An association between type A porcine endogenous retrovirus copy number and hematological parameters and gender in miniature pigs

Pig is the most promising species for transplantation of organs and cells into humans, although implementation of xenotransplantation in clinical practice has been hindered by the risk of infecting the recipient with zoonotic infectious diseases. Porcine endogenous retroviruses (PERV) are capable of incorporating copies of DNA into the genome of a host cell. Based on the nucleotide sequence of the envelope gene (env), three main types of pig retrovirus, PERV-A, PERV-B and PERV-C, have been recognized, with PERV-A and PERV-B having the capability of infecting human cell lines in vitro. Selection for animals with low copy number of retroviruses in the genome using simple phenotypic indications is required for the widespread implementation of xenotransplantation. The objective of this study was to evaluate the correlation between PERV-A env gene copy number and hematological parameters, gender and coat  color in miniature pigs of the Institute of Cytology and Genetics (ICG) SB RAS. Reference values for eighteen blood parameters of miniature pigs were determined, including white blood cell count (WBC), red blood cell count (RBC), platelet count (PLT), absolute (LYM#) and relative (LYM%)  lymphocyte counts, absolute (MID#) and relative (MID%) monocyte, basophil and eosinophil counts, absolute (GRA#) and relative (GRA%) granulocyte counts, hematocrit (HCT) and thrombocrit (PCT), mean cell volume (MCV) and mean platelet volume (MPV). Males had significantly higher reference values for WBC, MID#, GRA# and red cell distribution width (RDW-CV) as compared to females. The mean corpuscular hemoglobin  concentration (MCHC) and platelet distribution width (PDW-CV) were significantly higher in female animals. No correlation between PERV-A env gene copy number and the coat color of  animals was detected, suggesting that retroviral insertion sites and genes that determine the coat color of miniature pigs, namely KIT (chromosome 8) and MC1R (chromosome 6), are either located far apart on same chromosome or on different chromosomes. The copy number of PERV-A env gene in males was lower than in females. Presence of multiple copies of PERV-A on the X-chromosome is the most probable cause of such gender-related differences in miniature pigs. Thus, male miniature pigs of ICG SB RAS should be the source of material for xenotransplantation.

xenotransplantation, miniature pigs of ICG SB RAS, porcine endogenous retrovirus, PERV, envA gene, gender, coat color, blood test

1. Aitnazarov R.B., Ermolaev V.I., Nikitin S.V., Savina M.A., Kobzev V.F., Knyazev S.P., Goncharenko G.M., Bekenyov V.A., Yudin N.S. Association of endogenous retroviruses of different types with genetic markers in populations of domestic and wild pigs. Proc. of the Russ. Academy of Agricult. Sci. 2006;4:39-43. (in Russian)

2. Aitnazarov R.B., Yudin N.S., Kiril'chuk R.S., Kochnev N.N., Knyazev S.P., Voevoda M.I. Determination of the copy numbers of type A porcine endogenous retroviruses in domestic pigs and wild boars. Vavilovskii Zhurnal Genetiki i Selektsii = Vavilov Journal of Genetics and Breeding. 2016;20(6):756-761. DOI 10.18699/VJ16.192. (in Russian)

3. Aitnazarov R.B., Yudin N.S., Nikitin S.V., Ermolayev V.I., Voevo-da M.I. Identification of whole genomes of endogenous retroviruses in Siberian miniature pigs. Russ. J. Genet.: Appl. Res. 2014;4(6):523-525. DOI 10.1134/S2079059714060021.

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

5. Ekser B., Bianchi J., Ball S., Iwase H., Walters A., Ezzelarab M., Ve-roux M., Gridelli B., Wagner R., Ayares D., Cooper D.K. Comparison of hematologic, biochemical, and coagulation parameters in a1,3-galactosyltransferase gene-knockout pigs, wild-type pigs, and four primate species. Xenotransplantation. 2012;19(6):342-354. DOI 10.1111/xen.12007.

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

7. Garcia-Montojo M., Dominguez-Mozo M., Arias-Leal A., Garcia-Martinez A., De las Heras V, Casanova I., Faucard R., Gehin N., Madeira A., Arroyo R., Curtin F., Alvarez-Lafuente R., Perron H. The DNA copy number of human endogenous retrovirus-W (MSRV-type) is increased in multiple sclerosis patients and is influenced by gender and disease severity. PLoS ONE. 2013;8(1):e53623. DOI 10.1371/ journal.pone.0053623.

8. Godehardt A.W., Rodrigues Costa M., Tonjes 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.12154.11.

9. Jung W.Y., Kim J.E., Jung K.C., Jin D.I., Moran C., Park E.W., Jeon J.T., Lee J.H. Comparison of PERV genomic locations between Asian and European pigs. Anim. Genetics. 2010;41(1):89-92. DOI 10.1111/j.1365-2052.2009.01953.x.

10. Ka S., Kerje S., Bornold L., Liljegren U., Siegel P.B., Andersson L., Hallbook F. Proviral integrations and expression of endogenous avian leucosis virus during long term selection for high and low body weight in two chicken lines. Retrovirology. 2009;6:68. DOI 10.1186/1742-4690-6-68.

11. Kawaguchi H., Yamada T., Miura N., Takahashi Y., Yoshikawa T., Izu-mi H., Kawarasaki T., Miyoshi N., Tanimoto A. Reference values of hematological and biochemical parameters for the world smallest microminipigs. J. Vet. Med. Sci. 2012;74(7):933-936. DOI 10.1292/jvms.11-0571.

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. Kondrakhin S.P. (Editor). Methods of veterinary clinical laboratory diagnostics: Handbook. M.: KolosS Publ., 2004. (in Russian)

14. 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. Anim. Bio-technol. 2011;22(4):175-180. DOI 10.1080/10495398.2011.595294.

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

16. 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. Transplant. Proc. 2010;42(5):1949-1952. DOI 10.1016/j.transproceed.2010.01.054.

17. Mazurek U., Kimsa M.C., Strzalka-Mrozik B., Kimsa M.W., Adam-ska 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. Curr. Microbiol. 2013;67(4):505-514. DOI 10.1007/s00284-013-0397-3.12.

18. McMichael J.C., Stiers S., Coffin S. Prevalence of feline leukemia virus infection among adult cats at an animal control center: association of viremia with phenotype and season. Am. J. Vet. Res. 1986;47(4): 765-768.

19. McPhaul M.J., Matsumine H., Herbst M.A., Wilson J.D. Aromatase expression in extragonadal tissues of the Sebright chicken is controlled by a retroviral promoter. Trans. Assoc. Am. Physicians. 1991;104: 141-149.

20. Murphy W.G. The sex difference in haemoglobin levels in adults -mechanisms, causes, and consequences. Blood Rev. 2014;28(2):41-47. DOI 10.1016/j.blre.2013.12.003.

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

22. Nikitin S.V., Shatokhin K.S., Knyazev S.P., Goncharenko G.M., Zaporozhets V.I., Ermolaev V.I. Polymorphic loci of coat color in mini pigs. Vavilovskii Zhurnal Genetiki i Selektsii = Vavilov Journal of Genetics and Breeding. 2016;20:584-595. (in Russian)

23. Rispat G., Slaoui M., Weber D., Salemink P., Berthoux C., Shrivas-tava R. Haematological and plasma biochemical values for healthy Yucatan micropigs. Laboratory Animals. 1993;27(4):368-373.

24. Roca A.L., Nash W.G., Menninger J.C., Murphy W.J., O'Brien S.J. Inser-tional polymorphisms of endogenous feline leukemia viruses. J. Virol. 2005;79(7):3979-3986. DOI 10.1128/JVI.79.7.3979-3986.2005.

25. Sambrook J., Russell D.W. The Condensed Protocols from Molecular Cloning: A Laboratory Manual. N. Y.: Cold Spring Harbor Lab. Press, 2006.

26. Seperack P.K., Mercer J.A., Strobel M.C., Copeland N.G., Jenkins N.A. Retroviral sequences located within an intron of the dilute gene alter dilute expression in a tissue-specific manner. EMBO J. 1995;14(10):2326-2332.

27. Tandon R., Cattori V., Willi B., Meli M.L., Gomes-Keller M.A., Lutz H., Hofmann-Lehmann R. Copy number polymorphism of endogenous feline leukemia virus-like sequences. Mol. Cell. Probes. 2007;21(4):257-266. DOI 10.1016/j.mcp.2007.01.003.

28. Tikhonov V.N. Laboratory mini pigs, genetics and biomedical use. Novosibirsk: Publ. House SB RAS, 2010. (in Russian)

29. 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 Autom. Microbiol. 2007;15(2):199-205. DOI 10.1111/j.1745-4581.2007.00082.x.

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

31. 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 Cell. Dev. Biol. - Animal. 2010;46(5):408-410. DOI 10.1007/s11626-009-9264-8.