Detection and Characterisation of an Endogenous Betaretrovirus in Australian Wild Deer.

Endogenous retroviruses (ERVs) are the remnants of past retroviral infections that once invaded the host's germline and were vertically transmitted. ERV sequences have been reported in mammals, but their distribution and diversity in cervids are unclear. Using next-generation sequencing, we identified a nearly complete genome of an endogenous betaretrovirus in fallow deer (Dama dama). Further genomic analysis showed that this provirus, tentatively named cervid endogenous betaretrovirus 1 (CERV ß1), has typical betaretroviral genome features (gag-pro-pol-env) and the betaretrovirus-specific dUTPase domain. In addition, CERV ß1 pol sequences were detected by PCR in the six non-native deer species with wild populations in Australia. Phylogenetic analyses demonstrated that CERV ß1 sequences from subfamily Cervinae clustered as sister taxa to ERV-like sequences in species of subfamily Muntiacinae. These findings, therefore, suggest that CERV ß1 endogenisation occurred after the split of these two subfamilies (between 3.3 and 5 million years ago). Our results provide important insights into the evolution of betaretroviruses in cervids.

A human MMTV-like betaretrovirus linked to breast cancer has been present in humans at least since the copper age.

The betaretrovirus Mouse Mammary Tumor Virus (MMTV) is the well characterized etiological agent of mammary tumors in mice. In contrast, the etiology of sporadic human breast cancer (BC) is unknown, but accumulating data indicate a possible viral origin also for these malignancies. The presence of MMTVenv-like sequences (MMTVels) in the human salivary glands and saliva supports the latter as possible route of inter-human dissemination. In the absence of the demonstration of a mouse-man transmission of MMTV, we considered the possibility that a cross-species transmission could have occurred in ancient times. Therefore, we investigated MMTVels in the ancient dental calculus, which originates from saliva and is an excellent material for paleovirology. The calculus was collected from 36 ancient human skulls, excluding any possible mouse contamination. MMTV-like sequences were identified in the calculus of 6 individuals dated from the Copper Age to the 17th century. The MMTV-like sequences were compared with known human endogenous betaretroviruses and with animal exogenous betaretroviruses, confirming their exogenous origin and relation to MMTV. These data reveal that a human exogenous betaretrovirus similar to MMTV has existed at least since 4,500 years ago and indirectly support the hypothesis that it could play a role in human breast cancer.

Leukemic histiocytic sarcoma in a captive common squirrel monkey (Saimiri sciureus) with Saimiriine Gammaherpesvirus 2 (Rhadinovirus), Saimiri sciureus lymphocryptovirus 2 (Lymphocryptovirus) and Squirrel monkey retrovirus (ß-Retrovirus) coinfection.

Hematopoietic neoplasia other than lymphoma and leukemia is uncommon among non-human primates. Herein, we provide the first evidence of occurrence of leukemic histiocytic sarcoma in a captive common squirrel monkey with Saimiriine Gammaherpesvirus 2 (Rhadinovirus), Saimiri sciureus lymphocryptovirus 2 (Lymphocryptovirus), and Squirrel monkey retrovirus (ß-Retrovirus) coinfection.

In vitro and In vivo Susceptibility of Baboons (Papio sp.) to Infection with and Apparent Antibody Reactivity to Simian Betaretrovirus (SRV).

Despite the lack of confirmed reports of an exogenous Simian betaretrovirus (SRV) isolated from baboons (Papio sp.), reports of simian endogenous gammaretrovirus (SERV) in baboons with complete genomes suggest that such viruses may be potentially infectious. In addition, serologic tests have repeatedly demonstrated antibody reactivity to SRV in baboons from multiple colonies. These findings complicate the management and use of such animals for research. To provide further insight into this situation, we performed in vitro and in vivo studies to determine if baboons are or can be infected with SRV. In our initial experiment, we were not able to isolate SRV from 6 seropositive or sero-indeterminate baboons by coculturing their peripheral blood mononuclear cells (PBMC) with macaque PBMC or permissive cell lines. In a subsequent experiment, we found that baboon PBMC infected in vitro with high dose SRV were permissive to virus replication. To test in vivo infectibil- ity, groups of naive baboons were infused intravenously with either (i) the same SRV tissue culture virus stocks used for the in vitro studies, (ii) SRV antibody positive and PCR positive macaque blood, (iii) SRV antibody positive or indeterminate, but PCR negative baboon blood, or (iv) SRV antibody and PCR negative baboon blood. Sustained SRV infection, as defined by reproducible PCR detection and/or antibody seroconversion, was confirmed in 2 of 3 baboons receiving tissue culture virus but not in any recipients of transfused blood from seropositive macaques or baboons. In conclusion, the data indicate that even though baboon cells can be infected experimentally with high doses of tissue culture grown SRV, baboons that are repeatedly SRV antibody positive and PCR negative are unlikely to be infected with exogenous SRV and thus are unlikely to transmit a virus that would threaten the SPF status of captive baboon colonies.

Characterization of an integrated, endogenous mouse mammary tumor virus-like (MMTV) betaretrovirus genome in a black Syrian hamster (Mesocricetus auratus).

Retroviruses (family Retroviridae) are important agents of humans and animals. This study reports the detection and complete genome characterization of a novel endogenous retrovirus from the black Syrian hamster (Mesocricetus auratus) with a squamous cell skin tumor. The proviral genome, tentatively named black Syrian hamster retrovirus (BSHRV/2013/HUN, MK304634), was 8784 nucleotide in length with typical full-length betaretrovirus genome organization of 5'LTR-gag-pro-pol-env-3'LTR and with a characteristic mouse mammary tumor virus-like (MMTV) betaretrovirus dUTPase domain but without a sag gene. The BSHRV gag (534aa), pro/pol (~1099aa) and env (672aa) proteins had 56%/63%/50% aa identity to the corresponding proteins of MMTV (AF228552). The proviral DNA is detectable in tumor as well as in tumor-free cells by conventional PCR and qPCR but only visible in the tumor cells by in situ hybridization. Low level retroviral RNA expression was found only in the DNase-treated RNA tumor samples using RT/nested PCR. BSHRV/2013/HUN-like betaretrovirus DNA was also identified from a faecal and tissue samples from 1 of the further 3 tested individuals by nested-PCR and qPCR. Further research is needed to investigate the distribution, activity and etiological role of this novel MMTV-like betaretrovirus species in hamster.

First detection and genotypic analysis of goat enzootic nasal tumor virus 2 in Chongqing, China.

Enzootic nasal adenocarcinoma (ENA) of goats, characterized by transformation of epithelial cells of the ethmoid turbinates, is caused by enzootic nasal tumor virus 2 (ENTV-2). ENTV-2 belongs to the genus Betaretrovirus and has extended its distribution globally with a high prevalence; however, the genetic diversity and genotypic distribution for ENTV-2 have not been analyzed systematically due to the limited availability of sequence data. In this study, an infection by ENTV-2 was detected by RT-PCR in Chongqing in July 2018, and the complete sequence of one strain (CQ1) was determined. Phylogenetic analysis indicated a high degree of genetic heterogeneity among ENTV-2 sequences, with the existence of two main lineages. Lineage 1 and 2 were composed of ENTV-2 from China and the UK, respectively. Although CQ1 was closely related to recent ENTV-2 strains collected in the neighboring provinces of Chongqing (Shaanxi and Sichuan), it formed a separate sublineage of lineage 1 (sublineage 1.3). This report will enhance our understanding of the epidemiology of ENTV-2 in China.

EvaGreen-based real-time PCR assay for sensitive detection of enzootic nasal tumor virus 2.

Enzootic nasal tumor virus 2 (ENTV-2), the aetiological agent of enzootic nasal adenocarcinoma in goats, is prevalent in China; resulting in substantial economic losses to the goat-breeding industry. Therefore, it is necessary to establish an efficient detection method for the diagnosis and prevention of ENTV-2 infection. More recently, EvaGreen is emerging as a novel alternative fluorescent dye for quantitative real-time PCR because of its low cost, specific amplification and high resolution. In this study, we developed a specific, sensitive, and cost-effective detection method-an EvaGreen-based real-time PCR assay for the detection of ENTV-2. This assay exhibited high specificity and sensitivity and was able to detect ENTV-2 at concentrations as low as 3.0 × 101 copies, which was more sensitive than the conventional PCR method (detection limit, 3.0 × 102 copies). In addition, the reproducibility test indicated that EvaGreen dye in our assay had a good reproducibility. In conclusion, we report that a highly sensitive, specific, and cost-effective EvaGreen-based real-time PCR assay is successful for the rapid detection of ENTV-2.

Development of real-time PCR-based methods for the detection of enzootic nasal tumor virus 2 in goats.

Enzootic nasal adenocarcinoma (ENA) is a contagious neoplasm of sheep and goats, associated with the oncogenic retroviruses enzootic nasal tumor virus (ENTV) 1 and 2, respectively. It appears to be common in countries with substantial small ruminant-production. ENA diagnosis in goats is based on autopsy and histopathology, and there is no real-time PCR method available for ENTV-2 detection. Here, a novel one-tube real-time RT-PCR (RT-qPCR) method for the detection and quantification of ENTV-2 in nasal swabs is presented. The method targets the env gene/U3 region. For the design of ENTV-2-specific oligonucleotides, molecular characterization of seven Greek ENTV-2 strains was performed. Phylogenetic analysis revealed three distinct phylogenetic clades of ENTV-2 that correlate with the country of sample collection. Evaluation of the analytical performance of the RT-qPCR revealed an amplification efficiency of 92.8% and a linear range of quantification between 2 × 108 and 2 × 102 RNA transcripts. Analysis of nasal swabs from 23 histopathologically confirmed, naturally occurring ENA cases via RT-qPCR yielded positive results. Moreover, modification of the method for use in a real-time PCR (qPCR) assay enables detection of proviral DNA in tumor specimens. Both methods are highly specific and can be used for the confirmation of ENA-suspected cases. Future applications could include ante-mortem diagnosis, verification of the ENTV-2-free status in animal trade, disease surveillance, and control programs.

Is PBC a viral infectious disease?

The human betaretrovirus and the closely related mouse mammary tumor virus have been linked with the development of cholangitis and mitochondrial antibody production in patients with primary biliary cholangitis (PBC) and mouse models of autoimmune biliary disease, respectively. In vitro, betaretroviruses have been found to stimulate the expression of mitochondrial autoantigens on the cell surface of biliary epithelial cells. In vivo, both mitochondrial autoantigens and viral proteins have been shown to be co-expressed in biliary epithelium and lymphoid tissue. Notably, both mice and humans make poor antibody responses to betaretrovirus infection, whereas proinflammatory responses to viral proteins have been observed in T lymphocyte studies. Furthermore, proviral integration studies have confirmed the presence of human betaretrovirus in biliary epithelium of patients with PBC. Preliminary proof of principal studies using combination antiretroviral therapy have shown that suppression of viral expression is associated with sustained biochemical response. As the previous regimen used was poorly tolerated, further randomized controlled trials are planned to determine whether betaretrovirus infection plays an important role in the development of PBC.

Full-length genome sequence analysis of enzootic nasal tumor virus isolated from goats in China.

BACKGROUND: Enzootic nasal tumor virus (ENTV) is a betaretrovirus of sheep (ENTV-1) and goats (ENTV-2) associated with neoplastic transformation of epithelial cells of the ethmoid turbinate. Confirmation of the role of ENTV in the pathogenesis of enzootic nasal adenocarcinoma (ENA) has yet to be resolved due to the inability to culture the virus. Very little is known about the prevalence of this disease, particularly in China. METHODS: To evaluate the genetic diversity of ENTV-2 from Shaanxi province of China, the complete genome sequence of four isolates from Shaanxi province was determined by RT-PCR. These sequences were analyzed to evaluate their genetic relatedness with other small ruminant betaretroviruses. Phylogenetic analyses based on the gag gene and env gene were performed. RESULTS: The ENTV-2-Shaanxi1 genome shared 97.0% sequence identity with ENTV-2-SC (accession number HM104174.1), and 89.6% sequence identity with the ENTV-2 sequences (accession number AY197548.1). ENTV-2 is closely related to the ENTV-1 and jaagsiekte retrovirus (JSRV). The main sequence differences between these viruses reside in LTR, two small regions of Gag, Orf-x, and the transmembrane (TM) region of Env. A stretch of 6 consecutive proline residues exists in VR1 of the ENTV-2-Shaanxi1 ~ 4 isolates. All the ENTV-2-Shaanxi isolates have the YXXM motif in the cytoplasmic tail of the Env. Phylogenetic analysis by nucleotide sequences showed that ENTV-2-Shaanxi1 ~ 4 isolates were closest related to two ENTV-2 isolates published in NCBI, especially with ENTV-2-SC strain. CONCLUSIONS: This finding indicates that ENA most likely was introduced to Shaanxi province by the movement of contaminated goats from other areas in China. This study adds to understand the circulation, variation and distribution of ENTV-2, and may prove beneficial in future control or eradication programmes.

Genome-Wide Screening of Retroviral Envelope Genes in the Nine-Banded Armadillo (Dasypus novemcinctus, Xenarthra) Reveals an Unfixed Chimeric Endogenous Betaretrovirus Using the ASCT2 Receptor.

UNLABELLED: Retroviruses enter host cells through the interaction of their envelope (Env) protein with a cell surface receptor, which triggers the fusion of viral and cellular membranes. The sodium-dependent neutral amino acid transporter ASCT2 is the common receptor of the large RD114 retrovirus interference group, whose members display frequent env recombination events. Germ line retrovirus infections have led to numerous inherited endogenous retroviruses (ERVs) in vertebrate genomes, which provide useful insights into the coevolutionary history of retroviruses and their hosts. Rare ERV-derived genes display conserved viral functions, as illustrated by the fusogenic syncytin env genes involved in placentation. Here, we searched for functional env genes in the nine-banded armadillo (Dasypus novemcinctus) genome and identified dasy-env1.1, which clusters with RD114 interference group env genes and with two syncytin genes sharing ASCT2 receptor usage. Using ex vivo pseudotyping and cell-cell fusion assays, we demonstrated that the Dasy-Env1.1 protein is fusogenic and can use both human and armadillo ASCT2s as receptors. This gammaretroviral env gene belongs to a provirus with betaretrovirus-like features, suggesting acquisition through recombination. Provirus insertion was found in several Dasypus species, where it has not reached fixation, whereas related family members integrated before diversification of the genus Dasypus >12 million years ago (Mya). This newly described ERV lineage is potentially useful as a population genetic marker. Our results extend the usage of ASCT2 as a retrovirus receptor to the mammalian clade Xenarthra and suggest that the acquisition of an ASCT2-interacting env gene is a major selective force driving the emergence of numerous chimeric viruses in vertebrates. IMPORTANCE: Retroviral infection is initiated by the binding of the viral envelope glycoprotein to a host cell receptor(s), triggering membrane fusion. Ancient germ line infections have generated numerous endogenous retroviruses (ERVs) in nearly all vertebrate genomes. Here, we report a previously uncharacterized ERV lineage from the genome of a xenarthran species, the nine-banded armadillo (Dasypus novemcinctus). It entered the Dasypus genus >12 Mya, with one element being inserted more recently in some Dasypus species, where it could serve as a useful marker for population genetics. This element exhibits an env gene, acquired by recombination events, with conserved viral fusogenic properties through binding to ASCT2, a receptor used by a wide range of recombinant retroviruses infecting other vertebrate orders. This specifies the ASCT2 transporter as a successful receptor for ERV endogenization and suggests that ASCT2-binding env acquisition events have favored the emergence of numerous chimeric viruses in a wide range of species.

Specific pathogen free macaque colonies: a review of principles and recent advances for viral testing and colony management.

Specific pathogen free (SPF) macaques provide valuable animal models for biomedical research. In 1989, the National Center for Research Resources [now Office of Research Infrastructure Programs (ORIP)] of the National Institutes of Health initiated experimental research contracts to establish and maintain SPF colonies. The derivation and maintenance of SPF macaque colonies is a complex undertaking requiring knowledge of the biology of the agents for exclusion and normal physiology and behavior of macaques, application of the latest diagnostic technology, facilitiy management, and animal husbandry. This review provides information on the biology of the four viral agents targeted for exclusion in ORIP SPF macaque colonies, describes current state-of-the-art viral diagnostic algorithms, presents data from proficiency testing of diagnostic assays between laboratories at institutions participating in the ORIP SPF program, and outlines management strategies for maintaining the integrity of SPF colonies using results of diagnostic testing as a guide to decision making.

Combination antiretroviral studies for patients with primary biliary cirrhosis.

Following the characterization of a human betaretrovirus in patients with primary biliary cirrhosis (PBC), pilot studies using antiretroviral therapy have been conducted as proof of principal to establish a link of virus with disease and with the eventual aim to find better adjunct therapies for patients unresponsive to ursodeoxycholic acid. In the first open label pilot study, the reverse transcriptase inhibitor lamivudine had little demonstrable biochemical or histological effect after 1 year. Whereas, lamivudine in combination with zidovudine was associated with a significant reduction in alkaline phosphatase as well as improvement in necroinflammatory score, cholangitis and ductopenia over a 12 mo period. A double blind, multi-center randomized controlled trial using lamivudine with zidovudine for 6 mo confirmed a significant reduction in alkaline phosphatase, ALT and AST in patients on antiviral therapy. However, none of the patients achieved the stringent endpoint criteria for normalization of alkaline phosphatase. Furthermore, some patients developed biochemical rebound consistent with drug resistance. A major fault of these studies has been the inability to measure the viral load in peripheral blood and therefore, provide a direct correlation between improvement of hepatic biochemistry and reduction in viral load. Nevertheless, viral mutants to lamivudine with zidovudine were later characterized in the NOD.c3c4 mouse model of PBC that has been used to test other antiretroviral regimens to betaretrovirus. The combination of tenofovir and emtricitabine reverse transcriptase inhibitors and the HIV protease inhibitor, lopinavir were found to abrogate cholangitis in the NOD.c3c4 mouse model and the same regimen normalized the liver tests in a PBC patient with HIV and human betaretrovirus infection. This combination antiretroviral therapy has now been used in a double blind randomized controlled crossover study for patients with PBC followed by an open label extension study. Only a third of the PBC patients were able to tolerate the lopinavir but those maintained on tenofovir, emtricitabine and lopinavir experienced sustained and clinically meaningful reduction in hepatic biochemistry. While we await the histological and virological evaluation, it is clear that better tolerated regimens of antiretroviral treatment will be required in future clinical trials.

Construction of a molecular clone of ovine enzootic nasal tumor virus.

BACKGROUND: Enzootic nasal tumor virus (ENTV-1) is an ovine betaretrovirus that has been linked to enzootic nasal adenocarcinoma (ENA), a contagious tumor of the ethmoid turbinates of sheep. Transmission experiments performed using virus isolated from cell free nasal tumor homogenates suggest that ENTV-1 is the causative agent of ENA; however, this etiological relationship has not been conclusively proven due to the fact that the virus cannot be propagated in vitro nor is there an infectious molecular clone of the virus. METHODS: Here we report construction of a molecular clone of ENTV-1 and demonstrate that transfection of this molecular clone into HEK 293T cells produces mature virus particles. RESULTS: Analysis of recombinant virus particles derived from the initial molecular clone revealed a defect in the proteolytic processing of Gag; however, this defect could be corrected by co-expression of the Gag-Pro-Pol polyprotein from the highly related Jaagsiekte sheep retrovirus (JSRV) suggesting that the polyprotein cleavage sites in the ENTV-1 molecular clone were functional. Mutagenesis of the molecular clone to correct amino acid variants identified within the pro gene did not restore proteolytic processing; whereas deletion of one proline residue from a polyproline tract located in variable region 1 (VR1) of the matrix resulted in production of CA protein of the mature (cleaved) size strongly suggesting that normal virion morphogenesis and polyprotein cleavage took place. Finally, electron microscopy revealed the presence of spherical virus particles with an eccentric capsid and an average diameter of about 100 nm. CONCLUSION: In summary, we have constructed the first molecular clone of ENTV-1 from which mature virus particles can be produced. Future experiments using virus produced from this molecular clone can now be conducted to fulfill Koch's postulates and demonstrate that ENTV-1 is necessary and sufficient to induce ENA in sheep.

A novel endogenous betaretrovirus in the common vampire bat (Desmodus rotundus) suggests multiple independent infection and cross-species transmission events.

The Desmodus rotundus endogenous betaretrovirus (DrERV) is fixed in the vampire bat D. rotundus population and in other phyllostomid bats but is not present in all species from this family. DrERV is not phylogenetically related to Old World bat betaretroviruses but to betaretroviruses from rodents and New World primates, suggesting recent cross-species transmission. A recent integration age estimation of the provirus in some taxa indicates that an exogenous counterpart might have been in recent circulation.

Development of an ante-mortem diagnostic test for enzootic nasal tumor virus and detection of neutralizing antibodies in host serum.

Enzootic nasal adenocarcinoma (ENA) is a contagious neoplasm of the nasal mucosa of sheep and goats and is associated with enzootic nasal tumour virus (ENTV). As ENA is a common disease in North America and there are no vaccines against ENTV-1, diagnostic tests that can identify infected animals and assist with eradication and disease surveillance efforts are greatly needed. In this study, we endeavoured to develop a novel, non-invasive diagnostic tool that could be used not only to validate clinical signs of ENA but also to detect ENTV-1 infection prior to the onset of disease signs (i.e. pre-clinical diagnosis). Cytology, serology and reverse transcription (RT)-PCR-based diagnostic methods were investigated. Although the cytology-based assay was able to detect ENTV-1 infection in some animals, it had poor sensitivity and specificity and thus was not developed further as an ante-mortem diagnostic method. Three different assays, including ELISA, Western blotting and virus neutralization, were developed to detect the presence of ENTV-1-specific antibodies in sheep serum. Whilst a surprisingly large number of sheep mounted an antibody-mediated immune response against ENTV-1, and in some cases neutralizing, correlation with disease status was poor. In contrast, RT-PCR on RNA extracted from nasal swabs reliably detected exogenous ENTV-1 sequences, did not amplify endogenous ovine betaretroviral sequences, demonstrated high concordance with immunohistochemical staining for ENTV-1 envelope protein, and had perfect sensitivity and specificity. This report describes a practical and highly specific RT-PCR technique for the detection of clinical and pre-clinical ENA that may prove beneficial in future control or eradication programmes.

Experimental transmission of enzootic nasal adenocarcinoma in sheep.

Enzootic nasal adenocarcinoma (ENA) is a contagious neoplasm of the secretory epithelial cells of the nasal mucosa of sheep and goats. It is associated with the betaretrovirus, enzootic nasal tumor virus (ENTV), but a causative relationship has yet to be demonstrated. In this study, 14-day-old lambs were experimentally infected via nebulization with cell-free tumor filtrates derived from naturally occurring cases of ENA. At 12 weeks post-infection (wpi), one of the five infected lambs developed clinical signs, including continuous nasal discharge and open mouth breathing, and was euthanized. Necropsy revealed the presence of a large bilateral tumor occupying the nasal cavity. At 45 wpi, when the study was terminated, none of the remaining infected sheep showed evidence of tumors either by computed tomography or post-mortem examination. ENTV-1 proviral DNA was detected in the nose, lung, spleen, liver and kidney of the animal with experimentally induced ENA, however there was no evidence of viral protein expression in tissues other than the nose. Density gradient analysis of virus particles purified from the experimentally induced nasal tumor revealed a peak reverse transcriptase (RT) activity at a buoyant density of 1.22 g/mL which was higher than the 1.18 g/mL density of peak RT activity of virus purified from naturally induced ENA. While the 1.22 g/mL fraction contained primarily immature unprocessed virus particles, mature virus particles with a similar morphology to naturally occurring ENA could be identified by electron microscopy. Full-length sequence analysis of the ENTV-1 genome from the experimentally induced tumor revealed very few nucleotide changes relative to the original inoculum with only one conservative amino acid change. Taken together, these results demonstrate that ENTV-1 is associated with transmissible ENA in sheep and that under experimental conditions, lethal tumors are capable of developing in as little as 12 wpi demonstrating the acutely oncogenic nature of this ovine betaretrovirus.

A novel endogenous betaretrovirus group characterized from polar bears (Ursus maritimus) and giant pandas (Ailuropoda melanoleuca).

Transcriptome analysis of polar bears (Ursus maritimus) yielded sequences with highest similarity to the human endogenous retrovirus group HERV-K(HML-2). Further analysis of the polar bear draft genome identified an endogenous betaretrovirus group comprising 26 proviral copies and 231 solo LTRs. Molecular dating indicates the group originated before the divergence of bears from a common ancestor but is not present in all carnivores. Closely related sequences were identified in the giant panda (Ailuropoda melanoleuca) and characterized from its genome. We have designated the polar bear and giant panda sequences U. maritimus endogenous retrovirus (UmaERV) and A. melanoleuca endogenous retrovirus (AmeERV), respectively. Phylogenetic analysis demonstrated that the bear virus group is nested within the HERV-K supergroup among bovine and bat endogenous retroviruses suggesting a complex evolutionary history within the HERV-K group. All individual remnants of proviral sequences contain numerous frameshifts and stop codons and thus, the virus is likely non-infectious.

Identification of diverse full-length endogenous betaretroviruses in megabats and microbats.

BACKGROUND: Betaretroviruses infect a wide range of species including primates, rodents, ruminants, and marsupials. They exist in both endogenous and exogenous forms and are implicated in animal diseases such as lung cancer in sheep, and in human disease, with members of the human endogenous retrovirus-K (HERV-K) group of endogenous betaretroviruses (ßERVs) associated with human cancers and autoimmune diseases. To improve our understanding of betaretroviruses in an evolutionarily distinct host species, we characterized ßERVs present in the genomes and transcriptomes of mega- and microbats, which are an important reservoir of emerging viruses. RESULTS: A diverse range of full-length ßERVs were discovered in mega- and microbat genomes and transcriptomes including the first identified intact endogenous retrovirus in a bat. Our analysis revealed that the genus Betaretrovirus can be divided into eight distinct sub-groups with evidence of cross-species transmission. Betaretroviruses are revealed to be a complex retrovirus group, within which one sub-group has evolved from complex to simple genomic organization through the acquisition of an env gene from the genus Gammaretrovirus. Molecular dating suggests that bats have contended with betaretroviral infections for over 30 million years. CONCLUSIONS: Our study reveals that a diverse range of betaretroviruses have circulated in bats for most of their evolutionary history, and cluster with extant betaretroviruses of divergent mammalian lineages suggesting that their distribution may be largely unrestricted by host species barriers. The presence of ßERVs with the ability to transcribe active viral elements in a major animal reservoir for viral pathogens has potential implications for public health.

Characterization of a full-length endogenous beta-retrovirus, EqERV-beta1, in the genome of the horse (Equus caballus).

Information on endogenous retroviruses fixed in the horse (Equus caballus) genome is scarce. The recent availability of a draft sequence of the horse genome enables the detection of such integrated viruses by similarity search. Using translated nucleotide fragments from gamma-, beta-, and delta-retroviral genera for initial searches, a full-length beta-retrovirus genome was retrieved from a horse chromosome 5 contig. The provirus, tentatively named EqERV-beta1 (for the first equine endogenous beta-retrovirus), was 10434 nucleotide (nt) in length with the usual retroviral genome structure of 5'LTR-gag-pro-pol-env-3'LTR. The LTRs were 1361 nt long, and differed approximately 1% from each other, suggestive of a relatively recent integration. Coding sequences for gag, pro and pol were present in three different reading-frames, as common for beta-retroviruses, and the reading frames were completely open, except that the env gene was interrupted by a single stopcodon. No reading frame was apparent downstream of the env gene, suggesting that EqERV-beta1 does not encode a superantigen like mouse mammary tumor virus (MMTV). A second proviral genome of EqERV-beta1, with no stopcodon in env, is additionally integrated on chromosome 5 downstream of the first virus. Single EqERV-beta1 LTRs were abundantly present on all chromosomes except chromosome 24. Phylogenetically, EqERV-beta1 most closely resembles an unclassified retroviral sequence from cattle (Bos taurus), and the murine beta-retrovirus MMTV.