Vaccination against the Koala Retrovirus (KoRV): Problems and Strategies

The koala retrovirus (KoRV) is spreading in the koala population from the north to the south of Australia and is also in the process of endogenization into the koala genome. Virus infection is associated with tumorigenesis and immunodeficiency and is contributing to the decline of the animal population. Antibody production is an excellent marker of retrovirus infection; however, animals carrying endogenous KoRV are tolerant. Therefore, the therapeutic immunization of animals carrying endogenous KoRV seems to be ineffective. Using the recombinant transmembrane (TM) envelope protein of the KoRV, we immunized goats, rats and mice, obtaining in all cases neutralizing antibodies which recognize epitopes in the fusion peptide proximal region (FPPR), and in the membrane-proximal external region (MPER). Immunizing several animal species with the corresponding TM envelope protein of the closely related porcine endogenous retrovirus (PERV), as well as the feline leukemia virus (FeLV), we also induced neutralizing antibodies with similar epitopes. Immunizing with the TM envelope protein in addition to the surface envelope proteins of all three viruses resulted in higher titers of neutralizing antibodies. Immunizing KoRV-negative koalas with our vaccine (which is composed of both envelope proteins) may protect these animals from infection, and these may be the starting points of a virus-free population.

Geographic patterns of koala retrovirus genetic diversity, endogenization and subtype distributions

Koala retrovirus subtype A is the youngest endogenized retrovirus, providing a unique system to elucidate retroviral-host co-evolution. We characterised KoRV geography using faecal DNA from 192 samples across 20 populations throughout the koala’s range. We reveal an abrupt change in KoRV genetics and incidence at the Victoria/NSW state border. In northern koalas, pol gene copies were ubiquitously present at greater than 5 per-cell, consistent with endogenous KoRV. In southern koalas, pol copies were detected in only 25.8% of koalas and always at copy numbers less than one, while the env gene was detected in all animals and in a majority at copy numbers of greater than one per-cell. These results suggest that southern koalas carry partial endogenous KoRV-like sequences. Deep sequencing of the env hypervariable region revealed three putatively endogenous KoRV-A sequences in northern koalas and a single, distinct sequence present in all southern koalas. Among northern populations, env sequence diversity decreased with distance from the equator, suggesting infectious KoRV-A invaded the koala genome in northern Australia and then spread south. The previously described exogenous KoRV subtypes (B-K), two novel subtypes (L and M), and intermediate or hybrid subtypes were detected in all northern koala populations but strikingly absent from all southern animals tested. Apart from KoRV-D, these exogenous subtypes were generally locally prevalent but geographically restricted, producing KoRV genetic differentiation among northern populations. This suggests that sporadic evolution and local transmission of the exogenous subtypes has occurred within northern Australia, but this has not extended into animals within southern Australia.

Koala retrovirus genetic diversity and transmission dynamics within captive koala populations

Koala populations are currently in rapid decline across Australia, with infectious diseases being a contributing cause. The koala retrovirus (KoRV) is a gammaretrovirus present in both captive and wild koala colonies that presents an additional challenge for koala conservation in addition to habitat loss, climate change, and other factors. Currently, nine different subtypes (A to I) have been identified; however, KoRV genetic diversity analyses have been limited. KoRV is thought to be exogenously transmitted between individuals, with KoRV-A also being endogenous and transmitted through the germline. The mechanisms of exogenous KoRV transmission are yet to be extensively investigated. Here, deep sequencing was employed on 109 captive koalas of known pedigree, housed in two institutions from Southeast Queensland, to provide a detailed analysis of KoRV transmission dynamics and genetic diversity. The final dataset included 421 unique KoRV sequences, along with the finding of an additional subtype (KoRV-K). Our analysis suggests that exogenous transmission of KoRV occurs primarily between dam and joey, with evidence provided for multiple subtypes, including nonendogenized KoRV-A. No evidence of sexual transmission was observed, with mating partners found to share a similar number of sequences as unrelated koala pairs. Importantly, both distinct captive colonies showed similar trends. These findings indicate that breeding strategies or antiretroviral treatment of females could be employed as effective management approaches in combating KoRV transmission.

Molecular Diagnosis of Koala Retrovirus (KoRV) in South Australian Koalas (Phascolarctos cinereus)

Koala retrovirus, a recent discovery in Australian koalas, is endogenised in 100% of northern koalas but has lower prevalence in southern populations, with lower proviral and viral loads, and an undetermined level of endogenisation. KoRV has been associated with lymphoid neoplasia, e.g., lymphoma. Recent studies have revealed high complexity in southern koala retroviral infections, with a need to clarify what constitutes positive and negative cases. This study aimed to define KoRV infection status in Mount Lofty Ranges koalas in South Australia using RNA-seq and proviral analysis (n = 216). The basis for positivity of KoRV was deemed the presence of central regions of the KoRV genome (gag 2, pol, env 1, and env 2) and based on this, 41% (89/216) koalas were positive, 57% (124/216) negative, and 2% inconclusive. These genes showed higher expression in lymph node tissue from KoRV positive koalas with lymphoma compared with other KoRV positive koalas, which showed lower, fragmented expression. Terminal regions (LTRs, partial gag, and partial env) were present in SA koalas regardless of KoRV status, with almost all (99.5%, 215/216) koalas positive for gag 1 by proviral PCR. Further investigation is needed to understand the differences in KoRV infection in southern koala populations.

Concerns regarding vaccination as a management strategy against koala retrovirus

Koalas are of great cultural, ecological and economic significance to Australia. However, they are in steep decline throughout much of their geographic range, with the recently endogenized koala retrovirus (KoRV) presumed to be a contributing factor. Olagoke, et al. have proposed vaccination against KoRV as a suitable control mechanism to reduce the severity of KoRV infections and suggest that their recombinant vaccine is effective at invoking a neutralising immune response and significantly reducing viral loads. Here, we report the findings of our own attempts to detect antibodies specific to the KoRV envelope protein (Env) in infected animals. Antibodies against Env were undetectable in all animals, as would be expected in the case of an endogenous virus antigen which should proceed towards a pathway of self-tolerance. This finding calls into question the results of Olagoke, et al., and prior work by this team. Following a critical review of this work, we have identified a number of assay inaccuracies and possible over interpretations of the data and conclude that further work to develop and test a vaccine against KoRV in koalas is not supported by evidence.

Toll-Like Receptor and Cytokine Responses to Infection with Endogenous and Exogenous Koala Retrovirus, and Vaccination as a Control Strategy

Koala populations are currently declining and under threat from koala retrovirus (KoRV) infection both in the wild and in captivity. KoRV is assumed to cause immunosuppression and neoplastic diseases, favoring chlamydiosis in koalas. Currently, 10 KoRV subtypes have been identified, including an endogenous subtype (KoRV-A) and nine exogenous subtypes (KoRV-B to KoRV-J). The host’s immune response acts as a safeguard against pathogens. Therefore, a proper understanding of the immune response mechanisms against infection is of great importance for the host’s survival, as well as for the development of therapeutic and prophylactic interventions. A vaccine is an important protective as well as being a therapeutic tool against infectious disease, and several studies have shown promise for the development of an effective vaccine against KoRV. Moreover, CRISPR/Cas9-based genome editing has opened a new window for gene therapy, and it appears to be a potential therapeutic tool in many viral infections, which could also be investigated for the treatment of KoRV infection. Here, we discuss the recent advances made in the understanding of the immune response in KoRV infection, as well as the progress towards vaccine development against KoRV infection in koalas.

Toll-Like Receptor Expression Profiles in Koala (Phascolarctos cinereus) Peripheral Blood Mononuclear Cells Infected with Multiple KoRV Subtypes

Toll-like receptors (TLRs), evolutionarily conserved pattern recognition receptors, play an important role in innate immunity by recognizing microbial pathogen-associated molecular patterns. Koala retrovirus (KoRV), a major koala pathogen, exists in both endogenous (KoRV-A) and exogenous forms (KoRV-B to J). However, the expression profile of TLRs in koalas infected with KoRV-A and other subtypes is yet to characterize. Here, we investigated TLR expression profiles in koalas with a range of subtype infection profiles (KoRV-A only vs. KoRV-A with KoRV-B and/or -C). To this end, we cloned partial sequences for TLRs (TLR2–10 and TLR13), developed real-time PCR assays, and determined TLRs mRNA expression patterns in koala PBMCs and/or tissues. All the reported TLRs for koala were expressed in PBMCs, and variations in TLR expression were observed in koalas infected with exogenous subtypes (KoRV-B and KoRV-C) compared to the endogenous subtype (KoRV-A) only, which indicates the implications of TLRs in KoRV infection. TLRs were also found to be differentially expressed in koala tissues. This is the first report of TLR expression profiles in koala, which provides insights into koala’s immune response to KoRV infection that could be utilized for the future exploitation of TLR modulators in the maintenance of koala health.

Retroviral integrations contribute to elevated host cancer rates during germline invasion

Repeated retroviral infections of vertebrate germlines have made endogenous retroviruses ubiquitous features of mammalian genomes. However, millions of years of evolution obscure many of the immediate repercussions of retroviral endogenisation on host health. Here we examine retroviral endogenisation during its earliest stages in the koala (Phascolarctos cinereus), a species undergoing germline invasion by koala retrovirus (KoRV) and affected by high cancer prevalence. We characterise KoRV integration sites (IS) in tumour and healthy tissues from 10 koalas, detecting 1002 unique IS, with hotspots of integration occurring in the vicinity of known cancer genes. We find that tumours accumulate novel IS, with proximate genes over-represented for cancer associations. We detect dysregulation of genes containing IS and identify a highly-expressed transduced oncogene. Our data provide insights into the tremendous mutational load suffered by the host during active retroviral germline invasion, a process repeatedly experienced and overcome during the evolution of vertebrate lineages.

Koala Retrovirus in Northern Australia Shows a Mixture of Stable Endogenization and Exogenous Lineage Diversification within Fragmented Koala Populations

The koala population in northern Australia has become increasingly fragmented due to natural and man-made barriers and interventions. This situation has created a unique opportunity to study both endogenous and exogenous koala retrovirus (KoRV). To determine the impact that population isolation has had on KoRV diversity in Queensland, 272 koalas from six fragmented koala populations were profiled for their KoRV provirus across two natural biogeographical barriers (the St Lawrence Gap and the Brisbane Valley Barrier), one man-made geographical barrier (the city of Brisbane) and two translocation events (the single movement of koalas to an island and the repeated movement of koalas into a koala sanctuary). Analysis revealed that all koalas tested were KoRV-A positive, with 90 - 96% of the detected KoRV provirus from each koala representing a single, likely endogenous, KoRV-A strain. The next most abundant proviral sequence was a defective variant of the dominant KoRV-A strain, accounting for 3 – 10% of detected provirus. The remaining KoRV provirus represented expected exogenous strains of KoRV and included geographically localized patterns of KoRV-B, -C, -D, -F, -G, and -I. These results indicate that lineage diversification of exogenous KoRV is actively ongoing. In addition, comparison of KoRV provirus within known dam-sire-joey family groups from the koala sanctuary revealed that joeys consistently had KoRV proviral patterns more similar to their dams than their sires in KoRV-B, -C and -D provirus composition. Collectively, this study highlights both the consistency of endogenous KoRV and the diversity of exogenous KoRV across the fragmented koala populations in northern Australia. IMPORTANCE KoRV infection has become a permanent part of koalas in northern Australia. With KoRV presence and abundance linked to more severe chlamydial disease and neoplasia in these koalas, understanding how KoRV exists throughout an increasingly fragmented koala population is a key first step in designing conservation and management strategies. This survey of KoRV provirus in Queensland koalas indicates that endogenous KoRV provirus is ubiquitous and consistent throughout the state while exogenous KoRV provirus is diverse and distinct in fragmented koala populations. Understanding the prevalence and impact of both endogenous and exogenous KoRV will be needed to ensure a future for all koala populations.