Journal of Animal Science and Technology

Korean Society of Animal Sciences and Technology (한국축산학회)
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Detection and Classification of Porcine Endogenous Retroviruses by Polymerase Chain Reaction

중합효소 연쇄반응을 이용한 돼지 내인성 레트로 바이러스의 검출과 분류

  • Lee, D.H. (Department of Animal Biotechnology, College of Animal Bioscience & Technology, Konkuk University) ;
  • Lee, J.E. (Department of Animal Biotechnology, College of Animal Bioscience & Technology, Konkuk University) ;
  • Kim, H.M. (Department of Animal Biotechnology, College of Animal Bioscience & Technology, Konkuk University) ;
  • Kim, G.W. (Department of Animal Resources Science, College of Industrial Science, Kongju National University) ;
  • Park, H.Y. (Department of Animal Biotechnology, College of Animal Bioscience & Technology, Konkuk University) ;
  • Kim, Young-Bong (Department of Animal Biotechnology, College of Animal Bioscience & Technology, Konkuk University)
  • 이동희 (건국대학교 동물생명과학대학 동물생명공학과) ;
  • 이정은 (건국대학교 동물생명과학대학 동물생명공학과) ;
  • 김훈미 (건국대학교 동물생명과학대학 동물생명공학과) ;
  • 김계웅 (공주대학교 산업과학대학 동물자원학과) ;
  • 박홍양 (건국대학교 동물생명과학대학 동물생명공학과) ;
  • 김영봉 (건국대학교 동물생명과학대학 동물생명공학과)
  • Published : 2007.06.30
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Abstract

Pigs have been considered as an ideal source of donor organs because of their plentiful supply and their numerous anatomical and physiological similarities to the human in xenotransplantation. However, for the public health risks associated with the potential for porcine endogenous retrovirus(PERV) infection through xenograft from pig to human, the investigation of methods for elimination and/or control of PERV has been required. In this study we developed the detection and classification methods for PERV based on PCR using specific primers. PERV-A and PERV-B were found in all pigs including Berkshire, Duroc, Landrace, Yorkshire, miniature pig, and Korean native black pig from Jeju by PCR with type-specific primers for PERV. However, PERV-C was detected only from Duroc, miniature pig, and Korean native black pig from Jeju. PERV-A and PERV-B could be distinguished by PCR-RFLP with BamHI. These methods for PERV will be useful in rapid screening of safe organ for xenograft, furthermore, helpful in monitoring of PERV during and after xenotransplantation.

돼지의 내인성 레트로 바이러스인 PERV의 존재는 돼지의 무균 사육을 통해서도 제거할 수 없으며, 장기 이식 시 인수 공통 감염의 위험성을 내포하고 있으므로, 이종 간 장기 이식 시 면역 거부 반응과 더불어 해결해야 할 가장 큰 문제점 중의 하나이다. 본 연구에서는 국내에서 사육되고 있는 Berkshire, Duroc, Landrace, Yorkshire 종을 비롯하여 국내 미니 돼지 및 제주도 토종 흑돼지에서 PERV의 존재 유무 및 종류에 대하여 확인 및 검출하는 중합효소 연쇄반응(PCR) 기반 기법을 확립하였다. 사용된 모든 공시 돼지의 genomic DNA로부터 PERV- A 및 PERV-B가 모두 검출되었으며, PERV-C의 경우 음성대조구인 PK15 세포에서의 결과와 비교했을 때, Duroc종에서 매우 낮은 양의 PCR 산물이, 국내 미니돼지와 제주 토종 흑돼지에서는 상대적으로 높은 양의 PCR 산물이 검출되었다. 또한 PERV-A와 PERV-B의 경우 특이적 primer와 제한효소 BamHI을 이용한 PCR- RFLP를 통하여 확인할 수 있었다. 본 연구의 PERV 검출법은 이종 간 장기 이식 시 PERV에 대한 안전 검정 및 PERV의 분류 방법으로서 활용가능할 것으로 사료된다.

Keywords

References

  1. 김영봉, 박홍양, 이동희, 이정은. 2006. 무균돼지 내인성 레트로 바이러스의 엔벨로프 단백질 및 유전자. 특허 제 10-0657012호
  2. 김영봉, 유재영, 이종영, 김계웅, 박홍양. 2004. 국내 돼지에 존재하는 내인성 레트로 바이러스의 분포. 한국동물자원과학회지 46(3):307-314
  3. 이동희, 유재영, 이정은, 김계웅, 박홍양, 이훈택, 김영봉. 2005. 국내 돼지에 존재하는 내인성 레트로 바이러스의 엔밸로프 유전자 클로닝 및 분자 계통학적 분석. 한국동물자원과학회지 47(2):177-186
  4. 이동흐, 이정은, 권무식, 박홍양, 김영봉. 2004. 무균돼지에 존재하는 ㅐㄴ인성 레트로. 바이러스 PERV-A와 PERV-B Envelope 유전자 클로닝. 대한미생물학회지 34(4):373-380
  5. 이정은, 이동희, 유재영, 김계웅, 박홍양, 이훈택, 김영봉. 2006. 국내 돼지에서 분리한 내인성 레트로 바이러스 gag 유전자의 분자 생물학적 특성 분석. 대한미생물학회지 36(3):185-194 https://doi.org/10.4167/jbv.2006.36.3.185
  6. Breese, S. S. Jr. 1970. Virus-like particles occurring in cultures of stable pig kidney cell lines. Brief report. Arch. Gesamte. Virusforsch. 30(4):401-404 https://doi.org/10.1007/BF01258369
  7. Clemenceau, B., Jegou, D., Martignat, L. and Sai, P. 2002. Microchimerism and transmission of porcine endogenous retrovirus from a pig cell line or specific pathogen-free pig islets to mouse tissues and human cells during xenografts in nude mice. Diabetologia 45(6):914-923 https://doi.org/10.1007/s00125-002-0832-7
  8. Deng, Y. M., Tuch, B. E. and Rawlinson, W. D. 2000. Transmission of porcine endogenous retroviruses in severe combined immunodeficient mice xenotransplanted with fetal porcine pancreatic cells. Transplantation 70(7):1010-1016 https://doi.org/10.1097/00007890-200010150-00004
  9. Dinsmore, J. H., Manhart, C., Raineri, R., Jacoby, D. B. and Moore, A. 2000. No evidence for infection of human cells with porcine endogenous retrovirus (PERV) after exposure to porcine fetal neuronal cells. Transplantation 70(9):1382-1389 https://doi.org/10.1097/00007890-200011150-00020
  10. Dorling, A. 2002. Clinical xenotransplantation: pigs might fly? Am. J. Transplant. 2(8):695-700. https://doi.org/10.1034/j.1600-6143.2002.20803.x
  11. Ericsson, T. A., Takeuchi, Y., Templin, C., Quinn, G., Farhadian, S. F., Wood, J. C., Oldmixon, B. A, Suling, K. M., Ishii, J. K., Kitagawa, Y., Miyazawa, T., Salomon, D. R., Weiss, R. A. and Patience, C. 2003. Identification of receptors for pig endogenous retrovirus. Proc. Natl. Acad. Sci. USA 100(11):6759-6764
  12. Harrison, I., Takeuchi, Y., Bartosch, B. and Stoye, J. P. 2004. Determinants of high titer III recombinant porcine endogenous retroviruses. J. Virol. 78(24):13871-13879 https://doi.org/10.1128/JVI.78.24.13871-13879.2004
  13. Klymiuk, N., Muller, M., Brem, G. and Aigner, B. 2002. Characterization of porcine endogenous retrovirus gamma pro-pol nucleotide sequences. J. Virol. 76(22):11738-11743 https://doi.org/10.1128/JVI.76.22.11738-11743.2002
  14. Klymiuk, N., Muller, M., Brem, G. and Aigner, B. 2003. Recombination analysis of human-tropic porcine endogenous retroviruses. J. Gen. Virol. 84 (Pt 10):2729-2734 https://doi.org/10.1099/vir.0.19284-0
  15. Lee, D., Lee, J., Uhm, S. J., Lee, Y. S., Park, M. J., Park, H. Y, Kwon, M., Lee, H. T. and Kim, Y. B. 2006. Molecular characterization of the porcine endogenous retrovirus subclass A and B envelope gene from pigs. Transplant. Proc. 38(9):3066-3069 https://doi.org/10.1016/j.transproceed.2006.08.144
  16. Lee, J. H., Webb, G. C,, Allen, R. D. and Moran, C. 2002. Characterizing and mapping porcine endogenous retroviruses III Westran pigs. J. Virol. 76(11):5548-5556 https://doi.org/10.1128/JVI.76.11.5548-5556.2002
  17. Levy, M. F., Crippin, J., Sutton, S., Netto, G., McCormack, J., Curiel, T., Goldstein, R. M., Newman, J. T., Gonwa, T. A., Banchereau, J., Diamond, L. E., Byrne, G., Logan, J. and Klintmalm, G. B. 2000. Liver allotransplantation after extracorporeal hepatic support with transgenic (hCD55/hCD59) porcine livers: clinical results and lack of pig-to-human transmission of the porcine endogenous retrovirus. Transplantation 69(2):272-280 https://doi.org/10.1097/00007890-200001270-00013
  18. Magre, S., Takeuchi, Y. and Bartosch, B. 2003. Xenotransplantation and pig endogenous retroviruses. Rev. Moo. Virol. 13(5):311-329 https://doi.org/10.1002/rmv.404
  19. Mang, R., Maas, J., Chen, X, Goudsmit, J. and van Der Kuyl, A. C. 2001. Identification of a novel type C porcine endogenous retrovirus: evidence that copy number of endogenous retroviruses increases during host inbreeding. J. Gen. Virol. 82(Pt 8):1829-1834 https://doi.org/10.1099/0022-1317-82-8-1829
  20. Mollnes, T. E. and Fiane, A. E. 1999. Xenotransplantation: how to overcome the complement obstacle? Mol. Immunol. 36(4-5):269-276 https://doi.org/10.1016/S0161-5890(99)00050-4
  21. Niebert, M. and Tonjes, R. R. 2003. Analyses of prevalence and polymorphisms of six replicationcompetent and chromosomally assigned porcine endogenous retroviruses in individual pigs and pig subspecies. Virology 313(2):427-434 https://doi.org/10.1016/S0042-6822(03)00316-7
  22. Niebert, M. and Tonjes, R. R. 2005. Evolutionary spread and recombination of porcine endogenous retroviruses in the suiformes. J. Virol. 79(1):649654 https://doi.org/10.1128/JVI.79.1.649-654.2005
  23. Paradis, K., Langford, G., Long, Z., Heneine, W., Sandstrom, P., Switzer, W. M., Chapman, L. E., Lockey, C, Onions, D. and Otto, E. 1999. Search for cross-species transmission of porcine endogenous retrovirus in patients treated with living pig tissue. The XEN 111 Study Group. Science 285(5431):1236-1241 https://doi.org/10.1126/science.285.5431.1236
  24. Patience, C., Patton, G. S., Takeuchi, Y., Weiss, R. A., McClure, M. O., Rydberg, L. and Breimer, M. E. 1998. No evidence of pig DNA or retroviral infection in patients with short-term extracorporeal connection to pig kidneys. Lancet 352(9129):699-701 https://doi.org/10.1016/S0140-6736(98)04369-4
  25. Patience, C., Switzer, W. M., Takeuchi, Y., Griffiths, D. J., Goward, M. E., Heneine, W., Stoye, J. P. and Weiss, R. A. 2001. Multiple groups of novel retroviral genomes in pigs and related species. J. Virol. 75(6):2771-2775 https://doi.org/10.1128/JVI.75.6.2771-2775.2001
  26. Patience, C., Takeuchi, Y. and Weiss R. A. 1997. Infection of human cells by an endogenous retrovirus of pigs. Nat. Med. 3(3):282-286 https://doi.org/10.1038/nm0397-282
  27. Pitkin, Z. and Mullon, C. 1999. Evidence of absence of porcine endogenous retrovirus (PERV) infection in patients treated with a bioartificial liver support system. Artif Organs 23(9):829-833 https://doi.org/10.1046/j.1525-1594.1999.06444.x
  28. Specke, V., Plesker, R., Coulibaly, C., Boller, K., and Denner, J. 2002a. Productive infection of a mink cell line with porcine endogenous retroviruses (PERVs) but lack of transmission to minks in vivo. Arch. Virol. 147(2):305-319 https://doi.org/10.1007/s705-002-8321-z
  29. Specke, V., Schuurman, H. J., Plesker, R., Coulibaly, C., Ozel, M,. Langford, G., Kurth, R., and Dermer, J. 2002b. Virus safety in xenotransplantation: first exploratory in vivo studies in small laboratory animals and non-human primates. Transpl. Immunol. 9(2-4):281-288 https://doi.org/10.1016/S0966-3274(02)00039-4
  30. Swindle, M. M. 1996. Considerations of specific pathogen-free swine (SPF) in xenotransplantation. J. Invest. Surg. 9(4):267-271 https://doi.org/10.3109/08941939609012476
  31. Swindle, M. M. 1998. Defining appropriate health status and management programs for specificpathogen- free swim: for xenotransplantation. Ann. N. Y. Acad. Sci. 862:111-120 https://doi.org/10.1111/j.1749-6632.1998.tb09123.x
  32. Tacke, S. J., Kurth, R. and Denner, J. 2000. Porcine endogenous retroviruses inhibit human immune cell function: risk for xenotransplantation? Virology 268(1):87-93 https://doi.org/10.1006/viro.1999.0149
  33. Takeuchi Y, Patience c., Magre S., Weiss RA, Banerjee PT, Le Tissier P. and Stoye JP. 1998. Host range and interference studies of three classes of pig endogenous retrovirus. J Virol 72(12):9986 9991
  34. van der Laan, L. J., Lockey, C., Griffeth, B. C, Frasier, F. S., Wilson, e. A., Onions, D. E., Hering, B. J., Long, Z., Otto, E., Torbett, B. E. and Salomon, D. R. 2000. Infection by porcine endogenous retrovirus after islet xenotransplantation in SCID mice. Nature 407(6800):90-94 https://doi.org/10.1038/35024089
  35. Weiss, R. A., Magre, S. and Takeuchi, Y. 2000. Infection hazards of xenotransplantation. J. Infect. 40(1):21-25 https://doi.org/10.1053/jinf.1999.0604
  36. White, S. A. and Nicholson, M. L. 1999. Xenotransplantation. Br. J. Surg. 86(12):1499-1514 https://doi.org/10.1046/j.1365-2168.1999.01340.x
  37. Wilson, C. A., Wong, S., Muller, J., Davidson, C. E., Rose, T. M. and Burd, P. 1998. Type c retrovirus released from porcine primary peripheral blood mononuclear cells infects human cells. J. Virol. 72(4):3082-3087