiratory
syndrome virus 102Arg Pro Arg Phe Ser Ala Gln Ala Leu Ile Asp Arg Gly Gly
Pro Leu1 5 10 15Ala Asp
Val His Ala Lys Ile Lys His Arg Val Tyr Glu Gln Cys Leu 20
25 30Gln Ala Cys Glu Pro Gly Ser Arg Ala
Thr Pro Ala Thr Lys Lys Trp 35 40
45Leu Asp Lys Met Trp Asp Arg Val Asp Met Lys Thr Trp Arg Cys Thr 50
55 60Ser Gln Phe Gln Ala Gly Arg Ile Leu
Ala Ser Leu Lys Phe Leu Pro65 70 75
80Asp Met Ile Gln Asp Thr Pro Pro Pro Val Pro Arg Lys Asn
Arg Ala 85 90 95Ser Asp
Asn Ala Gly Leu Lys Gln Leu Val Ala Gln Trp Asp Arg Lys 100
105 110Leu Ser Gly Thr Pro Pro Pro Lys Ser
Ala Gly Leu Val Leu Asp Gln 115 120
125Thr Val Pro Pro 130103132PRTPorcine reproductive and respiratory
syndrome virus 103Arg Pro Arg Phe Ser Ala Gln Ala Leu Ile Asp Arg Gly Gly
Pro Leu1 5 10 15Ala Asp
Val His Ala Lys Ile Lys His Arg Val Tyr Glu Gln Cys Leu 20
25 30Gln Ala Cys Glu Pro Gly Ser Arg Ala
Thr Pro Ala Thr Lys Lys Trp 35 40
45Leu Asp Lys Met Trp Asp Arg Val Asp Met Lys Thr Trp Arg Cys Thr 50
55 60Ser Gln Phe Gln Ala Gly Arg Ile Leu
Ala Ser Leu Lys Phe Leu Pro65 70 75
80Asp Met Ile Gln Asp Thr Pro Pro Pro Val Pro Arg Lys Asn
Arg Ala 85 90 95Ser Asp
Asn Ala Gly Leu Lys Gln Leu Val Ala Gln Trp Asp Arg Lys 100
105 110Leu Ser Gly Thr Pro Pro Pro Lys Pro
Ala Gly Ser Val Leu Asp Gln 115 120
125Thr Val Pro Pro 130104132PRTPorcine reproductive and respiratory
syndrome virus 104Arg Pro Arg Phe Ser Ala Gln Ala Leu Ile Asp Arg Gly Gly
Pro Leu1 5 10 15Ala Asp
Val His Ala Lys Ile Lys His Arg Val Tyr Glu Gln Cys Leu 20
25 30Gln Ala Cys Glu Pro Gly Ser Arg Ala
Thr Pro Ala Thr Lys Lys Trp 35 40
45Leu Asp Lys Met Trp Asp Arg Val Asp Met Lys Thr Trp Arg Cys Thr 50
55 60Ser Gln Phe Gln Ala Gly Arg Ile Leu
Ala Ser Leu Lys Phe Leu Pro65 70 75
80Asp Met Ile Gln Asp Thr Pro Pro Pro Val Pro Arg Lys Asn
Arg Ala 85 90 95Ser Asp
Asn Ala Gly Leu Lys Gln Leu Val Ala Gln Trp Asp Arg Lys 100
105 110Leu Ser Gly Thr Pro Pro Pro Lys Pro
Ala Gly Ser Val Leu Asp Gln 115 120
125Thr Val Pro Pro 130105132PRTPorcine reproductive and respiratory
syndrome virus 105Arg Pro Arg Phe Ser Ala Gln Ala Leu Ile Asp Arg Gly Gly
Pro Leu1 5 10 15Ala Asp
Val His Ala Lys Ile Lys His Arg Val Tyr Glu Gln Cys Leu 20
25 30Gln Ala Cys Glu Pro Gly Ser Arg Ala
Thr Pro Ala Thr Lys Lys Trp 35 40
45Leu Asp Lys Met Trp Asp Arg Val Asp Met Lys Thr Trp Arg Cys Thr 50
55 60Ser Gln Phe Gln Ala Gly Arg Ile Leu
Ala Ser Leu Lys Phe Leu Pro65 70 75
80Asp Met Ile Gln Asp Thr Pro Pro Pro Val Pro Arg Lys Asn
Arg Ala 85 90 95Ser Asp
Asn Ala Gly Leu Lys Gln Leu Val Ala Gln Trp Asp Arg Lys 100
105 110Leu Ser Gly Thr Pro Pro Pro Lys Pro
Ala Gly Ser Val Phe Asp Gln 115 120
125Ala Val Pro Pro 130106132PRTPorcine reproductive and respiratory
syndrome virus 106Arg Pro Arg Phe Ser Ala Gln Ala Leu Ile Asp Arg Gly Gly
Pro Leu1 5 10 15Ala Asp
Val His Ala Lys Ile Lys His Arg Val Tyr Glu Gln Cys Leu 20
25 30Gln Ala Cys Glu Pro Gly Ser Arg Ala
Thr Pro Ala Thr Lys Lys Trp 35 40
45Leu Asp Lys Met Trp Asp Arg Val Asp Met Lys Thr Trp Arg Cys Thr 50
55 60Ser Gln Phe Gln Ala Gly Arg Ile Leu
Ala Ser Leu Lys Phe Leu Pro65 70 75
80Asp Met Ile Gln Asp Thr Pro Pro Pro Val Pro Arg Lys Asn
Arg Ala 85 90 95Ser Asp
Asn Ala Gly Leu Lys Gln Leu Val Ala Gln Trp Asp Arg Lys 100
105 110Leu Ser Gly Thr Pro Pro Pro Lys Pro
Ala Gly Ser Val Leu Asp Gln 115 120
125Ala Val Pro Pro 130107132PRTPorcine reproductive and respiratory
syndrome virus 107Arg Pro Arg Phe Ser Ala Gln Ala Leu Ile Asp Arg Gly Gly
Pro Leu1 5 10 15Ala Asp
Val His Ala Lys Ile Lys Asn Gln Val Tyr Glu Arg Cys Leu 20
25 30Gln Ala Cys Glu Pro Gly Ser Arg Ala
Thr Pro Ala Thr Arg Glu Trp 35 40
45Leu Asp Lys Met Trp Glu Arg Val Asp Met Lys Thr Trp Arg Cys Thr 50
55 60Ser Gln Phe Gln Ala Gly Arg Ile Leu
Ala Ser Leu Lys Phe Leu Pro65 70 75
80Asp Met Ile Gln Asp Thr Pro Pro Pro Val Pro Arg Lys Asn
Arg Ala 85 90 95Ser Asp
Asn Ala Gly Leu Lys Gln Leu Val Ala Gln Trp Asp Arg Lys 100
105 110Leu Ser Met Thr Ser Pro Gln Lys Pro
Val Gly Ser Val Leu Asp Gln 115 120
125Thr Ala Phe Pro 130108132PRTPorcine reproductive and respiratory
syndrome virus 108Arg Pro Arg Phe Ser Ala Gln Ala Leu Ile Asp Arg Gly Gly
Pro Leu1 5 10 15Ala Asp
Val His Ala Lys Ile Lys Asn Arg Val Tyr Glu Arg Cys Leu 20
25 30Gln Ala Cys Glu Pro Gly Ser Arg Ala
Thr Pro Ala Thr Lys Glu Trp 35 40
45Leu Asp Lys Met Trp Asp Arg Val Asp Met Lys Thr Trp Cys Cys Thr 50
55 60Ser Gln Phe Gln Ala Gly Arg Ile Leu
Ala Ser Leu Lys Phe Leu Pro65 70 75
80Asp Met Ile Gln Asp Thr Pro Pro Pro Val Pro Arg Lys Asn
Arg Ala 85 90 95Ser Asp
Asn Ala Asp Leu Lys Gln Leu Val Ala Gln Trp Asp Arg Lys 100
105 110Leu Ser Met Thr Pro Pro Gln Lys Pro
Val Glu Pro Val Leu Asp Gln 115 120
125Thr Val Ser Pro 130109132PRTPorcine reproductive and respiratory
syndrome virus 109Arg Pro Lys Tyr Ser Ala Gln Ala Ile Ile Asp Leu Gly Gly
Pro Cys1 5 10 15Ser Gly
His Leu Gln Arg Glu Lys Glu Ala Cys Leu Arg Ile Met Arg 20
25 30Glu Ala Cys Asp Ala Ala Lys Leu Ser
Asp Pro Ala Thr Gln Glu Trp 35 40
45Leu Ser Arg Met Trp Asp Arg Val Asp Met Leu Thr Trp Arg Asn Thr 50
55 60Ser Ala Tyr Gln Ala Phe Arg Thr Leu
Asp Gly Arg Phe Gly Phe Leu65 70 75
80Pro Lys Met Ile Leu Glu Thr Pro Pro Pro Tyr Pro Cys Gly
Phe Val 85 90 95Met Leu
Pro His Thr Pro Ala Pro Ser Val Ser Ala Glu Ser Asp Leu 100
105 110Thr Ile Gly Ser Val Ala Thr Glu Asp
Ile Pro Arg Ile Leu Gly Lys 115 120
125Ile Glu Asn Thr 130110132PRTPorcine reproductive and respiratory
syndrome virus 110Arg Pro Lys Tyr Ser Ala Gln Ala Ile Ile Asp Ser Gly Gly
Pro Cys1 5 10 15Ser Gly
His Leu Gln Lys Glu Lys Glu Ala Cys Leu Arg Ile Met Arg 20
25 30Glu Ala Cys Asp Ala Ala Arg Leu Gly
Asp Pro Ala Thr Gln Glu Trp 35 40
45Leu Ser His Met Trp Asp Arg Val Asp Val Leu Thr Trp Arg Asn Thr 50
55 60Ser Val Tyr Gln Ala Phe Arg Thr Leu
Asp Gly Arg Phe Gly Phe Leu65 70 75
80Pro Lys Met Ile Leu Glu Thr Pro Pro Pro Tyr Pro Cys Gly
Phe Val 85 90 95Met Leu
Pro His Thr Pro Thr Pro Ser Val Ser Ala Glu Ser Asp Leu 100
105 110Thr Ile Gly Ser Val Ala Thr Glu Asp
Val Pro Arg Ile Leu Gly Lys 115 120
125Thr Glu Asn Thr 130111132PRTPorcine reproductive and respiratory
syndrome virus 111Arg Pro Lys Tyr Ser Ala Gln Ala Ile Ile Asp Ser Gly Gly
Pro Cys1 5 10 15Ser Gly
His Leu Gln Arg Glu Lys Glu Ala Cys Leu Ser Ile Met Arg 20
25 30Glu Ala Cys Asp Ala Ala Lys Leu Ser
Asp Pro Ala Thr Gln Glu Trp 35 40
45Leu Ser Arg Met Trp Asp Arg Val Asp Met Leu Thr Trp Arg Asn Thr 50
55 60Ser Ala Tyr Gln Ala Leu His Thr Leu
Asp Gly Arg Ser Gly Phe Leu65 70 75
80Pro Lys Met Ile Leu Glu Thr Pro Pro Pro His Pro Cys Gly
Phe Val 85 90 95Met Leu
Pro His Thr Pro Ala Pro Ser Val Ser Ala Lys Ser Asp Leu 100
105 110Thr Ile Gly Ser Val Ala Thr Glu Asp
Val Pro Arg Ile Leu Gly Lys 115 120
125Ile Glu Asn Thr 130112132PRTPorcine reproductive and respiratory
syndrome virus 112Arg Pro Lys Tyr Ser Ala Gln Ala Ile Ile Asp Ser Gly Gly
Pro Cys1 5 10 15Ser Gly
His Leu Arg Arg Glu Lys Glu Ala Cys Leu Ser Ile Met Arg 20
25 30Lys Ala Cys Asp Ala Ala Lys Leu Ser
Asp Pro Ala Thr Gln Glu Trp 35 40
45Leu Ser Arg Met Trp Asp Arg Val Asp Met Leu Thr Trp Arg Asn Thr 50
55 60Ser Ala Tyr Gln Ala Leu Arg Val Leu
Asp Gly Arg Phe Gly Phe Leu65 70 75
80Pro Lys Met Ile Leu Glu Thr Pro Pro Pro Tyr Pro Cys Gly
Phe Val 85 90 95Met Leu
Pro His Thr Pro Ala Pro Ser Val Ser Ala Glu Ser Asp Leu 100
105 110Thr Ile Gly Ser Val Ala Thr Glu Asp
Val Pro Arg Ile Leu Gly Lys 115 120
125Thr Glu Asn Ala 130113132PRTPorcine reproductive and respiratory
syndrome virus 113Arg Pro Lys His Ser Ala Gln Ala Ile Ile Asp Ser Gly Gly
Pro Cys1 5 10 15Ser Gly
His Leu Arg Arg Glu Lys Glu Ala Cys Leu Ser Ile Met Arg 20
25 30Glu Ala Cys Asp Ala Ala Lys Leu Ser
Asp Pro Ala Thr Gln Glu Trp 35 40
45Leu Ser Arg Met Trp Asp Arg Val Asp Met Leu Thr Trp Arg Asn Thr 50
55 60Ser Ala Tyr Gln Ala Phe Arg Ile Leu
Asp Gly Arg Phe Glu Phe Leu65 70 75
80Pro Lys Met Ile Leu Glu Thr Pro Pro Pro Tyr Pro Cys Gly
Phe Val 85 90 95Met Leu
Pro His Thr Pro Ala Pro Ser Val Gly Ala Glu Ser Asp Leu 100
105 110Thr Ile Gly Ser Val Ala Thr Glu Asp
Val Pro Arg Ile Leu Gly Lys 115 120
125Ile Glu Asn Ala 130114132PRTPorcine reproductive and respiratory
syndrome virus 114Arg Pro Lys His Ser Ala Gln Ala Ile Ile Asp Ser Gly Gly
Pro Cys1 5 10 15Ser Gly
His Leu Arg Arg Glu Lys Glu Ala Cys Leu Ser Ile Met Arg 20
25 30Glu Ala Cys Asp Ala Ala Lys Leu Ser
Asp Pro Ala Thr Gln Glu Trp 35 40
45Leu Ser Arg Met Trp Asp Arg Val Asp Met Leu Thr Trp Arg Asn Thr 50
55 60Ser Ala Tyr Gln Ala Phe Arg Ile Leu
Asp Gly Arg Phe Glu Phe Leu65 70 75
80Pro Lys Met Ile Leu Glu Thr Pro Pro Pro Tyr Pro Cys Gly
Phe Val 85 90 95Met Leu
Pro His Thr Pro Ala Pro Ser Val Gly Ala Glu Ser Asp Leu 100
105 110Thr Ile Gly Ser Val Ala Thr Glu Asp
Val Pro Arg Ile Leu Gly Lys 115 120
125Ile Glu Asn Ala 130115132PRTPorcine reproductive and respiratory
syndrome virus 115Arg Pro Lys Tyr Ser Ala Gln Ala Ile Ile Asp Phe Gly Gly
Pro Cys1 5 10 15Ser Gly
His Leu Gln Lys Glu Lys Glu Ala Cys Leu Ser Ile Met Arg 20
25 30Glu Ala Cys Asp Ala Ser Lys Leu Ser
Asp Pro Ala Thr Gln Glu Trp 35 40
45Leu Ser Arg Met Trp Asp Arg Val Asp Met Leu Thr Trp Arg Asn Thr 50
55 60Ser Ala Tyr Gln Ala Phe Arg Thr Leu
Asn Gly Arg Phe Glu Phe Leu65 70 75
80Pro Lys Met Ile Leu Glu Thr Pro Pro Pro His Pro Cys Gly
Phe Val 85 90 95Met Leu
Pro His Thr Pro Ala Pro Ser Val Ser Ala Glu Ser Asp Leu 100
105 110Thr Ile Gly Ser Val Ala Thr Glu Asp
Val Pro Arg Ile Leu Gly Lys 115 120
125Ile Gly Asp Thr 130
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