Primate T-lymphotropic Viruses Research Articles

Absence of accessory genes in a divergent simian T-lymphotropic virus type 1 isolated from a bonnet macaque (Macaca radiata)

BackgroundPrimate T-lymphotropic viruses type 1 (PTLV-1) are complex retroviruses infecting both human (HTLV-1) and simian (STLV-1) hosts. They share common epidemiological, clinical and molecular features. In addition to the canonical gag, pol, env retroviral genes, PTLV-1 purportedly encodes regulatory (i.e. Tax, Rex, and HBZ) and accessory proteins (i.e. P12/8, P13, P30). The latter have been found essential for viral persistence in vivo.Methodology/Principal findingsWe have isolated a STLV-1 virus from a bonnet macaque (Macaca radiata–Mra18C9), a monkey from India. The complete sequence was obtained and phylogenetic analyses were performed. The Mra18C9 strain is highly divergent from the known PTLV-1 strains. Intriguingly, the Mra18C9 lacks the 3 accessory open reading frames. In order to determine if the absence of accessory proteins is specific to this particular strain, a comprehensive analysis of the complete PTLV-1 genomes available in Genbank was performed and found that the lack of one or many accessory ORF is common among PTLV-1.ConclusionThis study raises many questions regarding the actual nature, role and importance of accessory proteins in the PTLV-1 biology.

Detailed phylogenetic analysis of primate T-lymphotropic virus type 1 (PTLV-1) sequences from orangutans (Pongo pygmaeus) reveals new insights into the evolutionary history of PTLV-1 in Asia

While human T-lymphotropic virus type 1 (HTLV-1) originates from ancient cross-species transmission of simian T-lymphotropic virus type 1 (STLV-1) from infected nonhuman primates, much debate exists on whether the first HTLV-1 occurred in Africa, or in Asia during early human evolution and migration. This topic is complicated by a lack of representative Asian STLV-1 to infer PTLV-1 evolutionary histories. In this study we obtained new STLV-1 LTR and tax sequences from a wild-born Bornean orangutan (Pongo pygmaeus) and performed detailed phylogenetic analyses using both maximum likelihood and Bayesian inference of available Asian PTLV-1 and African STLV-1 sequences. Phylogenies, divergence dates and nucleotide substitution rates were co-inferred and compared using six different molecular clock calibrations in a Bayesian framework, including both archaeological and/or nucleotide substitution rate calibrations. We then combined our molecular results with paleobiogeographical and ecological data to infer the most likely evolutionary history of PTLV-1. Based on the preferred models our analyses robustly inferred an Asian source for PTLV-1 with cross-species transmission of STLV-1 likely from a macaque (Macaca sp.) to an orangutan about 37.9–48.9kya, and to humans between 20.3–25.5kya. An orangutan diversification of STLV-1 commenced approximately 6.4–7.3kya. Our analyses also inferred that HTLV-1 was first introduced into Australia ~3.1–3.7kya, corresponding to both genetic and archaeological changes occurring in Australia at that time. Finally, HTLV-1 appears in Melanesia at ~2.3–2.7kya corresponding to the migration of the Lapita peoples into the region. Our results also provide an important future reference for calibrating information essential for PTLV evolutionary timescale inference. Longer sequence data, or full genomes from a greater representation of Asian primates, including gibbons, leaf monkeys, and Sumatran orangutans are needed to fully elucidate these evolutionary dates and relationships using the model criteria suggested herein.

Search for primate T-lymphotropic virus type 4 in highly exposed human populations and gorillas from Central Africa

The primate T Lymphotropic viruses, which include HTLV-1/STLV-1, HTLV-2/STLV-2, HTLV-3/STLV-3 and HTLV-4/STLV-4 constitute a group of related human and simian retroviruses which share some common epidemiological, biological and molecular features. While HTLV-1 and HTLV-2 are relatively widespread, HTLV-3 has only been observed in few African individuals living in close contact with infected non-human primates (NHPs) and HTLV-4 has been identified in only one hunter living in South Cameroon. Recently, a molecular study conducted in Cameroon on 1107 NHPs of 21 different species detected and characterized STLV-4 infection in 6 gorillas. Therefore HTLV-4 could have emerged from a gorilla reservoir. In the context of our current ongoing studies on PTLV in Cameroon, we decided to look for PTLV-4 in a large series of blood DNA originating from persons at risk for such infection (hunters of NHPs), living in the same region than the index case, and also from several gorillas. HTLV WB serology was available for all the persons. We searched for HTLV-4/STLV-4 infection in humans and gorillas using two different nested PCR, one targeting the pol gene, the other one targeting the LTR. These methods were able to detect 1 copy in a background of 250 ng of DNA. We included in the first preliminary study: 1) 228 individuals from Cameroon that had reported a contact (mostly bite) with a NHP (80 of them specifically with a gorilla); 2) 21 gorillas (16 from Cameroon, 2 from Gabon and 3 from European zoos). Out of the 21 gorillas, one originating from Cameroon was found positive for both STLV-4 pol and LTR PCR. The screening of the human samples is still in progress and will be presented.

Discovery and characterization of auxiliary proteins encoded by type 3 simian T-cell lymphotropic viruses.

Human T-cell lymphotropic virus type 1 (HTLV-1) and HTLV-2 encode auxiliary proteins that play important roles in viral replication, viral latency, and immune escape. The presence of auxiliary protein-encoding open reading frames (ORFs) in HTLV-3, the latest HTLV to be discovered, is unknown. Simian T-cell lymphotropic virus type 3 (STLV-3) is almost identical to HTLV-3. Given the lack of HTLV-3-infected cell lines, we took advantage of STLV-3-infected cells and of an STLV-3 molecular clone to search for the presence of auxiliary transcripts. Using reverse transcriptase PCR (RT-PCR), we first uncovered the presence of three unknown viral mRNAs encoding putative proteins of 5, 8, and 9 kDa and confirmed the presence of the previously reported RorfII transcript. The existence of these viral mRNAs was confirmed by using splice site-specific RT-PCR with ex vivo samples. We showed that p5 is distributed throughout the cell and does not colocalize with a specific organelle. The p9 localization is similar to that of HTLV-1 p12 and induced a strong decrease in the calreticulin signal, similarly to HTLV-1 p12. Although p8, RorfII, and Rex-3 share an N-terminal sequence that is predicted to contain a nucleolar localization signal (NoLS), only p8 is found in the nucleolus. The p8 location in the nucleolus is linked to a bipartite NoLS. p8 and, to a lesser extent, p9 repressed viral expression but did not alter Rex-3-dependent mRNA export. Using a transformation assay, we finally showed that none of the STLV-3 auxiliary proteins had the ability to induce colony formation, while both Tax-3 and antisense protein of HTLV-3 (APH-3) promoted cellular transformation. Altogether, these results complete the characterization of the newly described primate T-lymphotropic virus type 3 (PTLV-3). Together with their simian counterparts, HTLVs form the primate T-lymphotropic viruses. HTLVs arose from interspecies transmission between nonhuman primates and humans. HTLV-1 and HTLV-2 encode auxiliary proteins that play important roles in viral replication, viral latency, and immune escape. The presence of ORFs encoding auxiliary proteins in HTLV-3 or STLV-3 genomes was unknown. Using in silico analyses, ex vivo samples, or in vitro experiments, we have uncovered the presence of 3 previously unknown viral mRNAs encoding putative proteins and confirmed the presence of a previously reported viral transcript. We characterized the intracellular localization of the four proteins. We showed that two of these proteins repress viral expression but that none of them have the ability to induce colony formation. However, both Tax and the antisense protein APH-3 promote cell transformation. Our results allowed us to characterize 4 new retroviral proteins for the first time.

Inhibition of Tax transformation activity using a small molecule targeting Tax/PDZ domain interactions

Primate T-lymphotropic virus species comprise four members (HTLV-1 to -4) that have been discovered in human. Only the HTLV-1 infection leads to adult T-cell leukemia/lymphoma (ATLL). All the four viruses share a similar genomic organization and encode transforming Tax oncoproteins. In contrast to HTLV-2 and 4, HTLV-1 and 3 Tax proteins contain a PSD-95/Drosophila Discs Large/Zona Occludens-I (PDZ) binding motif at their C-terminal that has been shown to play crucial roles in the distinct transforming properties of the Tax proteins. Here, we used a collection of human full-length protein-coding open reading frames (ORFeome v3.1) to identify novel PDZ domain containing proteins that specifically interact with HTLV-1 Tax. Novel Tax interactors include syntenin-1 and -2, LNX2, DVL3, GIPC2, INTU, PDLIM4 and -7, RADIL and RGS3. These proteins are involved in diverse biological processes including cell division, cell fate determination, and cell survival. We further characterized interaction between Tax and syntenins and showed that, FJ9 a small molecule able to disrupt Tax/PDZ interactions, could antagonize Tax-transformation activity in a Rat-1 model. Our study identify novel PDZ-containing proteins interacting with HTLV-1 Tax and provides the first example where Tax protein-protein interactions with PDZ containing proteins and Tax-transformation capacity could be inhibited by a small molecule.

Diversity of STLV-1 strains in wild chimpanzees ( Pan troglodytes verus) from Côte d’Ivoire

Simian T-lymphotropic viruses type 1 (STLV-1) are regarded as a highly conserved group of viruses with genotypes clustering according to geographic regions rather than to infected species. In free living West African chimpanzees we have described a variety of STLV-1 strains and suggested that this diversity results from interspecies transmissions. Here we present new data on STLV-1 infections in these chimpanzees with the presence of two new distinct clades, proposing the establishing of two new STLV-1 subtypes. Moreover, in one of the chimpanzees, the Central African STLV-1 subtype B was detected. The STLV-1 strains detected here display a much wider diversity than heretofore reported for STLV-1 with presence of three distinct subtypes in chimpanzees from one distinct geographic region. In conclusion, the hypothesis of primate T-lymphotropic virus type 1 (PTLV-1) clustering by geography rather than host should be reconsidered, at least regarding STLV-1 infections in chimpanzees.

Healthy hunting in central Africa

People in contact with wild animals, and particularly nonhuman primates, are acutely at risk of zoonosis and should take steps to avoid infections. Despite only limited testing, we already know that more than 1% of rural Cameroonians are infected with wild primate T-lymphotropic viruses (PTLVs; Wolfe et al., 2005) and another 1% are infected with wild primate spumaviruses or simian foamy virus (SFV; Wolfe et al., 2004). In south-east Asia, a preliminary risk assessment found that about five in 1000 temple visitors risk infections with SFV from bites from temple macaques (Engel et al., 2006). There are also numerous cases of accidental laboratory infections with non-human primate pathogens such as PTLV, SFV, simian immunodeficiency virus (SIV) and others. Cross-species transmission of non-human primate pathogens to humans are not rare events; however, to avoid infections simple steps can be followed and such steps have been elaborated for laboratory workers (Guidelines to prevent simian immunodeficiency virus infection in laboratory workers and animal handlers, http://www.cdc.gov/mmwr/ preview/mmwrhtml/00001303.htm) and in rules and signs for people visiting tourist sites where interactions with monkeys occur (Fuentes, 2006). For people living in rural central Africa, such information is obviously more difficult to come by despite the high number of exposed individuals and the diversity of pathogens from a diverse number of wild primates. While the risk of infection is apparent, the health implications, for at least some of the viruses, are less clear. We do know that the ebola and monkeypox viruses, which can be transmitted through hunting and butchering of wild animals, have serious health effects. Additional research is however urgently needed to determine the effects of a number of other viruses, in particular the diverse PTLVs, one of which human T-Lymphotropic virus 1 (HTLV-1) can cause a form of blood cancer and inflammatory disease in 2–5% of people infected. Given that little is known about many of these pathogens and their effects, the threat posed by an infection may not outweigh the potential value of bushmeat as a food source or as a source of revenue to a hunter or butcher (J. Brashares, this vol.; M. C. Monroe & A. S. Willcox, this vol.). However, some methods for avoiding infections are not costly. For example, simple and easy-to-practise measures (M. C. Monroe & A. S. Willcox, this vol.) include avoiding contact between open wounds and animal blood and washing any bites, scratches or knife injuries. Of course, the best way to avoid infection with wildlife pathogens is by avoiding contact with wild animals altogether, an option with a high cost that will not be adopted in many villages in southern Cameroon given the low level of domestic animal meat production and the lack of dietary replacements (Koppert et al., 1993). The avoidance of certain species is a second option, with animals of any species found dead in the forest being the most important as they may transmit acute and deadly pathogens such as Ebola or anthrax. Nonhuman primates, in particular the great apes, would also be an important group to avoid as they share many pathogens with humans given our similar physiologies. As non-human primates rarely make up the majority of animals hunted at any one locality (rodents and antelopes are much more regularly captured in central Africa), avoiding primates does not necessarily have to be so costly as the hunting effort can be redirected into hunting of other species. Such a redirection of effort would benefit the conservation of the threatened non-human primates, although the redirection may affect the sustainability of hunting other species. It is important to ensure that the hunting of threatened species is not inadvertently encouraged or legitimized by promoting healthy hunting (P. Daszak, this vol.; M. C. Monroe & A. S. Willcox, this vol.). Some people may even use this as an argument against communicating healthy hunting information altogether, as has occurred with other programmes of harm minimization for illegal activities such

Implications of the evolution pattern of human T-cell leukemia retroviruses on their pathogenic virulence (Review)

Simian retroviruses pose a serious threat to public health, as two human pathogenic retroviruses, HIV and HTLV, have been already proved to originate from such non-human viruses. Therefore, studying their natural prevalence among wild non-human primates is important for planning strategies to prevent the emergence of additional human retroviral pathogens. This article is focused on tracing the origin and evolution of the human T-cell leukemia viruses HTLV-I and HTLV-II in comparison to that of the simian lymphotropic viruses STLV-I, STLV-II and STLV-L, which are phylo-genically classified into a common group called primate T-lymphotropic viruses (PTLV). Thus, HTLV-I and STLV-I are referred to as PTLV-I and HTLV-II and STLV-II as PTLV-II, whereas STLV-L, which is highly divergent from both HTLV types, comprises a third subgroup called PTLV-L. The phylogeny of PTLV indicates that both, HTLV-I and HTLV-II emerged from a simian origin, but their subsequent evolution continued in different patterns. HTLV-I includes 6 subtypes which evolved from STLV-I through several times of different geographic interspecies transmission between simian and human hosts. These repeated invasions to new primate species are likely to give rise to viral strains with increasing pathogenic potential. On the other hand, HTLV-II includes 4 subtypes which appear to originate from a common human ancestor virus that emerged from only one simian to human transmission, whereas the subsequent evolution of HTLV-II and STLV-II strains continued separately only within the Homo sapiens and Pan paniscus species respectively, without repeated interspecies jumps. Such evolution pattern likely involves less genetic changes and selection of viral strains with low pathogenic virulence that could co-exist with their hosts for long time. These different evolution patterns can explain the much wider implication of HTLV-I with human clinical disorders than HTLV-II. Of note, however, more recently HTLV-II started spreading much more rapidly through intravenous drug users to many geographical regions, with a 150-350 fold higher mutation rate than that of its previous strictly endemic strains. This change in the mode of the virus spread creates a serious risk for emergence of HTLV-II strains with higher virulence.

Characterization of a new simian T-lymphocyte virus type 1 (STLV-1) in a wild living chimpanzee (Pan troglodytes verus) from Ivory Coast: evidence of a new STLV-1 group?

A new strain of simian T-lymphotropic virus type 1 in blood samples from a chimpanzee that lived in the tropical rainforest of Ivory Coast is described. The sequence obtained from the long terminal repeat region of the genome is significantly divergent from all known human and nonhuman primate T-lymphotropic virus type 1 strains (963% homology to the closest related strains from Central African subtype B) and clusters with none of the established clades. The tax sequences reveal two sequence differences that seem to be unique as they are not found in any of the HTLV-1 or STLV-1 tax sequences from the public databases.

Complete sequence of a novel highly divergent simian T-cell lymphotropic virus from wild-caught red-capped mangabeys (Cercocebus torquatus) from Cameroon: a new primate T-lymphotropic virus type 3 subtype.

Among 65 samples obtained from a primate rescue center located in Cameroon, two female adult red-capped mangabeys (Cercocebus torquatus) (CTO-602 and CTO-604), of wild-caught origin, had a peculiar human T-cell lymphotropic virus type 2 (HTLV-2)-like Western blot seroreactivity (p24, RGD21, +/-K55). Analyses of the simian T-cell lymphotropic virus type 3 (STLV-3)/CTO-604 complete proviral sequence (8,919 bp) indicated that this novel strain was highly divergent from HTLV-1 (60% nucleotide similarity), HTLV-2 (62%), or STLV-2 (62%) prototypes. It was, however, related to STLV-3/PH-969 (87%), a divergent STLV strain previously isolated from an Eritrean baboon. The STLV-3/CTO-604 sequence possesses the major open reading frames corresponding to the structural, enzymatic, and regulatory proteins. However, its long terminal repeat is shorter, with only two 21-bp repeats. Furthermore, as demonstrated by reverse transcriptase PCR, this new STLV exhibits significant differences from STLV-3/PH-969 at the mRNA splice junction position level. In all phylogenetic analyses, STLV-3/CTO-604 and STLV-3/PH-969 clustered in a highly supported single clade, indicating an evolutionary lineage independent from primate T-lymphotropic virus type 1 (PTLV-1) and PTLV-2. Nevertheless, the nucleotide divergence between STLV-3/PH-969 and STLV-3/CTO-604 is equivalent to or higher than the divergence observed between the different HTLV-1 or HTLV-2 subtypes. Thus, the STLV-3/CTO-604 strain can be considered the prototype of a second subtype in the PTLV-3 type. The presence of two related viruses in evolutionarily distantly related African monkeys species, living in two opposite ecosystems (rain forest versus desert), reinforces the possible African origin of PTLV and opens new avenues regarding the search for a possible human counterpart of these viruses in individuals exhibiting such HTLV-2-like seroreactivities.

Absence of evidence of infection with divergent primate T-lymphotropic viruses in United States blood donors who have seroindeterminate HTLV test results.

Recent identification of divergent simian or primate T-lymphotropic viruses (STLVs; PTLVs) in bonobos (formerly called pygmy chimpanzees; Pan paniscus; viruses: STLVpan-p and STLVpp1664) and a baboon (Papio hamadryas; viruses: STLVph969 or PTLV-L) have raised the possibility of human infection with these viruses. Divergent PTLV-infected primate sera show p24 bands on HTLV-I Western blots (WBs). It was investigated whether infection by divergent PTLV-like viruses could explain a subset of United States blood donors who reacted on HTLV-I EIAs and had indeterminate HTLV-I WBs with p24 bands. Epidemiologic characteristics of 1889 donors with HTLV-I-indeterminate WBs were compared to those of donors with confirmed retrovirus infections (393 with HIV, 201 with HTLV-I, 513 with HTLV-II) and 1.6 million donors with nonreactive screening tests. To directly probe for infection with divergent PTLVs, 2 HTLV-I-indeterminate donors born in Africa and 269 representative non-African-born donors with p24 bands on HTLV-I WBs (previously shown to be negative for HTLV-I and -II DNA by PCR) were selected for PTLV PCR analysis. DNA from peripheral blood MNC samples was tested for a proviral tax sequence by PCR using generic primers that amplify HTLV-I, HTLV-II, and the divergent PTLVs. Amplified tax sequences were detected by Southern blot hybridization to a (32)P-labeled generic PTLV probe. PCR-positive samples could then be typed by hybridization with virus-specific internal probes that differentiate HTLV-I, HTLV-II, PTLV-L, and STLVpan-p. In the epidemiologic analysis, HTLV-indeterminate status was independently associated with age of at least 25 years (OR = 2.19; 95% CI, 1.93-2.49), black (OR = 3.27; 95% CI, 2.90-3.67) or Hispanic (OR = 1.82; 95% CI, 1.52-2.16) race or ethnicity, and donation at one blood center (Baltimore) (OR = 1. 30; 95% CI, 1.11-1.53). None of the 271 HTLV-I WB-indeterminate samples tested positive by generic PTLV PCR analysis. Although the epidemiologic data suggest the possibility of undiagnosed HTLV-I, HTLV-II, or a cross-reactive virus such as PTLV among older, black, and Hispanic blood donors, the PCR data do not support the presence of divergent PTLV infection among US blood donors with HTLV-I-indeterminate results.

Tempo and mode of human and simian T-lymphotropic virus (HTLV/STLV) evolution revealed by analyses of full-genome sequences.

We investigated the tempo and mode of evolution of the primate T-lymphotropic viruses (PTLVs). Several different models of nucleotide substitution were tested on a general phylogenetic tree obtained using the 20 full-genome HTLV/STLV sequences available. The likelihood ratio test showed that the Tamura and Nei model with discrete gamma-distributed rates among sites is the best-fitting substitution model. The heterogeneity of nucleotide substitution rates along the PTLV genome was further investigated for different genes and at different codon positions (cdp's). Tests of rate constancy showed that different PTLV lineages evolve at different rates when first and second cdp's are considered, but the molecular-clock hypothesis holds for some PTLV lineages when the third cdp is used. Negative selection was evident throughout the genome. However, in the gp46 region, a small fragment subjected to positive selection was identified using a Monte Carlo simulation based on a likelihood method. Employing correlations of the virus divergence times with anthropologically documented migrations of their host, a possible timescale was estimated for each important node of the PTLV tree. The obtained results on these slow-evolving viruses could be used to fill gaps in the historical records of some of the host species. In particular, the HTLV-I/STLV-I history might suggest a simian migration from Asia to Africa not much earlier than 19,500-60,000 years ago.

Phylogenetic Analysis of Simian T Lymphotropic Virus Type I from Kenyan Olive Baboons (Papio anubis), Lowland Sykes Monkeys (Cercopithecus mitis), and Vervet Monkeys (Cercopithecus aethiops pygerythrus)

781 H UM AN T CELL LEU KEM IA/lymphotropic virus type I (HTLVI) strains have a worldwide distribution and are characterized by the genetic stability of their genome and their limited horizontal transmission. Simian T cell leukemia/lymphotropic virus type I (STLV-I) strains have been found in Old World nonhuman primates in Africa and Asia. Together, these two virus groups are referred to as primate T lymphotropic viruses (PTLVs) and have been found in all three Old World African primate families: the Cercopithecidae, 2±4 the Pongidae, and the Hominidae. These viruses cannot be separate d into distinct phyloge netic lineages according to the fam ilies or species of origin, but rather accordi ng to geographical regions , which suggest s multiple interspe cies transm issions in the past. Although uncertain, it is thought that the origina l virus infected nonhu man simian ancesto rs that eventua lly infected humans. 2 Understan ding the sequence diversit y among STLV-I strains may therefor e provide valuable insight into the evolutio n of the PTLV-I group. In this study, we analyze d the HTLV-I sequenc e of 13 nonhum an primates, using HTLV-I cross-r eacting antibod ies provide d with the HTLV-I Serodia particle aggluti nation assay (Fujirebio , Tokyo, Japan). The animals were either colony born (Institu te of Primate Research [IPR], Nairobi, Kenya) or trapped in Kibwezi (eastern Kenya) and in Gilgil and Namanga (Rift Valley Province) . The animals include d four vervet monkeys (Cercopit hecus aethiop s pygerythrus ), two lowland Sykes monkeys (Cercopit hecus mitis), and seven olive baboons (Papio anubis ) (Table 1). We have focused on the diverge nt long terminal repeat (LTR) region because it is more inform ative for sequence analysi s and subtyping, and because of the availab ility of inform ation on other PTLV-I LTR sequenc es. DNA was isolated from peripheral blood mononuclear cells (PBMCs) using a QiaAmp blood kit (Qiagen GmbH, Hilden, Germany). The LTR region was amplified by the polymerase chain reaction (PCR) with primer pairs HFL1/HFL6 and HFL5/HFL6, as described. 11 PCR products were cloned using the PCR-Script SK( 1 ) cloning kit (Stratagene, La Jolla, CA) according to the manufacturer instructions. Sequencing was performed using the Applied Biosystem s model 373 automatic DNA sequencer (Applied Biosystems/Perkin-Elmer , Foster City, CA). Nucleotide sequences were aligned with CLUSTAL V together with reference sequences from the GenBank database. Kimura distance calculation, 13 bootstrap analysis, 14 and tree construction were done with the TREECON software package. Nucleotide sequences were submitted to GenBank under accession numbers AF117282±AF117294. Comparison of the LTR region of the strains revealed that the elements that are critical for viral gene expression were well conserved. No nucleotide changes were observed in the polyadenylation signal (AATAAA), the TATA box (TATAAA), and the splice donor site (TAGGTAA). In the tree containing the LTR sequence (Fig. 1), STLV and HTLV-I clusters were identified as containing sequences representative of many different subtypes.

ESTIMATION OF THE DIVERGENCE TIME FOR THE MAJOR HUMAN AND SIMIAN T-LYMPHOTROPIC VIRUS (HTLV/STLV) LINEAGES BY ANALYSING FULL GENOME SEQUENCES.

P168 We investigated the tempo of evolution of the human and simian T-lymphotropic viruses (HTLV/STLVs). A general phylogenetic tree was obtained using the 20 full genome HTLV/STLV sequences available. As expected the tree can be divided a primate T-lymphotropic virus type I (PTLV-I) part, joining all the HTLV-I strains together with the STLV-I strain isolated from a macaque in Indonesia and a primate T-lymphotropic virus type II (PTLV-II) part, joining the HTLV-II strains together with the STLV-II strains from African pygmy chimpanzees. The third major lineage, STLV-L, isolated from an African baboon, is almost equidistant from PTLV-I and 2PTLV-II. Using BLV as outgroup and comparing several phylogenetic methods, the root of the PTLVs tree can be placed on the branch leading to PTLV-I. Correlations of the virus divergence times with anthropologically documented migrations of their host allowed us to set a time scale for each important node of the tree. The origin of the primate T-lymphotropic virus can be placed around 300,000-1,000,000 years ago. STLV-L diverged from the PTLV-II around 280,000-980,000, years ago, whereas HTLV-II separated from STLV-II 115,000 to 403,000 years ago, suggesting an ancient interspecies transmission. STLV-I TE4 strain diverged from the other PTLV-I strains 35-70,000 years ago, somewhere in Asia, whereas the simian ancestors of HTLV-Ib and HTLV-Ia African and Cosmopolitan subtypes diverged 9,6-19,200 years ago, a date which represents the explosive spread of PTLV-I in Africa. In particular the HTLV-I/STLV-I history suggests a simian migration from Asia to Africa between 9,600 and 60,000 years ago for which evidences could be investigated in the archaeological records.

Complete nucleotide sequence of the new simian T-lymphotropic virus, STLV-PH969 from a Hamadryas baboon, and unusual features of its long terminal repeat.

A third type of primate T-lymphotropic virus, PTLV-L, with STLV-PH969 as a prototype, has recently been isolated from an African baboon (Papio hamadryas). Classification of this virus has been based on partial sequence analysis of cDNA from a virus-producing cell line, PH969. We obtained the complete nucleotide sequence of this virus with a proviral genome of 8,916 bp. All major genes, homologous in all human T-cell lymphotropic virus (HTLV)-related viruses, and their corresponding mRNAs, including appropriate splicing, were identified. One additional nonhomologous open reading frame in the proximal pX region is accessible for translation through alternative splicing. Sequence comparison shows that STLV-PH969 is equidistantly related to HTLV type 1 (HTLV-1) and HTLV-2. In all coding regions, the similarity tends to be the lowest between STLV-PH969 and HTLV-1. However, in the long terminal repeat (LTR) region, the lowest similarity was found between STLV-PH969 and HTLV-2. The U3-R and R-U5 boundaries of the STLV-PH969 LTR were experimentally determined at nucleotides 268 and 524, respectively. This 695-bp LTR is 60 and 73 bp shorter than the LTRs of HTLV-1 and HTLV-2, respectively, but its general organization is similar to the one found in the HTLV-bovine leukemia virus genus. In the long region between the polyadenylation signal and the poly(A) site, sequence similarity with the HTLV-1 Rex-responsive element (RexRE) core and secondary structure prediction suggest the presence of a RexRE. The presence of three 21-bp repeats is conserved within the U3 region of HTLV-1, HTLV-2, and BLV. Only two direct repeats with similarity to these Tax-responsive elements were found in the STLV-PH969 LTR, which might suggest differences in the Tax-mediated transactivation of this virus. We conclude that STLV-PH969 has all the genes and genomic regions to suggest a replication cycle comparable to that of HTLV-1 and HTLV-2.

The presence of a divergent T-lymphotropic virus in a wild-caught pygmy chimpanzee (Pan paniscus) supports an African origin for the human T-lymphotropic/simian T-lymphotropic group of viruses

We isolated a divergent simian T-lymphotropic virus (STLV) (strain PP1664) from a wild-caught African bonobo (pygmy chimpanzee, Pan paniscus). Molecular and phylogenetic characterization of this virus show that it reliably separates from the two well-established primate T-lymphotropic virus types, HTLV-I/STLV-I (PTLV-I) and PTLV-II, and from a third type isolated from an African-born Papio hamadryas and designated by us as PTLV-L. Four of eight bonobos kept at the Antwerp Zoo, Belgium, showed an aberrant PTLV serology. We amplified and sequenced a 709 bp PTLV proviral tax/rex fragment from one of the reactive bonobos. It differs by about 25 % from the homologous nucleotide sequences of PTLV-I and PTLV-L and by about 17 % from PTLV-II. This is comparable to the differences among the three known types. Including the most divergent STLV-I strains sequenced to date, for example, strain PHSu1 sequenced here, the divergence in this region within PTLV-I is less than 11 % and within PTLV-II less than 4%. Although very divergent, this new bonobo STLV is the closest well-characterized simian relative of HTLV-II, raising the possibility of very divergent new HTLV strains. Our results show that the number of PTLV types should be considered open and that the variety of indigenous viruses in the PTLV group is greatest in Africa. Thus, as for the other primate retroviruses HIV and SIV, PTLV most probably has its origins in Africa.

Questions on the evolution of primate T-lymphotropic viruses raised by molecular and epidemiological studies of divergent strains.

In human and simian T-lymphotropic viruses (HTLV and STLV), collectively referred to as primate T-lymphotropic viruses (PTLV), four distinct clades can be distinguished: PTLV-I, PTLV-II, and the newly discovered divergent STLVs isolated from hamadryas baboons and from bonobos (pygmy chimpanzees). The hamadryas STLV is clearly distinct from types I and II, in terms both of sequence divergence and of genomic structure, and would qualify as a separate type, provisionally called PTLV-L. The bonobo STLV is closer to, although clearly distinct from, PTLV-II, at present known only in humans. While PTLV-II, PTLV-L, and the bonobo STLV appear presently to be species specific, PTLV-I has spread during its evolution through repeated interspecies transmissions between primates and is now present in many species of Old World monkeys and apes and in humans. The human subtypes of PTLV-I arose from at least three acquisitions from separate simian reservoirs.

The widening spectrum of primate T-lymphotropic viruses

The first identified and most widespread of the Primate Tlymhotropic viruses (PTLV) are the human HTLV-I and simian STLV-I, present in a large number of Old World monkey and ape species. The fact that molecular genetic differences are more related to geographic origin than to host species indicate several interspecies transmissions among primates including man. Genomic analysis indicates two main groups, an African and an Australo-Asian, with long independent evolutions (t). The African group contains two HTLV-I clusters, a cosmopolitan, which spread over the world population, and a more localized central African cluster. In Africa many STLV-I group with the one or the other of these clusters, but very divergent STLV-I are also present, e.g. the STLVTAN90. The Australo-Asian group consists of the Melanesian HTLV-I and a number of divergent Asian STLV-I. The second PTLV is represented by HTLV-II. First thought to be a typical New World virus HTLV-II has now also been detected in autochtonous populations of Africa (2,3) and Mongolia. We identified a new type of PTLV, provisionally named PTLV-L, in a hamadryas baboon of north-eastern Africa (4). Its differences in nucleotide sequences from the types I and II are similar to that between the two known types. The genome organization of the pX region of the genome also differs from the other types (5). Recently we isolated another divergent PTLV from bonobos (pygmy chimpanzees), a species only living on the left bank of the Zaire river (6). At present the analysis of part of its genome in the env and pX regions indicates that it is more related to HTLV-II than to the other types, but is still very divergent from HTLV-II (7). The picture of PTLV that is emerging is that of slowly evolving viruses who have been present in Old World simians during much of their evolution. In the present view PTLVI would appear as the more successful type which spread to many species, and HTLV-II as the most specifically human PTLV.

Primate T-Lymphotropic Virus Type I LTR Sequence Variation and Its Phylogenetic Analysis: Compatibility with an African Origin of PTLV-I

Due to the low evolutionary rate and the limited horizontal transmission of human T-lymphotrophic virus type I (HTLV-I), its phylogenetic analysis reveals the movements and contacts of ancient populations. Since simian strains cannot be distinguished from human strains by phylogenetic criteria, this virus has appropriately been called primate T-lymphotropic virus type I (PTLV-I). We sequenced the LTR of six PTLV-I strains: three HTLV-I strains from African patients with tropical spastic paraparesis (TSP) (Equateur, Zaire), two laboratory HTLV-I strains of Japanese origin, MT-2 and MT-4, and one STLV-I from a baboon of the primate center in Sukhumi, Georgia. We applied four phylogenetic inference methods: neighbor-joining (NJ), unweighted pair group method using arithmetic averages (UPGMA), Fitch and Wagner parsimony (pars), and maximum likelihood (ML), to these 6 LTR sequences and 18 published LTR sequences (cosmopolitan, African, and Melanesian HTLV-I strains and African and Asian STLV-I strains). Three major HTLV-I subtypes can be identified with all four methods: the cosmopolitan HTLV-Ia, the central African HTLV-Ib, clearly descendant from a STLV-I CH-like African ancestral simian strain, and the Melanesian HTLV-Ic, probably descendant from an Asian STLV-I strain. We observe a segregation of PTLV-I sequences according to their geographical origin and not according to host species. The Zairean strains form a cluster closely related to an STLV-I strain isolated from a chimpanzee (STLV-I CH) and distinct from western African strains, which belong to the cosmopolitan subtype of HTLV-I. The Sukhumi STLV-I strain found in a captive-born baboon was of Asian descent. We experienced rooting problems with UPGMA when using HTLV-II as an outgroup. Concordant results with all four methods were obtained by eliminating HTLV-II LTR sequence fragments with bad alignment to HTLV-I. This resulted in a HTLV-II root node on the African STLV-I TAN9O terminal branch (with bootstrap values above 92% for the NJ and pars methods) and not on the Asian STLV-I PtM3 branch, as has been derived by others based on their use of UPGMA. The results of the analyses also support a higher evolutionary rate of PTLV-I in Asia, implying that the trees obtained with the NJ and ML methods have a higher reliability. These results are more compatible with an ancient African origin of PTLV-I than with an Asian origin.

A primate T-lymphotropic virus, PTLV-L, different from human T-lymphotropic viruses types I and II, in a wild-caught baboon (Papio hamadryas).

Searching for clues to the evolution of the primate T-lymphotropic viruses (PTLVs), which include the human and the simian T-lymphotropic viruses (HTLV and STLV), we have identified another PTLV, which differs sufficiently from the known PTLV-I and PTLV-II types to be designated here PTLV-L. The virus was isolated from a wild-born baboon (Papio hamadryas) from Eritrea. In a cDNA library a 1802-bp-long fragment was identified that extends from the env region, including the complete transmembrane protein gene, to part of the tax/rex gene. Homologies at the nucleotide sequence level of PTLV-L, prototype simian T-lymphotropic virus-PH969, with HTLV-I and -II, respectively, were 62% and 64% overall, 65% and 70% in the env region, and 80% and 80% in the partial tax/rex sequence. In the 5' part of the pX region a significant homology was seen only with HTLV-II (52%). Phylogenetic analysis based on the gene encoding the transmembrane protein indicates that PTLV-L represents a PTLV type with a long independent evolution, longer than any strain within the PTLV-I or PTLV-II groups. The finding of another PTLV type in African baboons is further evidence of the wide variety of PTLV found on this continent. Whether PTLV-L resembles PTLV-I and PTLV-II in the extension of its host range to other primates, including humans, remains to be seen.