PATHOGENESIS
Our limited understanding of what appears likely to be the role of KoRV in oncogenesis or immunomodulation and disease in koalas is a key knowledge gap. Northern koalas, in particular, suffer frequently from a spectrum of diseases that includes: chlamydial infertility and death; a range of immunosuppression-like, opportunistic infectious diseases; blood and bone marrow disorders (myelodysplasia); and neoplasms (e.g.
lymphoma and leukaemia, mesothelial and craniofacial tumours). Among 296 wild- caught koalas from south-east Qld, 8% had lesions or syndromes considered consistent with retroviral disease in other species and, from longitudinal data, the incidence rate of these diseases in the population was 12.5% annually (Hanger and Loader 2014). Among koalas in managed care, bone marrow conditions (14%) and AIDSlike syndromes (20%) are reported to contribute significantly to mortality (Gillett 2014). This collection of diseases is sometimes collectively referred to as KIDS (koala immune deficiency syndrome) or presumptively labelled as ‘KoRV’, based on the similarity of these diseases to those seen in humans with AIDS and cats with FeLV (Denner and Young 2013). However, the multifactorial nature of disease and limitations across standardisation of case definitions, understanding of KoRV diversity and behaviour, development of immunological tools and access to the necessary sample populations, have all confounded the search for associative, let alone causative, relationships between KoRV and disease.Until recently, evidence for a role of KoRV in disease comprised the observation of type C retrovirus-like particles budding from koala leukaemic cells (Canfield et al. 1988), significantly greater KoRV plasma viral RNA load detected (by pol gene RT-qPCR) in koalas with lymphoid neoplasia compared with those without (Tarlinton et al.
2005) and equivocal study results regarding an association of chlamydial disease and plasma viral RNA load (Tarlin- ton et al. 2005; Simmons 2011). Interpretation of causative relationships associated with viral loads is problematic because cell activation, such as in active inflammation and leukaemia, can itself increase retroviral expression (Johnston et al. 2001). Although the greater prevalence of lymphoma and severe chlamydial disease is spatially associated with the greater KoRV prevalence in NSW and Qld, over the southern states of Vic. and SA (Gillett 2014) other differences exist between the populations, particularly with respect to koala immunogenetics (Lau et al. 2014) and chlamydial strains (Legione et al. 2016).Oncogenic retroviruses use a variety of mechanisms to induce neoplasia, but two common processes are involved: the capture of a cellular oncogene, which is inserted into the host cell’s genome, and activation of cellular oncogenes by insertional mutagenesis. Multiple oncogenes have been identified for closely related viruses such as FeLV and MuLV, and associations have been reported between KoRV integration sites adjacent to cellular oncogenes and neoplasia in koalas (McEwen et al. 2021). Alternatively, KoRV infection could cause neoplasia through immune suppression or dysregulation. An essential role of immune surveillance is removal of early neoplastic cells and immune dysregulation secondary to retroviral infection is a well-known cause of neoplasia in humans infected with HIV-1 and in cats infected with FIV. Immune dysregulation also comprises a feasible pathogenic mechanism for the wide range of supposed KoRV-associated diseases reported in koalas.
Support for a role for KoRV in immune dysregulation is more difficult to prove. Associations have been reported between various aspects of KoRV infection and cytokine expression and chlamydial disease thanks to advances in koala immunology (Mathew et al. 2013, 2014; Maher et al. 2014). In managed, KoRV A-infected NSW origin koalas, KoRV B infection was associated with reduced expression of interleukin-10 (IL10) genes at rest and greater upregulation of a range of cytokines in response to mitogen stimulation, in particular IL17, which is a cytokine associated with the florid inflammatory response that underpins damage in chlamydial disease (Maher and Higgins 2016).
An association of KoRV B infection with chlamydial disease (but not chlamydial infection) has since been found in a well characterised, longitudinally sampled, free-ranging group from southeast Qld (Waugh et al. 2017; Quigley et al. 2018). For presumed exogenous KoRV A infection in free ranging koalas in Vic., KoRV A-positive koalas had significantly lower resting expression of IL17A and interferon-gamma (IFNγ), as well as a decreased CD4:CD8 gene expression ratio (Maher et al. unpublished) and were more likely to be in poor condition and show evidence of urinary incontinence (Legione et al. 2016) than KoRV negative koalas. As expected, based on the complex pathogenesis of chlamydial and retroviral diseases, the heterogeneity of wildlife populations and the challenges associated with wildlife immunology, the story is not yet clear-cut. Causative relationships will be even more difficult to establish and will likely require a combination of in vitro mechanistic studies and detailed longitudinal surveys to elucidate.Most recent work suggests that disease is most strongly associated with greater KoRV transcription, regardless of sub-type; it is likely that likelihood of detection of subtypes in previous studies was a factor of load rather than presence/ absence, as PCR-based methods used are less sensitive than amplicon deep sequencing used in more recent studies. Thus, measures of transcription, such as env or pol mRNA quantification have the strongest association with disease (Blyton et al. 2022b). Progressing from associative studies to proving causation will likely need a series of longitudinal and mechanistic laboratory-based studies.
In theory, KoRV may impact the immune system though several means. All KoRV subtypes express a transmembrane envelope protein, p15E, which facilitates the fusion of the viral and the cellular membrane during infection. P15E has an immunosuppressive domain (ISD), designated CKS-17, that is identical in KoRV, GALV, MuLV and FeLV.
Although the mechanism of action of the ISD is still unclear, it has been shown to have many immunosuppressive effects in cells of other species. These include the downregulation in vitro of Thl (cellular immune response) cytokines (IFNγ, tumour necrosis factor-alpha (TNFα), IL2, IL12) and upregulation of the Th2 (humoral)-associated cytokine IL10 and inhibition of neutrophil function, macrophage accumulation at inflammatory foci in mice, human natural killer cell and monocyte functions (Haraguchi et al. 2008; Denner 2014). Human peripheral blood mononuclear cells incubated with purified KoRV had increased expression of the Th2 cytokines IL6, IL10, growth-related oncogene and monocyte chemotactic protein-1 (Fiebig et al. 2006), indicating that, providing the relevant koala receptors and cell pathways are compatible, the ISD could be playing a role in KoRV pathogenesis. Alternative mechanisms for immunomodulation include direct cytotoxicity as a result of infection, direct immunomodulation by the virus, or modulation of immune gene expression due to proviral integration in or adjacent to immune genes or their control regions.Although it has not yet been examined, the potential for interaction of KoRV with other co-infecting agents should be considered. Herpes simplex 2 (HSV-2) infection is associated with an increase in transmission rates of HIV-2 in humans (Freeman et al. 2006). Human herpes virus 8 (HHV8) is the underlying cause of Kaposi’s sarcoma in humans, a sarcoma frequently seen after immunosuppression secondary to HIV-1 infection. Herpes viruses are also implicated in the pathogenesis of multiple sclerosis in humans, whereby it has been proposed that herpes viruses may have a ‘triggering’ role as a co-factor and may be involved in inducing the expression of ERV elements (Perron et al. 1993). Immune modulation by KoRV could similarly lead to herpes viruses causing disease, or herpes viruses could cause disease by inducing expression of endogenous KoRV. Two gammaherpesviruses have been discovered in koalas (Vaz et al. 2011, 2012). The three individuals in whom the herpes viruses were detected were all suffering from comorbidities, however, the KoRV status was not determined (these were Vic. koalas so may have been KoRV positive or -negative).
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