Koala Retrovirus in Free-Ranging Populations — Prevalence

The prevalence of koala retrovirus (KoRV) provirus (DNA) and the average number of proviral insertions per cell vary in different free-ranging koala (Phascolarctos cinereus) populations across Australia. Populations in the northern states of Queensland and New South Wales have 100% proviral prevalence and mean proviral copy number of 140–165 per cell. In contrast, the proviral prevalence in the southern states of Victoria and South Australia differs among populations, with a mean prevalence in these states’ mainland populations of 73% and 38%, respectively and with the prevalence on southern island populations ranging from 0–50%. The proviral load in southern populations is comparatively low, with some populations having an average of less than 1 proviral copy per cell. The KoRV RNA load in plasma shows a similar discordance between northern and southern populations, with consistently high loads in northern koalas (103 to 1010 RNA copies per ml plasma), and loads ranging from 0 to 102 copies per ml in southern KoRV provirus-positive koalas. The variation in KoRV proviral prevalence and the disparity in proviral and viral loads between northern and southern koalas may reflect different types of infection in the two populations (endogenous versus exogenous). Alternatively, it is possible that KoRV has been present for a longer time period in northern populations resulting in differences in the host-virus relationship. Meers, Joanne, Greg Simmons, Kiersten Jones, Daniel T. W. Clarke, and Paul R. Young. 2014. Koala retrovirus in free-ranging populations—prevalence. In The Koala and its Retroviruses: Implications for Sustainability and Survival, ed. Geoffrey W. Pye, Rebecca N. Johnson and Alex D. Greenwood. Technical Reports of the Australian Museum, Online 24: 15–17. Koala retrovirus (KoRV) is a gammaretrovirus of koalas (Phascolarctos cinereus) that possesses features of both endogenous and exogenous viruses. In previous work, we demonstrated that KoRV is truly an endogenous virus in koalas in south-east Queensland, with proviral DNA present in every animal tested and also present in single sperm cells. We also showed evidence of specific proviral insertion inheritance in Queensland koalas (Tarlinton et al., 2006). However, KoRV is clearly not endogenous in all koala populations in Australia because our early work demonstrated mixed KoRV presence in some southern populations (Tarlinton et al., 2006). Despite its endogenous nature in Queensland koalas, KoRV also displays exogenous virus characteristics in these populations, with high levels of viral RNA present in the blood of every animal tested, indicating active transcription of the KoRV proviral elements (Tarlinton et al., 2005). There is also considerable variation in the number and sites of KoRV proviral insertions in individual koalas, which again is not typical for an endogenous virus where the conservation of a proviral 16 Technical Reports of the Australian Museum, Online (2014) No. 24 integration pattern is expected (Tarlinton et al., 2006). Taken together, these findings suggest that the virus is behaving as both an exogenous and endogenous virus in different koala populations, with transmission of the virus possibly occurring by inherited and/or horizontal routes. KoRV is closely related to gibbon ape leukaemia virus (GALV), a pathogenic exogenous virus of gibbons (Hanger et al., 2000; Tarlinton et al., 2008). The genetic similarity between KoRV and GALV led to speculation that the two viruses diverged only recently (Bromham, 2002). The sequence similarity between KoRV and GALV is so close across the complete proviral genome that either recent crossspecies transmission of virus between koalas and gibbons, or transmission from an intermediate host species is likely (Hanger et al., 2000; Martin et al., 1999). Given that koalas and gibbons do not exist in the same geographical locations in nature and that to date GALV has only been detected in captive gibbons, direct cross-species transmission in the wild appears unlikely. The most probable explanation for the close genetic similarity between KoRV and GALV is that they each were derived from a common virus hosted by a third species of animal whose distribution encompasses that of both gibbons and koalas. To better understand the endogenous/exogenous nature of KoRV infection in different koala populations, and potentially to gain insight into the origins of the virus, we are conducting on-going studies into the prevalence of KoRV infection across the species’ range, including investigation of KoRV proviral DNA load and viral RNA load in these different populations. The samples tested in our studies are collected from a range of sources including koalas presented to veterinary clinics because of illness or trauma, koalas trapped in other research projects, archival samples stored by other researchers and koalas undergoing sterilisation procedures on Kangaroo Island. Related to the diversity in source of samples, the sample type also covered a spectrum including blood, internal organs from euthanized animals, and ear punch biopsies. Methods used to detect and quantify KoRV proviral DNA and viral RNA comprise standard PCR using pol gene primers (Tarlinton et al., 2006), nested PCR using internal primers (Simmons et al., 2012), real-time PCR (qPCR) (Tarlinton et al., 2005), and reverse transcriptase real-time PCR (RT-qPCR). Prevalence of KoRV provirus in free-ranging koala populations The prevalence of KoRV provirus in different koala populations ranges from 100% in the northern states of Queensland and New South Wales (NSW) to 0% on one of the southern off-shore islands (Phillip Island) (Simmons et al., 2012). Previously published and recent work has shown that the prevalence in populations on mainland Victoria and South Australia falls between these two extremes, with a mean prevalence on mainland Victoria and South Australia of 73% and 38%, respectively (Simmons et al., 2012; Jones, unpublished data). The prevalence on other southern islands varied considerably, with 50% of koalas tested being proviruspositive on Snake Island, 35% on Raymond Island, 21% on French Island and 15% on Kangaroo Island (in 2007), although the small number of samples tested from some of these islands limits the strength of these data (Simmons et al., 2012). In contrast, the KoRV proviral prevalence on islands off the coast of Queensland is 100% (Jones, unpublished data). To investigate an apparent change in the dynamics of KoRV infection on Kangaroo Island, we are conducting a temporal and spatial study of KoRV proviral prevalence on the island, which is off the coast of the state of South Australia. In our initial study of blood samples collected from koalas on the island in 2004, none of 26 koalas was provirus-positive (Tarlinton et al., 2006). Twenty-four of 162 (15%) blood samples collected in 2007 were proviruspositive (Simmons et al., 2012), and 19 of 50 (38%) and 10 of 38 (26%) samples collected in 2009 and 2011, respectively were provirus-positive (Jones, unpublished data). There does not appear to be a clear geographic segregation of KoRV provirus-positive and –negative animals on Kangaroo Island. However, koalas in the central region of the island are over-represented in each of the four years of our sample collection, thus interpretation of these data must be guarded. The samples used in our study have been collected from koalas caught for a governmentfunded sterilisation program, which aims to address the over-abundance of the introduced koala population on the island. In the years we obtained samples, this program was focused on the central region of the island, with only limited trapping activities in the western and eastern parts of the island. To date, the small number of animals tested from the western part of the island has been KoRV provirus-negative, but further samples from that part of the island should be tested to confirm these findings. KoRV proviral and viral loads We established methods to quantify KoRV proviral DNA and viral RNA loads in cells and plasma, respectively. Initially we used these methods to investigate relationships between either proviral or viral load and disease in koalas, in particular lymphoid neoplasia and chlamydial disease. We found a significant association between KoRV viral load in plasma and the presence of lymphoid neoplasia (Tarlinton et al., 2005). Although there was a trend towards increasing viral load and severity of chlamydiosis, the relationship was not significant. Using qPCR, we then investigated the levels of KoRV proviral load amongst different koala populations in Australia. The proviral copy number per cell was estimated either from the quantification of DNA concentration in the sample or from comparison to beta-actin copy number (Delta-Delta Ct). Using the first of these approaches, the proviral copy number per cell from DNA extracted from ear punch biopsies of Queensland koalas was markedly higher than that of koalas in southern states, with a mean of 165 copies per cell in Queensland koalas compared to means of 1.5, 0.00153 and 0.000129 copies per cell in three different populations of Victorian koalas (Simmons et al., 2012). Similarly, using the second approach, the means of proviral copies per cell from DNA extracted from blood cell pellets varied from 140 in Queensland koalas, 10 in Kangaroo Island koalas and 1.3 in South Australian mainland koalas (Jones, unpublished data). KoRV viral RNA levels were determined using RT-qPCR on plasma samples. The viral load in plasma of Queensland koalas is consistently high, ranging from 4.3×103 to 8.2×1010 copies/ml plasma (Simmons, 2011). In contrast, plasma samples from koalas on Kangaroo Island range from only 1.1×102 to 4.3×102 copies/ml plasma (Jones, unpublished data) and not all KoRV provirus-positive koalas have detectable viral RNA in plasma. Meers et al.: Prevalence of KoRV 17