Enterovirus 3C Protease Cleaves TRIM7 To Dampen Its Antiviral Activity.

Mammalian TRIM7 is an antiviral protein that inhibits multiple human enteroviruses by degrading the viral 2BC protein. Whether TRIM7 is reciprocally targeted by enteroviruses is not known. Here, we report that the 3C protease (3Cpro) from two enteroviruses, coxsackievirus B3 (CVB3) and poliovirus, targets TRIM7 for cleavage. CVB3 3Cpro cleaves TRIM7 at glutamine 24 (Q24), resulting in a truncated TRIM7 that fails to inhibit CVB3 due to dampened E3 ubiquitin ligase activity. TRIM7 Q24 is highly conserved across mammals, except in marsupials, which instead have a naturally occurring histidine (H24) that is not subject to 3Cpro cleavage. Marsupials also express two isoforms of TRIM7, and the two proteins from koalas have distinct antiviral activities. The longer isoform contains an additional exon due to alternate splice site usage. This additional exon contains a unique 3Cpro cleavage site, suggesting that certain enteroviruses may have evolved to target marsupial TRIM7 even if the canonical Q24 is missing. Combined with computational analyses indicating that TRIM7 is rapidly evolving, our data raise the possibility that TRIM7 may be targeted by enterovirus evasion strategies and that evolution of TRIM7 across mammals may have conferred unique antiviral properties. IMPORTANCE Enteroviruses are significant human pathogens that cause viral myocarditis, pancreatitis, and meningitis. Knowing how the host controls these viruses and how the viruses may evade host restriction is important for understanding fundamental concepts in antiviral immunity and for informing potential therapeutic interventions. In this study, we demonstrate that coxsackievirus B3 uses its virally encoded protease to target the host antiviral protein TRIM7 for cleavage, suggesting a potential mechanism of viral immune evasion. We additionally show that TRIM7 has evolved in certain mammalian lineages to express protein variants with distinct antiviral activities and susceptibilities to viral protease-mediated cleavage.

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

Koala retrovirus (KoRV) subtype A (KoRV-A) is currently in transition from exogenous virus to endogenous viral element, providing an ideal system to elucidate retroviral-host coevolution. We characterized KoRV geography using fecal 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/New South Wales state border. In northern koalas, pol gene copies were ubiquitously present at above five per cell, consistent with endogenous KoRV. In southern koalas, pol copies were detected in only 25.8% of koalas and always at copy numbers below one, while the env gene was detected in all animals and in a majority at copy numbers above 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 exogenous KoRV subtypes (B to K), two novel subtypes, and intermediate subtypes were detected in all northern koala populations but were strikingly absent from all southern animals tested. Apart from KoRV subtype 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 have occurred within northern Australia, but this has not extended into animals within southern Australia.

Expansion of a retrovirus lineage in the koala genome.

Retroviruses have left their legacy in host genomes over millions of years as endogenous retroviruses (ERVs), and their structure, diversity, and prevalence provide insights into the historical dynamics of retrovirus-host interactions. In bioinformatic analyses of koala (Phascolarctos cinereus) whole-genome sequences, we identify a recently expanded ERV lineage (phaCin-ß) that is related to the New World squirrel monkey retrovirus. This ERV expansion shares many parallels with the ongoing koala retrovirus (KoRV) invasion of the koala genome, including highly similar and mostly intact sequences, and polymorphic ERV loci in the sampled koala population. The recent phaCin-ß ERV colonization of the koala genome appears to predate the current KoRV invasion, but polymorphic ERVs and divergence comparisons between these two lineages predict a currently uncharacterized, possibly still extant, phaCin-ß retrovirus. The genomics approach to ERV-guided discovery of novel retroviruses in host species provides a strong incentive to search for phaCin-ß retroviruses in the Australasian fauna.

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.

Koala retrovirus (KoRV) subtypes and their impact on captive koala (Phascolarctos cinereus) health.

Koala retrovirus (KoRV), a major pathogen of koalas, exists in both endogenous (KoRV-A) and exogenous forms (KoRV-B to J). However, the impact of infection with multiple subtypes is not well understood. Accordingly, in this study, we surveyed a representative sample from a Japanese zoo population to determine the infection status for three KoRV subtypes (KoRV-A, B, and C) and to investigate the proviral and RNA load profiles in animals with single- and multiple-subtype infections, using peripheral blood mononuclear cells (PBMCs) and plasma. Six koalas were evaluated in the study; all were infected with KoRV-A, and two koalas were coinfected with non-A subtypes (KoRV-B and/or KoRV-C). The highest KoRV total RNA and viral loads in PBMCs and plasma were found in a koala infected with multiple subtypes (KoRV-A, -B and -C). The other koala infected with multiple subtypes (KoRV-A and B) showed the highest proviral PBMC load but the lowest RNA copy number in PBMC and plasma. PBMCs from this animal were cultured for further investigation, and KoRV RNA was detected in the cells and culture supernatant after 7 and/or 14 days. The koalas harboring multiple subtypes had a higher white blood cell count than those harboring only KoRV-A and were judged to be leukemic, and they subsequently died due to lymphoma. Accordingly, we conclude that coinfection with multiple KoRV subtypes may be linked to more-severe disease. In a sequence alignment, the detected KoRV-A env gene showed 100% sequence identity to the reference gene, whereas the KoRV-B and -C env genes varied from their reference sequences.

Comparative analysis reveals the species-specific genetic determinants of ACE2 required for SARS-CoV-2 entry.

Coronavirus interaction with its viral receptor is a primary genetic determinant of host range and tissue tropism. SARS-CoV-2 utilizes ACE2 as the receptor to enter host cell in a species-specific manner. We and others have previously shown that ACE2 orthologs from New World monkey, koala and mouse cannot interact with SARS-CoV-2 to mediate viral entry, and this defect can be restored by humanization of the restrictive residues in New World monkey ACE2. To better understand the genetic determinants behind the ability of ACE2 orthologs to support viral entry, we compared koala and mouse ACE2 sequences with that of human and identified the key residues in koala and mouse ACE2 that restrict viral receptor activity. Humanization of these critical residues rendered both koala and mouse ACE2 capable of binding the spike protein and facilitating viral entry. Our study shed more lights into the genetic determinants of ACE2 as the functional receptor of SARS-CoV-2, which facilitates our understanding of viral entry.

The environmental and ecological determinants of elevated Ross River Virus exposure in koalas residing in urban coastal landscapes.

Koala populations in many areas of Australia have declined sharply in response to habitat loss, disease and the effects of climate change. Koalas may face further morbidity from endemic mosquito-borne viruses, but the impact of such viruses is currently unknown. Few seroprevalence studies in the wild exist and little is known of the determinants of exposure. Here, we exploited a large, spatially and temporally explicit koala survey to define the intensity of Ross River Virus (RRV) exposure in koalas residing in urban coastal environments in southeast Queensland, Australia. We demonstrate that RRV exposure in koalas is much higher (> 80%) than reported in other sero-surveys and that exposure is uniform across the urban coastal landscape. Uniformity in exposure is related to the presence of the major RRV mosquito vector, Culex annulirostris, and similarities in animal movement, tree use, and age-dependent increases in exposure risk. Elevated exposure ultimately appears to result from the confinement of remaining coastal koala habitat to the edges of permanent wetlands unsuitable for urban development and which produce large numbers of competent mosquito vectors. The results further illustrate that koalas and other RRV-susceptible vertebrates may serve as useful sentinels of human urban exposure in endemic areas.

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.

CD4, CD8b, and Cytokines Expression Profiles in Peripheral Blood Mononuclear Cells Infected with Different Subtypes of KoRV from Koalas (Phascolarctos cinereus) in a Japanese Zoo.

Koala retrovirus (KoRV) poses a major threat to koala health and conservation, and currently has 10 identified subtypes: an endogenous subtype (KoRV-A) and nine exogenous subtypes (KoRV-B to KoRV-J). However, subtype-related variations in koala immune response to KoRV are uncharacterized. In this study, we investigated KoRV-related immunophenotypic changes in a captive koala population (Hirakawa zoo, Japan) with a range of subtype infection profiles (KoRV-A only vs. KoRV-A with KoRV-B and/or -C), based on qPCR measurements of CD4, CD8b, IL-6, IL-10 and IL-17A mRNA expression in unstimulated and concanavalin (Con)-A-stimulated peripheral blood mononuclear cells (PBMCs). Although CD4, CD8b, and IL-17A expression did not differ between KoRV subtype infection profiles, IL-6 expression was higher in koalas with exogenous infections (both KoRV-B and KoRV-C) than those with the endogenous subtype only. IL-10 expression did not significantly differ between subtype infection profiles but did show a marked increase-accompanying decreased CD4:CD8b ratio-in a koala with lymphoma and co-infected with KoRV-A and -B, thus suggesting immunosuppression. Taken together, the findings of this study provide insights into koala immune response to multiple KoRV subtypes, which can be exploited for the development of prophylactic and therapeutic interventions for this iconic marsupial species.

Koalas vaccinated against Koala retrovirus respond by producing increased levels of interferon-gamma.

Koala retrovirus (KoRV) is believed to be in an active state of endogenization into the koala genome. KoRV is present as both an endogenous and exogenous infection in all koalas in northern Australia. KoRV has been linked to koala pathologies including neoplasia and increased susceptibility to Chlamydia. A KoRV vaccine recently trialled in 10 northern koalas improved antibody response and reduced viral load. This communication reports the expression of key immune genes underlining the innate and adaptive immune response to vaccination in these northern koalas. The results showed that prior to vaccination, IL-8 was expressed at the highest levels, with at least 200-fold greater expression compared to other cytokines, while CD8 mRNA expression was significantly higher than CD4 mRNA expression level. Interferon-γ was up-regulated at both 4- and 8-weeks post-vaccination while IL-8 was down-regulated at 8-weeks post-vaccination.

Koala retrovirus diversity, transmissibility, and disease associations.

BACKGROUND: Koalas 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. RESULTS: All 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. CONCLUSIONS: Our 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.

Identification and Prevalence of Phascolarctid Gammaherpesvirus Types 1 and 2 in South Australian Koala Populations.

To determine Phascolarctid gammaherpesviruses (PhaHV) infection in South Australian koala populations, 80 oropharyngeal swabs from wild-caught and 87 oropharyngeal spleen samples and swabs from euthanased koalas were tested using two specific PCR assays developed to detect PhaHV-1 and PhaHV-2. In wild-caught koalas, active shedding of PhaHV was determined by positive oropharyngeal samples in 72.5% (58/80) of animals, of which 44.8% (26/58) had PhaHV-1, 20.7% (12/58) PhaHV-2 and 34.5% (20/58) both viral subtypes. In the euthanased koalas, systemic infection was determined by positive PCR in spleen samples and found in 72.4% (63/87) of koalas. Active shedding was determined by positive oropharyngeal results and found in 54.0% (47/87) of koalas. Koalas infected and actively shedding PhaHV-1 alone, PhaHV-2 alone or shedding both viral subtypes were 48.9% (23/47), 14.9% (7/47) and 36.2% (17/47), respectively. Only 45.9% (40/87) were not actively shedding, of which 40.0% (16/40) of these had systemic infections. Both wild-caught and euthanased koalas actively shedding PhaHV-2 were significantly more likely to be actively shedding both viral subtypes. Active shedding of PhaHV-2 had a significant negative correlation with BCS in the euthanased cohort, and active shedding of PhaHV-1 had a significant positive relationship with age in both wild-caught and euthanased cohorts.

Koala retrovirus epidemiology, transmission mode, pathogenesis, and host immune response in koalas (Phascolarctos cinereus): a review.

Koala retrovirus (KoRV) is a major threat to koala health and conservation. It also represents a series of challenges across the fields of virology, immunology, and epidemiology that are of great potential interest to any researcher in the field of retroviral diseases. KoRV is a gammaretrovirus that is present in both endogenous and exogenous forms in koala populations, with a still-active endogenization process. KoRV may induce immunosuppression and neoplastic conditions such as lymphoma and leukemia and play a role in chlamydiosis and other diseases in koalas. KoRV transmission modes, pathogenesis, and host immune response still remain unclear, and a clear understanding of these areas is critical for devising effective preventative and therapeutic strategies. Research on KoRV is clearly critical for koala conservation. In this review, we provide an overview of the current understanding and future challenges related to KoRV epidemiology, transmission mode, pathogenesis, and host immune response and discuss prospects for therapeutic and preventive vaccines.

A micro-PRNT for the detection of Ross River virus antibodies in mosquito blood meals: A useful tool for inferring transmission pathways.

INTRODUCTION: Many arboviruses of public health significance are maintained in zoonotic cycles with complex transmission pathways. The presence of serum antibody against arboviruses in vertebrates provides evidence of their historical exposure but reveals nothing about the vector-reservoir relationship. Moreover, collecting blood or tissue samples from vertebrate hosts is ethically and logistically challenging. We developed a novel approach for screening the immune status of vertebrates against Ross River virus that allows us to implicate the vectors that form the transmission pathways for this commonly notified Australian arboviral disease. METHODS: A micro-plaque reduction neutralisation test (micro-PRNT) was developed and validated on koala (Phascolarctos cinereus) sera against a standard PRNT. The ability of the micro-PRNT to detect RRV antibodies in mosquito blood meals was then tested using two mosquito models. Laboratory-reared Aedes aegypti were fed, via a membrane, on sheep blood supplemented with RRV seropositive and seronegative human sera. Aedes notoscriptus were fed on RRV seropositive and seronegative human volunteers. Blood-fed mosquitoes were harvested at various time points after feeding and their blood meals analysed for the presence of RRV neutralising antibodies using the micro-PRNT. RESULTS: There was significant agreement of the plaque neutralisation resulting from the micro-PRNT and standard PRNT techniques (R2 = 0.65; P<0.0001) when applied to RRV antibody detection in koala sera. Sensitivity and specificity of the micro-PRNT assay were 88.2% and 96%, respectively, in comparison with the standard PRNT. Blood meals from mosquitoes fed on sheep blood supplemented with RRV antibodies, and on blood from RRV seropositive humans neutralised the virus by ≥50% until 48 hr post feeding. The vertebrate origin of the blood meal was also ascertained for the same samples, in parallel, using established molecular techniques. CONCLUSIONS: The small volumes of blood present in mosquito abdomens can be used to identify RRV antibodies and therefore host exposure to arbovirus infection. In tandem with the accurate identification of the mosquito, and diagnostics for the host origin of the blood meal, this technique has tremendous potential for exploring RRV transmission pathways. It can be adapted for similar studies on other mosquito borne zoonoses.

Helping koalas battle disease - Recent advances in Chlamydia and koala retrovirus (KoRV) disease understanding and treatment in koalas.

The iconic Australian marsupial, the koala (Phascolarctos cinereus), has suffered dramatic population declines as a result of habitat loss and fragmentation, disease, vehicle collision mortality, dog attacks, bushfires and climate change. In 2012, koalas were officially declared vulnerable by the Australian government and listed as a threatened species. In response, research into diseases affecting koalas has expanded rapidly. The two major pathogens affecting koalas are Chlamydia pecorum, leading to chlamydial disease and koala retrovirus (KoRV). In the last eight years, these pathogens and their diseases have received focused study regarding their sources, genetics, prevalence, disease presentation and transmission. This has led to vast improvements in pathogen detection and treatment, including the ongoing development of vaccines for each as a management and control strategy. This review will summarize and highlight the important advances made in understanding and combating C. pecorum and KoRV in koalas, since they were declared a threatened species. With complementary advances having also been made from the koala genome sequence and in our understanding of the koala immune system, we are primed to make a significant positive impact on koala health into the future.

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.

Infectious KoRV-related retroviruses circulating in Australian bats.

Bats are reservoirs of emerging viruses that are highly pathogenic to other mammals, including humans. Despite the diversity and abundance of bat viruses, to date they have not been shown to harbor exogenous retroviruses. Here we report the discovery and characterization of a group of koala retrovirus-related (KoRV-related) gammaretroviruses in Australian and Asian bats. These include the Hervey pteropid gammaretrovirus (HPG), identified in the scat of the Australian black flying fox (Pteropus alecto), which is the first reproduction-competent retrovirus found in bats. HPG is a close relative of KoRV and the gibbon ape leukemia virus (GALV), with virion morphology and Mn2+-dependent virion-associated reverse transcriptase activity typical of a gammaretrovirus. In vitro, HPG is capable of infecting bat and human cells, but not mouse cells, and displays a similar pattern of cell tropism as KoRV-A and GALV. Population studies reveal the presence of HPG and KoRV-related sequences in several locations across northeast Australia, as well as serologic evidence for HPG in multiple pteropid bat species, while phylogenetic analysis places these bat viruses as the basal group within the KoRV-related retroviruses. Taken together, these results reveal bats to be important reservoirs of exogenous KoRV-related gammaretroviruses.

Transmission of Koala Retrovirus from Parent Koalas to a Joey in a Japanese Zoo.

Koala retrovirus (KoRV) is of an interest to virologists due to its currently active endogenization into the koala (Phascolarctos cinereus) genome. Although KoRV has frequently been isolated in wild and captive koala populations, its pathogenesis and transmission remain to be fully characterized, and most previous research has concentrated on adult koalas rather than on joeys. Here, we characterized KoRV isolates obtained from a deceased male joey and its parents (animals reared in a Japanese zoo) to investigate KoRV transmission mode and pathogenesis. We sequenced the KoRV long terminal repeat (LTR) and envelope genes isolated from the joey and its parents and found KoRV-A and KoRV-C in genomic DNA from both the parents and the joey. Notably, both parents were also positive for KoRV-B, whereas the joey was KoRV-B negative, further confirming that KoRV-B is an exogenous strain. The KoRV LTR sequence of the joey was considerably closer to that of its sire than its dam. For further characterization, total KoRV, KoRV-A, KoRV-B, and KoRV-C proviral loads were quantified in peripheral blood mononuclear cells from the parents and in blood samples from the joey. Total KoRV, KoRV-A, and KoRV-C proviral loads were also quantified for different tissues (bone, liver, kidney, lung, spleen, heart, and muscle) from the joey, revealing differences suggestive of a distinct tissue tropism (highest total KoRV proviral load in the spleen and lowest in bone). The amount of KoRV-C in the parents was less than that in the joey. Our findings contribute to an improved understanding of KoRV pathogenesis and transmission mode and highlight useful areas for future research.IMPORTANCE KoRV is unique among retroviruses in that one strain (KoRV-A) is undergoing endogenization, whereas the other main subtype (KoRV-B) and another subtype (KoRV-C) are reportedly exogenous strains. Its transmission and pathogenesis are of interest in the study of retroviruses and are crucial for any conservation strategy geared toward koala health. This study provides new evidence on the modes of KoRV transmission from parent koalas to their joey. We found vertical transmission of KoRV-A, confirming its endogenization, but with closer conservation between the joey and its sire than its dam (previous reports on joeys are rare but have postulated dam-to-joey vertical transmission). This is also the first report of a KoRV-B-negative joey from KoRV-B-positive parents, contrasting with the few previous reports of 100% transmission of KoRV-B from dams to joeys. Thus, the results in this study give some novel insights for the transmission mode of KoRV.

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.

The piRNA Response to Retroviral Invasion of the Koala Genome.

Antisense Piwi-interacting RNAs (piRNAs) guide silencing of established transposons during germline development, and sense piRNAs drive ping-pong amplification of the antisense pool, but how the germline responds to genome invasion is not understood. The KoRV-A gammaretrovirus infects the soma and germline and is sweeping through wild koalas by a combination of horizontal and vertical transfer, allowing direct analysis of retroviral invasion of the germline genome. Gammaretroviruses produce spliced Env mRNAs and unspliced transcripts encoding Gag, Pol, and the viral genome, but KoRV-A piRNAs are almost exclusively derived from unspliced genomic transcripts and are strongly sense-strand biased. Significantly, selective piRNA processing of unspliced proviral transcripts is conserved from insects to placental mammals. We speculate that bypassed splicing generates a conserved molecular pattern that directs proviral genomic transcripts to the piRNA biogenesis machinery and that this "innate" piRNA response suppresses transposition until antisense piRNAs are produced, establishing sequence-specific adaptive immunity.