Invasion and Amplification of Endogenous Retroviruses in Dasyuridae Marsupial Genomes.

Retroviruses are an ancient viral family that have globally coevolved with vertebrates and impacted their evolution. In Australia, a continent that has been geographically isolated for millions of years, little is known about retroviruses in wildlife, despite the devastating impacts of a retrovirus on endangered koala populations. We therefore sought to identify and characterize Australian retroviruses through reconstruction of endogenous retroviruses from marsupial genomes, in particular the Tasmanian devil due to its high cancer incidence. We screened 19 marsupial genomes and identified over 80,000 endogenous retrovirus fragments which we classified into eight retrovirus clades. The retroviruses were similar to either Betaretrovirus (5/8) or Gammaretrovirus (3/8) retroviruses, but formed distinct phylogenetic clades compared to extant retroviruses. One of the clades (MEBrv 3) lost an envelope but retained retrotranspositional activity, subsequently amplifying throughout all Dasyuridae genomes. Overall, we provide insights into Australian retrovirus evolution and identify a highly active endogenous retrovirus within Dasyuridae genomes.

Relatives of rubella virus in diverse mammals.

Since 1814, when rubella was first described, the origins of the disease and its causative agent, rubella virus (Matonaviridae: Rubivirus), have remained unclear1. Here we describe ruhugu virus and rustrela virus in Africa and Europe, respectively, which are, to our knowledge, the first known relatives of rubella virus. Ruhugu virus, which is the closest relative of rubella virus, was found in apparently healthy cyclops leaf-nosed bats (Hipposideros cyclops) in Uganda. Rustrela virus, which is an outgroup to the clade that comprises rubella and ruhugu viruses, was found in acutely encephalitic placental and marsupial animals at a zoo in Germany and in wild yellow-necked field mice (Apodemus flavicollis) at and near the zoo. Ruhugu and rustrela viruses share an identical genomic architecture with rubella virus2,3. The amino acid sequences of four putative B cell epitopes in the fusion (E1) protein of the rubella, ruhugu and rustrela viruses and two putative T cell epitopes in the capsid protein of the rubella and ruhugu viruses are moderately to highly conserved4-6. Modelling of E1 homotrimers in the post-fusion state predicts that ruhugu and rubella viruses have a similar capacity for fusion with the host-cell membrane5. Together, these findings show that some members of the family Matonaviridae can cross substantial barriers between host species and that rubella virus probably has a zoonotic origin. Our findings raise concerns about future zoonotic transmission of rubella-like viruses, but will facilitate comparative studies and animal models of rubella and congenital rubella syndrome.

Changes in Endogenous and Exogenous Koala Retrovirus Subtype Expression over Time Reflect Koala Health Outcomes.

Koala retrovirus (KoRV) is unique in that it exists as both an exogenous and actively endogenizing gamma retrovirus of koalas. While nine subtypes of KoRV have been recognized, focused study of these subtypes in koalas over time and with different health outcomes has been lacking. Therefore, in this study, three wild koala cohorts were established and monitored to examine KoRV proviral and expression data from koalas that either remained healthy over time, began healthy before developing chlamydial cystitis, or presented with chlamydial cystitis and were treated with antibiotics. Deep sequencing of the proviral KoRV envelope gene revealed KoRV-A, -B, -D, and -F to be the major subtypes in this population and allowed for subtype-specific assays to be created. Quantification of KoRV transcripts revealed that KoRV-D expression mirrored the total KoRV expression levels (106 copies/ml of plasma), with KoRV-A and KoRV-F expression being ∼10-fold less and KoRV-B expression being ∼100-fold less, when detected. Strikingly, there was significantly higher expression of KoRV-D in healthy koalas than in koalas that developed chlamydial cystitis, with healthy koalas expressing a major KoRV-D/minor KoRV-A profile, whereas koalas that developed cystitis had variable KoRV expression profiles. Total anti-KoRV IgG antibody levels were found not to correlate with the expression of total KoRV or any individual KoRV subtype. Finally, KoRV expression was consistent between systemic and mucosal body sites and during antibiotic treatment. Collectively, this gives a comprehensive picture of KoRV dynamics during several important koala health states.IMPORTANCE The long-term survival of the koala is under serious threat, with this iconic marsupial being declared "vulnerable" by the Australian Government and officially listed as a threatened species. KoRV is clearly contributing to the overall health status of koalas, and research into this virus has been lacking detailed study of the multiple subtypes at both the proviral and expressed viral levels over time. By designing new subtype-specific assays and following well-defined koala cohorts over time, this study has generated a new more complete picture of KoRV and its relationship to koala health outcomes in the wild. Only by building a comprehensive picture of KoRV during both koala health and disease can we bring meaningful koala health interventions into better focus.

Convergent Co-option of the Retroviral gag Gene during the Early Evolution of Mammals.

Endogenous retroviruses, records of past retroviral infections, are ubiquitous in vertebrate genomes. On occasion, vertebrate hosts have co-opted retroviral genes for their own biological functions. Here, we perform a phylogenomic survey of retroviral gag gene homologs within vertebrate genomes and identify two ancient co-opted retroviral gag genes, designated wucaishi1 (wcs1) and wucaishi2 (wcs2), in mammals. Conserved synteny and evolutionary analyses suggest that the wcs1 and wcs2 co-options occurred before the origin of modern placental mammals (∼100 million years ago) and before the origin of modern marsupials (∼80 million years ago), respectively. We found that the wcs genes were lost or pseudogenized multiple times during the evolutionary course of mammals. While the wcs1 gene is mainly subject to negative selection in placental mammals (except in Perissodactyla), the wcs2 gene underwent positive selection in marsupials. Moreover, analyses of transcriptome-sequencing (RNA-seq) data suggest that the wcs1 and the wcs2 genes are expressed in a wide range of tissues. The convergent wcs co-option in mammals implies the retroviral gag gene might have been repurposed more frequently than previously thought.IMPORTANCE Retroviruses occasionally can infect host germ lines, forming endogenous retroviruses. Vertebrates, in turn, recruited retroviral genes for their own biological functions, a process formally known as co-option or exaptation. To date, co-opted retroviral gag genes have rarely been reported. In this study, we identified two co-opted retroviral gag genes, designated wucaishi1 (wcs1) and wucaishi2 (wcs2), in mammals. The co-option of wcs1 and wcs2 occurred before the origin of modern placentals and before the origin of modern marsupials, respectively. Our study indicates that retroviral gag gene co-option might have occurred more frequently than previously thought during the evolutionary course of vertebrates.

Fecal Viral Diversity of Captive and Wild Tasmanian Devils Characterized Using Virion-Enriched Metagenomics and Metatranscriptomics.

The Tasmanian devil is an endangered carnivorous marsupial threatened by devil facial tumor disease (DFTD). While research on DFTD has been extensive, little is known about viruses in devils and whether any are of potential conservation relevance for this endangered species. Using both metagenomics based on virion enrichment and sequence-independent amplification (virion-enriched metagenomics) and metatranscriptomics based on bulk RNA sequencing, we characterized and compared the fecal viromes of captive and wild devils. A total of 54 fecal samples collected from two captive and four wild populations were processed for virome characterization using both approaches. In total, 24 novel marsupial-related viruses, comprising a sapelovirus, astroviruses, rotaviruses, picobirnaviruses, parvoviruses, papillomaviruses, polyomaviruses, and a gammaherpesvirus, were identified, as well as known mammalian pathogens such as rabbit hemorrhagic disease virus 2. Captive devils showed significantly lower viral diversity than wild devils. Comparison of the two virus discovery approaches revealed substantial differences in the number and types of viruses detected, with metatranscriptomics better suited for RNA viruses and virion-enriched metagenomics largely identifying more DNA viruses. Thus, the viral communities revealed by virion-enriched metagenomics and metatranscriptomics were not interchangeable and neither approach was able to detect all viruses present. An integrated approach using both virion-enriched metagenomics and metatranscriptomics constitutes a powerful tool for obtaining a complete overview of both the taxonomic and functional profiles of viral communities within a sample.IMPORTANCE The Tasmanian devil is an iconic Australian marsupial that has suffered an 80% population decline due to a contagious cancer, devil facial tumor disease, along with other threats. Until now, viral discovery in this species has been confined to one gammaherpesvirus (dasyurid herpesvirus 2 [DaHV-2]), for which captivity was identified as a significant risk factor. Our discovery of 24 novel marsupial-associated RNA and DNA viruses, and that viral diversity is lower in captive than in wild devils, has greatly expanded our knowledge of gut-associated viruses in devils and provides important baseline information that will contribute to the conservation and captive management of this endangered species. Our results also revealed that a combination of virion-enriched metagenomics and metatranscriptomics may be a more comprehensive approach for virome characterization than either method alone. Our results thus provide a springboard for continuous improvements in the way we study complex viral communities.

Koala and Wombat Gammaherpesviruses Encode the First Known Viral NTPDase Homologs and Are Phylogenetically Divergent from All Known Gammaherpesviruses.

There is a large taxonomic gap in our understanding of mammalian herpesvirus genetics and evolution corresponding to those herpesviruses that infect marsupials, which diverged from eutherian mammals approximately 150 million years ago (mya). We compare the genomes of two marsupial gammaherpesviruses, Phascolarctid gammaherpesvirus 1 (PhaHV1) and Vombatid gammaherpesvirus 1 (VoHV1), which infect koalas (Phascolarctos cinereus) and wombats (Vombatus ursinus), respectively. The core viral genomes were approximately 117 kbp and 110 kbp in length, respectively, sharing 69% pairwise nucleotide sequence identity. Phylogenetic analyses showed that PhaHV1 and VoHV1 formed a separate branch, which may indicate a new gammaherpesvirus genus. The genomes contained 60 predicted open reading frames (ORFs) homologous to those in eutherian herpesviruses and 20 ORFs not yet found in any other herpesvirus. Seven of these ORFs were shared by the two viruses, indicating that they were probably acquired prespeciation, approximately 30 to 40 mya. One of these shared genes encodes a putative nucleoside triphosphate diphosphohydrolase (NTPDase). NTPDases are usually found in mammals and higher-order eukaryotes, with a very small number being found in bacteria. This is the first time that an NTPDase has been identified in any viral genome. Interrogation of public transcriptomic data sets from two koalas identified PhaHV1-specific transcripts in multiple host tissues, including transcripts for the novel NTPDase. PhaHV1 ATPase activity was also demonstrated in vitro, suggesting that the encoded NTPDase is functional during viral infection. In mammals, NTPDases are important in downregulation of the inflammatory and immune responses, but the role of the PhaHV1 NTPDase during viral infection remains to be determined.IMPORTANCE The genome sequences of the koala and wombat gammaherpesviruses show that the viruses form a distinct branch, indicative of a novel genus within the Gammaherpesvirinae Their genomes contain several new ORFs, including ORFs encoding a ß-galactoside α-2,6-sialyltransferase that is phylogenetically closest to poxvirus and insect homologs and the first reported viral NTPDase. NTPDases are ubiquitously expressed in mammals and are also present in several parasitic, fungal, and bacterial pathogens. In mammals, these cell surface-localized NTPDases play essential roles in thromboregulation, inflammation, and immune suppression. In this study, we demonstrate that the virus-encoded NTPDase is enzymatically active and is transcribed during natural infection of the host. Understanding how these enzymes benefit viruses can help to inform how they may cause disease or evade host immune defenses.

A Novel Marsupial Hepatitis A Virus Corroborates Complex Evolutionary Patterns Shaping the Genus Hepatovirus.

The discovery of highly diverse nonprimate hepatoviruses illuminated the evolutionary origins of hepatitis A virus (HAV) ancestors in mammals other than primates. Marsupials are ancient mammals that diverged from other Eutheria during the Jurassic. Viruses from marsupials may thus provide important insight into virus evolution. To investigate Hepatovirus macroevolutionary patterns, we sampled 112 opossums in northeastern Brazil. A novel marsupial HAV (MHAV) in the Brazilian common opossum (Didelphis aurita) was detected by nested reverse transcription-PCR (RT-PCR). MHAV concentration in the liver was high, at 2.5 × 109 RNA copies/g, and at least 300-fold higher than those in other solid organs, suggesting hepatotropism. Hepatovirus seroprevalence in D. aurita was 26.6% as determined using an enzyme-linked immunosorbent assay (ELISA). Endpoint titers in confirmatory immunofluorescence assays were high, and marsupial antibodies colocalized with anti-HAV control sera, suggesting specificity of serological detection and considerable antigenic relatedness between HAV and MHAV. MHAV showed all genomic hallmarks defining hepatoviruses, including late-domain motifs likely involved in quasi-envelope acquisition, a predicted C-terminal pX extension of VP1, strong avoidance of CpG dinucleotides, and a type 3 internal ribosomal entry site. Translated polyprotein gene sequence distances of at least 23.7% from other hepatoviruses suggested that MHAV represents a novel Hepatovirus species. Conserved predicted cleavage sites suggested similarities in polyprotein processing between HAV and MHAV. MHAV was nested within rodent hepatoviruses in phylogenetic reconstructions, suggesting an ancestral hepatovirus host switch from rodents into marsupials. Cophylogenetic reconciliations of host and hepatovirus phylogenies confirmed that host-independent macroevolutionary patterns shaped the phylogenetic relationships of extant hepatoviruses. Although marsupials are synanthropic and consumed as wild game in Brazil, HAV community protective immunity may limit the zoonotic potential of MHAV.IMPORTANCE Hepatitis A virus (HAV) is a ubiquitous cause of acute hepatitis in humans. Recent findings revealed the evolutionary origins of HAV and the genus Hepatovirus defined by HAV in mammals other than primates in general and in small mammals in particular. The factors shaping the genealogy of extant hepatoviruses are unclear. We sampled marsupials, one of the most ancient mammalian lineages, and identified a novel marsupial HAV (MHAV). The novel MHAV shared specific features with HAV, including hepatotropism, antigenicity, genome structure, and a common ancestor in phylogenetic reconstructions. Coevolutionary analyses revealed that host-independent evolutionary patterns contributed most to the current phylogeny of hepatoviruses and that MHAV was the most drastic example of a cross-order host switch of any hepatovirus observed so far. The divergence of marsupials from other mammals offers unique opportunities to investigate HAV species barriers and whether mechanisms of HAV immune control are evolutionarily conserved.

Novel adenovirus detected in kowari (Dasyuroides byrnei) with pneumonia.

A male kowari (Dasyuroides byrnei) originating from a zoo facility was delivered for post mortem evaluation in Hungary. Acute lobar pneumonia with histopathologic changes resembling an adenovirus (AdV) infection was detected by light microscopic examination. The presence of an AdV was confirmed by obtaining partial sequence data from the adenoviral DNA-dependent DNA-polymerase. Although the exact taxonomic position of this novel marsupial origin virus could not be determined, pairwise identity analyses and phylogenetic calculations revealed that it is distantly related to other members in the family Adenoviridae.

Germline viral "fossils" guide in silico reconstruction of a mid-Cenozoic era marsupial adeno-associated virus.

Germline endogenous viral elements (EVEs) genetically preserve viral nucleotide sequences useful to the study of viral evolution, gene mutation, and the phylogenetic relationships among host organisms. Here, we describe a lineage-specific, adeno-associated virus (AAV)-derived endogenous viral element (mAAV-EVE1) found within the germline of numerous closely related marsupial species. Molecular screening of a marsupial DNA panel indicated that mAAV-EVE1 occurs specifically within the marsupial suborder Macropodiformes (present-day kangaroos, wallabies, and related macropodoids), to the exclusion of other Diprotodontian lineages. Orthologous mAAV-EVE1 locus sequences from sixteen macropodoid species, representing a speciation history spanning an estimated 30 million years, facilitated compilation of an inferred ancestral sequence that recapitulates the genome of an ancient marsupial AAV that circulated among Australian metatherian fauna sometime during the late Eocene to early Oligocene. In silico gene reconstruction and molecular modelling indicate remarkable conservation of viral structure over a geologic timescale. Characterisation of AAV-EVE loci among disparate species affords insight into AAV evolution and, in the case of macropodoid species, may offer an additional genetic basis for assignment of phylogenetic relationships among the Macropodoidea. From an applied perspective, the identified AAV "fossils" provide novel capsid sequences for use in translational research and clinical applications.

Full genome characterization of the culicoides-borne marsupial orbiviruses: Wallal virus, Mudjinbarry virus and Warrego viruses.

Viruses belonging to the species Wallal virus and Warrego virus of the genus Orbivirus were identified as causative agents of blindness in marsupials in Australia during 1994/5. Recent comparisons of nucleotide (nt) and amino acid (aa) sequences have provided a basis for the grouping and classification of orbivirus isolates. However, full-genome sequence data are not available for representatives of all Orbivirus species. We report full-genome sequence data for three additional orbiviruses: Wallal virus (WALV); Mudjinabarry virus (MUDV) and Warrego virus (WARV). Comparisons of conserved polymerase (Pol), sub-core-shell 'T2' and core-surface 'T13' proteins show that these viruses group with other Culicoides borne orbiviruses, clustering with Eubenangee virus (EUBV), another orbivirus infecting marsupials. WARV shares <70% aa identity in all three conserved proteins (Pol, T2 and T13) with other orbiviruses, consistent with its classification within a distinct Orbivirus species. Although WALV and MUDV share <72.86%/67.93% aa/nt identity with other orbiviruses in Pol, T2 and T13, they share >99%/90% aa/nt identities with each other (consistent with membership of the same virus species - Wallal virus). However, WALV and MUDV share <68% aa identity in their larger outer capsid protein VP2(OC1), consistent with membership of different serotypes within the species - WALV-1 and WALV-2 respectively.

Detection and identification of a gammaherpesvirus in Antechinus spp. in Australia.

We detected herpesvirus infection in a male yellow-footed antechinus (Antechinus flavipes) and male agile antechinus (Antechinus agilis) during the period of postmating male antechinus immunosuppression and mortality. Histopathologic examination of tissues revealed lesions consistent with herpesvirus infection in the prostate of both animals. Herpesvirus virions were observed by transmission electron microscopy in the prostate tissue collected from the male yellow-footed antechinus. Herpesvirus DNA was detected in prostate, liver, lung, kidney, spleen, and ocular/nasal tissues using a pan-herpesvirus PCR targeting the viral DNA polymerase. Nucleotide sequencing identified a novel herpesvirus from the Gammaherpesvirinae subfamily that we have tentatively designated dasyurid herpesvirus 1 (DaHV-1).

Insights into Polyomaviridae microRNA function derived from study of the bandicoot papillomatosis carcinomatosis viruses.

Several different members of the Polyomaviridae, including some human pathogens, encode microRNAs (miRNAs) that lie antisense with respect to the early gene products, the tumor (T) antigens. These miRNAs negatively regulate T antigen expression by directing small interfering RNA (siRNA)-like cleavage of the early transcripts. miRNA mutant viruses of some members of the Polyomaviridae express increased levels of early proteins during lytic infection. However, the importance of miRNA-mediated negative regulation of the T antigens remains uncertain. Bandicoot papillomatosis carcinomatosis virus type 1 (BPCV1) is associated with papillomas and carcinomas in the endangered marsupial the western barred bandicoot (Perameles bougainville). BPCV1 is the founding member of a new group of viruses that remarkably share distinct properties in common with both the polyomavirus and papillomavirus families. Here, we show that BPCV1 encodes, in the same orientation as the papillomavirus-like transcripts, a miRNA located within a long noncoding region (NCR) of the genome. Furthermore, this NCR serves the function of both promoter and template for the primary transcript that gives rise to the miRNA. Unlike the polyomavirus miRNAs, the BPCV1 miRNA is not encoded antisense to the T antigen transcripts but rather lies in a separate, proximal region of the genome. We have mapped the 3' untranslated region (UTR) of the BPCV1 large T antigen early transcript and identified a functional miRNA target site that is imperfectly complementary to the BPCV1 miRNA. Chimeric reporters containing the entire BPCV1 T antigen 3' UTR undergo negative regulation when coexpressed with the BPCV1 miRNA. Notably, the degree of negative regulation observed is equivalent to that of an identical reporter that is engineered to bind to the BPCV1 miRNA with perfect complementarity. We also show that this miRNA and this novel mode of early gene regulation are conserved with the related BPCV2. Finally, papillomatous lesions from a western barred bandicoot express readily detectable levels of this miRNA, stressing its likely importance in vivo. Combined, the alternative mechanisms of negative regulation of T antigen expression between the BPCVs and the polyomaviruses support the importance of miRNA-mediated autoregulation in the life cycles of some divergent polyomaviruses and polyomavirus-like viruses.

Vigilância sorológica para arbovírus em Juruti, Pará, Brasil / Serological survey for arboviruses in Juruti, Pará State, Brazil

O objetivo foi avaliar a prevalência de anticorpos para arbovírus na área de influência do Projeto Juruti antes da exploração mineral local. Foram examinados 1.597 soros humanos e 85 de animais silvestres. A pesquisa de anticorpos foi realizada pelo teste de inibição da hemaglutinação contra antígenos dos arbovírus mais prevalentes na Amazônia brasileira e IgM-ELISA para dengue e febre amarela. Soros humanos inibidores da hemaglutinação positivos para Mayaro e Oropouche foram também testados por IgM-ELISA. Anticorpos inibidores da hemaglutinação para alfavírus, orthobunyavirus e/ou flavivírus foram detectados, sendo 28,7 por cento reações monotípicas. Infecções recentes para Oropouche (n = 23), Mayaro (n = 5), e dengue (n = 20) foram confirmadas por IgM-ELISA. A prevalência de anticorpos inibidores da hemaglutinação em soros de animais silvestres foi de 8,2 por cento para flavivírus. Os resultados obtidos mostram que houve circulação ativa dos vírus Mayaro, Oropouche e dengue no período do estudo em humanos em Juruti, Pará, Brasil, e de diversos arbovírus em animais silvestres, indicando presença desses vírus em áreas do Município de Juruti.

This study aimed to measure the prevalence of antibodies to arboviruses in the area affected by the Juruti Project before local mining operations. A total of 1,597 human and 85 wild animal sera were examined, using the hemagglutination inhibition test against 19 antigens from the most prevalent arboviruses in the Brazilian Amazon and IgM-ELISA for dengue and yellow fever. Positive sera for Mayaro and Oropouche viruses were also tested by IgM-ELISA. Prevalence of hemagglutination inhibition antibodies to alphaviruses, flaviviruses, and orthobunyaviruses in humans was 28.7 percent, while for wild animals it was 8.2 percent for flaviviruses. Recent infections based on presence of IgM was confirmed for Mayaro (n = 5), Oropouche (n = 23), and dengue (n = 20). The results showed active circulation of the dengue, Mayaro, and Oropouche viruses in Juruti, as well as flaviviruses in wild animals, suggesting the circulation of these viruses in the Municipality of Juruti.

Demography, disease and the devil: life-history changes in a disease-affected population of Tasmanian devils (Sarcophilus harrisii).

1. Examining the demographic responses of populations to disease epidemics and the nature of compensatory responses to perturbation from epidemics is critical to our understanding of the processes affecting population dynamics and our ability to conserve threatened species. Such knowledge is currently available for few systems. 2. We examined changes to the demography and life-history traits of a population of Tasmanian devils (Sarcophilus harrisii) following the arrival of a debilitating infectious disease, devil facial tumour disease (DFTD), and investigated the population's ability to compensate for the severe population perturbation caused by this epizootic. 3. There was a significant change to the age structure following the arrival of DFTD to the Freycinet Peninsula. This shift to a younger population was caused by the loss of older individuals from the population as a direct consequence of DFTD-driven declines in adult survival rates. 4. Offspring sex ratios of disease mothers were more female biased than those of healthy mothers, indicating that devils may facultatively adjust offspring sex ratios in response to disease-induced changes in maternal condition. 5. We detected evidence of reproductive compensation in response to disease impacts via a reduction in the age of sexual maturity of females (an increase in precocial breeding) over time. 6. The strength of this compensatory response appeared to be limited by factors that constrain the ability of individuals to reach a critical size for sexual maturity in their first year, because of the time limit dictated by the annual breeding season. 7. The ongoing devastating impacts of this disease for adult survival and the apparent reliance of precocial breeding on rapid early growth provide the opportunity for evolution to favour of this new life-history pattern, highlighting the potential for novel infectious diseases to be strong selective forces on life-history evolution.

Genomic characterization of a novel virus found in papillomatous lesions from a southern brown bandicoot (Isoodon obesulus) in Western Australia.

The genome of a novel virus, tentatively named bandicoot papillomatosis carcinomatosis virus type 2 (BPCV2), obtained from multicentric papillomatous lesions from an adult male southern brown bandicoot (Isoodon obesulus) was sequenced in its entirety. BPCV2 had a circular double-stranded DNA genome consisting of 7277 bp and open reading frames encoding putative L1 and L2 structural proteins and putative large T antigen and small t antigen transforming proteins. These genomic features, intermediate between Papillomaviridae and Polyomaviridae are most similar to BPCV1, recently described from papillomas and carcinomas in the endangered western barred bandicoot (Perameles bougainville). This study also employed in situ hybridization to definitively demonstrate BPCV2 DNA within lesion biopsies. The discovery of BPCV2 provides evidence of virus-host co-speciation between BPCVs and marsupial bandicoots and has important implications for the phylogeny and taxonomy of circular double-stranded DNA viruses infecting vertebrates.

In situ hybridization to detect bandicoot papillomatosis carcinomatosis virus type 1 in biopsies from endangered western barred bandicoots (Perameles bougainville).

The western barred bandicoot (Perameles bougainville) is an endangered Australian marsupial species in which a papillomatosis and carcinomatosis syndrome occurs. Bandicoot papillomatosis carcinomatosis virus type 1 (BPCV1) is associated with the lesions of this progressively debilitating syndrome. Five digoxigenin-labelled DNA probes were generated for in situ hybridization (ISH) and the technique was optimized and performed on formalin-fixed paraffin-embedded (FFPE) biopsies. Staining of keratinocyte and sebocyte nuclei within lesions was achieved with all five probes. The sensitivity of ISH (76.9%) surpassed that of PCR (30.8%) for FFPE samples. The sensitivity of ISH varied from 81% (papillomas) and 70% (carcinoma in situ) to 29% (squamous cell carcinomas). The specificity of the test was confirmed using an irrelevant probe and papillomas from other species. These results strengthen the association between BPCV1 and the western barred bandicoot papillomatosis and carcinomatosis syndrome and give insight into the biology of the virus-host interaction.

A novel virus detected in papillomas and carcinomas of the endangered western barred bandicoot (Perameles bougainville) exhibits genomic features of both the Papillomaviridae and Polyomaviridae.

Conservation efforts to prevent the extinction of the endangered western barred bandicoot (Perameles bougainville) are currently hindered by a progressively debilitating cutaneous and mucocutaneous papillomatosis and carcinomatosis syndrome observed in captive and wild populations. In this study, we detected a novel virus, designated the bandicoot papillomatosis carcinomatosis virus type 1 (BPCV1), in lesional tissue from affected western barred bandicoots using multiply primed rolling-circle amplification and PCR with the cutaneotropic papillomavirus primer pairs FAP59/FAP64 and AR-L1F8/AR-L1R9. Sequencing of the BPCV1 genome revealed a novel prototype virus exhibiting genomic properties of both the Papillomaviridae and the Polyomaviridae. Papillomaviral properties included a large genome size ( approximately 7.3 kb) and the presence of open reading frames (ORFs) encoding canonical L1 and L2 structural proteins. The genomic organization in which structural and nonstructural proteins were encoded on different strands of the double-stranded genome and the presence of ORFs encoding the nonstructural proteins large T and small t antigens were, on the other hand, typical polyomaviral features. BPCV1 may represent the first member of a novel virus family, descended from a common ancestor of the papillomaviruses and polyomaviruses recognized today. Alternatively, it may represent the product of ancient recombination between members of these two virus families. The discovery of this virus could have implications for the current taxonomic classification of Papillomaviridae and Polyomaviridae and can provide further insight into the evolution of these ancient virus families.

The nucleotide sequence of koala (Phascolarctos cinereus) retrovirus: a novel type C endogenous virus related to Gibbon ape leukemia virus.

A novel retrovirus, morphologically consistent with mammalian C-type retroviruses, was detected by electron microscopy in mitogen-stimulated peripheral blood mononuclear cell cultures from 163 koalas and in lymphoma tissue from 3 koalas. PCR amplified provirus from the blood and tissues of 17 wild and captive koalas, and reverse transcriptase-PCR demonstrated viral mRNA, viral genomic RNA, and reverse transcriptase activity in koala serum and cell culture supernatants. Comparison of viral sequences derived from genomic DNA and mRNA showed identity indicative of a single retroviral species-here designated koala retrovirus (KoRV). Southern blot analysis of koala tissue genomic DNA using labelled KoRV probes demonstrated banding consistent with an endogenous retrovirus. Complete and apparently truncated proviruses were detected in DNA of both clinically normal koalas and those with hematopoietic disease. KoRV-related viruses were not detected in other marsupials, and phylogenetic analysis showed that KoRV paradoxically clusters with gibbon ape leukemia virus (GALV). The strong similarity between GALV and KoRV suggests that these viruses are closely related and that recent cross-host transmission has occurred. The complete proviral DNA sequence of KoRV is reported.

Analysis of the thymidine kinase genes of macropodid herpesviruses 1 and 2.

The nucleotide sequences of the entire protein coding regions of the thymidine kinase (TK) genes of macropodid herpesvirus type 1 (MaHV-1) and type 2 (MaHV-2) were determined. The coding region of the MaHV-1 TK gene was 984 bp long and was predicted to encode a polypeptide of 327 amino acids. The coding region of the MaHV-2 TK gene was 1020 bp long and encoded a polypeptide of 340 amino acids. Comparisons of their deduced amino acid sequences with those of fifteen other herpesviruses revealed close homology to those of other alphaherpesviruses, particularly to human herpesvirus type 1 (HHV-1) and type 2 (HHV-2).

Human endogenous retrovirus type I-related viruses have an apparently widespread distribution within vertebrates.

Retroviruses from lower vertebrate hosts have been poorly characterized to date. Few sequences have been isolated, and those which have been reported are all highly divergent when compared to the retroviruses known to be harbored by mammals and birds. Here we show that retroviruses with significant homology to the human endogenous retrovirus type I (HERV-I) are present within the genomes of fish, reptiles, birds, and mammals and that they may well be widespread within many vertebrates. Phylogenetic analysis of nucleotide sequences strongly supported the inclusion of viruses from each of these vertebrate classes into one monophyletic group. This analysis also demonstrated that the HERV-I-related viruses are more closely related to retroviruses belonging to the murine leukemia virus genus than to members of the other retroviral genera. The presence of HERV-I-related retroviruses in so many disparate vertebrate hosts suggests that other endogenous human retroviruses may also have a much wider distribution than is currently appreciated.