Determination of the copy numbers of type A porcine endogenous retroviruses in domestic pigs and wild boars

Modern transplantology is in need of transplants. To solve this problem, the use of animal organs and tissues for grafting to humans (xenografts) has been proposed. However, the progress in this direction is hampered by the risk of zoogenous infection of recipients. With regard to economic and ethical criteria and to the anatomical and physiological similarity to humans, the pig is the best source of xenografts. The pig genome harbors type A porcine endogenous retroviruses (PERV), which can infect human cell lines in vitro. A population of Siberian minipigs was raised at the Institute of Cytology and Genetics just for xenografting. The goal of the present study is to analyze the copy numbers of PERV A in Siberian minipigs, their founder breeds Landrace and Large White, and wild boars. The copy numbers of PERVs have been determined by absolute measurement with SYBR Green dye. End-point dilutions of a sample with a known copy number have been used for reference. The PERV A copy numbers in standard samples of Siberian minipig DNA are 2.4, 3.6, and 4.9 per cell, which is consistent with data obtained by other scientists. Minipigs and wild boars show a significant difference in retrovirus copy numbers. Thus, the Siberian minipig genome has a considerable number of type A PERVs, conceivably pathogenic to humans. It is necessary to select animals with minimum PERV numbers in the genome for xenografting. The method of PERV A quantitation with SYBR Green allows detection of such animals and selection of Siberian minipigs for reduction of this index.

1. Aitnazarov R.B., Nikitin S.V., Knyazev S.P., Yudin N.S. The saturation of genome of the pig by porcine endogenous retroviruses: the influence of heredity and environment. Innovatsii i prodovolstvennaya bezopasnost = Innovations and food security. 2014;2:41-49. (in Russian)

2. Aitnazarov R.B., Yudin N.S., Nikitin S.V., Ermolayev V.I., Voevoda M.I. Identification of whole genomes of endogenous retroviruses in Siberian miniature pigs. Rus. J. Genetics: Applied Research. 2014;4(6):523-525. DOI 10.1134/S2079059714060021.

3. Akiyoshi D.E., Denaro M., Zhu H., Greenstein J.L., Banerjee P., Fishman J.A. Identification of a full-length cDNA for an endogenous retrovirus of miniature swine. J. Virology. 1998;72(5):4503-4507.

4. Bosch S., Arnauld C., Jestin A. Study of full-length porcine endogenous retrovirus genomes with envelope gene polymorphism in a specificpathogen- free Large White swine herd. J. Virology. 2000;74(18): 8575-8581.

5. Denner J. Recombinant porcine endogenous retroviruses (PERV-A/C): a new risk for xenotransplantation? Archives Virology. 2008;153(8): 1421-1426. DOI 10.1007/s00705-008- 0141-7.

6. Denner J. How active are porcine endogenous retroviruses (PERVs)? Viruses. 2016;8(8):E215. DOI 10.3390/v8080215.

7. Ekser B., Cooper D.K., Tector A.J. The need for xenotransplantation as a source of organs and cells for clinical transplantation. Intern. J. Surgery. 2015;23:199-204. DOI 10.1016/j.ijsu.2015.06.066.

8. Frühauf J.H., Mertsching H., Giri S., Frühauf N.R., Bader A. Porcine endogenous retrovirus released by a bioartificial liver infects primary human cells. Liver International. 2009;29(10):1553-1561. DOI 10.1111/j.1478-3231.2009.02087.x.

9. Godehardt A.W., Rodrigues Costa M., Tönjes R.R. Review on porcine endogenous retrovirus detection assays–impact on quality and safety of xenotransplants. Xenotransplantation. 2015;22(2):95-101. DOI 10.1111/xen.12154.

10. Herring C., Quinn G., Bower R., Parsons N., Logan N.A., Brawley A., Elsome K., Whittam A., Fernandez-Suarez X.M., Cunningham D., Onions D., Langford G., Scobie L. Mapping full- length porcine endogenous retroviruses in a large white pig. J. Virology. 2001;75(24): 12252-12265.

11. Karlas A., Irgang M., Votteler J., Specke V., Ozel M., Kurth R., Denner J. Characterisation of a human cell-adapted porcine endogenous retrovirus PERV-A/C. Ann. Transplantation. 2010;15(2):45-54.

12. Kimsa M.C., Strzalka-Mrozik B., Kimsa M.W., Gola J., Nicholson P., Lopata K., Mazurek U. Porcine endogenous retroviruses in xenotransplantation – molecular aspects. Viruses. 2014;6(5):2062-2083. DOI 10.3390/v6052062.

13. Le Tissier P., Stoye J.P., Takeuchi Y., Patience C., Weiss R.A. Two sets of human-tropic pig retrovirus. Nature. 1997;389(6652):681-682.

14. Lee D., Kim N.Y., Bae G.E., Lee H.J., Kwon M., Kim S.S., Lee H.T., Yang J.M., Kim Y.B. Transmissible infection of human 293T cells with porcine endogenous retroviruses subgroup А from NIH-miniature pig. Transplantation Proceed. 2008;40(10):3742-3745. DOI 10.1016/j.transproceed.2008.09.035.

15. Lee D., Lee J., Yoon J.K., Kim N.Y., Kim G.W., Park C., Oh Y.K., Kim Y.B. Rapid determination of PERV copy number from porcine genomic DNA by real-time polymerase chain reaction. Animal Biotechnol. 2011;22(4):175-180. DOI 10.1080/10495398.2011.595294.

16. Li Z., Ping Y., Shengfu L., Yangzhi Z., Jingqiu C., Youping L., Hong B. Variation of host cell tropism of porcine endogenous retroviruses expressed in chinese Banna minipig inbred. Intervirology. 2006;49(4): 185-191.

17. Liu G., Li Z., Pan M., Ge M., Wang Y., Gao Y. Genetic prevalence of porcine endogenous retrovirus in chinese experimental miniature pigs. Transplantation Proceed. 2011;43(7):2762-2769. DOI 10.1016/j.transproceed.2011.06.061.

18. Ma Y., Yang Y., Lv M., Yan Q., Zheng L., Ding F., Wu J., Tian K., Zhang J. Real-time quantitative polymerase chain reaction with SYBR green I detection for estimating copy numbers of porcine endogenous retrovirus from Chinese miniature pigs. Transplantation Proceed. 2010;42(5):1949-1952. DOI 10.1016/j.transproceed. 2010.01.054.

19. Mang R., Maas J., Chen X., Goudsmit J., van der Kuyl A.C. Identification of a novel type C porcine endogenous retrovirus: evidence that copy number of endogenous retroviruses increases during host inbreeding. J. General Virology. 2001;82(Pt. 8):1829-1834.

20. Marcucci K.T., Argaw T., Wilson C.A., Salomon D.R. Identification of two distinct structural regions in a human porcine endogenous retrovirus receptor, HuPAR2, contributing to function for viral entry. Retrovirology. 2009;6(3):1-15. DOI 10.1186/1742-4690-6-3.

21. Mazurek U., Kimsa M.C., Strzalka-Mrozik B., Kimsa M.W., Adamska J., Lipinski D., Zeyland J., Szalata M., Slomski R., Jura J., Smorag Z., Nowak R., Gola J. Quantitative analysis of porcine endogenous retroviruses in different organs of transgenic pigs generated for xenotransplantation. Current Microbiology. 2013;67(4):505-514. DOI 10.1007/s00284-013- 0397-3.

22. Morozov V.A., Morozov A.V., Rotem A., Barkai U., Bornstein S., Denner J. Extended microbiological characterization of Göttingen minipigs in the context of xenotransplantation: detection and vertical transmission of hepatitis E virus. PLoS One. 2015;10(10):e0139893. DOI 10.1371/journal.pone.0139893.

23. Nakaya Y., Shojima T., Yasuda J., Imakawa K., Miyazawa T. Epigenetic regulation on the 5’- proximal CpG island of human porcine endogenous retrovirus subgroup A receptor 2/GPR172B. Microbes Infect. 2011;13(1):49-57. DOI 10.1016/j.micinf.2010.09.014.

24. Niebert M., Tonjes R.R. Evolutionary spread and recombination of porcine endogenous retroviruses in suiformes. J. Virology. 2005;79(1): 649-654.

25. Nikitin S.V., Yudin N.S., Knyazev S.P., Aitnazarov R.B., Kobzev V.F., Bekenev V.A., Savvina M.A., Ermolaev V.I. Frequency of chromosomes carrying endogenous retroviruses in the populations of domestic pig and wild boar. Genetika = Genetics (Moscow). 2008;44(6):789- 797. (in Russian)

26. Quereda J.J., Herrero-Medrano J.M., Abellaneda J.M., García-Nicolás O., Martínez-Alarcón L., Pallarés F.J., Ramírez P., Muñoz A., Ramis G. Porcine endogenous retrovirus copy number in different pig breeds is not related to genetic diversity. Zoonoses Public Health. 2012; 59(6):401-407. DOI 10.1111/j.1863-2378.2012.01467.x.

27. Rebrikov D.V., Samatov G.A., Trofimov D.Y., Semenov P.A., Savilova A.M., Kofiadi I.A., Abramov D.D. PTsR «v real’nom vremeni» [Real- time PCR]. Moscow, BINOM. Laboratoriya znaniy Publ., 2009. (in Russian)

28. Sedlak R.H., Jerome K.R. Viral diagnostics in the era of digital polymerase chain reaction. Diagnostic Microbiology Infectious Disease. 2013;75(1):1-4. DOI 10.1016/j.diagmicrobio.2012.10.009.

29. Shimatsu Y., Yamada K., Horii W., Hirakata A., Sakamoto Y., Waki S., Sano J., Saitoh T., Sahara H., Shimizu A., Yazawa H., Sachs D.H., Nunoya T. Production of cloned NIBS (Nippon Institute for Biological Science) and α-1, 3-galactosyltransferase knockout MGH miniature pigs by somatic cell nuclear transfer using the NIBS breed as surrogates. Xenotransplantation. 2013;20(3):157-164. DOI 10.1111/xen.12031.

30. Specke V., Rubant S., Denner J. Productive infection of human primary cells and cell lines with porcine endogenous retroviruses. Virology. 2001;285(2):177-180.

31. Tikhonov V.N. Laboratornye mini-svin’i, genetika i mediko-biologicheskoe ispolzovanie [Laboratory Mini-pigs: Genetics and Biomedical Use]. Novosibirsk, SB RAS Publ., 2010. (in Russian)

32. Yang L., Güell M., Niu D., George H., Lesha E., Grishin D., Aach J., Shrock E., Xu W., Poci J., Cortazio R., Wilkinson R.A., Fishman J.A., Church G. Genome-wide inactivation of porcine endogenous retroviruses (PERVs). Science. 2015;350(6264):1101-1104. DOI 10.1126/science.aad1191.

33. Yu P., Zhang L., Li S.F., Cheng J.Q., Lu Y.R., Zeng Y.Z., Li Y.P., Bu H. A rapid method for detection of the copy number of porcine endogenous in swine. J. Rapid Methods Automation Microbiology. 2007; 15:199-205.

34. Yu P., Zhang P., Zhang L., Li S.F., Cheng J.Q., Lu Y.R., Li Y.P., Bu H. Studies on long-term infection of human cells with porcine endogenous retrovirus. Acta Virologica. 2009;53(3):169-174.

35. Yudin N.S., Aitnazarov R.B., Ermolaev V.I. Porcine endogenous retroviruses: what are the risks of infection transmission in xenotransplantation? Rus. J. Genet. Appl. Res. 2011;1(6):532-539. DOI 10.1134/S207905971106013X.

36. Zhang P., Yu P., Wang W., Zhang L., Li S., Bu H. An effective method for the quantitative detection of porcine endogenous retrovirus in pig tissues. In Vitro Cellular & Develop. Biology – Animal. 2010; 46(5):408-410. DOI 10.1007/s11626-009-9264-8.