Coinfection with koala retrovirus subtypes A and B and its impact on captive koalas in Japanese zoos


Koala retrovirus (KoRV) is unique among endogenous retroviruses because its endogenization is still active. Two major KoRV subtypes, KoRV-A and B, have been described, and KoRV-B is associated with disease and poses a health threat to koalas. Here, we investigated the co-prevalence of KoRV-A and KoRV-B, detected by type-specific PCR and sequencing, and their impact on the health of koalas in three Japanese zoos. We also investigated KoRV proviral loads and found varying amounts of genomic DNA (gDNA) in peripheral blood mononuclear cells (PBMCs). We found that 100% of the koalas examined were infected with KoRV-A and 60% (12/20) were coinfected with KoRV-B. The KoRV-A sequence was highly conserved, whereas the KoRV-B sequence varied among individuals. Interestingly, we observed possible vertical transmission of KoRV-B in one offspring in which the KoRV-B sequence was similar to that of the father but not the mother. Moreover, we characterized the KoRV growth patterns in concanavalin-A-stimulated PBMCs isolated from KoRV-B-coinfected or KoRV-B-uninfected koalas. We quantified the KoRV provirus in gDNA and the KoRV RNA copy numbers in cells and culture supernatants by real-time PCR at days 4, 7, and 14 post-seeding. As the study population is housed in captivity, a longitudinal study of these koalas may provide an opportunity to study the transmission mode of KoRV-B. In addition, we characterized KoRV isolates by infecting tupaia cells. The results suggested that tupaia may be used as an infection model for KoRV. Thus, this study may enhance our understanding of KoRV-B coinfection and transmission in the captive koalas.

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  1. 1.

    Hanger JJ, Bromham LD, McKee JJ, O’Brien TM, Robinson WF (2000) The nucleotide sequence of Koala (Phascolactos cinereus) retrovirus: a novel type C endogenous virus related to Gibbon ape leukemia virus. J Virol 74:4264–4272

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  2. 2.

    Fiebig U, Hartmann MG, Bannert N, Kurth R, Denner J (2006) Transspecies transmission of the endogenous koala retrovirus. J Virol 80:5651–5654

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  3. 3.

    Miyazawa T, Shojima T, Yoshikawa R, Ohata T (2011) Isolation of koala retroviruses from koalas in Japan. J Vet Med Sci 73:65–70

    CAS  Article  PubMed  Google Scholar 

  4. 4.

    Shojima T, Hoshino S, Abe M, Yasuda J, Shogen H, Kobayashi T, Miyazawa T (2013) Construction and characterization of an infectious molecular clone of koala retrovirus. J Virol 87:5081–5088

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  5. 5.

    Xu W, Stadler CK, Gorman K, Jensen N, Kim D, Zheng H, Tang S, Switzer WM, Pye GW, Eiden MV (2013) An exogenous retrovirus isolated from koalas with malignant neoplasias in a US zoo. Proc Natl Acad Sci USA 110(28):11547–11552

    CAS  Article  PubMed  Google Scholar 

  6. 6.

    Fiebig U, Keller M, Denner J (2016) Detection of koala retrovirus subgroup B (KoRV-B) in animals housed at European zoos. Arch Virol 161:3549–3553

    CAS  Article  PubMed  Google Scholar 

  7. 7.

    Kayesh MEH, Yamato O, Rahman MM, Hashem MA, Maetani F, Eiei T, Mochizuki K, Sakurai H, Tsukiyama-Kohara K (2019) Molecular dynamics of koala retrovirus infection in captive koalas in Japan. Arch Virol 164:757–765

    CAS  Article  PubMed  Google Scholar 

  8. 8.

    Cui J, Tachedjian G, Tachedjian M, Holmes EC, Zhang S, Wang LF (2012) Identification of diverse groups of endogenous gammaretroviruses in mega- and microbats. J Gen Virol 93:2037–2045

    CAS  Article  PubMed  Google Scholar 

  9. 9.

    Denner J, Young PR (2013) Koala retroviruses: characterization and impact on the life of koalas. Retrovirology 10:108

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. 10.

    Gifford R, Tristem M (2003) The evolution, distribution and diversity of endogenous retroviruses. Virus Genes 26:291–315

    CAS  Article  PubMed  Google Scholar 

  11. 11.

    Bock M, Stoye JP (2000) Endogenous retroviruses and the human germline. Curr Opin Genet Dev 10:651–655

    CAS  Article  PubMed  Google Scholar 

  12. 12.

    Boeke JD, Stoye JP (1997) Retrotransposons, endogenous retroviruses, and the evolution of retroelements. In: Coffin JM, Hughes SH, Varmus HE (eds) Retroviruses. Cold Spring Harbor Laboratory, Cold Spring Harbor, pp 343–346

    Google Scholar 

  13. 13.

    Quigley BL, Ong VA, Hanger J, Timms P (2018) Molecular dynamics and mode of transmission of Koala Retrovirus as it invades and spreads through a wild Queensland koala population. J Virol 92:e01871-17.

    Article  PubMed  PubMed Central  Google Scholar 

  14. 14.

    Tarlinton R, Meers J, Young P (2006) Retroviral invasion of the koala genome. Nature 422:79–81

    Article  CAS  Google Scholar 

  15. 15.

    Stoye JP (2006) Koala retrovirus: a genome invasion in real time. Genome Biol 7:241

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. 16.

    Kinney ME, Pye GW (2016) Koala retrovirus: a review. J Zoo Wildl Med 47:387–396

    Article  PubMed  Google Scholar 

  17. 17.

    Maher IE, Higgins DP (2016) Altered immune cytokine expression associated with KoRV B infection and season in captive Koalas. PloS One 11:e0163780

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. 18.

    Waugh CA, Hanger J, Loader J, King A, Hobbs M, Johnson R, Timms P (2017) Infection with koala retrovirus subgroup B (KoRV-B), but not KoRV-A, is associated with chlamydial disease in free-ranging koalas (Phascolarctos cinereus). Sci Rep 7:134

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. 19.

    Saitou N, Nei M (1987) The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol 4:406–425

    CAS  PubMed  Google Scholar 

  20. 20.

    Nei M, Kumar S (2000) Molecular evolution and phylogenetics. Oxford University Press, New York

    Google Scholar 

  21. 21.

    Kumar S, Stecher G, Tamura K (2016) MEGA7: Molecular evolutionary genetics analysis version 7.0 for bigger datasets. Mol Biol Evol 33:1870–1874

    CAS  Article  Google Scholar 

  22. 22.

    Tarlinton R, Meers J, Hanger J, Young P (2005) Real-time reverse transcriptase PCR for the endogenous koala retrovirus reveals an association between plasma viral load and neoplastic disease in koalas. J Gen Virol 86:783–787

    CAS  Article  PubMed  Google Scholar 

  23. 23.

    Taketomi M, Nishi Y, Ohkawa Y, Inui N (1986) Establishment of lung fibroblastic cell lines from a non-human primate Tupaia belangeri and their use in a forward gene mutation assay at the hypoxanthine-guanine phosphoribosyl transferase locus. Mutagenesis 1:359–365

    CAS  Article  PubMed  Google Scholar 

  24. 24.

    Kayesh MEH, Kitab B, Sanada T, Hayasaka D, Morita K, Kohara M, Tsukiyama-Kohara K (2017) Susceptibility and initial immune response of Tupaia belangeri cells to dengue virus infection. Infect Genet Evol 51:203–210

    CAS  Article  PubMed  Google Scholar 

  25. 25.

    Simmons GS, Young PR, Hanger JJ, Jones K, Clarke D, McKee JJ, Meers J (2012) Prevalence of koala retrovirus in geographically diverse populations in Australia. Aust Vet J 90:404–409

    CAS  Article  PubMed  Google Scholar 

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This work was supported by grants from the Ministry of Education, Science, and Culture, Japan.

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Correspondence to Kyoko Tsukiyama-Kohara.

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Hashem, M.A., Kayesh, M.E.H., Yamato, O. et al. Coinfection with koala retrovirus subtypes A and B and its impact on captive koalas in Japanese zoos. Arch Virol 164, 2735–2745 (2019).

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