An investigation of the role of retroviruses in leukaemia and related diseases in koalas

Abstract

Lymphoid neoplasia is the most common form of cancer affecting koalas. It has been reported in free-living koalas, and is a very common cause of mortality in captive koalas in south-east Queensland. The wide age range of koalas affected by this disease, and its similarities with retrovirus-associated diseases in other species, particularly cats, suggested a possible retroviral aetiology in koalas. Furthermore, retrovirus-like particles had been detected in tissues from a koala with lymphoid leukaemia, and retroviral sequences had reportedly been detected in koalas with the disease. This study aimed to further investigate the role of retroviruses in leukaemia and related diseases in koalas.Over the study period, veterinary and necropsy examinations were performed on free-living koalas presented to a volunteer wildlife rescue organisation (WILDCARE), and captive koalas held at two fauna parks in the Gold Coast region A variety of diseases, potentially with retroviral aetiology were observed, including lymphoid neoplasia, other haematopoietic neoplasms, aplastic anaemia, myelodysplasia and opportunistic infections, possibly associated with specific immunodeficiencies. Within the two captive koala populations, these diseases accounted for approximately 90% of mortalities. A significant rate of mortality of pouch joeys was also observed, as well as miscellaneous conditions in adult koalas of unknown aetiology. These included plantar and palmar focal proliferative hyperkeratosis, microscopic pulmonary adenomatosis, osteochondroma and oral ulceration.A genus-specific primer pair was used to amplify partial C-type mammalian retrovirus sequences from koala genomic DNA by the polymerase chain reaction (PCR). Comparison of the product sequence with other known retroviral sequences, demonstrated closest homology with simian C-type retroviruses including gibbon ape leukaemia virus (GALV) and the truncated, oncogene-containing simian sarcoma virus (SSV). More specific primers were designed based upon this sequence similarity and were successful in amplifying complete proviral sequences from the genomic DNA o f koalas. A number of clones of the provirus were sequenced and showed homology indicative of a single retroviral species. These sequences had approximately 80% homology to GALV across the complete genome, and appeared to be complete proviral sequences with intact open reading frames for gag, pol and env retrovirus genes. The novel provirus was designated koala retrovirus (KoRV).In addition to the complete provirus, truncated proviruses were detected in all koala tissues tested. In vivo transcription of KoRV provirus was demonstrated in tissues including tumour tissue, blood and bone marrow in a number of koalas. Virus particles were demonstrated electron microscopically in both tumour tissue and peripheral blood mononuclear cell cultures, but these were unable to be purified and isolated.The genetic similarity of KoRV with GALV may be most easily explained by a recent host jump, possibly involving a third host species. A number of mammalian and avian species have geographical distributions or migratory patterns that overlap those of gibbons and koalas. This host jump also may have been a contemporary event, winch occurred since European settlement o f Australia, possibly in a zoo situation. Novel retroviral sequences were detected in domestic dogs, an Eastern Grey kangaroo and a Black Flying-fox, but these were not helpful in determining the origin of KoRV.A number of lines o f evidence suggested that. KoRV exists as an endogenous retrovirus, at least in some koalas, but did not exclude the possibility that exogenous forms also occur. Firstly, KoRV provirus was detected in all tissues from all koalas tested, including free-living koalas in a geographically isolated population. Secondly, the spectrum of PCR bands representing truncated proviruses was identical between tissues in individual koalas. Thirdly, Southern blot hybridisation of koala genomic DNA with a KoRV-specific probe, demonstrated the monoclonal integration of multiple proviruses in DNA from a range of tissues in different koalas.More research is needed to further characterise KoRV, particularly its ability to produce infectious virions. The involvement of this virus in haematopoietic and other diseases of koalas has not yet been defined, therefore its importance in the ecology of the koala is also unknown. It is possible that this virus poses a greater threat to koalas than chlamydiosis by causing specific immunodeficiency syndromes. If this is the case, the urgency to conserve wild koala populations and their habitat becomes far greater. Clearly then, defining the pathogenic significance of KoRV should be a research priority.