This application claims priority of United States Provisional Application 60/136,880, filed 1 June 1999, the disclosure of which is hereby incorporated by reference in its entirety.

FIELD OF THE INVENTION The invention relates to newly identified polynucleotides, polypeptides, polypeptides encoded by porcine endogenous retroviral sequences, peptides encoded by porcine endogenous retroviral sequences, and methods of using the porcine endogenous retroviral nucleic acids and peptides.

BACKGROUND OF THE INVENTION Organ procurement currently poses one of the major problems in solid organ transplantation, as the number of patients requiring transplants far exceeds the number of organs available. A path for eliminating the shortage of donor organs for allotransplantation is to develop the technologies required to transplant non-human organs into humans, i. e., xenotransplantation. The development of clinical xenotransplantation will also allow for the transplantation of non-human cells and tissues.

The species considered being the primary potential source of such xenogeneic organs is the pig. In particular, a certain strain of the domesticated pig, denoted miniature swine, is ideal because of its similar size to humans (see below). Furthermore, the proposed use of pigs as organ donors in xenotransplantation would obviate problems associated with the consideration of non-human primates as donors. Xenografts from non-human primates present considerable risk of transmission of pathogens and the consequent development of emerging infections. Several pathogens that cause disease are known to infect both humans and non-human primates, for example, in the transmission of HIV from the chimpanzee to humans. Furthermore, chimpanzees and orangutans, the closest non-human primates phylogenetically, are endangered species and far too rare to be considered for organ transplantation purposes. Baboons are too small to be an appropriate donor for most organ transplants. Even the largest baboons weigh less than 40 kg. In addition, the gestation times and productivity of primates would not allow a commercially significant generation of source animals.

The physiology of many organ systems of pigs has been shown to be highly similar to the human counterparts (Sachs, D. H. 1994. Veterinary Immunology & Immunopathology 43: 185- 191). The breed of pigs described as miniature swine has a variety of advantages as potential xenograft donors. They achieve adult weights of approximately 100-150 kg, a size that is more compatible to human weights than that of the domestic pig, which reaches weights of over 500 kg. Through a selective breeding program over the past 20 years, partially inbred, miniature swine have been produced (Sachs et al. 1976. Transplantation 22: 559-567; Sachs, D. H. 1992.

In Swine as models in biomedical research, eds M. Swindle, D. Moody, and L. Phillips, pp. 3-15. Ames Iowa State Univ.

Press; Sachs, 1994. Veterinary Immunology & Immunopathology 43: 185-191). This breeding program has resulted in herds of animals that are genetically well characterized and inbred at the major histocompatibility complex (MHC). These animals have been used in large animal model studies for many years and have, like their domestic counterparts, very favorable breeding characteristics for being used as donors of organs in xenotransplantation.

A central concern regarding xenotransplantation is the risk of xenosis, infection by organisms transferred with the xenograft into both the transplant recipient and the general population. In particular,"emerging infections"caused by new previously unknown infectious agents with altered pathogenicity, have to be considered as a potential risk associated with xenotransplantation. The risk of viral infection is increased in transplantation by the presence of factors commonly associated with viral activation, e. g., immune suppression, graft-versus-host disease, graft rejection, viral co-infection, and cytotoxic therapies.

Since endogenous retroviruses are potential sources of infection not always susceptible to conventional breeding practices or barrier elimination, other means for assessing the risk of endogenous retroviruses in general clinical practices and in xenotransplantation are needed.

Retroviruses constitute a large family of enveloped animal viruses with single stranded, positive sense RNA genomes (Table 1). Weiss et al. 1984. In RNA Tumor Viruses, New York: Cold Spring Harbor Press; Levy, 1992- 1995. In The Retroviridae, eds, F. C. Heinz and R. R. Wagner.

New York: Plenum Press). The defining characteristic of retroviruses is that following infection of a target cell, the genomic RNA is converted to a double-stranded DNA form which becomes stably integrated into the chromosomal DNA of

the host cell. This DNA provirus encodes viral proteins using cellular mechanisms for transcription and translation.

Full-length RNA transcripts are packaged into progeny virions.

Table 1 Characteristic features of retroviral groups Virus Type B-Type C-Type D-Type A-Type Lentiviruses Spumaviruses Typical MMTV MLV MPMV MuIAP HIV HFV members HTLV HuIAP Visna SFV Particle 125-130 80-110 100-120 60-90 80-130 90-110 diameter (nm) Mature core dense dense dense lucent truncated cone lucent acentric central cylindrical Envelope prominent small small not present prominent prominent spikes Core cytoplasm plasma cytoplasm cytoplasm plasma ER or cis- assembly membrane or ER membrane Golgi Infectious yes yes yes no yes yes members Endogenous yes yes yes yes no distantly members related

Retroviruses can be classified into two categories depending on their mode of replication. Firstly they can be termed exogenous, being horizontally transmitted from an animal to permissive cells of another animal by infectious routes. Secondly, retroviruses can be termed endogenous, being inherited according to Mendelian expectations by subsequent generations as a normal part of the germline DNA. These vertically transmitted endogenous proviruses are subject to the same biological regulation as the rest of the chromosomal DNA that constitutes the genome. By definition, endogenous retroviruses are present in the genomes of all cells of an organism. Despite the diversity of exogenous and endogenous retroviruses, they share a common structure, genome organization and many life-cycle features. The persistence of exogenous retroviruses may, in comparison with endogenous persistence, represent only a minor and relatively speaking, an insignificant part of the biological spectrum of retroviral genes. Importantly, complex interactions between exogenous and endogenous persistence between retroviral sequences exist (Deinhardt F. 1982. In Viral Oncology, ed. G. Klein, pp. 357-398.

New York: Raven Press). Some strains of retroviruses are endogenous in one species and exogenous in other species.

During these interactions, advantageous cycles between vertical and horizontal transmission within a particular species and between species could occur. Of particular importance for the potential risks associated with retroviruses and xenotransplantation, certain retroviruses change their pathogenicity following interspecies transmission and may result in emerging infections. Thus, certain endogenous retroviruses may be parasitic in one host and symbiotic in another host.

Also, retroviruses have evolved efficient mechanisms for

survival and persistence in a wide host range. It has been shown that endogenous retroviruses influence a diversity of biological phenomena and that they are etiological agents in the development of multigenic disorders such as cancer (reviewed in Larsson and Andersson. 1998. Scand. J. Immunol. 48: 329-338).

Although there are variations in particle morphology among members of different retroviral groups, they all conform to the same general plan (Bolognesi et al. 1978.

Science 199: 183-186). Retrovirus particles are 80-130 nm in diameter and are composed of a nucleoprotein core surrounded by a phospholipid envelope. The core of the virion contains two molecules of genomic RNA closely associated with nucleocapsid protein (NC) (Leis et al.

1988. J. Virol. 62: 1808-1809) and a tRNA molecule that is required as a primer for initiation of reverse transcription. The viral genomic RNA resembles eukaryotic mRNA with a 3'polyadenylated tail and a 5' methylguanosine cap.

The size of full-length retroviral genomes varies between 8-13 kilobase pairs (kb). All retroviruses share a common genome organization in which the protein coding regions are central and the regulatory sequences are predominantly located in the terminal regions. Retroviral RNAs are flanked by untranslated sequences denoted RU5 and U3R. Following reverse transcription of the retroviral RNA and subsequent integration of the DNA, a duplication of these sequences occurs creating two long terminal repeat (LTR) elements. These LTRs consist of 5'- U3-R-U5-3'sequences at both ends of the resulting proviral DNA. Activation of retroviral transcription is dependent on the 5'-LTR.

Infectious retroviruses have at least three genes, designated gag, pol and env. The gag gene encodes the internal structural proteins of the virion, namely the matrix protein (MA), the major capsid protein (CA) and the NC. The pol gene encodes the viral enzymes which are contained within the capsid and include an aspartic protease (PR), an RNA-directed DNA polymerase (reverse transcriptase, RT) and an endonuclease (IN). The env gene encodes the envelope transmembrane (TM) and surface (SU) glycoproteins. Full length ERVs show the same basic genome organization of infectious retroviruses. However, as the selective pressure to code for infectious particles does not apply to their replication cycle, their open reading frames (ORFs) have often become mutated and deleted rendering the genomes replication defective.

On the basis of their associated diseases, exogenous retroviruses have been classified into three groups: oncoviruses which cause tumors and immunodeficiency diseases, lentiviruses (slow viruses) which cause chronic progressive diseases, and spumaviruses which cause vacuolation of cells in culture but which have yet to be shown to produce disease. The oncoviruses have been further sub-divided on the basis of morphological appearance by electron microscopy into types A-D (Bernhard, 1960. Cancer Res. 20: 712-727; Fine and Schochetman. 1978. Cancer Res. 38: 3123-3139). More recently, the relationships between the different retroviral groups have been further defined by phylogenetic analyses of retroviral nucleotide sequences.

The International Committee on Taxonomy of Viruses (ICTV) has recognized seven distinct genera within the family of Retroviridae (Coffin JM. 1992. In The Retroviridae, ed.

J. A. Levy, pp 19-49. New York: Plenum Press). Recently,

retroviruses have been assigned a new nomenclature (van Regenmortel et al. 2000. In Virus Taxonomy, VIIth ICTV Report, pp 1104. Academic Press, San Diego, CA).

Different abbreviations have been used for endogenous retroviruses (ERVs) (Lower et al. 1996. Proc.

Natl. Acad. Sci. USA, 93: 5177-84). The designation PERV, which is an abbreviation of porcine endogenous retrovirus, has been used for the endogenous retroviruses in the pig genome. The first described PERVs were denoted PERV-A,-B, and-C. The novel PERVs described in this invention are identified as PERV-MSN1 and PERV-MSN4 (Porcine Endogenous Retrovirus from Miniature Swine New).

Future classification of endogenous retroviruses should be based on their sequence similarity to the currently recognized seven retroviral genera within the family of Retroviridae.

As discussed above, endogenous retroviruses (ERVs) may be transmitted from parent to offspring as a provirus in the germ line DNA (reviewed by Coffin, 1982, Endogenous Retroviruses, in RNA Tumor Viruses, eds. R.

Weiss, N. Teich, H. Varmus and J. Coffin. New York: Cold Spring Harbor Laboratory Press; Stoye and Coffin, 1985, Endogenous Retroviruses, in RNA Tumor Viruses, eds. R.

Weiss, N. Teich, H. Varmus and J. Coffin. New York: Cold Spring Harbor Laboratory Press; Wilkinson et al. 1994, Endogenous human retroviruses, in The Retroviridae, J.

Levy, ed., pp 465-535. New York: Plenum Press). ERV can also replicate within cells by intracellular transposition, i. e., by the integration of a provirus at a new site in the genome of the cell (Tchenio and Heidmann, 1991. J. Virol. 69: 1079-1084). Although intracellular transpositions are rare events, over evolutionary time periods, these events have contributed

towards the amplification of many ERV families. The amplification of defective ERVs presumably requires gag and pol activities to be supplied in trans from functional retroviral elements. ERV can also be replicated during chromosomal DNA amplification events.

Human endogenous retroviruses (HERVs) have been classified based on their sequence similarities to animal exogenous retroviruses (Wilkinson et al. 1994).

Endogenous human retroviruses. In The Retroviridae, J.

Levy, ed., pp 465-535. New York: Plenum Press).

Phylogenetic analyses of ERVs from other species have shown that this classification is practical to adopt for all animal endogenous retroviruses. Class II families exhibit substantial sequence similarity to mammalian type B and D retrovirus strains.

Type C retroviruses from cells of swine origin have been characterized (Arida, E. and Hultin, T. 1977. Am. J.

Public Health 67: 380; Armstrong et al. 1971. J. Gen.

Virol. 10: 195-198; Benveniste, R. E. and Todaro, G. J.

1973. Proc. Natl. Acad. Sci. USA 70: 3316-3320; Bouilant et al. 1975. J. Gen. Virol. 27: 173-180; Frazier, M. E.

1985. Arch. Virol. 83: 83-97; Lieber et al. 1975.

Virology 66: 616-619; Susuka et al. 1985. FEBS Lett. 183: 124-128; Susuka et al. 1986. FEBS Lett. 198: 339-343; Todaro et al. 1974. Virology 58: 65-74; Woods et al.

1973. J. Virol. 12: 1184-1186; Akiyoshi et al. 1998. J.

Virol. 72: 4503-4507); as yet, no disease following infection by these viruses has been identified. A recent report demonstrated that a virus from PK15 (porcine kidney-derived) cells can infect human cells in vitro (Patience et al. 1997. Nature Medicine 3: 276-282).

Characterization of swine cells and cell lines has resulted in the identification of at least three type C

pig endogenous retrovirus sequences (PERV-A,-B,-C), (WO 97/40167; WO 97/21836; Le Tissier et al. 1997. Nature 389: 681-682; Czauderna et al. 1998: Genbank Accession Number Y17013). These sequences have distinct envelope (env) genes but share highly conserved sequences in the rest of the genome. Southern blot analysis of genomic DNA prepared from different pig tissues and cell lines (Patience et al. 1997. Nature Medicine 3: 276-282) showed the presence of numerous loci in genomic DNA extracted from normal pig hearts and from pig cell lines. The Southern blot banding profile for DNA prepared from normal pig hearts is similar to that obtained from DNA of the pig cell lines and is typical of an endogenous inherited retrovirus suggesting heterogeneity with approximately 50 integration sites. These results were confirmed and extended to analysis of MHC-inbred miniature swine where the numbers of potentially full- length provirus copies are approximately 8 to 15 per genome for inbred and outbred swine and 10 to 20 in PK15 cells (Akiyoshi et al. 1998. J. Virol., 72: 4503-4507).

The envelope determines host range and cell tropism.

The envelopes of PERV-A,-B, and-C are distinct (see above). In particular, a high degree of amino acid differences in the VRA, VRB, and PRO regions in the SU glycoproteins was evident upon sequence comparison. Host range analyses using retrovirus vectors bearing corresponding envelope proteins showed that PERV-A and PERV-B envelopes have wider host range including several human cell lines as compared to PERV-C envelope, which has been shown to infect only two pig cell lines and a single human cell line. All three strains of type C PERVs have been shown to infect pig cells. Receptors for PERV-A and PERV-B have been shown to be present on cells of some other species, including mink, rat, mouse and

dog. Interference studies showed that the three PERV strains use distinct receptors to each other and to a number of other type C mammalian retroviruses (Takeuchi et al. 1998. J. Virol., 72: 9986-9991).

The pol genes of type B and D retroviruses are highly similar and in phylogenetic analyses these viruses cluster together. However, the env genes of type D viruses, are more closely related to the MLV-like group of retroviruses, whereas the rest of the type D genome more closely resembles mammalian type B viruses. This feature implies that a recombination event occurred involving conversion of the env gene from an MLV-like virus into one resembling MMTV at some point during evolution of the type D group of viruses (Doolittle et al. 1989. Quant. Rev. Biol. 64 (1): 1-30).

Mouse mammary tumor virus (MMTV) is considered the prototype type B retrovirus. MMTVs exist both in endogenous and infectious exogenous forms. Moreover, the role of MMTV integration and the development of mammary carcinoma are well-established features of MMTV (for review see Tekmal and Keshava. 1997. Front. Biosci. 2: 519-526).

Thus, a potential risk associated with type B/D PERVs is that they might be involved in pathogenesis of the transplanted organ. Like type C PERVs, they might also be transmitted during xenotransplantation. Indeed, since a large number of human endogenous retroviruses are type-B, they are more likely to recombine with the PERV- type B retroviruses and hence pose a greater risk than the PERV-type C. Therefore, methods to monitor for their expression and possible retrotransposition in immunocompromised recipients of xenografts is advantageous in insuring that these retroelements do not

contribute to the development of disease. The identification and nucleotide sequence determination of novel PERV-MSN1-like and PERV-MSN4-like sequences by the PCR-based (where PCR means"polymerase chain reaction") system described in the present invention disclosure should provide a more complete arsenal of reagents and assays to monitor for the presence and expression of potentially hazardous PERVs.

SUMMARY OF THE INVENTION It is an object of the present invention to provide a method for the detection of PERV sequences.

Further objects of the present invention are to provide various novel polynucleotide sequences called PERV-MSN1 and PERV-MSN4. The new sequences may be derived from genomic DNA of swine peripheral blood lymphocytes, porcine tissues, cells, and organs.

Another object of the present invention is to provide a method for detecting PERV-MSN1-like and PERV- MSN4-like retroviral sequences in a sample of porcine or primate tissues, as well as probes and primers that may be utilized in such a method. The porcine or primate tissues may include primary porcine tissue and primate cell lines which have been cultivated in the presence of a porcine cell line, or primate tissues which are from a human or non-human primate who has received a xenotransplant. Nucleic acid (e. g., mRNA, total RNA, DNA or total nucleic acid) from the tissues or cells may be probed directly or, if desired, retroviral sequences may be amplified using primers suitable for amplifying retroviral sequences in general prior to detecting PERV-

MSN1-like and PERV-MSN4-like sequences of the invention.

Using the sequences described herein full-length sequences of proviral genomes representing the PERV-MSN1 and PERV-MSN4 retroviral sequences (as well as sequences closely related to these sequences) can be detected and identified. As used herein, the term"closely related"is deemed to encompass sequences showing at least 65% sequence identity, or sequence homology, preferably 75% sequence identity, especially preferable being at least 90% sequence identity, and most preferably 98% sequence identity, or sequence homology, with the sequences disclosed according to the present invention.

It is also an object of the present invention to provide isolated PERV polypeptides, and immunologically and/or physiologically active fragments thereof, especially for use as vaccines and vaccine compositions, the latter also being part of the invention.

An additional object of the invention relates to antibodies specific for the polypeptides disclosed herein, such antibodies also being useful in identifying polypeptides of the invention, especially where this serves to diagnose the presence of viruses within an animal or a sample taken from an animal.

BRIEF DESCRIPTION OF THE DRAWINGS The following drawings are illustrative of embodiments of the invention and are not meant to limit the scope of the invention as encompassed by the claims.

Figure 1 shows alignment of DNA sequences from PERV- MSN1 clones BTP-4, BTP-5, and BTP-6, each containing porcine endogenous retroviral pol region sequences.

Figure 2 shows alignment of DNA sequences from PERV- MSN4 clones BTP-9, BTP-10, and BTP-11, each containing porcine endogenous retroviral pol region sequences Figure 3 shows alignment of DNA sequences from PERV- MSN1 (BTP-4) and PERV-MSN4 (BTP-9) containing porcine endogenous retroviral pol region sequences. Nucleotides that are the same in the alignment are shown while a dash (-) indicates non-alignment.

Figure 4 shows a comparison of the PERV-MSN1 sequence BTP-4 with HERV-K10 nucleotides 4001-4700.

Comparison was performed using GeneWorks software with the parameters set at: cost to open a gap = 2, cost to lengthen a gap = 4, Minimum Diagonal Length = 6, and Maximal Diagonal Offset = 10.

Figure 5 shows a comparison of the PERV-MSN1 sequence BTP-4 to PERV-A nucleotides 1961-2620 using GeneWorks software with the parameters set as in Figure 4. Nucleotides that are the same are shown below the two sequences.

Figure 6 shows a comparison of the PERV-MSN1 sequence BTP-4 to PERV-B nucleotides 1620-2270 using GeneWorks software with the parameters set as in Figure 4. Nucleotides that are the same are shown below the two sequences.

Figure 7 shows a comparison of the PERV-MSN1 sequence BTP-4 to PERV-C nucleotides 1950-2610. using GeneWorks software with the parameters set as in Figure 4. Nucleotides that are the same are shown below the two sequences.

Figure 8 shows a comparison of the PERV-MSN4 sequence BTP-9 with HIV-1 nucleotides 2500-3100 using GeneWorks software with the parameters set as in Figure 4. Nucleotides that are the same are shown below the two sequences.

Figure 9 shows the results of a comparison of the PERV-MSN4 sequence BTP-9 with PERV-A nucleotides 2000- 2640 using GeneWorks software with the parameters set as in Figure 4. Nucleotides that are the same are shown below the two sequences.

Figure 10 shows the results of a comparison of the PERV-MSN4 sequence BTP-9 with PERV-B nucleotides 1620- 2386 using GeneWorks software with the parameters set as in Figure 4. Nucleotides that are the same are shown below the two sequences.

Figure 11 shows the results of a comparison of the PERV-MSN4 sequence BTP-9 with PERV-C nucleotides 2097- 2668 using GeneWorks software with the parameters set as in Figure 4. Nucleotides that are the same are shown below the two sequences.

Figure 12 shows the results of a hypothetical translation of PERV-MSN1 sequence BTP-4 and showing all three reading frames.

Figure 13 shows the results of a hypothetical translation of PERV-MSN4 sequence BTP-9 showing all three reading frames.

Figure 14 shows a Southern blot analysis of porcine genomic DNAs using PERV-MSN1 sequence as the probe.

Samples of the DNA were cleaved using EcoRI. The autoradiograph was exposed for 10 days. Lane 1 contains partially digested miniature swine genomic DNA, lanes 2- 16 contain 15 miniature swine genomic DNA samples, lane 4 is genomic DNA from an SLA c/c haplotype while the others are of the d/d haplotype, and lane 17 contains a domestic outbred pig DNA sample.

Figure 15 shows a Southern blot analysis of porcine genomic DNAs using PERV-MSN4 sequence as the probe.

Samples of the DNA were cleaved using EcoRI. The autoradiograph was exposed for 10 days. Lane 1 contains partially digested miniature swine genomic DNA, lanes 2- 16 contain 15 miniature swine genomic c DNA samples, lane 4 is genomic DNA from an SLA c/c haplotype while the others are of the d/d haplotype, and lane 17 contains a domestic outbred pig DNA sample.

Figure 16 shows the results of RT-PCR of PERV-MSN1 and PERV-MSN4 sequences. Lanes are identified as per the legend at the right of the gel. Figure 16 (A) shows the results for PERV-MSN1 specific RT-PCR; 16 (B) shows the results for PERV-MSN4 specific RT-PCR.

Figure 17 shows the specificity of PERV-MSN1 and PERV-MSN4. Here, a PCR was done using the primers specific for PERV-MSN1 and PERV-MSN4 to evaluate the specificity of the primers and the PCR conditions. For this procedure, 4 X 105 copies of a PERV-C clone, 100 ng human genomic DNA (LCL721.221 EBV B cell line) and PK-15 cDNA (Frohman, M. A., Dush, M. K., and Martin, G. R., Proc.

Natl. Acad. Sci. USA 85 (23): 8998-9002 (1988) were tested with both PERV-MSN1 and PERV-MSN4 primer pairs; all results were negative. As a positive control, 100 ng of

PK-15 genomic DNA was also tested with both primer pairs.

The expected PCR products were obtained. Here, lanes 1, 9, and 17 represent the 100 bp ladder; lanes 2 and 10 are blanks; lanes 3 and 11 are Neptune-6 (PERV-C); lanes 4 and 12 are LCL 721.221; lanes 5 and 13 are PK-15 cDNA at 1: 50 dilution; lanes 6 and 14 are PK-15 cDNA (PERV-MSN1); lane 16 is 2 x 106 copies of PERV-MSN1; lane 16 is 2 x 106 copies of PERV-MSN4.

DETAILED SUMMARY OF THE INVENTION In accordance with the present invention, there are provided isolated nucleic acids (polynucleotides) that correspond to porcine endogenous retroviral pol region sequences.

Polynucleotide sequences of the present invention have been isolated from genomic DNA isolated from miniature swine. The polynucleotide sequences of the present invention show low sequence identity with other known retroviral sequences, including the PERV-A, B, or- C sequences of porcine endogenous retroviruses.

"Polynucleotide sequences"as used herein refers to a chain of nucleotides such as deoxyribose nucleic acid (DNA) and transcription products thereof, such as RNA.

The polynucleotides of the present invention may be in the form of RNA which includes retroviral RNA present in infected cells or in virus particles, or in the form of DNA, which DNA includes cDNA, genomic DNA, and synthetic DNA.

In accordance with the present invention, the term "percent identity"or"percent identical,"when referring

to a sequence, means that a sequence is compared to a claimed or described sequence after alignment of the sequence to be compared (the"Compared Sequence") with the described or claimed sequence (the"Reference Sequence").

The Percent Identity is then determined according to the following formula: Percent Identity = 100 [1- (C/R)] wherein C is the number of differences between the Reference Sequence and the Compared Sequence over the length of alignment between the Reference Sequence and the Compared Sequence wherein (i) each base or amino acid in the Reference Sequence that does not have a corresponding aligned base or amino acid in the Compared Sequence and (ii) each gap in the Reference Sequence and (iii) each aligned base or amino acid in the Reference Sequence that is different from an aligned base or amino acid in the Compared Sequence, constitutes a difference; and R is the number of bases or amino acids in the Reference Sequence over the length of the alignment with the Compared Sequence with any gap created in the Reference Sequence also being counted as a base or amino acid.

If an alignment exists between the Compared Sequence and the Reference Sequence for which the percent identity as calculated above is about equal to or greater than a specified minimum Percent Identity then the Compared Sequence has the specified minimum percent identity to the Reference Sequence even though alignments may exist in which the hereinabove calculated Percent Identity is less than the specified Percent Identity.

In accordance with a further aspect of the present invention, the nucleic acid sequences according to SEQ ID Nos: 3,4,5 and 6 or appropriate fragments thereof may be utilized under stringent hybridization conditions to isolate from porcine tissue by procedures known in the art, the DNA corresponding to PERV-MSN pol region and thereafter more complete PERV-MSN sequences. Likewise, polypeptides corresponding to peptides encoded by PERV- MSN sequences may be utilized to generate antibodies that could detect the presence of PERV-MSN polypeptides when they are expressed in porcine tissues.

The present invention further relates to an isolated polypeptide, including functional derivatives thereof, wherein said isolated polypeptide has pol PERV physiological and/or immunogenic activity. For example, such isolated polypeptide is commonly a PERV B/D polypeptide. In a specific embodiment, such an isolated polypeptide is a recombinant polypeptide and may be produced by any means available to chemists and molecular biologists. Thus, the polypeptides of the present invention may be prepared by in vitro expression systems, using a polynucleotide sequence, including a cDNA, that encodes the polypeptide. In addition, said polynucleotide may commonly be present in an expression vector, which itself has been inserted into a cell, either prokaryotic or eukaryotic, which cell then expresses the polynucleotide as a polypeptide that may eventually be secreted into the medium and collected, or may be collected by collecting and disrupting the cells so as to facilitate isolation of the polypeptide. Such polypeptides may also be prepared, where feasible, by direct chemical synthesis, for example, using solid phase synthesis, such as protocols involving automated synthesis of proteins.

The present invention further relates to a polypeptide, as well as fragments, analogs and derivatives of such polypeptide.

The terms"fragment,""derivative"and"analog"when referring to a polypeptide, mean a polypeptide which retains essentially the same biological function or activity as such polypeptide. Thus, an analog includes a proprotein which can be activated by cleavage of the proprotein portion to produce an active mature polypeptide. Such fragments, derivatives and analogs must have sufficient similarity to the polypeptide of SEQ ID NO: 4 so that activity of the native polypeptide is retained.

The polypeptide of the present invention may be a recombinant polypeptide, a natural polypeptide or a synthetic polypeptide, preferably a recombinant polypeptide.

The fragment, derivative or analog of the polypeptide may be (i) one in which one or more of the amino acid residues are substituted with a conserved or non-conserved amino acid residue (preferably a conserved amino acid residue) and such substituted amino acid residue may or may not be one encoded by the genetic code, or (ii) one in which one or more of the amino acid residues includes a substituent group, or (iii) one in which the mature polypeptide is fused with another compound, such as a compound to increase the half-life of the polypeptide (for example, polyethylene glycol), or (iv) one in which the additional amino acids are fused to the mature polypeptide, such as a leader or secretory sequence or a

sequence which is employed for purification of the mature polypeptide or a proprotein sequence. Such fragments, derivatives and analogs are deemed to be within the scope of those skilled in the art from the teachings herein.

The polypeptides and polynucleotides of the present invention are preferably provided in an isolated form, and preferably are purified to homogeneity.

The term"isolated"means that the material is removed from its original environment (e. g., the natural environment if it is naturally occurring). For example, a naturally-occurring polynucleotide or polypeptide present in a living animal is not isolated, but the same polynucleotide or polypeptide, separated from some or all of the coexisting materials in the natural system, is isolated. Such polynucleotides could be part of a vector and/or such polynucleotides or polypeptides could be part of a composition, and still be isolated in that such vector or composition is not part of its natural environment.

As known in the art"similarity"between two polypeptides is determined by comparing the amino acid sequence and its conserved amino acid substitutes of one polypeptide to the sequence of a second polypeptide.

Fragments or portions of the polypeptides of the present invention may be employed for producing the corresponding full-length polypeptide by peptide synthesis; therefore, the fragments may be employed as intermediates for producing the full-length polypeptides.

Fragments or portions of the polynucleotides of the

present invention may be used to synthesize full-length polynucleotides of the present invention.

The isolated polypeptides according to the present invention include polypeptides comprise an amino acid sequence having at least 95% sequence identity to the amino acid sequence of Figure 12, including functionally active fragments thereof. In a separate embodiment, the isolated polypeptide of the invention comprise an amino acid sequence having at least 95% sequence identity to the amino acid sequence of Figure 13, including functionally active fragments thereof.

Such polypeptides are also useful as vaccines and vaccine compositions. A vaccine according to the invention comprises a member selected from the group consisting of: (a) the recombinant polypeptide of claim 17; and (b) an inactivated PERV virus, wherein said member is suspended in a pharmacologically acceptable carrier.

Generally, vaccines are prepared as injectables, in the form of aqueous solutions or suspensions. Vaccines in an oil base are also well known such as for inhaling.

Solid forms which are dissolved or suspended prior to use may also be formulated. Pharmaceutically acceptable carriers, diluents and excipients are generally added that are compatible with the active ingredients and acceptable for pharmaceutical use.

The pharmaceutical compositions useful herein also contain a pharmaceutically acceptable carrier, including any suitable diluent or excipient, which includes any pharmaceutical agent that does not itself induce the production of antibodies harmful to the individual receiving the composition, and which may be administered without undue toxicity. Pharmaceutically acceptable carriers include, but are not limited to, liquids such as water, saline, glycerol and ethanol, and the like, including carriers useful in forming sprays for nasal and other respiratory tract delivery or for delivery to the ophthalmic system. A thorough discussion of pharmaceutically acceptable carriers, diluents, and other excipients is presented in REMINGTON'S PHARMACEUTICAL SCIENCES (Mack Pub. Co., N. J. current edition).

Vaccine compositions may further incorporate additional substances to stabilize pH, or to function as adjuvants, wetting agents, or emulsifying agents, which can serve to improve the effectiveness of the vaccine.

Vaccines are generally formulated for parenteral administration and are injected either subcutaneously or intramuscularly. Such vaccines can also be formulated as suppositories or for oral administration, using methods known in the art, or for administration through nasal or respiratory routes.

The amount of vaccine sufficient to confer immunity to pathogenic bacteria, viruses, or other microbes is determined by methods well known to those skilled in the art. This quantity will be determined based upon the characteristics of the vaccine recipient and the level of immunity required. Where vaccines are administered by

subcutaneous or intramuscular injection, a range of. 5 to 500 jug purified protein may be given. As useful in the present invention, such dosages are commonly sufficient to provide about 1 J. g, possibly 10 g, even 50 pg, and as much as 100 pg, up to 500 pg of immunogenic protein, or immunogenic polypeptide, or immunogenically active fragments thereof. In addition, more than one such active material may be present in the vaccine. Thus, more than one antigenic structure may be used in formulating the vaccine, or vaccine composition to use in the methods disclosed herein. This may include two or more individually immunogenic proteins or polypeptides, proteins or polypeptides showing immunogenic activity only when in combination, either quantitatively equal in their respective concentrations or formulated to be present in some ratio, either definite or indefinite. Thus, a vaccine composition for use in the processes disclosed herein may include one or more immunogenic proteins, one or more immunogenic polypeptides, and/or one or more immunogenically active immunogens comprising antigenic fragments of said immunogenic proteins and polypeptides, the latter fragments being present in any proportions selected by the use of the present invention. The exact components, and their respective quantities, making up the vaccines, and vaccine compositions, useful in the methods of the present invention are determined, inter alia, by the nature of the disease to be treated or prevented, the severity of such condition where it already exists, the age, sex, and general health of the recipient.

The vaccines according to the invention may also include inactivated virus, either alone or in combination with the polypeptides and/or active fragments thereof, where such virus has been inactivated by chemical or

physical attenuation, including where said virus has been merely weakened or where it has been killed.

The present invention also relates to antibodies specific for one or more of the polypeptides disclosed herein as being part of the invention. Such antibodies may include fragments of such antibodies so long as they retain immunological specificity and activity, especially where said antibodies are neutralizing antibodies.

With the advent of methods of molecular biology and recombinant technology, it is now possible to produce antibody molecules by recombinant means and thereby generate gene sequences that code for specific amino acid sequences found in the polypeptide structure of the antibodies. Such antibodies can be produced by either cloning the gene sequences encoding the polypeptide chains of said antibodies or by direct synthesis of said polypeptide chains, with in vitro assembly of the synthesized chains to form active tetrameric (H2L2) structures with affinity for specific epitopes and antigenic determinants. This has permitted the ready production of antibodies having sequences characteristic of neutralizing antibodies from different species and sources.

Regardless of the source of the antibodies, or how they are recombinantly constructed, or how they are synthesized, in vitro or in vivo, using transgenic animals, such as cows, goats and sheep, using large cell cultures of laboratory or commercial size, in bioreactors or by direct chemical synthesis employing no living organisms at any stage of the process, all antibodies have a similar overall 3-dimensional structure. This structure is often given as H2L2 and refers to the fact

that antibodies commonly comprise 2 light (L) amino acid chains and 2 heavy (H) amino acid chains. Both chains have regions capable of interacting with a structurally complementary antigenic target. The regions interacting with the target are referred to as"variable"or"V" regions and are characterized by differences in amino acid sequence from antibodies of different antigenic specificity.

The variable regions of either H or L chains contains the amino acid sequences capable of specifically binding to antigenic targets. Within these sequences are smaller sequences dubbed"hypervariable"because of their extreme variability between antibodies of differing specificity. Such hypervariable regions are also referred to as"complementarity determining regions"or"CDRs".

These CDRs account for the basic specificity of the antibody for a particular antigenic determinant structure.

The CDRs represent non-contiguous stretches of amino acids within the variable regions but, regardless of species, the positional locations of these critical amino acid sequences within the variable heavy and light chain regions have been found to have similar locations within the amino acid sequences of the variable chains. The variable heavy and light chains of all antibodies each have 3 CDRs, each non-contiguous with the others (termed L1, L2, L3, Hl, H2, H3) for the respective light (L) and heavy (H) chains. The accepted CDR regions have been described by Kabat et al, J. Biol. Chem. 252: 6609-6616 (1977). The numbering scheme is shown in the figures, where the CDRs are underlined and the numbers follow the Kabat scheme.

In all mammalian species, antibody polypeptides contain constant (i. e., highly conserved) and variable regions, and, within the latter, there are the CDRs and the so-called"framework regions"made up of amino acid sequences within the variable region of the heavy or light chain but outside the CDRs.

Still another aspect the present invention relates to a method of using one or more antibodies (monoclonal or polyclonal, natural or recombinant, and regardless of how prepared, i. e., by purification from a natural source, or generated by cloning or by direct chemical synthesis), preferably, but not necessarily, specific for one or more antigenic determinants present in the vaccine, or vaccine composition selected for use in the methods of the present invention.

Many useful methods for practicing the invention disclosed herein are to be found in the molecular biology literature, especially in Sambrook, et al., Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor, N. Y., (1989), Wu et al, Methods in Gene Biotechnology (CRC Press, New York, NY, 1997), and Recombinant Gene Expression Protocols, in Methods in Molecular Biology, Vol. 62, (Tuan, ed., Humana Press, Totowa, NJ, 1997), the disclosures of which are hereby incorporated by reference.

Fragments of the polynucleotide sequences of the present invention may be used as hybridization probes for a cDNA or DNA library to isolate the full-length retroviral sequence or fragments thereof. Probes of this

type preferably have at least 20 bases, preferably 30 bases and may contain, for example, 50 or more, perhaps as many as 75, bases total. In addition, such probes might conceivably possess as little as 65% sequence identity with the target sequence, preferably 75%, more preferably 90%, especially preferable being 95% and most preferably 98% sequence identity, as defined herein.

The present invention further provides an isolated PERV polynucleotide fragment that is capable of stringently hybridizing to a PERV polynucleotide sequence. In this manner, the present invention provides probes and/or primers for use in ex vivo PERV detection studies. Typical detection studies include PCR, sequence analysis, and hybridization studies. Stringent hybridization can be effected at a temperature of between 50°C and 70°C in 2x SSC (1X SSC is 17.5 g NaCl, 8.8 g of sodium citrate in 800 ml of H20, the pH is adjusted to 7.4 with NaOH and the volume adjusted to one liter), containing 0.1% sodium dodecyl sulfate (SDS). It is most preferred that the sample and probes be sufficiently similar that the hybridization is unaffected by being treated with 0.1 X SSC and 0.1% SDS at 65°C. PERV-MSN specific oligonucleotides may be designed to specifically hybridize to PERV nucleic acid. They may be synthesized by known techniques and used as primers in PCR or sequencing reactions or as probes in hybridizations designed to detect the presence of PERV material in a sample. The oligonucleotides may be labeled by suitable labels known in the art, such as, radioactive labels, chemiluminescent labels or fluorescent labels and the like. Thus, the present invention also provides PERV specific oligonucleotide probes and primers.

The term"oligonucleotide"encompasses nucleotides of preferably at least 15 bases (e. g. 15 bases to 600 bases) in length, more preferably 15 bases to 100 bases and most preferably 15 bases to 50 bases.

The PERV specific oligonucleotides may be determined from the PERV sequences of the present invention and may be synthesized according to known techniques. They may have substantial sequence identity (e. g., at least 75%, at least 90% or at least 95% sequence identity) with one of the strands shown herein or an RNA equivalent, or with part of such a strand.

Typically, the melting temperature (Tm) of an oligonucleotide less than 30 bases may be calculated according to the formula: Tm = 86. 35-0.41 [% (G+C)]-600/N where N = Chain Length (i. e., number of base pairs) EXAMPLE 1 Isolation and DNA sequence analysis of PERV-MSN1 and PERV-MSN4 Genomic DNA preparation Template for the PCR reactions constituted genomic DNA prepared from peripheral blood lymphocytes (PBLs) taken from miniature swine (obtained from the Transplantation Biology Research Center (TBRC) at Massachusetts General Hospital, Boston, MA). Cells were isolated from whole blood by centrifugation with Histopaque 1077 (Sigma Chemical Co, St. Louis, MO), and genomic DNA was extracted using QIAGEN's Genomic-tip

500/G (Qiagen, Inc. Stanford Clarita, CA) or Gentra System's PureGenes DNA Isolation Kit (Gentra Systems, Inc.

Minneapolis, MN).

Polymerase chain reaction (PCR) PCR amplification (Saiki et al. 1988. Science 239: 487- 491) of pol sequences from miniature swine genomic DNA was performed using the 5'-MOP-2 (SEQ ID NO 1) and 3'- MOP-2 (SEQ ID NO: 2) degenerate oligonucleotide primers.

Nucleotide sequence of the primers (see below) and PCR conditions were derived from (Li et al. 1996. Virology 217 (1): 1-10). Genosys Biotechnologies, Inc (Houston, TX) synthesized the PCR primers described by Li et al (supra). The nucleotide sequences of the primers are shown below.

SEQ ID NO: 1 5'-MOP-2 5'CCW TGG AAT ACT CCY RTW TT 3' SEQ ID NO: 2 3'-MOP-2 5'GTC KGA ACC AAT TWA TAT YYC C 3' R = A or G Y = C or T K = G or T W = A or T Either 1 ig or 500 ng of miniature swine genomic DNA was added to a 100 pl reaction volume. The final reagent mixture included 50 mM KC1,10 mM Tris-HCl (pH 8.3), 1.5 mM MgCl2,0.2 mM dNTP and 2.5 units of Amplitaq Gold (Perkin-Elmer Corporation, Norwalk, CT). (SEQ. ID. NO.: 1) 5'-MOP-2 and (SEQ. ID. NO.: 2). 3'-MOP-2 primers were present at a concentration of 0.5 pM each. The

reactions were amplified in a Perkin-Elmer GeneAmp<D 9600 thermal cycler. The initial denaturing step was 9 min at 95°C (also required to activate the"hot-start"Amplitaq Golf') followed by 40 cycles of 94°C for 30 s, 45°C for 30 s and 72°C for 30 s. Thermal cycling was followed by a final extension step at 72°C for 5 min and subsequently brought down to 4°C.

Purification and cloning of PCR products The approximately 650 bp PCR products were purified following agarose gel electrophoresis either using the QIAEX 110 extraction Kit (Qiagen) or purified using a Microspin G-50 column (Amersham Pharmacia Biotech, Arlington Heights, I1). The purified PCR products were TA-ligated into the pCR2'2.1-TOPO vector (Invitrogen, Carlsbad, CA). The ligation reactions were then used to transform competent TOP10 E. coli (F-, mcrA= (mrr-hsdRMS- mcrBC) 0801acZMl5-lacX74 deoR recA1 araD139 = (ara- leu) 7697 galUgalK rpsL (StrR) endAl nupG, Invitrogen, Carlsbad, CA). The cells were incubated on LB agar plates containing 50 pg/ml carbenicillin (Gibco Life Technologies, Baltimore, MD) 40 jul 100 mM IPTG (Gibco Life Technologies)/1600 g X-gal (Amresco, Inc, Solon, OH). White colonies were selected for further analysis.

Colony PCRs using 5'-MOP-2 and 3'-MOP-2 followed by restriction enzyme digestion using AluI, DdeI and HaeIII (New England Biolabs, Beverly, MA) were used to screen 84 colonies. From the restriction digest pattern the colonies were assigned to one of seven different groups.

Representative colonies were grown up in LB broth.

Plasmid DNAs were extracted using the Wizards Plus Miniprep DNA purification kit (Promega, Madison, WI).

DNA sequencing The plasmid DNA preparations were sequenced by standard methodologies.

Comparisons of nucleotide sequences and phylogenetic and statistical analyses Phylogenetic analyses were performed using the MEGA phylogenetic software (version 1. 01, (Kumar et al. 1993.

MEGA: Molecular Evolutionary Genetics Analysis, Version 1.01. The Pennsylvania State University, University Park, PA). The neighbor-joining tree building method (Saitou and Nei 1987. Mol. Biol. Evol. 4: 406-425), was used applying the method of Jukes and Cantor for correction for multiple hits (Jukes and Cantor. 1969. In Mammalian Protein Metabolism, ed. H. N. Munro pp. 21-132. New York: Academic Press). To address the liability of the obtained phylogenetic trees, bootstrap analysis was done using the MEGA program. Evolutionary distances of these retroviral pol sequences were calculated using the methods described in Tajima and Nei (1984. Mol Biol Evol. 1: 269-285) and Tamura (1992. Mol Biol Evol 9: 678-687). Moreover, confidence probability values were calculated to confirm the statistical significance for the obtained differences.

DNA Sequence Analysis The restriction digest pattern of the colony DNAs indicated that seven different groups were identified.

Sequence analysis established that five of the seven

groups contained sequences corresponding to non-PERV sequences but rather to retroelements and as such will not be discussed further. Sixty-one colonies out of the 84 screened belonged to PERV-MSN1 and 14 belonged to PERV-MSN4. Restriction analysis of a small number of PERV-MSN1 clones showed approximately equal numbers of BTP-4/5 and BTP-6 varieties.

PERV-MSN1 The nucleotide sequences of a total of three PERV- MSN1 pol region clones (BTP-4,-5, and-6) were determined which originated from three miniature swine (11216 c/c, 12525 d/d and 11842 d/d, respectively). BTP-4 is shown as SEQ ID NO 5; BTP-5 is shown as SEQ ID NO 6; BTP-6 is shown as SEQ ID NO 7.

SEQ ID NO: 3 BTP-4 sequence TGTAAGAAAA AAGAAATCTG GAAAATGGAG AATGTTAACA GATTTAAGGA AAGTTAATAG 60 TAGCATGGAA CCTACGGGGG CCTACAGCCA GGACTCCCGT CACCTGCTGT GATACGTGAA 120 CAATGGCCAA TTCTTATTAT TGATCTTAAA GATTGTTTTT ATACTGTACC ATTACATGAA 180 AAGGATATTT CTAGATTTGC ATTTACAGTT CCATCCCTAA ATAATAAAAA ACCTGTTAAA 240 AGATACACCT GGAAAGTTCT ACCTCAAGGG ATGATAAACT GCCCTACTTT ATGCCAAACT 300 TTTGTAGCAA CGCCTTTACA GAATATTCGA CGACAATTTC CAGATGCATA TGTCACTCAT 360 TACACGGATG ATATTTTGCT GCCTCATAAA GATCCTTTAA AGAAAATATA TTTACAAACA 420 GGTTGAATTT ACTAAATTTG GATTAAAGAT AGCACCTGAT AAAACACAAG ATGCTGAGCC 480 TTTCACTCAC TTAGGATACC TTATGGCCAA AGGGTGTTTT AAACCACAAA AGGTACAAAT 540 TCGTACAGAA CATCTTAAAA CCTGAAACGA CTTTCAGAAA TTATTG 586 SEQ ID NO: 4 BTP-5 Sequence TGTAAGAAAA AAGAAATCTG GAAAATGGAG AATGTTAACA GATTTAAGGA 50 AAGTTAATAG TAGCATGGAA CCTACGGGGG CCTACAGCCA GGACTCCCGT 100

CACCTGCTGT GATACGTGAA CAATGGCCAA TTCTTATTAT TGATCTTAAA 150 GATTGTTTTT ATACTGTACC ATTACATGAA AAGGATATTT CTAGATTTGC 200 ATTTACAGTT CCATCCCTAA ATAATAAAAA ACCTGTTAAA AGATACACCT 250 GGAAAGTTCT ACCTCAAGGG ATGATAAACT GCCCTACTTT ATGCCAAACT 300 TTTGTAGCAA CGCCTTTACA GAATATTCGA CGACAATTTC CAGATGCATA 350 TGTCACTCAT TACACGGATG ATATTTTGCT GCCTCATAAA GATCCTTTAA 400 AGAAAATATA TTTACAAACA GGTTGAATTT ACTAAATTTG GATTAAAGAT 450 AGCACCTGAT AAAACACAAG ATGCTGAGCC TTTCACTCAC TTAGGATACC 500 TTATGGCCAA AGGGTGTTTT AAACCACAAA AGGTACAAAT TCGTACAGAA 550 CATCTTAAAA CCTGAAACGA CTTTCAGAAA TTATT 585 SEQ ID NO: 5 BTP-6 Sequence TGTAAGAAAA AAGAAATCTG GAAAATGGAG AATGTTAACA GATTTAAGGA 50 AAGTTAATAG TAGCATGGAA CCTACGGGGG CCTACAGCCA GGACTCCCGT 100 CACCTGCTGT GATACGTGAA CAATGGCCAA TTCTTATTAT TGATCTTAAA 150 GATTGTTTTT ATACTGTACC ATTACATGAA AAGGAGAGTC CTAGATTTGC 200 ATTTACAGTT CCATCCCTAA ATAATAAAAA AAACCTGTTA AAAGATACAC 250 CTGGAAAGTT CTACCTCAAG GGATGATAAA CAGCCCTACT TTATGCCAAA 300 CTTTTGTAGC AACGCCTTTA CAGAATATTC GACGACAATT TCCAGATGCA 350 TATGTCACTC ATTACACGGA TGATATTTTG CTGGCTCATA AAGATCCTTT 400 AAAGAAAATA TATTTACAAA CAGAGGTTGA ATTTACTAAA TTTGGATTAA 450 AGATAGCACC TGATAAAACA CAAGATGCTG AGCCTTTCAC TCACTTAGGA 500 TATCTTATGG CCAAAGGGAG TTTTAAACCA CAAAAGGTAC AAATTCGTAC 550 AGAACATCTT AAAACCTGAA ACGACTTTCA GAAATTATTG 590 Alignment of the PERV-MSN1 sequences (Figure 1) showed that BTP-4 and BTP-5 were identical and that BTP-6

displayed differences at 11 positions (seven nucleotide substitutions and two 2-bp insertions).

The identical nucleotide sequences of BTP-4 and BTP- 5 clones were used for comparative analysis (Table 2).

Nucleotide sequence comparison and phylogenetic analyses showed similarities to several pol genes from different types of retroviruses including Type-B,-D and lentiviruses (Table 2). Recently Type-A, B,-D retroviruses have been assigned to a family of retroviruses denoted betaretroviruses (van Regenmortel et al. 2000. In Virus Taxonomy, VIIth ICTV Report, pp 1104.

Academic Press, San Diego, CA).

The pol genes of PERV-MSN1 and-MSN4 are related to Betaretroviruses as described below.

Phylogenetic alignments were performed based on translated sequences from the amino terminus of pol using either a stretch from the motif AINA and its analogs to two amino acids before the super-conserved motif YVDD, or a more narrow one, from motif AINA to motif VKRY and its analogs. The first alignment had 129 columns, while the latter had 84. The phylogenetic tree was derived from the longer alignment. The shorter alignment contained a more complete representation of human endogenous Betaretrovirus sequences (from the NMWV clones, which end at VKRY) than the longer one. In the shorter alignment, the sequences most similar to PERV-MSN1 were MMTV and HERV-K10 while PERV-MSN4 was most similar to JSRV. In the longer alignment, PERV-MSN1 wasbetween PERV-MSN4 and HML- 6, while PERV-MSN4 was between JSRV and PERV-MSN1. In trees based on the two alignments, these two PERV sequences branched together with the HML sequences, with only HRV-5 and the IAPs being clearly more ancestral relative to the rest of Retroviridae. We conclude that

the two novel PERV sequences belong to genus Betaretrovirus, and that although they are unique they are related to several human MMTV-like sequences.

Furthermore, the results show that Retroviridae has a much greater spectrum of members than previously understood. The known extant exogenous retroviruses, which previously dominated our view of Retroviridae only represent a minor portion of the family of retroviruses.

PERVs span a similar sequence spectrum as HERVs. This indicates that there may be a pool of common ERVs in vertebrates, created by extensive interspecies transfers during vertebrate evolution.

Phylogenetic tree analysis using the Neighbor-Joining method of tree building showed that PERV-MSN1 was most similar to the human endogenous retrovirus HML-6 (Medstrand and Blomberg. 1993. J Virol. 67: 6778-6787), which clusters with Betaretrovirus (type ABD) sequences.

Based on the obtained nucleotide sequence of PERV-MSN4 pol, it was possible to assign this sequence phylogenetically also to the Betaretrovirus genus (see above).

Phylogenetic trees made with MEGA using DNA sequences showed that PERV-MSN1 was mostly similar to Type-B and-D retroviruses. Both Maximum Parsimony and Neighbor- Joining methods of tree building showed that PERV-MSN1 was most similar to MMTV and HERV-K10 (Genbank Accession Numbers AF033807 and M14123, respectively) BTP-4 is most identical to HERV-K10 at 62%. The longest stretch of nucleotides that are identical is 11.

Other lengths of sequence and % identity are as follows: 11 mer: 100% 20 mer: 17 out of 20 = 85%

30 mer: 21=70% 32: 25 out of 32=78% Table 2 Percent Nucleotide Sequence Identity of PERV-MSN1 pol to Known Retroviruses Retrovirus Genus Genbank % Identity to Ace. # PERV-MSN1 HERV-K10 Type-B M14123 62 % MMTV Type-B AF033807 60 % JSRV Type-D M80216 60 % MPMV Type-D M12349 57% GaLV Mam. Type-C M26927 39 % PERV-A Mam. Type-C AF038601 39 % PERV-B Mam. Type-C Y17013 40 % PERV-C Mam. Type-C AF038600 38 % PERV-MSN4 Betaretroviruses----44% HERV-K family members show conservation of complete and intact retroviral genes (Ono et al. 1986. J. Virol.

60: 589-598; Mayer et al. 1999. Nature Genetics 21: 257- 258). Moreover, HERV-K members contain complete genomes with ORFs encoding gag, pol, and env proteins. Enzymatic activities for the HERV-K dUTPase, protease, and endonuclease have been reported (Harris et al. 1997.

Biochem. Cell. Biol. 75: 143-151; Kitamura et al. 1996.

J. Virol. 70: 3302-3306; Schommer et al. 1996. J. Gen.

Virol. 77 : 375-379). Recently, it was shown that several HERV-K members encode also functional RT polymerase and RNase H domain (Berkhout et al. 1999. J. Virol. 73: 2365- 2375). Thus, the human genome harbors functional copies of type B endogenous retroviruses. It is possible that these retroelements are actively retrotranscribed and mobile. Therefore, it cannot be excluded that they could also be infective and transmitted between hosts. The identification in the porcine genome of PERV-MSN1, and

its phylogenetic relationship to the HERV-K family of endogenous retroviruses, raise the possibility of biologically active PERV-MSN1 relatives in the porcine genome.

PERV-MSN4 Three PERV-MSN4 clones were sequenced which originated from three miniature swine from the d haplotype (12525 d/d, 11797 d/d and 11842 d/d). All three clones were identical and have the sequence shown as SEQ ID NO: 6 SEQ ID NO: 6 BTP-9 Sequence TACTATCCCC CCAAAATCAG GTAAATGGAG GCTGTTGCAT TATTTCAGAA AAATTAATAA 60 AACAACGTAC ACTATGAGAG CCTTACAAAC CTATGACTTG CCTTCTCCTA CTGCAATTCC 120 CTTAGACTGG GCTTTAATTG TTACAGATTT AAAGGATTGT ATTTCACCAT ACCTTATCCC 180 CTCAAGATAA ATGTAGATTT GCTTTCTCTG TTCCTGCATT AAATAATAAA GCACCTATGA 240 AATGATATCA GTGGAAAGTG TTGCCGCAGG GCATGAAAAC AGCCCTACCA TGTGCCAAGA 300 ATTTGTGGAT CAAGCCTTAG CGCCTGTTAG ACTTAAATAC CCTGAGGCAT ATATTATTCA 360 CTACATGGAT GATATTCTCT TCTCTGATCC AAAAGACCAA CACGTTTTTA ACATCTTGCT 420 AGATACCAAA GTATGTCTTA ATAAAAGGGG GCTTATTACA GCCCCAGATA AAATTCAAAA 480 AACCCCTCCA TTTCAACATT TAGCAACTTC GATAAAAGTC TGTACTATTC GGCCACAGAA 540 AAAATGCAAA TTTTCAAGGA CCAATTGAAT ACATTAAATG ATTTTCAAAA ATTATTG 597 Nucleotide sequence comparison showed similarities to several types of retroviruses (Table 3).

Alignment of PERV-MSN1 and PERV-MSN4 (BTP-9) sequences (Figure 3) indicates no significant sequence identity. However, in phylogenetic analyses using the longer sequences (see above), PERV-MSN1 cluster between PERV-MSN4 and HML-6 whereas PERV-MSN4 cluster between

JSRV and PERV-MSN1. Thus, both PERVs described here were assigned to the betaretrovirus genus. Even though the overall % nucleotide sequence identity was higher to HIV- 1 and Type-C viruses (approximately 50%), closer phylogenetic relationship was found between PERV-MSN4 and Jaagsiekte sheep retrovirus (JSRV) which had 44% identity (Table 3).

Table 3 Percent Nucleotide Sequence Identity of PERV-MSN4 pol to Known Retroviruses Retrovirus Genus Genbank % Identity to Ace. # PERV-MSN4 HIV-I Lentivirus M62320 52 % HTLV-1 HTLV-type J02029 50 % PERV-A Mam. Type-C AF038601 49 % PERV-B Mam. Type-C Y17013 50 % PERV-C Mam. Type-C AF038600 51 % PERV-MSN 1 Type-B/D-----------44 % HERV-K10 Type-B M14123 46 % JSRV Type-D M80216 44% Hypothetical Protein Sequences for PERV-MSN1 and PERV- MSN4 The hypothetical reading frames of PERV-MSN1 and PERV-MSN4 nucleotide sequences are shown in Figure 12 and Figure 13, respectively. Using the three reading frames for each nucleotide sequence, protein alignments were made to published protein sequences using GeneWorks 2.5.

PERV-MSN1 was mostly similar to type B and D retroviruses (Betaretroviruses).

Translation in silico (within a computer-simulated environment) of the PERV sequences described in the present invention disclosure revealed them to be defective. However, among the proviral sequences present

in the genome of the MHC inbred miniature swine, there may be highly similar sequences with the potential to be expressed as proteins. Identification of such expressed polypeptides and the sequences of such may subsequently be used to generate vaccines and antibodies specific for type B/D PERVs.

EXAMPLE 2 Southern Blot Analysis of Porcine Genomes for PERV-MSN1 and PERV-MSN4 Sequences Genomic DNA was prepared from peripheral blood lymphocytes (PBLs) of 15 individual miniature swine using Qiagen's (Valencia, CA) QIAmp Bloods Kit. Genomic DNA from domestic pig was obtained from Clontech (Palo Alto, CA). Either 7 or 5 pg of DNA were digested with 350 units of EcoRI (New England Biolabs, Beverly, MA) for 16 h and run on a 0.8% agarose gel for 18 h. After base denaturation and neutralization, the DNA was then transferred to a nylon membrane using Schleicher and Schuell's (Keene, NH) Turboblot kit. The Southern probes were made using Ready-to-go Beads (D random priming kit (Amersham Pharmacia Biotech, Newark, NJ) and 32P dCTP (NEN Life Sciences, Pittsburgh, PA). 40 ng of PCR product from both a PERV-MSN1 or a PERV-MSN4 clone using 5'-MOP-2 and 3'-MOP-2 primers was used as a template for a probe. Approximately 5 x 106 cpm of probe were added to 10 ml of ExpressHybe (Clontech). The hybridization was conducted at 60°C for PERV-MSN1 and at 57°C for PERV-MSN4 for 1.5 h. The membranes were washed three times for 5 min each at room temperature with 2X SSC/0.1% SDS. The room temperature washes were followed by two subsequent 10 min high-stringency washes with 0.2X SSC/0.1% SDS at

60°C for PERV-MSN1 and at 57°C for PERV-MSN4. The membranes were finally washed with 2X SSC and exposed for autoradiography at-70°C for several days before development.

A single membrane was used and initially hybridized using the PERV-MSN1 probe as described. After development, the hybridized probe was removed by boiling for 15 min in 0.1X SSC/0.5% SDS. The membrane was then hybridized with the PERV-MSN4 probe.

PERV-MSN1 Figure 14, lane 1 contains partially digested miniature swine genomic DNA, followed by 15 lanes of 15 individual miniature swine genomic DNA samples. Lane 17 contains domestic pig genomic DNA. The Southern blot shows at least 1 to 2 copies of PERV-MSN1 exist in the swine genome. Several polymorphisms are evident. Lane 4 is genomic DNA from a SLA c/c haplotype miniature swine.

All other miniature swine samples are of the d/d haplotype. The SLA c/c swine produces a 6 kb band rather than the 7 kb band found in the d/d swine. Several samples show an intense band at 4 kb that is not evident in others. The domestic pig has the intense band at 7 kb and a second faint band at 6 kb.

PERV-MSN4 The Southern blot (Figure 15) shows at least a single copy PERV-MSN4 locus in the miniature swine genome. The faint bands may represent divergent copies of PERV-MSN4 loci or cross-hybridization to related or similar sequences. No apparent RFLPs are evident from this experiment. The domestic pig also appears to contain PERV-MSN4 loci in its genome.

EXAMPLE 3 Transcription of PERV-MSN1 Type B/D and PERV-MSN4 Porcine Endogenous Retroviral Sequences RNA preparation and cDNA synthesis Total RNA was extracted from tissues or cells using the TRIzol kit (Gibco Life Technologies, Inc) according to the manufacturers'instructions.

The cDNA was synthesized using the Gibco Life Technologies Superscript Preamplification System for First Strand cDNA Synthesis. Between 0.5 to 1 yg of RNA was added to a 16.5 yl reaction volume containing 15mM Tris-HCl (pH 8.4), 75mM KC1,3.8mM MgCl2,1 unit of DNase I. Samples were incubated for 10 min at 37°C. The reaction was terminated for 5 min at 75°C.

200 ng of Random Hexamers (Genosys Biotechnologies, Inc. (The Woodlands, TX), 0.6mM dNTP, 8mM DTT were added to the reaction. Samples were incubated for 5 min at room temperature and then 200 units of Superscriptt3 reverse transcriptase (RT) were added resulting in a 25 jul total reaction volume. The samples were incubated at room temperature for 10 min, then at 42°C for 50 min.

Heating at 75°C for 15 min terminated the reaction.

Two units of RNase H was added to the sample and incubated at 37°C for 20 min.

Amplifying the sample with an internal competitive standard, 18S ribosomal RNA, tested the quality of the obtained cDNA.

PERV-MSN1 and-MSN4 Specific RT-PCR 2.5 Ill of the cDNA sample was added to a 50 yl reaction volume. The final reagent mixture included 50 mM KC1,10 mM Tris-HC1 (pH 8.3), 1.5 mM MgCl2,0.2 mM dNTP and 1.25 units of Amplitaq Gold (Perkin-Elmer Corporation, Foster City, CA). The primers were present at a concentration of 0.2 M each.

The reactions were amplified in a Perkin-Elmer GeneAmp 96005 thermal cycler. The initial denaturing step was 9 min at 95° C followed by 35 cycles of 96°C for 10 sec, 59°C for 30 sec and 72°C for 30 sec. Cycling was followed by incubation for five min at 72°C and brought down to 4°C.

Primers specific for the pol gene of PERV-MSN1 and PERV-MSN4, respectively, were developed using GeneWorks 2.5 (IntelliGenetics, Inc., Mountain View, CA) and synthesized by Genosys Biotechnologies, Inc. (The Woodlands, TX). The nucleotide sequences of the forward (MSN 1F, SEQ ID NO: 7), reverse (MSN 1R, SEQ ID NO: 8), forward (MSN 4F, SEQ ID NO: 9), and reverse (MSN 4R, SEQ ID NO: 10) primers are shown below: SEQ ID NO: 7 MSN 1F 5'-GCATGGAACC TACGGGG-3' SEQ ID NO: 8 MSN 1R 5'-CACAAGATGC TGAGCCTTTC-3' SEQ ID NO: 9 MSN 4 F 5'-TGCAATTCCC TTAGACTGGG-3' SEQ ID NO: 10 MSN 4 R 5'-TCAGTGGAAA GTGTTGCCG-3'

Agarose gel electrophoresis of RT-PCR products obtained from the cell and tissue types from which the RNA was prepared are shown in Figures 16A and 16B. The cKit+ and whole bone marrow (Figure 16, A and B, lanes 1 and 2) RNAs were prepared from cells obtained from an a/d heterozygous miniature swine. The remaining samples (Figures 16A and 16B, lanes 3 to 7) were prepared from spleen, liver, thymus, PBLs, and lung cells obtained from an a/a homozygous miniature swine. A heart sample from an a/a miniature swine was analyzed only for PERV-MSN4 expression (Figure 16B, lane 8). Negative controls without the presence of RT are shown (Figure 16A, lanes 8 to 14; Figure 16B, lanes 9 to 16). Negative blank controls are also shown (Figure 16A, lane 16; Figure 16B, lane 19). Positive control amplifications using PERV-MSN1 and-MSN4 PCR products (Figure 16A, lane 15; Figure 16B, lane 17), and miniature swine genomic DNA (Figure 16A, lane 17; Figure 16B, lane 18) are shown.

The expression of PERV-MSN1 and-MSN4 pol in porcine bone marrow samples, as determined by RT-PCR (see above) mimics the expression pattern of biologically active HERV-K (Berkhout et al. 1999. J. Virol 73: 2365-2375).

Therefore, of particular importance for this invention disclosure, is the availability of nucleotide sequence information of porcine relatives of biologically active retroelements in the human (xenotransplant recipient) genome. Moreover, the expression of active HERV-K in human bone marrow as well as expression of both PERV-MSN1 and-MSN4 pol mRNA in porcine bone marrow might be significant with respect to potential recombination and cross-packaging of these retroelements. Currently we have no evidence for expression of biologically active PERV-MSN1 or-MSN4.

EXAMPLE 4 Method of obtaining the env gene, LTRs, and full length PERV-MSN1 and PERV-MSN4 sequences In order to obtain the env gene, LTRs and ultimately full-length cDNA clones corresponding to PERV-MSN1 and PERV-MSN4,3'-RACE technology (Frohman, M. A., Dush, M. K. and Martin, G. R., Proc. Natl. Acad. Sci. USA, 85 (23): 8998-9002 (1988)) can be performed using specific primers for PERV-MSN1 and PERV-MSN4, respectively. cDNA will be made from tissues expressing PERV-MSN1 and PERV- MSN4.

Nucleotide sequence determination of the env gene and LTRs in obtained cDNAs will allow the design of primers specific for the env genes and LTRs of PERV-MSN1 and PERV-MSN4, respectively. Using such primers, amplification of full-length cDNAs will subsequently be performed.

Currently no evidence is available for the existence of expressed full-length loci encoding either PERV-MSN1 or PERV-MSN4. The outlined technique can be used to determine whether or not such full-length sequences are expressed in the miniature swine. Moreover, this methodology allows identification and sequence determination of the env genes of both PERV-MSN1 and PERV-MSN4. Subsequent analyses of infectivity and cell tropism of these endogenous retroviruses is therefore facilitated by the information available.

Efforts to identify functional PERV-MSN1 and PERV- MSN4 viruses were performed using the Product-Enhanced Reverse Transcription (PERT) assay (Silver et al. 1993.

Nucleic Acids Research 21: 3593-3594; Pyra et al. 1994.

Proc. Natl. Acad. Sci. USA. 91: 1544-1548; Lugert et al.

1996. Biotechniques 20: 2-4). In general, sucrose gradient separation of supernatants taken from lysed pig bone marrow cells as well as supernatants from bone marrow cells that have been cultured four days. Fractions containing putative retroviral particles are isolated and analyzed for RT activity. The PERT assay is the most sensitive method currently available to detect enzymatic activity of endogenous retrovirus RT genes and can detect RT activity present in single virus particles. The cDNA produced is then amplified using PERV-MSN1 and PERV-MSN4 specific primer pairs. An example of a specific protocol used was as follows.

Sucrose density gradient centrifugation. For analysis of cell culture supernatants 3-4 x 106 cells were incubated overnight at 37°C with approximately 4 ml of culture medium (DMEM supplemented with 10% FBS). The culture medium was filtered through 0.45 ym ACRODISC filters (Gelman Sciences, Ann Arbor, MI), prior to loading onto 20-65% sucrose gradients.

Sucrose gradients were prepared light end first using a gradient-pourer and buffered with 100 mM NaCl, 10 mM Tris-HC1 pH 7.5,1 mM EDTA and centrifuged at 100,000Xg for 16 h at 4°C (27,000 rpm, Beckman SW41TI rotor).

Gradient fractions (1 ml/fraction) were collected by gravity flow from the bottom of the gradient. The density of each gradient fraction was then measured using a refractometer (Atago, Tokyo, Japan).

PCR-based reverse transcriptase assay. The assay used is an adaptation of that reported by Silver et al.,

(1994. Nucleic Acids Research. 21: 3593-3594) and has been described in detail (Patience et al. 1996. J. Virol.

70: 2654-2657). Briefly, virus particles present in 0.5 y1 aliquots of sucrose gradient fractions were lysed by the addition of 4.5 y1 of 0.22% Triton X-100.

Alternatively, cell line culture supernatant samples were filtered through 0.45 ym filters (ACRODISC) and 250 il samples were centrifuged at 355, OOOXg for 15 min at 4°C (Beckman TLA 100 rotor, 100,000 rpm). The supernatant was removed and the virus pellet was solubilized in 10 yl of 0.2 % Triton X-100 of which 5 jil was used in the RT reactions. Either of the virus preparations was subjected to the RT assay. In order to increase sensitivity, following agarose electrophoresis and capillary blotting, the products were probed with an internal 32P-endlabeled oligonucleotide SEQ ID NO 11 (5'-GCCTTTGAGAGTTACTTCTTTG- 3') and hybridization was tested by autoradiography at- 70°C Results: We used the PERT assay (Pyra et al. 1994. Proc. Natl.

Acad. Sci. USA, 91: 1544-1548; Lugert et al. 1996.

Biotechniques 20: 210-217) for detection of functional RT enzymatic activity. Supernatants from both salivary gland and thymus, cells that were shown to be positive for both PMSN-1 and PMSN-4 mRNA expression by RT-PCR, were isolated and subjected to sucrose gradient centrifugation. PERT assays were then performed on aliquots taken from the sucrose gradients. RT activity was not detected by this method even following radioactive probing. The sensitivity of the assay was beyond 10-4 Units of RT. From these experiments we can conclude that expression of loci competent for the

formation of retrovirus particles was not detected in cells expressing PMSN-1 and PMSN-4.

EXAMPLE 5 Hybridization conditions that distinguishes PERV-MSN1 and PERV-MSN4 sequences from other known PERVs.

The specificity and selectivity of the PCR amplifications and the primer hybridization conditions used with the degenerate 5'-MOP-2 and 3'-MOP-2 primers were analyzed. No amplification products corresponding to type C PERVs were generated by the PCR (Figure 17). Thus, these primers appear to be biased to amplify type B and D endogenous retroviruses.

Further specificity was obtained using the PERV-MSN1 and PERV-MSN4 specific primers described in Example 3.

PCR using the Group 1F (SEQ ID NO: 7) and Group 1R (SEQ ID NO: 8) selectively amplified PERV-MSN1 DNA templates but not PERV-MSN4 or type-C PERV templates. PCR using the Group 4F (SEQ ID NO: 9) and Group 4R (SEQ ID NO: 10) selectively amplified PERV-MSN4 DNA templates but not PERV-MSN1 or type-C PERV templates. Furthermore, neither primer pair amplified human genomic DNA. SEQ ID NOS: 7- 10 are also useful as probes.

In the RT-PCR assays described in Example 3, we were unable to detect any PERV-MSN1 or PERV-MSN4 products in RNA prepared from cells and tissues that express abundant amounts of type-C PERVs. Thus, specificity for the selective amplification of PERV-MSN1 and PERV-MSN4, respectively, was also observed in the RT-PCR assays.

Thus, specificity and selectivity for these primers and PCR conditions have been shown.