Molecular Analysis of the Complete Genome of a Simian Foamy Virus Infecting Hylobates pileatus (pileated gibbon) Reveals Ancient Co-Evolution with Lesser Apes.

Foamy viruses (FVs) are complex retroviruses present in many mammals, including nonhuman primates, where they are called simian foamy viruses (SFVs). SFVs can zoonotically infect humans, but very few complete SFV genomes are available, hampering the design of diagnostic assays. Gibbons are lesser apes widespread across Southeast Asia that can be infected with SFV, but only two partial SFV sequences are currently available. We used a metagenomics approach with next-generation sequencing of nucleic acid extracted from the cell culture of a blood specimen from a lesser ape, the pileated gibbon (Hylobates pileatus), to obtain the complete SFVhpi_SAM106 genome. We used Bayesian analysis to co-infer phylogenetic relationships and divergence dates. SFVhpi_SAM106 is ancestral to other ape SFVs with a divergence date of ~20.6 million years ago, reflecting ancient co-evolution of the host and SFVhpi_SAM106. Analysis of the complete SFVhpi_SAM106 genome shows that it has the same genetic architecture as other SFVs but has the longest recorded genome (13,885-nt) due to a longer long terminal repeat region (2,071 bp). The complete sequence of the SFVhpi_SAM106 genome fills an important knowledge gap in SFV genetics and will facilitate future studies of FV infection, transmission, and evolutionary history.

A novel Australian flying-fox retrovirus shares an evolutionary ancestor with Koala, Gibbon and Melomys gamma-retroviruses.

A novel gamma-retroviral sequence (7912 bp), inclusive of both partial 5' and 3' long terminal repeat regions, was identified from the brain of a black flying-fox (Pteropus alecto), Queensland, Australia. The sequence was distinct from other retroviral sequences identified in bats and showed greater identity to Koala, Gibbon ape leukaemia, Melomys burtoni and Woolly monkey retroviruses, forming their own phylogenetic clade. This finding suggests that these retroviruses may have an unknown common ancestor and that further investigation into the diversity of gamma-retroviruses in Australian Pteropus species may elucidate their evolutionary origins.

A new look at the origins of gibbon ape leukemia virus.

Is the origin of gibbon ape leukemia virus (GALV) human after all? When GALV was discovered and found to cause neoplastic disease in gibbons, it stimulated a great deal of research including investigations into the origins of this virus. A number of publications have suggested that the GALV progenitor was a retrovirus present in one of several species of South East Asian rodents that had close contact with captive gibbons. However, there are no published retroviral sequences from any South East Asian species to support this view. Here we present an alternative hypothesis that the origin of GALV is a virus closely related to Melomys burtoni retrovirus, and that this virus infected human patients in Papua New Guinea from whom biological material was obtained or in some way contaminated these samples. This material we propose contained infectious MbRV-related virus that was then unwittingly introduced into gibbons which subsequently developed GALV infections.

Episodic Diversifying Selection Shaped the Genomes of Gibbon Ape Leukemia Virus and Related Gammaretroviruses.

UNLABELLED: Gibbon ape leukemia viruses (GALVs) are part of a larger group of pathogenic gammaretroviruses present across phylogenetically diverse host species of Australasian mammals. Despite the biomedical utility of GALVs as viral vectors and in cancer gene therapy, full genome sequences have not been determined for all of the five identified GALV strains, nor has a comprehensive evolutionary analysis been performed. We therefore generated complete genomic sequences for each GALV strain using hybridization capture and high-throughput sequencing. The four strains of GALV isolated from gibbons formed a monophyletic clade that was closely related to the woolly monkey virus (WMV), which is a GALV strain that likely originated in a gibbon host. The GALV-WMV clade in turn formed a sister group to the koala retroviruses (KoRVs). Genomic signatures of episodic diversifying selection were detected among the gammaretroviruses with concentration in the env gene across the GALV strains that were particularly oncogenic and KoRV strains that were potentially exogenous, likely reflecting their adaptation to the host immune system. In vitro studies involving vectors chimeric between GALV and KoRV-B established that variable regions A and B of the surface unit of the envelope determine which receptor is used by a viral strain to enter host cells. IMPORTANCE: The gibbon ape leukemia viruses (GALVs) are among the most medically relevant retroviruses due to their use as viral vectors for gene transfer and in cancer gene therapy. Despite their importance, full genome sequences have not been determined for the majority of primate isolates, nor has comprehensive evolutionary analysis been performed, despite evidence that the viruses are facing complex selective pressures associated with cross-species transmission. Using hybridization capture and high-throughput sequencing, we report here the full genome sequences of all the GALV strains and demonstrate that diversifying selection is acting on them, particularly in the envelope gene in functionally important domains, suggesting that host immune pressure is shaping GALV evolution.

Full genome characterization and phylogenetic analysis of hepatitis B virus in gibbons and a caretaker in Central Kalimantan, Indonesia.

Hepatitis B virus (HBV) from gibbons was characterized, and the possibility of horizontal transmission between gibbons and humans was examined in a gibbon rehabilitation center in Central Kalimantan, Indonesia. Ten gibbons that were positive for the hepatitis B surface antigen (HBsAg) on arrival and 13 caretakers for those gibbons were included in this study. The duration of stay at the rehabilitation center ranged from 1 to 10 years. Serological and molecular analyses were performed. Six gibbons were positive for HBsAg, whereas HBV DNA was detected in all ten of the gibbons sampled. On the other hand, HBsAg was detected in only 1 of the 13 caretakers. HBV samples from seven gibbons and from the one infected human were chosen for complete genome sequencing. A phylogenetic analysis revealed that the cluster of gibbon strains in this study was distinct from strains previously reported from other countries. In the pre-S1 region, we found a unique amino acid residue substitution (P89K), three insertions between T87 and L88 in the genomes of three gibbons, and a 33-nucleotide deletion at the start of pre-S1 that is common in non-human primates. The caretaker sample was identified as HBV subgenotype B3, the most common type in Indonesia. For the complete HBV sequences, the similarity between gibbons in this study and other non-human primate and human HBV isolates was 90-91.9 % and 85.5-89.6 %, respectively. In conclusion, the gibbon HBV genotype was influenced by geographic location and species. To the best of our knowledge, this is the first report characterizing the HBV genes and genomes of indigenous gibbons in Indonesia.

Discovery of a novel retrovirus sequence in an Australian native rodent (Melomys burtoni): a putative link between gibbon ape leukemia virus and koala retrovirus.

Gibbon ape leukaemia virus (GALV) and koala retrovirus (KoRV) share a remarkably close sequence identity despite the fact that they occur in distantly related mammals on different continents. It has previously been suggested that infection of their respective hosts may have occurred as a result of a species jump from another, as yet unidentified vertebrate host. To investigate possible sources of these retroviruses in the Australian context, DNA samples were obtained from 42 vertebrate species and screened using PCR in order to detect proviral sequences closely related to KoRV and GALV. Four proviral partial sequences totalling 2880 bases which share a strong similarity with KoRV and GALV were detected in DNA from a native Australian rodent, the grassland melomys, Melomys burtoni. We have designated this novel gammaretrovirus Melomys burtoni retrovirus (MbRV). The concatenated nucleotide sequence of MbRV shares 93% identity with the corresponding sequence from GALV-SEATO and 83% identity with KoRV. The geographic ranges of the grassland melomys and of the koala partially overlap. Thus a species jump by MbRV from melomys to koalas is conceivable. However the genus Melomys does not occur in mainland South East Asia and so it appears most likely that another as yet unidentified host was the source of GALV.

Identification of novel inter-genotypic recombinants of human hepatitis B viruses by large-scale phylogenetic analysis.

Recombination plays an important role in the evolutionary history of Hepatitis B virus (HBV). We performed a phylogenetic analysis of 3403 full-length HBV genome sequences isolated from humans to define the genotype. The genome sequences were divided into 13 sub-datasets, each approximately 250 bp in length. Genotype designations obtained from the sub-datasets that differed from the genotype defined by the whole genome were assigned as putative recombinants. Our results showed that 3379 out of 3403 sequences belonged to the previously described and putative genotypes A to J respectively, with 315 sequences defined in this analysis. The remaining 24 viruses had sequence divergence of less than 8% with both genotypes B and C and were provisionally assigned genotype "BC". 1047 out of 3403 sequences were identified to be putative recombinants, of which 72 were identified to be novel recombinants. Notably, all viruses of the herein described genotype "BC" were identified to be B/C recombinants.

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).

Prevalence and molecular characterization of the polymerase gene of gibbon lymphocryptovirus.

BACKGROUND: Lymphocryptovirus (LCV) is found in various non-human primates. As a herpesvirus naturally infecting gibbons it is closely related to human Epstein-Barr virus (EBV) with which it shares considerable genetic, biological and epidemiologic features. METHODS: We collected blood samples from 70 gibbons (51 Hylobates lar, 18 Hylobates pileatus and 1 Hylobates agilis) for further separation into serum and peripheral blood mononuclear cells (PBMC). RESULTS: Only 13 of 70 (18.6%) sera were serologically positive for human EBV IgG but 64 of 70 (91.4%) PBMCs yielded the partial LCV DNA polymerase gene by semi-nested PCR, which we subjected to direct sequencing. All sequences showed 84% nucleic acid and 91% amino acid identity to human EBV. Phylogenetic tree analysis demonstrated gibbon LCVs clustered separately from other gammaherpesvirinae but closely related to LCV of other species. CONCLUSIONS: Based on LCV DNA detection, we discovered a high prevalence of LCV infection among gibbons. Further characterization of non-human primate LCV might thus provide new insight into both evolution and pathogenicity of gammaherpesvirinae.

Frequent infection of Hylobates pileatus (pileated gibbon) with species-associated variants of hepatitis B virus in Cambodia.

As well as being distributed widely in human populations, hepatitis B virus (HBV) infections occur frequently in chimpanzee, gibbon and other ape populations in sub-Saharan Africa and South-East Asia. To investigate the frequency and genetic relationships of HBV infecting gibbons in Cambodia, pileated gibbons (Hylobates pileatus) that were originally wild-caught were screened for surface antigen. Twelve of 26 (46 %) were positive, of which 11 were positive for HBV DNA. Phylogenetic analysis of complete genome sequences revealed two distinct genetic groups in the gibbon/orangutan clade. Three were similar to previously described variants infecting H. pileatus in Thailand and eight formed a distinct clade, potentially representing distinct strains of HBV circulating in geographically separated populations in South-East Asia. Because of the ability of HBV to cross species barriers, large reservoirs of infection in gibbons may hamper ongoing attempts at permanent eradication of HBV infection from human populations in South-East Asia through immunization.

Novel gamma-2-herpesvirus of the Rhadinovirus 2 lineage in gibbons.

We obtained 475 nucleotides of the DNA polymerase gene of a novel human herpesvirus 8 homolog sequence in a gibbon. The finding of this new gibbon virus, which clusters with a related chimpanzee virus in the rhadinovirus 2 genogroup, suggests the existence of a novel gamma-2-herpesvirus in humans.

Complete nucleotide sequence and phylogenetic analyses of hepatitis B virus isolated from two pileated gibbons.

We analyzed full-length sequence of hepatitis B virus (HBV) recovered from two pileated gibbons (Hylobates pileatus) originally born in East Asia. Two animals possessed a viral genome of 3182 nt in length with a 33 nt deletion in the pre-S1 region, and designated HBV PG-Makiko and HBV PG-Yohko, respectively. Both sequences had 65-90% similarity to type A-G of human HBV isolates. Phylogenetic analysis demonstrated that both isolates were distinct from the human and other nonhuman primate HBV isolates, but could be classified into gibbon isolates that were previously reported by others. Small spherical and tubular particles and large particles with outer envelopes were observed in the serum under immunoelectron microscopic examination. By immunohistochemical staining, HBsAg and HBcAg were detected in the cytoplasm and nuclei of hepatocytes, respectively. Our results suggested that HBV found in these animals is indigenous to their respective hosts and not recent acquisitions from human.

Geographic and species association of hepatitis B virus genotypes in non-human primates.

Infection with hepatitis B virus (HBV) has been detected in human populations throughout the world, as well as in a number of ape species (Pan troglodytes, Gorilla gorilla, gibbons [Nomascus and Hylobates species] and Pongo pygmaeus). To investigate the distribution of naturally occurring HBV infection in these species and other African Old World monkey species (Cercopithecidae), we screened 137 plasma samples from mainly wild caught animals by polymerase chain reaction (PCR) using several of highly conserved primers from the HB surface (HBs) gene, and for HBs antigen (HBsAg) by ELISA. None of the 93 Cercopithecidae screened (6 species) showed PCR or serology evidence for HBV infection; in contrast 2 from 8 chimpanzees and 5 from 22 gibbons were PCR-positive with each set of primers. Complete genome sequences from each of the positive apes were obtained and compared with all previously published complete and surface gene sequences. This extended phylogenetic analysis indicated that HBV variants from orangutans were interspersed by with HBV variants from southerly distributed gibbon species (H. agilis and H. moloch) occupying overlapping or adjacent habitat ranges with orangutans; in contrast, HBV variants from gibbon species in mainland Asia were phylogenetically distinct. A geographical rather than (sub)species association of HBV would account for the distribution of HBV variants in different subspecies of chimpanzees in Africa, and explain the inlier position of the previously described lowland gorilla sequence in the chimpanzee clade. These new findings have a number of implication for understanding the origins and epidemiology of HBV infection in non-human primates.

Molecular epidemiology of gibbon hepatitis B virus transmission.

Although transmission of human hepatitis B virus (HBV) variants to nonhuman primates is well documented, it remains to be elucidated whether nonhuman primate HBV is transmissible to humans. The prevalence and transmission routes of gibbon HBV were analysed in 101 captive gibbons in Thailand. Approximately 40 % of these animals showed at least one marker of HBV infection; 19 animals were chronic HBV carriers, characterized by elevated levels of alanine amino transferase and the presence of HBV DNA. Some of the chronic animals were found to be anti-HBc (HBV core antigen) negative (4 of 19), while precore promoter point mutations (nt 1762 or 1764) were determined in four animals by RFLP analysis. Phylogenetic tree analysis of the complete surface gene sequences revealed that gibbon viruses clustered separately from hepadnaviruses of other hosts. Evidence for horizontal and vertical transmission in captive gibbons was obtained. HBV DNA was also detected in the saliva of HBV carrier gibbons. Although some of the animal caretakers at the Krabok Koo Wildlife Breeding Centre were found to be chronic HBV carriers, genotype and sequence analysis did not reveal any evidence for zoonotic disease transmission.

Primate hepatitis B viruses - genetic diversity, geography and evolution.

There are six well characterised genotypes (A-F) of human hepatitis B virus that have distinct geographic ranges which generally relate to chronic HBV infection. A seventh human genotype (G) has recently been described, but there is limited information on ethnic and geographic distribution. Despite the fact that early studies indicated that HBV antigens were present in other primates, the prevailing dogma that HBV was a human disease precluded alternative explanations. Within the past 5 years, hepatitis B viruses have been characterised from all the Old World great apes (orangutan, gibbons, gorillas and chimpanzees) and from a New World woolly monkey. Each group of non-human primates appears to have a distinct strain of hepatitis B virus that can be distinguished from human sequences based upon the nucleotide sequence and selected amino acid changes in the viral proteins. The woolly monkey HBV is most divergent from other primate and human sequences, while the great ape HBV sequences cluster together with separate branches for each group.

Paired chimpanzee hepatitis B virus (ChHBV) and mtDNA sequences suggest different ChHBV genetic variants are found in geographically distinct chimpanzee subspecies.

The surface antigen gene region from five chronic hepatitis B virus (HBV) infected chimpanzees was amplified by PCR and the sequence determined. Sequence comparison confirmed that all of the sequences were chimpanzee hepatitis B virus (chHBV) and they appeared to represent three distinct clusters or branches. To address the question of whether the three branches represented recently identified subspecies of chimpanzees, we determined the sequence of the mitochondrial DNA hypervariable D loop from hair samples obtained from these five chimpanzees. The results indicated that the three chHBV branches reflected three distinct subspecies of chimpanzees that are from different geographic regions in West Africa. The complete HBV sequence from members of the Pan troglodytes troglodytes cluster and the Pan troglodytes verus cluster are in the published literature; we determined the complete genome sequence for the third branch of HBV present in Pan troglodytes vellerosus.

TT virus infection in gibbons.

TT virus (TTV) is not only an infectious agent of worldwide distribution but has also been demonstrated in various non-human primates in addition to humans. In the present study, we subjected the sera of 67 gibbons to PCR and nucleotide sequencing, with subsequent phylogenetic analysis to determine the nature of the relationship between TTV found in humans and non-human primates. We discovered the virus in 9/67 (13.4%) of the gibbon sera and subjected 6 of those to direct sequencing. The phylogenetic tree constructed encompassed all TTV species known to date, revealing a close proximity between the gibbon virus and those detected in Thai individuals, whereas the chimpanzee strains were phylogenetically more remote.

Hepadnavirus infection in captive gibbons.

The recent isolation of a nonhuman primate hepadnavirus from woolly monkeys prompted an examination of other primates for potentially new hepadnaviruses. A serological analysis of 30 captive gibbons revealed that 47% were positive for at least one marker of ongoing or previous infection with a hepatitis B virus (HBV). The amino acid sequences of the core and surface genes of human and gibbon virus isolates were very similar. Phylogenetic analysis indicated that the gibbon isolates lie within the human HBV family, indicating that these HBV isolates most likely stem from infection of gibbons from a human source.

Complete sequencing of a gibbon hepatitis B virus genome reveals a unique genotype distantly related to the chimpanzee hepatitis B virus.

We have sequenced the complete genome of a hepatitis B virus (HBV) strain that was transmitted from a gibbon with chronic hepatitis B to a chimpanzee that subsequently developed acute hepatitis B. The genome was 3,182 nucleotides long and had a genetic organization identical to and including the characteristics of other mammalian hepadnaviruses. Thus, the regulatory elements, the direct repeats, and the four open reading frames (ORFs) of this virus were all maintained, although there were amino acid substitutions affecting all the ORFs. Within the S gene encoding for the hepatitis B surface antigen (HBsAg), the subtype could be deduced as ayw3 in accordance with previous serological results. There were 25 amino acid substitutions affecting the P gene, 12 of which were within the spacer region. This region, which was the most divergent part of the genome compared to other HBV strains, also encodes for the pre-S proteins. A comparison with sequences of other hepadnaviruses revealed that the genome of gibbon HBV was unique as compared to previously described HBV genotypes. It was most similar to the chimpanzee HBV strain with which it shared 90.3% nucleic acid homology at the level of the complete genome and 96.3% homology at the level of the S-gene region corresponding to HBsAg, although being a distinct genotype as compared to the latter virus. Analyses performed using five different algorithms for phylogenetic tree construction showed more than 99% bootstrap support for the gibbon and the chimpanzee HBV to be grouped within the human HBV strains and that they represented later offshoots than the HBV strains of genotype F. However, in most of the dendrograms both the gibbon and the chimpanzee strains represented early lineages, indicating that these viruses are indigenous to their respective hosts and not recent acquisitions from man.