Koala Retrovirus Infection Research Articles

A preliminary study of gene expression changes in Koalas Infected with Koala Retrovirus (KoRV) and identification of potential biomarkers for KoRV pathogenesis

BackgroundKoala retrovirus (KoRV), a major pathogen of koalas, exists in both endogenous (KoRV-A) and exogenous forms (KoRV-A to I and K to M) and causes multiple disease phenotypes, including carcinomas and immunosuppression. However, the direct association between the different KoRV subtypes and carcinogenesis remains unknown. Differentially expressed gene (DEG) analysis of peripheral blood mononuclear cells (PBMCs) of koalas carrying both endogenous (KoRV-A) and exogenous (KoRV-A, B, and C) subtypes was performed using a high-throughput RNA-seq approach. PBMCs were obtained from three healthy koalas: one infected with endogenous (KoRV-A; Group I) and two infected with exogenous (KoRV-B and/or KoRV-C; Group II) subtypes. Additionally, spleen samples (n = 6) from six KoRV-infected deceased koalas (K1- K6) and blood samples (n = 1) from a live koala (K7) were collected and examined to validate the findings.ResultsAll koalas were positive for the endogenous KoRV-A subtype, and eight koalas were positive for KoRV-B and/or KoRV-C. Transcription of KoRV gag, pol, and env genes was detected in all koalas. Upregulation of cytokine and immunosuppressive genes was observed in koalas infected with KoRV-B or KoRV-B and -C subtypes, compared to koalas infected with only KoRV-A. We found 550 DEG signatures with significant (absolute p < 0.05, and absolute log2 Fold Change (FC) > 1.5) dysregulation, out of which 77.6% and 22.4% DEGs were upregulated (log2FC > 1.5) and downregulated (log2FC < − 1.5), and downregulated (log2 FC < − 1), respectively. We identified 17 unique hub genes (82.3% upregulated and 17.7% down-regulated), with KIF23, CCNB2, POLR3F, and RSL24D1 detected as the potential hub genes modified with KoRV infection. Real-time RT-qPCR was performed on seven koalas to ascertain the expression levels of four potential hub genes, which were subsequently normalized to actin copies. Notably, all seven koalas exhibited distinct expression signatures for the hub genes, especially, KIF23 and CCNB2 show the highest expression in healthy koala PBMC, and POLR3F shows the highest expression in koala with lymphoma (K1).ConclusionThus, it can be concluded that multiple KoRV subtypes affect disease progression in koalas and that the predicted hub genes could be promising prognostic biomarkers for pathogenesis.

Koala retrovirus infection and disease in South Australian koala (Phascolarctos cinereus) populations. In Proceedings of the Second Koala Retrovirus Workshop, ed. D. E. Alquezar-Planas, D. P. Higgins, C. L. Singleton, and A. D. Greenwood

Koala retrovirus (KoRV) infection, endogenous in all northern koalas (Phascolarctos cinereus), has been found to occur at lower, but increasing, prevalence in the Kangaroo Island and Mount Lofty Ranges koala populations in South Australia. Proviral and viral loads are also lower than in Queensland koalas, which may be due to exogenous spread of infection, or may be related to the variable presence of viral genes and fragmented expression that has been found in positive Mount Lofty Ranges koalas. However, high proviral loads and full expression across the KoRV genome in South Australian koalas has been found in individuals with neoplasia, particularly lymphoma, which can be as extensive and as severe as that observed in northern koalas. KoRV-A is the predominant subtype and no association with chlamydial status has been found except that high viral loads correlate with severity of chlamydiosis. Based on the complexity of KoRV infections in South Australian koalas, further research is needed to understand the differences in transmission and pathogenesis that occur.

Putative koala retrovirus-associated diseases in the Japanese captive koala (Phascolarctos cinereus) population. In Proceedings of the Second Koala Retrovirus Workshop, ed. D. E. Alquezar-Planas, D. P. Higgins, C. L. Singleton, and A. D. Greenwood

Japan began housing koalas (Phascolarctos cinereus) in 1984, increasing from six individuals in 1984 to a peak of 96 koalas in 1997. However, the number of koalas has almost halved since and as of 2020, 54 koalas remain in zoos in Japan. Although records of 330 koala deaths have been accumulated over 37 years, there have been no comprehensive reports on the relationship between the causes of death and koala retrovirus (KoRV) in the Japanese captive population. Based on the koala studbook updated by the Japanese Association of Zoos and Aquariums, we have investigated causes of death in the Japanese captive koala population. The most common cause of death was joeys falling. When combined with stunted joey growth, one-third of the koalas died within a year of birth. Deaths due to malignant neoplasms and opportunistic infections cannot be directly associated with KoRV infection because no test for KoRV had been performed before or during disease onset. It is suspected that KoRV may be associated with deaths due to the large number of cases of neoplasms, which accounted for 16.4% of all deaths.

Defining putative koala retrovirus-associated disease in koalas. In Proceedings of the Second Koala Retrovirus Workshop, ed. D. E. Alquezar-Planas, D. P. Higgins, C. L. Singleton, and A. D. Greenwood

Koalas suffer from a wide range of diseases and illness, some of which are well understood, and others that are observed but have unclear aetiologies. A largely undescribed and poorly defined area in koala health is diseases presumed to be associated with koala retrovirus (KoRV) infection. Disease conditions putatively linked to KoRV infection are defined here as “putative KoRV-associated diseases” (PKAD). These include neoplasia, severe dermatological and oral conditions, life-threatening fungal and opportunistic infections, haematological disorders, chronic ill-thrift or poor body condition of undefined cause and other conditions suggestive of immune dysfunction. Multiple conditions are usually present at once and koalas invariably die despite treatment. The multifactorial nature of PKAD and the lack of clarity around KoRV’s role in many conditions means that developing a standard case definition encompassing all presentations is difficult. As such, presenting conditions have been defined as dysplastic/neoplastic versus those associated with immune dysfunction (putative immune dysfunction disorders—PIDDS).

An overview of koala retrovirus epidemiology in Australia. In Proceedings of the Second Koala Retrovirus Workshop, ed. D. E. Alquezar-Planas, D. P. Higgins, C. L. Singleton, and A. D. Greenwood

Koala retrovirus (KoRV) epidemiology varies across koala (Phascolarctos cinereus) populations with distinct differences in viral prevalence, sequence diversity, and disease impact. Curiously the more genetically restricted southern populations are less impacted by KoRV with the virus not endogenized in its replication competent form in these animals. These southern animals do, however, have replication defective recKoRV variants in their genomes indicating historical exposure to KoRV and recKoRV. Whether southern animals are inherently resistant to KoRV infection and endogenization is not clear. It is also not clear whether the current regional epidemiological patterns will persist or whether exposure to animals with infectious KoRV or cross-breeding between different genetic populations will change the KoRV prevalence with time.

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.

Simple SummaryKoala retrovirus (KoRV) is a major pathogen of koala. Toll-like receptors (TLRs) are important innate immune component that are evolutionary conserved and play a crucial role in the early defense against invading pathogens. The expression profile of TLRs in KoRV infection in koalas is not characterized yet. Therefore, in this study, we characterized TLR expression patterns in koalas infected with KoRV-A only vs. KoRV-A with KoRV-B and/or -C. Using qRT-PCR, we measured TLR2–10 and TLR13 mRNA expression in peripheral blood mononuclear cells (PBMCs) and/or tissues from captive koalas in Japanese zoos. We observed variations in TLR expression in koalas with a range of subtype infection profiles (KoRV-A only vs. KoRV-A with KoRV-B and/or -C). The findings of this study might improve our current understanding of koala’s immune response to KoRV infection.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.

Koala retrovirus diversity, transmissibility, and disease associations

BackgroundKoalas are infected with the koala retrovirus (KoRV) that exists as exogenous or endogenous viruses. KoRV is genetically diverse with co-infection with up to ten envelope subtypes (A-J) possible; KoRV-A is the prototype endogenous form. KoRV-B, first found in a small number of koalas with an increased leukemia prevalence at one US zoo, has been associated with other cancers and increased chlamydial disease. To better understand the molecular epidemiology of KoRV variants and the effect of increased viral loads (VLs) on transmissibility and pathogenicity we developed subtype-specific quantitative PCR (qPCR) assays and tested blood and tissue samples from koalas at US zoos (n = 78), two Australian zoos (n = 27) and wild-caught (n = 21) in Australia. We analyzed PCR results with available clinical, demographic, and pedigree data.ResultsAll koalas were KoRV-A-infected. A small number of koalas (10.3%) at one US zoo were also infected with non-A subtypes, while a higher non-A subtype prevalence (59.3%) was found in koalas at Australian zoos. Wild koalas from one location were only infected with KoRV-A. We observed a significant association of infection and plasma VLs of non-A subtypes in koalas that died of leukemia/lymphoma and other neoplasias and report cancer diagnoses in KoRV-A-positive animals. Infection and VLs of non-A subtypes was not associated with age or sex. Transmission of non-A subtypes occurred from dam-to-offspring and likely following adult-to-adult contact, but associations with contact type were not evaluated. Brief antiretroviral treatment of one leukemic koala infected with high plasma levels of KoRV-A, -B, and -F did not affect VL or disease progression.ConclusionsOur results show a significant association of non-A KoRV infection and plasma VLs with leukemia and other cancers. Although we confirm dam-to-offspring transmission of these variants, we also show other routes are possible. Our validated qPCR assays will be useful to further understand KoRV epidemiology and its zoonotic transmission potential for humans exposed to koalas because KoRV can infect human cells.

Pathological Findings in Koala Retrovirus-positive Koalas (Phascolarctos cinereus) from Northern and Southern Australia

Koala retrovirus (KoRV) infection shows differences in prevalence and load between northern and southern Australian koala populations; however, the effect of this on diseases such as lymphoma and chlamydial disease is unclear. This study compared clinicopathological findings, haematology and splenic lymphoid area of KoRV-positive koalas from northern (Queensland [Qld], n=67) and southern (South Australia [SA], n=92) populations in order to provide further insight into KoRV pathogenesis. Blood was collected for routine haematology and for measurement of KoRV proviral load by quantitative polymerase chain reaction (qPCR). Plasma samples were assessed for KoRV viral load by reverse transcriptase qPCR and conjunctival and cloacal swabs were collected for measurement of the load of Chlamydia pecorum (qPCR). During necropsy examination, spleen was collected for lymphoid area analysis. Lymphoma was morphologically similar between the populations and occurred in koalas with the highest KoRV proviral and viral loads. Severe ocular chlamydial disease was observed in both populations, but urinary tract disease was more severe in Qld, despite similar C. pecorum loads. No associations between KoRV and chlamydial disease severity or load were observed, except in SA where viral load correlated positively with chlamydial disease severity. In both populations, proviral and viral loads correlated positively with lymphocyte and metarubricyte counts and correlated negatively with erythrocyte and neutrophil counts. Splenic lymphoid area was correlated positively with viral load. This study has shown further evidence for KoRV-induced oncogenesis and highlighted that lymphocytes and splenic lymphoid tissue may be key sites for KoRV replication. However, KoRV infection appears to be highly complex and continued investigation is required to fully understand its pathogenesis.

Haematological reference intervals of wild southern Australian koalas (Phascolarctos cinereus).

Current haematology reference intervals (RIs) for koalas were developed in northern Australian koalas, using low numbers and/or individuals of unknown Chlamydia pecorum and koala retrovirus (KoRV) status. This study developed haematological RIs for wild, clinically healthy southern Australian koalas of known C. pecorum and KoRV infection status and investigated the effects of population, age and sex. Haematological RIs were determined for 138 clinically healthy South Australian koalas (Mount Lofty Ranges [MLR], n = 68; Kangaroo Island, n = 70) examined in April 2016 and February 2017, respectively. C. pecorum and KoRV status were determined by PCR. RIs for southern koala haematological parameters were established for all koalas based on the finding that there were limited differences in haematological values in koalas with subclinical C. pecorum or KoRV infections (P > 0.05), except KoRV-infected koalas had a lower haematocrit than noninfected koalas. MLR koalas had significantly lower erythrocyte mass and leucocyte counts than Kangaroo Island koalas. Young koalas had significantly lower haemoglobin, haematocrit and higher mean cellular haemoglobin concentration and lymphocyte counts than adult koalas. MLR male koalas had elevated erythrocyte, leucocyte and neutrophil counts compared with MLR females. The haematological RIs developed in this study are based on a large number of clinically healthy koalas, where subclinical C. pecorum and KoRV infections had no effect on haematological values and will be a valuable tool during clinical examination and disease investigation by veterinarians and researchers Australia-wide.

Periodontal disease in free-ranging koalas (Phascolarctos cinereus) from the Mount Lofty Ranges, South Australia, and its association with koala retrovirus infection.

In northern Australian koala populations (Queensland and New South Wales), periodontal disease (gingivitis and periodontitis) is common while koala retrovirus subtype A is endogenous, with other subtypes transmitted exogenously. Koala retrovirus has been hypothesised to cause immune suppression and may predispose koalas to diseases caused by concurrent infections. In southern Australia populations (Victoria and South Australia) periodontal disease has not been investigated, and koala retrovirus is presumably exogenously transmitted. This study described oral health in South Australian koalas and investigated if an association between periodontal disease and koala retrovirus exists. Oral health was examined for wild-caught koalas from the Mount Lofty Ranges (n = 75). Koala retrovirus provirus was detected in whole blood using nested PCR and proviral load determined with qPCR. Periodontal disease severity was recorded and used to calculate the Final Oral Health Index (0-normal, 24-severe).Results Periodontal disease was observed in 84% (63/75) of koalas; 77% had gingivitis (58/75) and 65% (49/75) had periodontitis. The average Final Oral Health Index was 5.47 (s.d 3.13). Most cases of periodontal disease were associated with the incisors. Koala retrovirus-infected koalas were more likely to present with periodontitis (p = 0.042) and the Final Oral Health Index was negatively correlated with proviral load (ρ = -0.353, p = 0.017). South Australian koalas had a high prevalence of gingivitis and periodontitis. Periodontal disease was more prevalent in the incisors. Exogenous koala retrovirus infection may also facilitate the development of periodontitis by modulation of the immune response to concurrent oral bacterial infections.

Novel insights into viral infection and oncogenesis from koala retrovirus (KoRV) infection of HEK293T cells

Koala retrovirus is thought to be an underlying cause of high levels of neoplasia and immunosuppression in koalas. While epidemiology studies suggest a strong link between KoRV and disease it has been difficult to prove causality because of the complex nature of the virus, which exists in both endogenous and exogenous forms. It has been difficult to identify koalas completely free of KoRV, and infection studies in koalas or koala cells are fraught with ethical and technical difficulties, respectively. This study uses KoRV infection of the susceptible human cell line HEK293T and RNAseq to demonstrate gene networks differentially regulated upon KoRV infection. Many of the pathways identified are those associated with viral infection, such as cytokine receptor interactions and interferon signalling pathways, as well as viral oncogenesis pathways. This study provides strong evidence that KoRV does indeed behave similarly to infectious retroviruses in stimulating antiviral and oncogenic cellular responses. In addition, it provides novel insights into KoRV oncogenesis with the identification of a group of histone family genes that are part of several oncogenic pathways as upregulated in KoRV infection.

Koala retrovirus viral load and disease burden in distinct northern and southern koala populations

Koala retrovirus (KoRV) displays features of both an endogenous and exogenous virus and is linked to neoplasia and immunosuppression in koalas. This study explores the apparent differences in the nature and impact of KoRV infection between geographically and genetically separated “northern” and “southern” koala populations, by investigating the disease status, completeness of the KoRV genome and the proviral (DNA) and viral (RNA) loads of 71 northern and 97 southern koalas. All northern animals were positive for all KoRV genes (gag, pro-pol and env) in both DNA and RNA forms, whereas many southern animals were missing one or more KoRV genes. There was a significant relationship between the completeness of the KoRV genome and clinical status in this population. The proviral and viral loads of the northern population were significantly higher than those of the southern population (P < 0.0001), and many provirus-positive southern animals failed to express any detectable KoRV RNA. Across both populations there was a positive association between proviral load and neoplasia (P = 0.009). Potential reasons for the differences in the nature of KoRV infection between the two populations are discussed.

Antibody response against koala retrovirus (KoRV) in koalas harboring KoRV-A in the presence or absence of KoRV-B

Koala retrovirus (KoRV) is in the process of endogenization into the koala (Phascolarctos cinereus) genome and is currently spreading through the Australian koala population. Understanding how the koala’s immune system responds to KoRV infection is critical for developing an efficacious vaccine to protect koalas. To this end, we analyzed the antibody response of 235 wild koalas, sampled longitudinally over a four-year period, that harbored KoRV-A, and with or without KoRV-B. We found that the majority of the sampled koalas were able to make anti-KoRV antibodies, and that there was a linear increase in anti-KoRV IgG levels in koalas up to approximately seven years of age and then a gradual decrease thereafter. Koalas infected with both KoRV-A and KoRV-B were found to have slightly higher anti-KoRV IgG titers than koalas with KoRV-A alone and there was an inverse relationship between anti-KoRV IgG levels and circulating KoRV viral load. Finally, we identified distinct epitopes on the KoRV envelope protein that were recognized by antibodies. Together, these findings provide insight into the koala’s immune response to KoRV and may be useful in the development of a therapeutic KoRV vaccine.

Altered immune parameters associated with Koala Retrovirus (KoRV) and Chlamydial infection in free ranging Victorian koalas (Phascolarctos cinereus)

Koala Retrovirus (KoRV) has been widely speculated to cause immune suppression in koalas (Phascolarctos cinereus) and to underlie the koala’s susceptibility to infectious disease, however evidence for immunomodulation is limited. The aim of this study is to determine whether immunophenotypic changes are associated with KoRV infection in free ranging Victorian koalas. qPCR was used to examine mRNA expression for Th1 (IFNγ), Th2-promoting (IL6, IL10) and Th17 (IL17A) cytokines, along with CD4 and CD8 in whole blood of koalas (n = 74) from Mt Eccles and Raymond Island in Victoria, Australia, with and without natural chlamydial infection. KoRV positive koalas had significantly lower levels of IL17A (p`0.023) and IFNγ (p = 0.044) gene expression along with a decreased CD4:CD8 gene expression ratio (p = 0.025) compared to negative koalas. No effect of chlamydial infection or combined effect of KoRV and chlamydial infection was detected in these populations. The decreased expression of IFNγ could make KoRV infected koalas more susceptible to persistent chlamydial infection, and a decrease in IL17A could make them more susceptible to gram negative bacterial, fungal and mycobacterial infection; but more tolerant of chlamydial infection.

Molecular dynamics of koala retrovirus infection in captive koalas in Japan

Koala retrovirus (KoRV) is a gammaretrovirus that is becoming endogenous in koalas. Here, we explored the dynamics of KoRV infection in captive koalas in Japan. We isolated peripheral blood mononuclear cells (PBMCs) from 11 koalas, from which we extracted the KoRV genome. We found the prevalence of KoRV provirus in the koalas to be 100%, and the copy number of KoRV proviral DNA in genomic DNA isolated from PBMCs was variable. The KoRV envelope genes from 11 koalas were sequenced and all were found to be KoRV type A. Nucleotide substitution analysis revealed differences in the KoRV env gene sequences of parents and their offspring. Although no viral KoRV RNA was detected in plasma of healthy koalas, a high copy number was found in plasma of a diseased koala (#6). Hematological analysis showed a high white blood cell (WBC) count in the blood of koala #6. Notably, when retested ~ 5 months later, koala #6 was found to be negative for KoRV in plasma, and the WBC count was within the normal range. Therefore, KoRV in the plasma could be a possible indicator of koala health. We also investigated KoRV growth in concanavalin-A-stimulated koala PBMCs by measuring the KoRV provirus copy number in gDNA and the KoRV RNA copy number in cells and culture supernatants by real-time PCR at days 4, 7, and 14 post-culture. We also observed that KoRV isolates were able to infect HEK293T cells. These findings could enhance our understanding of the dynamics of KoRV and its pathogenesis in koalas.

Induction of neutralizing antibody response against koala retrovirus (KoRV) and reduction in viral load in koalas following vaccination with recombinant KoRV envelope protein

Koala retrovirus (KoRV) infects the majority of Australia’s koalas (Phascolarctos cinereus) and has been linked to several life-threatening diseases such as lymphoma and leukemia, as well as Chlamydia and thus poses a threat to the continued survival of this species. While quarantine and antiretroviral drug treatment are possible control measures, they are impractical, leaving vaccination as the only realistic option. In this study, we examined the effect of a recombinant envelope protein-based anti-KoRV vaccine in two groups of South Australian koalas: KoRV infected or KoRV free. We report a successful vaccination response in the koalas with no vaccine-associated side effects. The vaccine induced a significant humoral immune response as well as the production of neutralizing antibodies in both groups of koalas. We also identified B-cell epitopes that were differentially recognized in KoRV-infected versus KoRV-free koalas following vaccination. Importantly, we also showed that vaccination had a therapeutic effect on koalas infected exogenously with KoRV by reducing their circulating viral load. Together, this study highlights the possibility of successfully developing a vaccine against KoRV infection in koalas.

Lymphoma, Koala Retrovirus Infection and Reproductive Chlamydiosis in a Koala (Phascolarctos cinereus)

Koala retrovirus (KoRV) infection, thought to be associated with lymphoid neoplasia, and Chlamydia pecorum-related ocular and urogenital disease are both highly prevalent in eastern Australian koala (Phascolarctos cinereus) populations. However, in South Australian koalas, little is known about KoRV infection and C. pecorum-associated disease. We report the first South Australian case of lymphoma in a KoRV-A-positive female koala also affected by severe reproductive chlamydiosis. The koala was from the Mount Lofty Ranges population and was presented with hindlimb lameness. Clinical examination identified right stifle crepitus, enlarged superficial lymph nodes and paraovarian cysts. Necropsy examination revealed extensive cartilage degeneration and loss over the medial femoral condyle, solid femoral bone marrow, mesenteric and ovarian tumours, paraovarian cysts and purulent metritis. Histopathology confirmed lymphoma in the bone marrow, mesenteric lymph nodes and ovary, with infiltration and parenchymal effacement in the pancreas, adrenal glands and other tissues. Lymphoma, KoRV and chlamydiosis are being investigated further in this population.

Koala retrovirus genotyping analyses reveal a low prevalence of KoRV-A in Victorian koalas and an association with clinical disease.

Koala retrovirus (KoRV) is undergoing endogenization into the genome of koalas in Australia, providing an opportunity to assess the effect of retrovirus infection on the health of a population. The prevalence of KoRV in north-eastern Australia (Queensland and New South Wales) is 100 %, whereas previous preliminary investigations in south-eastern Australia (Victoria) suggested KoRV is present at a lower prevalence, although the values have varied widely. Here, we describe a large study of free-ranging koalas in Victoria to estimate the prevalence of KoRV and assess the clinical significance of KoRV infection in wild koalas. Blood or spleen samples from 648 koalas where tested for KoRV provirus, and subsequently genotyped, using PCRs to detect the pol and env genes respectively. Clinical data was also recorded where possible and analysed in comparison to infection status. The prevalence of KoRV was 24.7 % (160/648). KoRV-A was detected in 141/160 cases, but KoRV-B, a genotype associated with neoplasia in captive koalas, was not detected. The genotype in 19 cases could not be determined. Genomic differences between KoRV in Victoria and type strains may have impacted genotyping. Factors associated with KoRV infection, based on multivariable analysis, were low body condition score, region sampled, and 'wet bottom' (a staining of the fur around the rump associated with chronic urinary incontinence). Koalas with wet bottom were nearly twice as likely to have KoRV provirus detected than those without wet bottom (odds ratio=1.90, 95 % confidence interval 1.21, 2.98). Our findings have important implications for the conservation of this iconic species, particularly regarding translocation potential of Victorian koalas.

KOALA RETROVIRUS: A REVIEW

Koala retrovirus (KoRV) is a gammaretrovirus that has been identified in both captive and free-ranging koalas ( Phascolarctos cinereus ) with variable geographic distribution in Australia. KoRV is capable of both exogenous and endogenous transmission, which provides an interesting research platform for scientists to study active retrovirus endogenization into a host genome and offers veterinary scientists an opportunity to examine the clinical consequences of KoRV infection in koalas. Causation between KoRV and frequently recognized clinical conditions associated with immune suppression and neoplasia in koalas has not been definitively established, however research continues to evaluate a potential association. Three KoRV variants, KoRV-A, KoRV-B, and KoRV-J, have been the most thoroughly described and preliminary evidence suggests KoRV variability may be fundamental in host pathogenicity. In addition to reviewing what is currently known about KoRV, this article discusses treatment, management, and future research directions.