Pathology, coinfections and oncogenesis in South Australian koalas (Phascolarctos cinereus) and their association with koala retrovirus (KoRV)

Abstract

Koalas are a vulnerable iconic species, for which disease is a significant threat to populations around Australia. Lymphoid neoplasia and immunomodulation in koalas have been associated with an important pathogen, koala retrovirus (KoRV). The prevalence and transmission of KoRV differs in northern koalas, from Queensland and New South Wales, compared to southern koalas, from Victoria and South Australia. Northern koalas inherit and horizontally transmit KoRV via endogenous and exogenous mechanisms, respectively with all koalas infected. Southern koalas are hypothesised to only have exogenous transmission, and within these populations the prevalence is less. Both proviral load (inserted viral genome into host DNA) and viral load (extracellular RNA viral genome) are significantly higher in northern koalas in comparison to southern koalas, representing active transcription in a greater number of cells. Mechanisms for KoRV-associated oncogenesis are still unknown, along with the prevalence of lymphoid neoplasia in the Mount Lofty Ranges population, South Australia. Increased susceptibility of disease has been inferred from studies exploring KoRV coinfection with Chlamydia pecorum, a bacterium which causes both ocular and urogenital disease in koalas. KoRV and C. pecorum are well-studied key pathogens of koalas, although the significance of another infectious agent Phascolarctid gammaherpesviruses, is unknown. Avian and human studies have shown increased incidence of neoplasia when coinfected with a retrovirus and gammaherpesvirus, but there is no current evidence for this in koalas. Association of diseases is based on a confident diagnosis of infectious agents, for which koalas in South Australia have shown variations in proviral analysis creating complexity in the diagnosis of KoRV. The KoRV viral genome consists of three genes, gag, pol and env, flanked by long terminal repeats, LTRs. The pol gene qPCR has been a standard diagnostic tool, but more investigation across multiple genome targets has shown variance in SA koala PCR and qPCR results. In this study, PCR and qPCR methods were used against two targets in the gag gene, one in the pol gene and two in the env gene. Koalas for which all proviral targets were positive were designated KoRV positive and koalas for which one gag target, pol target and env targets were negative were designated KoRV negative. There were 41.2% (89/216) KoRV positive, 56.9% (123/216) KoRV negative koalas and only 1.9% (4/216) deemed inconclusive. Viral gene expression analysed by qPCR was found to be present in 10/10 KoRV positive koalas, and absent in 5/5 KoRV negative koalas. RNAseq analysis revealed transcription of sequences homologous to terminal regions of the KoRV genome in all koalas, verified by the presence of one gag gene target in almost all (215/216) koalas tested by PCR. The presence of these regions in South Australian koalas without the presence of the full KoRV genome, suggests an endogenised retroviral element, potentially within the koala genome prior to KoRV. Lymphomic koalas showed high expression of KoRV and higher proviral loads compared to KoRV positive koalas without lymphoma. Lymphoma was found in 1.2% (3/240) of koalas and these cases were collated with previous South Australian lymphoma cases for classification. All cases had abdominal involvement, were intermediate to large cell and of non-T cell origin. RNAseq data was compared from lymphomic lymph nodes, lymph nodes from KoRV positive and lymph nodes of KoRV negative koalas. High KoRV transcription was found in lymphomic tissue, 1207 genes showed differential expression between KoRV positive koalas diagnosed with lymphoma and KoRV negative koalas and 939 genes between KoRV positive koalas diagnosed with lymphoma and KoRV positive koalas. Oncogenes MYB, MYCL and FLT3 were significantly upregulated and possible candidates in the incitement of oncogenesis. Dysregulation in IL10, and pathways associated with NF-kB also support the role of immunosuppression in lymphoma pathogenesis. Based on theories of KoRV-associated immunosuppression leading to opportunistic infections and augmentation of disease with pathogens, coinfections and comorbidities were investigated in 247 necropsied koalas. KoRV was not found to be associated with C. pecorum or disease severity. However, C. pecorum was associated with another recently discovered infectious agent, Phascolarctid gammaherpesvirus (PhaHV), and in this cohort PhaHV was associated with the presence of paraovarian cysts, regardless of chlamydial status, similar to Victorian koalas. Also, KoRV and PhaHV-2 coinfection was associated with neoplasia and warrants further investigation. Disease and infectious agents were negatively correlated with victims of road traffic accidents showing the potential importance of this group of koalas. Novel pathologies were found in this koala necropsy cohort. Pulmonary actinomycosis, a new presentation of respiratory disease of koalas, with a subset of these koalas presenting with secondary hypertrophic osteopathy, a second novel pathology in koalas. Fifteen koalas were found to have pyogranulomatous lobar pneumonia, predominantly affecting the left caudal lung lobe. Histological examination showed Splendore Hoeppli phenomenon with associated Gram-positive or Gram-variable, non-acid fast, filamentous bacteria. The pathogen was identified as a novel Actinomyces sp. and through 16S rRNA gene sequencing was closest to A. timonensis. Collaboration with other wildlife veterinarians found that two of the study koalas also presented with another undescribed pathology in koalas, hypertrophic osteopathy, found secondary to the pulmonary lesions. These discoveries highlight the necessity for ongoing necropsy studies in koalas to increase the knowledge of disease presentations in this iconic species. Overall, this thesis aimed to examine the association of disease with several pathogens, with particular focus on the association between KoRV and lymphoma. Advancements were made in recommendations for KoRV diagnosis in South Australia, gene dysregulation in KoRVassociated lymphoma, interactions with other key pathogens; C. pecorum and PhaHV, and the report of novel disease presentations. The increased knowledge of infectious disease and interaction of disease in South Australian koalas can help management strategies protecting the health and welfare of these koalas. Also, identification of dysregulated genes has increased the knowledge of oncogenesis by koala retrovirus.