Evolutionarily Young African Rhinoceros Gammaretroviruses.

High-throughput sequences were generated from DNA and cDNA from four Southern white rhinoceros (Ceratotherium simum simum) located in the Taronga Western Plain Zoo in Australia. Virome analysis identified reads that were similar to Mus caroli endogenous gammaretrovirus (McERV). Previous analysis of perissodactyl genomes did not recover gammaretroviruses. Our analysis, including the screening of the updated white rhinoceros (Ceratotherium simum) and black rhinoceros (Diceros bicornis) draft genomes identified high-copy orthologous gammaretroviral ERVs. Screening of Asian rhinoceros, extinct rhinoceros, domestic horse, and tapir genomes did not identify related gammaretroviral sequences in these species. The newly identified proviral sequences were designated SimumERV and DicerosERV for the white and black rhinoceros retroviruses, respectively. Two long terminal repeat (LTR) variants (LTR-A and LTR-B) were identified in the black rhinoceros, with different copy numbers associated with each (n = 101 and 373, respectively). Only the LTR-A lineage (n = 467) was found in the white rhinoceros. The African and Asian rhinoceros lineages diverged approximately 16 million years ago. Divergence age estimation of the identified proviruses suggests that the exogenous retroviral ancestor of the African rhinoceros ERVs colonized their genomes within the last 8 million years, a result consistent with the absence of these gammaretroviruses from Asian rhinoceros and other perissodactyls. The black rhinoceros germ line was colonized by two lineages of closely related retroviruses and white rhinoceros by one. Phylogenetic analysis indicates a close evolutionary relationship with ERVs of rodents including sympatric African rats, suggesting a possible African origin of the identified rhinoceros gammaretroviruses. IMPORTANCE Rhinoceros genomes were thought to be devoid of gammaretroviruses, as has been determined for other perissodactyls (horses, tapirs, and rhinoceros). While this may be true of most rhinoceros, the African white and black rhinoceros genomes have been colonized by evolutionarily young gammaretroviruses (SimumERV and DicerosERV for the white and black rhinoceros, respectively). These high-copy endogenous retroviruses (ERVs) may have expanded in multiple waves. The closest relative of SimumERV and DicerosERV is found in rodents, including African endemic species. Restriction of the ERVs to African rhinoceros suggests an African origin for the rhinoceros gammaretroviruses.

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.

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.

Distribution of infectious endogenous retroviruses in mixed-breed and purebred cats.

Endogenous retroviruses of domestic cats (ERV-DCs) are members of the genus Gammaretrovirus that infect domestic cats (Felis silvestris catus). Uniquely, domestic cats harbor replication-competent proviruses such as ERV-DC10 (ERV-DC18) and ERV-DC14 (xenotropic and nonecotropic viruses, respectively). The purpose of this study was to assess invasion by two distinct infectious ERV-DCs, ERV-DC10 and ERV-DC14, in domestic cats. Of a total sample of 1646 cats, 568 animals (34.5%) were positive for ERV-DC10 (heterozygous: 377; homozygous: 191), 68 animals (4.1%) were positive for ERV-DC14 (heterozygous: 67; homozygous: 1), and 10 animals (0.6%) were positive for both ERV-DC10 and ERV-DC14. ERV-DC10 and ERV-DC14 were detected in domestic cats in Japan as well as in Tanzania, Sri Lanka, Vietnam, South Korea and Spain. Breeding cats, including Singapura, Norwegian Forest and Ragdoll cats, showed high frequencies of ERV-DC10 (60-100%). By contrast, ERV-DC14 was detected at low frequency in breeding cats. Our results suggest that ERV-DC10 is widely distributed while ERV-DC14 is maintained in a minor population of cats. Thus, ERV-DC10 and ERV-DC14 have invaded cat populations independently.

Molecular Dynamics and Mode of Transmission of Koala Retrovirus as It Invades and Spreads through a Wild Queensland Koala Population

The recent acquisition of a novel retrovirus (KoRV) by koalas (Phascolarctos cinereus) has created new opportunities for retroviral research and new challenges for koala conservation. There are currently two major subtypes of KoRV: KoRV-A, which is believed to be endogenous only in koalas from the northern part of Australia, and KoRV-B, which appears to be exogenous. Understanding and management of these subtypes require population level studies of their prevalence and diversity, especially when coinfected in the same population, and investigations of their modes of transmission in the wild. Toward this end, we studied a wild Queensland koala population of 290 animals over a 5-year period and investigated the prevalence, diversity and mode of transmission of KoRV-A and KoRV-B. We found KoRV-A to have an infection level of 100% in the population, with all animals sharing the same dominant envelope protein sequence. In contrast, the KoRV-B infection prevalence was only 24%, with 21 different envelope protein sequence variants found in the 83 KoRV-B-positive animals. Linked to severe disease outcomes, a significant association between KoRV-B positivity and both chlamydial disease and neoplasia was found in the population. Transmission of KoRV-B was found at a rate of 3% via adult-to-adult contact per year, while there was a 100% rate of KoRV-B-positive mothers transmitting the virus to their joeys. Collectively, these findings demonstrate KoRV-B as the pathogenic subtype in this wild koala population and inform future intervention strategies with subtype variation and transmission data. IMPORTANCE KoRV represents a unique opportunity to study a relatively young retrovirus as it goes through its molecular evolution in both an endogenous form and a more recently evolved exogenous form. The endogenous form, KoRV-A, now appears to have stably and completely established itself in Northern Australian koala populations and is progressing south. Conversely, the exogenous form, KoRV-B, is undergoing continuous mutation and spread in the north and, as yet, has not reached all southern koala populations. We can now link KoRV-B to neoplasia and chlamydial disease in both wild and captive koalas, making it an imminent threat to this already vulnerable species. This work represents the largest study of koalas in a wild population with respect to KoRV-A/KoRV-B-infected/coinfected animals and the linkage of this infection to chlamydial disease, neoplasia, viral evolution, and spread.

Molecular dynamics of koala retrovirus infection in captive koalas in Japan.

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

Coinfection with koala retrovirus subtypes A and B and its impact on captive koalas in Japanese zoos.

Koala retrovirus (KoRV) is unique among endogenous retroviruses because its endogenization is still active. Two major KoRV subtypes, KoRV-A and B, have been described, and KoRV-B is associated with disease and poses a health threat to koalas. Here, we investigated the co-prevalence of KoRV-A and KoRV-B, detected by type-specific PCR and sequencing, and their impact on the health of koalas in three Japanese zoos. We also investigated KoRV proviral loads and found varying amounts of genomic DNA (gDNA) in peripheral blood mononuclear cells (PBMCs). We found that 100% of the koalas examined were infected with KoRV-A and 60% (12/20) were coinfected with KoRV-B. The KoRV-A sequence was highly conserved, whereas the KoRV-B sequence varied among individuals. Interestingly, we observed possible vertical transmission of KoRV-B in one offspring in which the KoRV-B sequence was similar to that of the father but not the mother. Moreover, we characterized the KoRV growth patterns in concanavalin-A-stimulated PBMCs isolated from KoRV-B-coinfected or KoRV-B-uninfected koalas. We quantified the KoRV provirus in gDNA and the KoRV RNA copy numbers in cells and culture supernatants by real-time PCR at days 4, 7, and 14 post-seeding. As the study population is housed in captivity, a longitudinal study of these koalas may provide an opportunity to study the transmission mode of KoRV-B. In addition, we characterized KoRV isolates by infecting tupaia cells. The results suggested that tupaia may be used as an infection model for KoRV. Thus, this study may enhance our understanding of KoRV-B coinfection and transmission in the captive koalas.

Phylogenetic Diversity of Koala Retrovirus within a Wild Koala Population.

Koala populations are in serious decline across many areas of mainland Australia, with infectious disease a contributing factor. Koala retrovirus (KoRV) is a gammaretrovirus present in most wild koala populations and captive colonies. Five subtypes of KoRV (A to E) have been identified based on amino acid sequence divergence in a hypervariable region of the receptor binding domain of the envelope protein. However, analysis of viral genetic diversity has been conducted primarily on KoRV in captive koalas housed in zoos in Japan, the United States, and Germany. Wild koalas within Australia have not been comparably assessed. Here we report a detailed analysis of KoRV genetic diversity in samples collected from 18 wild koalas from southeast Queensland. By employing deep sequencing we identified 108 novel KoRV envelope sequences and determined their phylogenetic diversity. Genetic diversity in KoRV was abundant and fell into three major groups; two comprised the previously identified subtypes A and B, while the third contained the remaining hypervariable region subtypes (C, D, and E) as well as four hypervariable region subtypes that we newly define here (F, G, H, and I). In addition to the ubiquitous presence of KoRV-A, which may represent an exclusively endogenous variant, subtypes B, D, and F were found to be at high prevalence, while subtypes G, H, and I were present in a smaller number of animals. IMPORTANCE: Koala retrovirus (KoRV) is thought to be a significant contributor to koala disease and population decline across mainland Australia. This study is the first to determine KoRV subtype prevalence among a wild koala population, and it significantly expands the total number of KoRV sequences available, providing a more precise picture of genetic diversity. This understanding of KoRV subtype prevalence and genetic diversity will be important for conservation efforts attempting to limit the spread of KoRV. Furthermore, KoRV is one of the only retroviruses shown to exist in both endogenous (transmitted vertically to offspring in the germ line DNA) and exogenous (horizontally transmitted between infected individuals) forms, a division of fundamental evolutionary importance.

Koala retroviruses: characterization and impact on the life of koalas.

Koala retroviruses (KoRV) have been isolated from wild and captive koalas in Australia as well as from koala populations held in zoos in other countries. They are members of the genus Gammaretrovirus, are most closely related to gibbon ape leukemia virus (GaLV), feline leukemia virus (FeLV) and porcine endogenous retrovirus (PERV) and are likely the result of a relatively recent trans-species transmission from rodents or bats. The first KoRV to be isolated, KoRV-A, is widely distributed in the koala population in both integrated endogenous and infectious exogenous forms with evidence from museum specimens older than 150 years, indicating a relatively long engagement with the koala population. More recently, additional subtypes of KoRV that are not endogenized have been identified based on sequence differences and host cell receptor specificity (KoRV-B and KoRV-J). A specific association with fatal lymphoma and leukemia has been recently suggested for KoRV-B. In addition, it has been proposed that the high viral loads found in many animals may lead to immunomodulation resulting in a higher incidence of diseases such as chlamydiosis. Although the molecular basis of this immunomodulation is still unclear, purified KoRV particles and a peptide corresponding to a highly conserved domain in the envelope protein have been shown to modulate cytokine expression in vitro, similar to that induced by other gammaretroviruses. While much is still to be learned, KoRV induced lymphoma/leukemia and opportunistic disease arising as a consequence of immunomodulation are likely to play an important role in the stability of koala populations both in the wild and in captivity.

Discovery of retroviral homologs in bats: implications for the origin of mammalian gammaretroviruses.

Gammaretroviruses infect a wide range of vertebrate species where they are associated with leukemias, neurological diseases and immunodeficiencies. However, the origin of these infectious agents is unknown. Through a phylogenetic analysis of viral gene sequences, we show that bats harbor an especially diverse set of gammaretroviruses. In particular, phylogenetic analysis places Rhinolophus ferrumequinum retrovirus (RfRV), a new gammaretrovirus identified by de novo analysis of the Rhinolophus ferrumequinum transcriptome, and six other gammaretroviruses from different bat species, as basal to other mammalian gammaretroviruses. An analysis of the similarity in the phylogenetic history between the gammaretroviruses and their bat hosts provided evidence for both host-virus codivergence and cross-species transmission. Taken together, these data provide new insights into the origin of the mammalian gammaretroviruses.

Polymorphic integrations of an endogenous gammaretrovirus in the mule deer genome.

Endogenous retroviruses constitute a significant genomic fraction in all mammalian species. Typically they are evolutionarily old and fixed in the host species population. Here we report on a novel endogenous gammaretrovirus (CrERVγ; for cervid endogenous gammaretrovirus) in the mule deer (Odocoileus hemionus) that is insertionally polymorphic among individuals from the same geographical location, suggesting that it has a more recent evolutionary origin. Using PCR-based methods, we identified seven CrERVγ proviruses and demonstrated that they show various levels of insertional polymorphism in mule deer individuals. One CrERVγ provirus was detected in all mule deer sampled but was absent from white-tailed deer, indicating that this virus originally integrated after the split of the two species, which occurred approximately one million years ago. There are, on average, 100 CrERVγ copies in the mule deer genome based on quantitative PCR analysis. A CrERVγ provirus was sequenced and contained intact open reading frames (ORFs) for three virus genes. Transcripts were identified covering the entire provirus. CrERVγ forms a distinct branch of the gammaretrovirus phylogeny, with the closest relatives of CrERVγ being endogenous gammaretroviruses from sheep and pig. We demonstrated that white-tailed deer (Odocoileus virginianus) and elk (Cervus canadensis) DNA contain proviruses that are closely related to mule deer CrERVγ in a conserved region of pol; more distantly related sequences can be identified in the genome of another member of the Cervidae, the muntjac (Muntiacus muntjak). The discovery of a novel transcriptionally active and insertionally polymorphic retrovirus in mammals could provide a useful model system to study the dynamic interaction between the host genome and an invading retrovirus.

Identification of diverse groups of endogenous gammaretroviruses in mega- and microbats.

A previous phylogenetic study suggested that mammalian gammaretroviruses may have originated in bats. Here we report the discovery of RNA transcripts from two putative endogenous gammaretroviruses in frugivorous (Rousettus leschenaultii retrovirus, RlRV) and insectivorous (Megaderma lyra retrovirus, MlRV) bat species. Both genomes possess a large deletion in pol, indicating that they are defective retroviruses. Phylogenetic analysis places RlRV and MlRV within the diversity of mammalian gammaretroviruses, with the former falling closer to porcine endogenous retroviruses and the latter to Mus dunni endogenous virus, koala retrovirus and gibbon ape leukemia virus. Additional genomic mining suggests that both microbat (Myotis lucifugus) and megabat (Pteropus vampyrus) genomes harbour many copies of endogenous retroviral forms related to RlRV and MlRV. Furthermore, phylogenetic analysis reveals the presence of three genetically diverse groups of endogenous gammaretroviruses in bat genomes, with M. lucifugus possessing members of all three groups. Taken together, this study indicates that bats harbour distinct gammaretroviruses and may have played an important role as reservoir hosts during the diversification of mammalian gammaretroviruses.

Evolution of functional and sequence variants of the mammalian XPR1 receptor for mouse xenotropic gammaretroviruses and the human-derived retrovirus XMRV.

Genetic conflicts between retroviruses and their receptors result in the evolution of novel host entry restrictions and novel virus envelopes, and such variants can influence trans-species transmission. We screened rodents and other mammals for sequence variation in the Xpr1 receptor for the mouse xenotropic or polytropic mouse leukemia viruses (X-MLVs or P-MLVs, respectively) of the gammaretrovirus family and for susceptibility to mouse-derived X/P-MLVs and to XMRV (xenotropic murine leukemia virus-related virus), an X-MLV-like virus isolated from humans with prostate cancer and chronic fatigue syndrome. We identified multiple distinct susceptibility phenotypes; these include the four known Xpr1 variants in Mus and a novel fifth Xpr1 gene found in Mus molossinus and Mus musculus. We describe the geographic and species distribution of the Mus Xpr1 variants but failed to find the X-MLV-restrictive laboratory mouse allele in any wild mouse. We used mutagenesis and phylogenetic analysis to evaluate the functional contributions made by constrained, variable, and deleted residues. Rodent Xpr1 is under positive selection, indicating a history of host-pathogen conflicts; several codons under selection have known roles in virus entry. All non-Mus mammals are susceptible to mouse X-MLVs, but some restrict other members of the X/P-MLV family, and the resistance of hamster and gerbil cells to XMRV indicates that XMRV has unique receptor requirements. We show that the hypervariable fourth extracellular XPR1 loop (ECL4) contains three evolutionarily constrained residues that do not contribute to receptor function, we identify two novel residues important for virus entry (I579 and T583), and we describe a unique pattern of ECL4 variation in the three virus-restrictive Xpr1 variants found in MLV-infected house mice; these mice carry different deletions in ECL4, suggesting either that these sites or loop size affects receptor function.

Molecular characterization of a novel gammaretrovirus in killer whales (Orcinus orca).

There are currently no published data documenting the presence of retroviruses in cetaceans, though the occurrences of cancers and immunodeficiency states suggest the potential. We examined tissues from adult killer whales and detected a novel gammaretrovirus by degenerate PCR. Reverse transcription-PCR also demonstrated tissue and serum expression of retroviral mRNA. The full-length sequence of the provirus was obtained by PCR, and a TaqMan-based copy number assay did not demonstrate evidence of productive infection. PCR on blood samples from 11 healthy captive killer whales and tissues from 3 free-ranging animals detected the proviral DNA in all tissues examined from all animals. A survey of multiple cetacean species by PCR for gag, pol, and env sequences showed homologs of this virus in the DNA of eight species of delphinids, pygmy and dwarf sperm whales, and harbor porpoises, but not in beluga or fin whales. Analysis of the bottlenose dolphin genome revealed two full-length proviral sequences with 97.4% and 96.9% nucleotide identity to the killer whale gammaretrovirus. The results of single-cell PCR on killer whale sperm and Southern blotting are also consistent with the conclusion that the provirus is endogenous. We suggest that this gammaretrovirus entered the delphinoid ancestor's genome before the divergence of modern dolphins or that an exogenous variant existed following divergence that was ultimately endogenized. However, the transcriptional activity demonstrated in tissues and the nearly intact viral genome suggest a more recent integration into the killer whale genome, favoring the latter hypothesis. The proposed name for this retrovirus is killer whale endogenous retrovirus.

Biology and evolution of the endogenous koala retrovirus.

Although endogenous retroviruses are ubiquitous features of all mammalian genomes, the process of initial germ line invasion and subsequent inactivation from a pathogenic element has not yet been observed in a wild species. Koala retrovirus (KoRV) provides a unique opportunity to study this process of endogenisation in action as it still appears to be spreading through the koala population. Ongoing expression of the endogenous sequence and consequent high levels of viraemia have been linked to neoplasia and immunosuppression in koalas. This apparently recent invader of the koala genome shares a remarkably close sequence relationship with the pathogenic exogenous Gibbon ape leukaemia virus (GALV), and comparative analyses of KoRV and GALVare helping to shed light on how retroviruses in general adapt to a relatively benign or at least less pathogenic existence within a new host genome. (Part of a multi-author review).

Porcine endogenous retroviruses and xenotransplantation.

Xenotransplantation is defined by the PHS as any procedure that involves the transplantation, implantation or infusion into a human recipient of either (a) live cells, tissues or organs from a nonhuman animal source, or (b) human body fluids, cells, tissues or organs that have had ex vivo contact with live nonhuman animal cells, tissues or organs (Public Health Service Guideline on Infectious Disease Issues in Xenotransplantation). Use of pigs for human xenotransplantation raises concerns about the risks of transfer of infectious agents from the pig cells to xenotransplantation recipients. The observation that the porcine germline harbors genetic loci encoding porcine endogenous retroviruses (PERVs) that are in some cases infectious for human cells has resulted in renewed scientific interest in PERVs. However, in spite of the past 10 years of investigation, the actual risk for PERV infection, replication, and pathogenic outcome in human recipients of xenotransplantation products is still undefined. (Part of a multi-author review).

Murine endogenous retroviruses.

Up to 10% of the mouse genome is comprised of endogenous retrovirus (ERV) sequences, and most represent the remains of ancient germ line infections. Our knowledge of the three distinct classes of ERVs is inversely correlated with their copy number, and their characterization has benefited from the availability of divergent wild mouse species and subspecies, and from ongoing analysis of the Mus genome sequence. In contrast to human ERVs, which are nearly all extinct, active mouse ERVs can still be found in all three ERV classes. The distribution and diversity of ERVs has been shaped by host-virus interactions over the course of evolution, but ERVs have also been pivotal in shaping the mouse genome by altering host genes through insertional mutagenesis, by adding novel regulatory and coding sequences, and by their co-option by host cells as retroviral resistance genes. We review mechanisms by which an adaptive coexistence has evolved. (Part of a multi-author review).

Transmission, Evolution, and Endogenization: Lessons Learned from Recent Retroviral Invasions.

Viruses of the subfamily Orthoretrovirinae are defined by the ability to reverse transcribe an RNA genome into DNA that integrates into the host cell genome during the intracellular virus life cycle. Exogenous retroviruses (XRVs) are horizontally transmitted between host individuals, with disease outcome depending on interactions between the retrovirus and the host organism. When retroviruses infect germ line cells of the host, they may become endogenous retroviruses (ERVs), which are permanent elements in the host germ line that are subject to vertical transmission. These ERVs sometimes remain infectious and can themselves give rise to XRVs. This review integrates recent developments in the phylogenetic classification of retroviruses and the identification of retroviral receptors to elucidate the origins and evolution of XRVs and ERVs. We consider whether ERVs may recurrently pressure XRVs to shift receptor usage to sidestep ERV interference. We discuss how related retroviruses undergo alternative fates in different host lineages after endogenization, with koala retrovirus (KoRV) receiving notable interest as a recent invader of its host germ line. KoRV is heritable but also infectious, which provides insights into the early stages of germ line invasions as well as XRV generation from ERVs. The relationship of KoRV to primate and other retroviruses is placed in the context of host biogeography and the potential role of bats and rodents as vectors for interspecies viral transmission. Combining studies of extant XRVs and "fossil" endogenous retroviruses in koalas and other Australasian species has broadened our understanding of the evolution of retroviruses and host-retrovirus interactions.

Three cysteine residues of SLC52A1, a receptor for the porcine endogenous retrovirus-A (PERV-A), play a critical role in cell surface expression and infectivity.

Porcine endogenous retrovirus-A (PERV-A), a gammaretrovirus, infects human cells in vitro, thus raising the potential risk of cross-species transmission in xenotransplantation. Two members of the solute carrier family 52 (SLC52A1 and SLC52A2) are PERV-A receptors. Site-directed mutagenesis of the cDNA encoding SLC52A1 identified that only one of two putative glycosylation signals is occupied by glycans. In addition, we showed that glycosylation of SLC52A1 is not necessary for PERV-A receptor function. We also identified that at a minimum, three cysteine residues are sufficient for SLC52A1 cell surface expression. Mutation of cysteine at position 365 and either of the two cysteine residues in the C-terminal tail at positions 442 or 446 reduced SLC52A1 surface expression and PERV-A infection suggesting that these residues may contribute to overall structural stability and receptor function. Understanding interactions between PERV-A and its cellular receptor may provide novel strategies to prevent zoonotic infection in the setting of xenotransplantation.

Molecular and phylogenetic analyses of a new amphotropic murine leukemia virus (MuLV-1313).

BACKGROUND: The amphotropic murine leukemia viruses (MuLV-A's) are naturally occurring, exogenously acquired gammaretroviruses that are indigenous to the Southern California wild mice. These viruses replicate in a wide range of cell types including human cells in vitro and they can cause both hematological and neurological disorders in feral as well as in the inbred laboratory mice. Since MuLV-A's also exhibit discrete interference and neutralization properties, the envelope proteins of these viruses have been extremely useful for studying virus-host cell interactions and as vehicles for transfer of foreign genes into a variety of hosts including human cells. However, the genomic structure of any of the several known MuLV-A's has not been established and the evolutionary relationship of amphotropic retroviruses to the numerous exogenous or endogenous MuLV strains remains elusive. Herein we present a complete genetic structure of a novel amphotropic virus designated MuLV-1313 and demonstrate that this retrovirus together with other MuLV-A's belongs to a distinct molecular, biological and phylogenetic class among the MuLV strains isolated from a large number of the laboratory inbred or feral mice. RESULTS: The host range of MuLV-1313 is similar to the previously isolated MuLV-A's except that this virus replicates efficiently in mammalian as well as in chicken cells. Compared to ENV proteins of other MuLV-A's (4070A, 1504A and 10A-1), the gp70 protein of MuLV-1313 exhibits differences in its signal peptides and the proline-rich hinge regions. However, the MuLV-1313 envelope protein is totally unrelated to those present in a broad range of murine retroviruses that have been isolated from various inbred and feral mice globally. Genetic analysis of the entire MuLV-1313 genome by dot plot analyses, which compares each nucleotide of one genome with the corresponding nucleotide of another, revealed that the genome of this virus, with the exception of the env gene, is more closely related to the biologically distinct wild mouse ecotropic retrovirus (Cas-Br-E) isolated from another region of the Southern California, than to any of the 15 MuLV strains whose full-length sequences are present in the GenBank. This finding was corroborated by phylogenetic analyses and hierarchical clustering of the entire genomic sequence of MuLV-1313, which also placed all MULV-A's in a genetically distinct category among the large family of retroviruses isolated from numerous mouse strains globally. Likewise, construction of separate dendrograms for each of the Gag, Pol and Env proteins of MuLV-1313 demonstrated that the amphotropic retroviruses belong to a phylogenetically exclusive group of gammaretroviruses compared to all known MuLV strains. CONCLUSION: The molecular, biological and phylogenetic properties of amphotropic retroviruses including MuLV-1313 are distinct compared to a large family of exogenously- or endogenously-transmitted ecotropic, polytropic and xenotropic MuLV strains of the laboratory and feral mice. Further, both the naturally occurring amphotropic and a biologically discrete ecotropic retrovirus of the Southern California wild mice are more closely related to each other on the evolutionary tree than any other mammalian gammaretrovirus indicating a common origin of these viruses. This is the first report of a complete genomic analysis of a unique group of phylogenetically distinct amphotropic virus.