Molecular sequences of swine retroviruses method of using

Details Molecular sequences of swine retroviruses method of using Molecular sequences of swine retroviruses method of using

1996-12-13

2001-02-20

Riley, Jezia (Department: 1656)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving virus or bacteriophage

C435S006120, C536S022100, C536S023100, C536S023500, C536S024300, C536S024310

Reexamination Certificate

active

06190861

ABSTRACT:

FIELD OF THE INVENTION
The invention relates to porcine retroviral sequences, peptides encoded by porcine retroviral sequences, and methods of using the porcine retroviral nucleic acids and peptides.
BACKGROUND OF THE INVENTION
Advances in solid organ transplantation and a chronic shortage of suitable organ donors have made xenotransplantation an attractive alternative to the use of human allografts. However, the potential for introduction of a new group of infectious diseases from donor animals into the human population is a concern with the use of these methods.
The term applied to the natural acquisition by humans of infectious agents carried by other species is zoonosis. The transplantation of infection from nonhuman species into humans is best termed “direct zoonosis” or “xenosis.”
Nonhuman primates and swine have been considered the main potential sources of organs for xenotransplantation (Niekrasz et al. (1992)
Transplant Proc
24:625; Starzl et al. (1993)
Lancet
341:65; Murphy et al. (1970)
Trans Proc
4:546; Brede and Murphy (1972)
Primates Med
7:18; Cooper et al. In Xenotransplantation: The Transplantation of Organs and Tissues between Species, eds. Cooper et al. (1991) p. 457; R Y Calne (1970)
Transplant Proc
2:550; H. Auchincloss, Jr. (1988)
Transplantation
46:1; and Chiche et al. (1993)
Transplantation
6:1418). The infectious disease issues for primates and swine are similar to those of human donors. The prevention of infection depends on the ability to predict, to recognize, and to prevent common infections in the immunocompromised transplantation recipient (Rubin et al. (1993)
Antimicrob Agents Chemother
37:619). Because of the potential carriage by nonhuman primates of pathogens easily adopted to humans, ethical concerns, and the cost of maintaining large colonies of primates, other species have received consideration as organ donors (Brede and Murphy (1972)
Primates Med
7:18; Van Der Riet et al. (1987)
Transplant Proc
19:4069; Katler In Xenotransplantation: The Transplantation of Organs and Tissues between Species, eds. Cooper et al. (1991) p. 457; Metzger et al. (1981)
J Immunol
127:769; McClure et al. (1987)
Nature
330:487; Letvin et al. (1987)
J Infect Dis
156:406; Castro et al. (1991)
Virology
184:219; Benveniste and Todaro (1973)
Proc Natl Acad Sci USA
70:3316; and Teich, in RNA Tumor viruses, eds. Weiss et. al. (1985) p. 25) The economic importance of swine and experience in studies of transplantation in the miniature swine model have allowed some of the potential pathogens associated with these animals to be defined (Niekrasz et al. (1992)
Transplant Proc
24:625; Cooper et al. In Xenotransplantation: The Transplantation of Organs and Tissues between Species, eds. Cooper et al. (1991) p. 457; and Leman et al. (1992) Diseases of Swine, 7th ed. Ames, Iowa:Iowa State University). Miniature swine have received consideration as organ donors because of a number of features of the species. The structure and function of the main pig organs are comparable to those of man. Swine attain body weights and organ sizes adequate to the provision of organs for human use. Lastly, veterinarians and commercial breeders have developed approaches to creation of specific-pathogen-free (SPF) swine with the ability to eliminate known pathogens from breeding colonies (Alexander et al. (1980)
Proc
6
th Int Congr Pig Vet Soc,
Copenhagen; Betts (1961)
Vet Rec
73:1349; Betts et al. (1960)
Vet Rec
72:461; Caldwell etal. (1959) J Am Vet Med Assoc 135:504; and Yong (1964)
Adv Vet Sci
9:61).
Concern exists over the transfer of porcine retroviruses by xenotransplantation (Smith (1993)
N Engl J Med
328:141). Many of the unique properties of the retroviruses are due to the synthesis of a complementary DNA copy from the RNA template (by reverse transcriptase), and integration of this DNA into the host genome. The integrated retroviral copy (which is referred to as an endogenous copy or “provirus”) can be transmitted via the germ line.
SUMMARY OF THE INVENTION
In general, the invention features a purified swine or miniature swine retroviral nucleic acid, e.g., a Tsukuba nucleic acid, a purified miniature swine retroviral nucleic acid sequence of SEQ ID NO:1 or its complement, SEQ ID NO:2 or its complement, or SEQ ID NO:3 or its complement, and methods of their use in detecting the presence of porcine, e.g., miniature swine, retroviral sequences.
In another aspect, the invention features a purified nucleic acid, e.g., a probe or primer, which can specifically hybridize with a purified swine or miniature swine retroviral genome, e.g., a Tsukuba genome, the sequence of SEQ ID NO:1 or its complement, SEQ ID NO:2 or its complement, or SEQ ID NO:3 or its complement.
In preferred embodiments the nucleic acid is other than the entire retroviral genome of SEQ ID NO:1 or its complement, SEQ ID NO:2 or its complement, or SEQ ID NO:3 or its complement, e.g., it is at least 1 nucleotide longer, or at least 1 nucleotide shorter, or differs in sequence at at least one position, e.g., the nucleic acid is a fragment of the sequence of SEQ ID NO:1 or its complement SEQ ID NO:2 or its complement, or SEQ ID NO:3 or its complement, or it includes sequence additional to that of SEQ ID NO:1, or its complement, SEQ ID NO:2 or its complement, or SEQ ID NO:3 or its complement.
In preferred embodiments, the nucleic acid has at least 60%, 70%, 72%, more preferably at least 85%, more preferably at least 90%, more preferably at least 95%, most preferably at least 98%, 99% or 100% sequence identity or homology with a sequence from SEQ ID NO:1 or its complement, SEQ ID NO:2 or its complement, or SEQ ID NO:3 or its complement.
In other embodiments: the sequence of the nucleic acid differs from the corresponding sequence of SEQ ID NO:1 or its complement, SEQ ID NO:2 or its complement, or SEQ ID NO:3 or its complement, by 1, 2, 3, 4, or 5 base pairs; the sequence of the nucleic acid differs from the corresponding sequence of SEQ ID NO:1 or its complement, SEQ ID NO:2 or its complement, or SEQ ID NO:3 or its complement, by at least 1, 2,3,4, or 5 base pairs but less than 6, 7, 8, 9, or 10 base pairs.
In other preferred embodiments: the nucleic acid is at least 10, more preferably at least 15, more preferably at least 20, most preferably at least 25, 30, 50, 100, 1000, 2000, 4000, 6000, or 8060 nucleotides in length; the nucleic acid is less than 15, more preferably less than 20, most preferably less than 25, 30, 50, 100, 1000, 2000, 4000, 6000, or 8060 nucleotides in length.
In yet other preferred embodiments: the nucleic acid can specifically hybridize with a translatable region of a miniature swine retroviral genome, e.g., the retroviral genome of SEQ ID NO:1, or its complement, SEQ ID NO:2 or its complement, or SEQ ID NO:3 or its complement, e.g., a region from the gag, pol, or env gene; the probe or primer can specifically hybridize with an untranslated region of a miniature swine retroviral genome, e.g., the retroviral genome of SEQ ID NO:1, or its complement SEQ ID NO:2 or its complement, or SEQ ID NO:3 or its complement; the probe or primer can specifically hybridize with a non-conserved region of a miniature swine retroviral genome, e.g., the retroviral genome of SEQ ID NO:1, or its complement, SEQ ID NO:2 or its complement, or SEQ ID NO:3 or its complement; the probe or primer can specifically hybridize with the highly conserved regions of a miniature swine retroviral genome, e.g., the retroviral genome of SEQ ID NO:1, or its complement, SEQ ID NO:2 or its complement, or SEQ ID NO:3 or its complement.
In preferred embodiments, the primer is selected from the group consisting of SEQ ID NOs:4-74.
In preferred embodiments, hybridization of the probe to retroviral sequences can be detected by standard methods, e.g., by radiolabeled probes or by probes bearing nonradioactive markers such as enzymes or antibody binding sites. For example, a probe can be conjugated with an enzyme such as horseradish peroxidase, where the enzymatic activity of the conjugated enzyme is used as a signal

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