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SIVs infecting wild-living apes in west central Africa have crossed the species barrier to humans on at least four different occasions, one of which spawned the AIDS pandemic. Although the chimpanzee precursor of pandemic HIV-1 strains must have been able to infect humans, the capacity of SIVcpz strains to replicate in human lymphoid tissues (HLTs) is not known. Here, we show that SIVcpz strains from two chimpanzee subspecies are capable of replicating in human tonsillary explant cultures, albeit only at low titers. However, SIVcpz replication in HLT was significantly improved after introduction of a previously identified human-specific adaptation at position 30 in the viral Gag matrix protein. An Arg or Lys at this position significantly increased SIVcpz replication in HLT, while the same mutation reduced viral replication in chimpanzee-derived CD4(+) T cells. Thus, naturally occurring SIVcpz strains are capable of infecting HLTs, the major site of HIV-1 replication in vivo. However, efficient replication requires the acquisition of a host-specific adaptation in the viral matrix protein. These results identify Gag matrix as a major determinant of SIVcpz replication fitness in humans and suggest a critical role in the emergence of HIV/AIDS.

Understanding HIV-1 evolution is crucial for tracing its ape origin as well as considering genetic variation within and among hosts. Here, we review the current evidence documenting cross-species transmission from chimpanzees and gorillas to human. First, each HIV-1 groups M, N, O and P arose through 4 different cross-species transmissions of SIVcpz and SIVgor. Second, in vitro studies demonstrate that SIVcpz and SIVgor have many of the biological properties necessary for establishing a persistent infection in humans.

Chimpanzees and gorillas are the only nonhuman primates known to harbor viruses closely related to HIV-1. Phylogenetic analyses showed that gorillas acquired the simian immunodeficiency virus SIVgor from chimpanzees, and viruses from the SIVcpz/SIVgor lineage have been transmitted to humans on at least four occasions, leading to HIV-1 groups M, N, O, and P. To determine the geographic distribution, prevalence, and species association of SIVgor, we conducted a comprehensive molecular epidemiological survey of wild gorillas in Central Africa. Gorilla fecal samples were collected in the range of western lowland gorillas (n = 2,367) and eastern Grauer gorillas (n = 183) and tested for SIVgor antibodies and nucleic acids. SIVgor antibody-positive samples were identified at 2 sites in Cameroon, with no evidence of infection at 19 other sites, including 3 in the range of the Eastern gorillas. In Cameroon, based on DNA and microsatellite analyses of a subset of samples, we estimated the prevalence of SIVgor to be 1.6% (range, 0% to 4.6%), which is significantly lower than the prevalence of SIVcpzPtt in chimpanzees (5.9%; range, 0% to 32%). All newly identified SIVgor strains formed a monophyletic lineage within the SIVcpz radiation, closely related to HIV-1 groups O and P, and clustered according to their field site of origin. At one site, there was evidence for intergroup transmission and a high intragroup prevalence. These isolated hot spots of SIVgor-infected gorilla communities could serve as a source for human infection. The overall low prevalence and sporadic distribution of SIVgor could suggest a decline of SIVgor in wild populations, but it cannot be excluded that SIVgor is still more prevalent in other parts of the geographical range of gorillas.

Vpu proteins of pandemic HIV-1 M strains degrade the viral receptor CD4 and antagonize human tetherin to promote viral release and replication. We show that Vpus from SIVgsn, SIVmus, and SIVmon infecting Cercopithecus primate species also degrade CD4 and antagonize tetherin. In contrast, SIVcpz, the immediate precursor of HIV-1, whose Vpu shares a common ancestry with SIVgsn/mus/mon Vpu, uses Nef rather than Vpu to counteract chimpanzee tetherin. Human tetherin, however, is resistant to Nef and thus poses a significant barrier to zoonotic transmission of SIVcpz to humans. Remarkably, Vpus from nonpandemic HIV-1 O strains are poor tetherin antagonists, whereas those from the rare group N viruses do not degrade CD4. Thus, only HIV-1 M evolved a fully functional Vpu following the three independent cross-species transmissions that resulted in HIV-1 groups M, N, and O. This may explain why group M viruses are almost entirely responsible for the global HIV/AIDS pandemic.

Western lowland gorillas (Gorilla gorilla gorilla) are infected with a simian immunodeficiency virus (SIVgor) that is closely related to chimpanzee and human immunodeficiency viruses (SIVcpz and HIV-1, respectively) in west central Africa. Although existing data suggest a chimpanzee origin for SIVgor, a paucity of available sequences has precluded definitive conclusions. Here, we report the molecular characterization of one partial (BQ664) and three full-length (CP684, CP2135, and CP2139) SIVgor genomes amplified from fecal RNAs of wild-living gorillas at two field sites in Cameroon. Phylogenetic analyses showed that all SIVgor strains clustered together, forming a monophyletic lineage throughout their genomes. Interestingly, the closest relatives of SIVgor were not SIVcpzPtt strains from west central African chimpanzees (Pan troglodytes troglodytes) but human viruses belonging to HIV-1 group O. In trees derived from most genomic regions, SIVgor and HIV-1 group O formed a sister clade to the SIVcpzPtt lineage. However, in a tree derived from 5' pol sequences ( approximately 900 bp), SIVgor and HIV-1 group O fell within the SIVcpzPtt radiation. The latter was due to two SIVcpzPtt strains that contained mosaic pol sequences, pointing to the existence of a divergent SIVcpzPtt lineage that gave rise to SIVgor and HIV-1 group O. Gorillas appear to have acquired this lineage at least 100 to 200 years ago. To examine the biological properties of SIVgor, we synthesized a full-length provirus from fecal consensus sequences. Transfection of the resulting clone (CP2139.287) into 293T cells yielded infectious virus that replicated efficiently in both human and chimpanzee CD4(+) T cells and used CCR5 as the coreceptor for viral entry. Together, these results provide strong evidence that P. t. troglodytes apes were the source of SIVgor. These same apes may also have spawned the group O epidemic; however, the possibility that gorillas served as an intermediary host cannot be excluded.

It is now well established that the clade of simian immunodeficiency viruses (SIVs) infecting west central African chimpanzees (Pan troglodytes troglodytes) and western gorillas (Gorilla gorilla gorilla) comprises the progenitors of human immunodeficiency virus type 1 (HIV-1). In this study, we have greatly expanded our previous molecular epidemiological survey of SIVcpz in wild chimpanzees in Cameroon. The new results confirm a wide but uneven distribution of SIVcpzPtt in P. t. troglodytes throughout southern Cameroon and indicate the absence of SIVcpz infection in Pan troglodytes vellerosus. Analyzing 725 fecal samples from 15 field sites, we obtained partial nucleotide sequences from 16 new SIVcpzPtt strains and determined full-length sequences for two of these. Phylogenetic analyses of these new viruses confirmed the previously reported phylogeographic clustering of SIVcpzPtt lineages, with viruses related to the ancestors of HIV-1 groups M and N circulating exclusively in southeastern and south central P. t. troglodytes communities, respectively. Importantly, the SIVcpzPtt strains from the southeastern corner of Cameroon represent a relatively isolated clade indicating a defined geographic origin of the chimpanzee precursor of HIV-1 group M. Since contacts between humans and apes continue, the possibility of ongoing transmissions of SIV from chimpanzees (or gorillas) to humans has to be considered. In this context, our finding of distinct SIVcpzPtt envelope V3 sequence clades suggests that these peptides may be useful for the serological differentiation of SIVcpzPtt and HIV-1 infections, and thus the diagnosis of new cross-species transmissions if they occurred.

Human immunodeficiency virus type 1 (HIV-1) originated from three independent cross-species transmissions of simian immunodeficiency virus (SIVcpzPtt) infecting chimpanzees (Pan troglodytes troglodytes) in west central Africa, giving rise to pandemic (group M) and non-pandemic (groups N and O) clades of HIV-1. To identify host-specific adaptations in HIV-1 we compared the inferred ancestral sequences of HIV-1 groups M, N and O to 12 full length genome sequences of SIVcpzPtt and four of the outlying but closely related SIVcpzPts (from P. t. schweinfurthii). This analysis revealed a single site that was completely conserved among SIVcpzPtt strains but different (due to the same change) in all three groups of HIV-1. This site, Gag-30, lies within p17, the gag-encoded matrix protein. It is Met in SIVcpzPtt, underwent a conservative replacement by Leu in one lineage of SIVcpzPts but changed radically to Arg on all three lineages leading to HIV-1. During subsequent diversification this site has been conserved as a basic residue (Arg or Lys) in most lineages of HIV-1. Retrospective analysis revealed that Gag-30 had reverted to Met in a previous experiment in which HIV-1 was passaged through chimpanzees. To examine whether this substitution conferred a species specific growth advantage, we used site-directed mutagenesis to generate variants of these chimpanzee-adapted HIV-1 strains with Lys at Gag-30, and tested their replication in both human and chimpanzee CD4+ T lymphocytes. Remarkably, viruses encoding Met replicated to higher titers than viruses encoding Lys in chimpanzee T cells, but the opposite was found in human T cells. Taken together, these observations provide compelling evidence for host-specific adaptation during the emergence of HIV-1 and identify the viral matrix protein as a modulator of viral fitness following transmission to the new human host.

Studies of simian immunodeficiency viruses (SIVs) in their endangered primate hosts are of obvious medical and public health importance, but technically challenging. Although SIV-specific antibodies and nucleic acids have been detected in primate fecal samples, recovery of replication-competent virus from such samples has not been achieved. Here, we report the construction of infectious molecular clones of SIVcpz from fecal viral consensus sequences. Subgenomic fragments comprising a complete provirus were amplified from fecal RNA of three wild-living chimpanzees and sequenced directly. One set of amplicons was concatenated using overlap extension PCR. The resulting clone (TAN1.24) contained intact genes and regulatory regions but was replication defective. It also differed from the fecal consensus sequence by 76 nucleotides. Stepwise elimination of all missense mutations generated several constructs with restored replication potential. The clone that yielded the most infectious virus (TAN1.910) was identical to the consensus sequence in both protein and long terminal repeat sequences. Two additional SIVcpz clones were constructed by direct synthesis of fecal consensus sequences. One of these (TAN3.1) yielded fully infectious virus, while the second one (TAN2.69) required modification at one ambiguous site in the viral pol gene for biological activity. All three reconstructed proviruses produced infectious virions that replicated in human and chimpanzee CD4(+) T cells, were CCR5 tropic, and resembled primary human immunodeficiency virus type 1 isolates in their neutralization phenotype. These results provide the first direct evidence that naturally occurring SIVcpz strains already have many of the biological properties required for persistent infection of humans, including CD4 and CCR5 dependence and neutralization resistance. Moreover, they outline a new strategy for obtaining medically important "SIV isolates" that have thus far eluded investigation. Such isolates are needed to identify viral determinants that contribute to cross-species transmission and host adaptation.

Human immunodeficiency virus type 1 (HIV-1), the cause of human acquired immunodeficiency syndrome (AIDS), is a zoonotic infection of staggering proportions and social impact. Yet uncertainty persists regarding its natural reservoir. The virus most closely related to HIV-1 is a simian immunodeficiency virus (SIV) thus far identified only in captive members of the chimpanzee subspecies Pan troglodytes troglodytes. Here we report the detection of SIVcpz antibodies and nucleic acids in fecal samples from wild-living P. t. troglodytes apes in southern Cameroon, where prevalence rates in some communities reached 29 to 35%. By sequence analysis of endemic SIVcpz strains, we could trace the origins of pandemic (group M) and nonpandemic (group N) HIV-1 to distinct, geographically isolated chimpanzee communities. These findings establish P. t. troglodytes as a natural reservoir of HIV-1.

In order to understand primate lentivirus evolution, characterization of additional simian immunodeficiency virus (SIV) strains is essential. Here, an SIV from a black mangabey (Lophocebus aterrimus) originating from the Democratic Republic of Congo was analysed phylogenetically. The monkey had cross-reactive antibodies against human immunodeficiency virus type 1 (HIV-1) and HIV-2. The viral pol region sequence was amplified by nested PCR and sequence analysis confirmed that it was related to known SIV sequences. This is the first report to characterize genetically an SIV from the monkey genus Lophocebus. Phylogenetic analysis of the pol region revealed that this novel SIV, designated SIVbkm, fell into the SIVsyk and SIVgsn virus group, containing viruses isolated from the genus Cercopithecus, and suggests that cross-species transmission has occurred between species of the genera Lophocebus and Cercopithecus.

To monitor the presence of genotypic HIV-1 variants circulating in eastern Cameroon, blood samples from 57 HIV-1-infected individuals attending 3 local health centers in the bordering rural villages with Central African Republic (CAR) were collected and analyzed phylogenetically. Out of the 40 HIV-1 strains with positive polymerase chain reaction (PCR) profile for both gag and env-C2V3,12 (30.0%) had discordant subtype or CRF designation: 2 subtype B/A (gag/env), 1 B/CRF01, 2 B/CRF02, 1 CRF01/CRF01.A, 2 CRF11/CRF01, 1 CRF13/A, 1 CRF13/CRF01, 1 CRF13/CRF11, and 1 G/U (unclassified). Twenty-eight strains (70.0%) had concordant subtypes or CRF designation between gag and env: 27 subtype A and 1 F2. Out of the remaining 17HIV-1 strains negative for PCR with the env-C2V3 primers used, 10 (58.8%) had discordant subtype or CRF, and 7 (41.2%) had concordant one based on gag/pol/env-gp41 analysis. Altogether, a high proportion (22/57, 38.6%) of the isolates were found to be recombinant strains. In addition, an emergence of new forms of HIV-1 strains, such as subtype B/A (gag/env), B/CRF01 and B/CRF02, was identified. The epidemiologic pattern of HIV-1 in eastern Cameroon, relatively low and high prevalence of CRF02 and CRF11, respectively, was more closely related to those of CAR and Chad than that of other regions of Cameroon, where CRF02 is the most predominant HIV-1 strain. These findings strongly suggest that this part of Cameroon is a potential hotspot of HIV-1 recombination, with a likelihood of an active generation of new forms of HIV-1 variants, though epidemiologic significance of new HIV-1 forms is unknown.

To investigate the prevalence of subtypes A and C, and the existence of recombinants of both subtypes in the southeast of the Democratic Republic of Congo (DRC), blood samples were collected from 27 HIV-infected individuals in Likasi, located in an area bordering close to Zambia, and analyzed phylogenetically. Out of the 24 strains with a positive PCR profile for pol-IN and env-C2V3, 15 (62.5%) had a discordant subtype or CRF designation: one subtype A/G (pol/env), four A/U (unclassified), three G/A, one G/CRF01, three H/A, one J/C, one CRF02 (G)/A, and one U/A. Nine (37.5%) strains had a concordant subtype or CRF designation: five subtype A, two C, one D, and one CRF02/G. The remaining three samples negative for PCR with env-C2V3 primers used in this study were further analyzed with env-gp41 primers and revealed the presence of two profiles: two J/J (pol-IN/env-gp41) and one C/G. These data highlight the presence of a high proportion (16/27, 59.3%) of recombinant strains and a low prevalence (4.1 and 7.4%) of subtype C based on env-C2V3 and pol-IN analyses, respectively, in Likasi. In addition, this is the first report that CRF02_AG exists in DRC, though the epidemiological significance of the existence of CRF02_AG in DRC remains unknown.

To investigate the route of zoonotic transmission of HIV-1, we isolated three and seven HIV-1 strains from 449 Pygmy hunter gatherers and 169 neighboring Bantu, respectively, in southern Cameroon. Phylogenetic analysis based on pol-integrase and env-C2V3 sequences revealed that strains from Pygmies were 1CRF02_AG/CRF02_AG, 1 subtype G/CRF02 AG (pol/env), and 1 CRFll_cpx/CRF11_cpx, and that those from Bantu were 2 CRF02_AG/CRF02_AG, 1 CRF02_AG/CRF01_AE/A, 1 CRF02_AG/subtype A, 1 G/A, 1G/CRF02_AG, and 1 unclassified fH. CRF02_AG and CRF11_cpx have been identified in Cameroon. The results suggest that HIV-1 has been introduced into Pygmies through their neighboring Bantu rather than directly from nonhuman primates.

To investigate the changes of neutralizing antibodies and viruses during simian and human immunodeficiency virus (SHIV) infection, we examined the cross-neutralizing ability of sequential sera from three macaques infected with SHIV, NM-3rN, and analyzed the sensitivity of the reisolate to neutralizing antibodies. Neutralizing activities of macaques' sera against the parental HIV-1 showed a persistent increase. Neutralizing activities were highly strain specific, but the spectrum of the neutralizing activity expanded against various clades of primary HIV-1s at 3 years after infection in one of the three macaques. The reisolate from an NM3-rN-infected macaque at 56 wpi, designated as R4356, was neutralized by sera from this macaque at a much lower titer than NM-3rN, even by the sera collected 2 years after the reisolation. Sera from macaques that were newly infected with R4356 also did not neutralize R4356 despite neutralizing NM-3rN strongly. These results suggested that long-term persistent infection with SHIV induced neutralizing antibodies with a broad spectrum. However, a virus resistant to the neutralizing antibodies emerged in the persistently infected macaque.

To investigate the HIV-1 subtypes prevalent in the Republic of Congo, we isolated 28 HIV-1 strains from Congolese AIDS patients in 1996 and 1997, and analyzed them phylogenetically. Phylogenetic analysis based on part of the 5' tat-env (vpu) and env sequences revealed that only 13 (46.4%) of the 28 isolates belonged to the same subtype in the vpu tree as in the env tree; the remaining 15 (53.6%) strains showed discordant subtypes between vpu and env with 6 different profiles; that is, 1 A/A (vpu/env), 1 D/D, 5 G/G, 4 H/H, 2 unclassified (U)/U, 9 G/A, 2 G/H, 1 G/J, 1 H/G, 1 U/A, and 1 U/J. Thus, 9 of the 15 discordant HIV-1s were of the G/A (vpu/env) type, and did not form any subcluster within the subtype G lineage in the vpu-based phylogenetic tree. In addition, CRF02_AG (IbNG), which is a G/A (vpu/env) type, was not found in the Republic of Congo. These data suggest that the majority of HIV-1 subtypes circulating in the Republic of Congo have mosaic structures and may have been derived from independent recombinational events.

We found a novel primate lentivirus in mandrill (Mandrillus sphinx). To clarify the evolutionary relationships and transmission patterns of human/simian immunodeficiency virus (HIV/SIV), we screened blood samples from 30 wild-born healthy Cameroonian mandrills. Five (16.7%) of them were seropositive for SIV. Three SIV strains were isolated from the five seropositive mandrills by cocultivation of their peripheral blood mononuclear cells (PBMCs) with PBMCs of rhesus macaques, a human T cell line (M8166), and/or a cynomolgus macaque T cell line (HSC-F). One of the newly isolated SIV strains was intravenously inoculated into two rhesus macaques and resulted in chronic infection. In the SIV-infected macaques at 45 weeks after inoculation, we observed a mild decline in the number of peripheral CD4(+) lymphocytes, lymphadenopathy, and blastic follicular dendritic cells with mild follicular hyperplasia in the peripheral lymph nodes. A phylogenetic analysis based on the pol sequence showed that the newly found SIVs from Cameroonian mandrills did not cluster with SIVmndGB1, which is the former representative strain of SIVmnd. The SIVmnds from Cameroon formed a new, independent lineage that branched before the root of the HIV-1/SIVcpz lineage with 996 of 1000 bootstrap replications. They clustered host specifically, and exhibited about 16.9% diversity at the level of nucleotide sequence among Cameroonian SIVmnd strains. These results indicate that the SIVmnds isolated in Cameroon are a novel type of SIVmnd and have infected Cameroonian mandrills for a long time. We therefore designated the Cameroonian SIVmnd as SIVmnd type 2 and redesignated SIVmndGB1 as SIVmnd type 1. To date, M. sphinx is the only primate species other than humans that is naturally infected with two different types of SIV.

To assess the molecular epidemiology of HIV-1 in Republic of Congo (Congo), we investigated 29 HIV-1s obtained from 82 Congolese AIDS and ARC patients in 1996 and 1997. Part of the env region including the V3 loop was phylogenetically analyzed. The genotypes observed were varied: of 29 specimens, 12 (41 %) were subtype A, 1 (3%) was subtype D, 6 (21%) were subtype G, 6 (21%) were subtype H, 2 (7%) were subtype J, and 2 (7%) could not be classified as any known subtypes (U, unclassified). The heterogeneous profile of HIV-1 infection was different from the profiles of neighboring Central African countries. These data show that subtypes G and H as well as subtype A were circulating with high prevalence. The fact that new genetic subtypes (J and U) are circulating indicates a need for a greater surveillance for these subtypes both in Congo as well as in other parts of the world.

To better understand the origin of human T-cell leukemia virus type l (HTLV-l) in South America, we conducted a phylogenetic study on 27 new HTLV-ls in Brazil. These were obtained from Brazilians of various ethnic origins, such as Japanese immigrants, whites, blacks and mulattos. We amplified and sequenced proviral DNAs of a part of the long terminal repeats. Phylogenetic trees revealed that all but 6 of the new isolates were not only similar to each other but also similar to HTLV-ls of other South American countries, including those from Amerindians. However, the isolates differed from the HTLV-ls of Africa and Japan. The other six isolates were from Japanese immigrants and were phylogenetically almost identical to HTLV-ls in Japan but different from the majority of South American HTLV-ls, including the other new Brazilian HTLV-ls. These findings indicate that the recent introduction of HTLV-1 from Japan is limited to Japanese immigrants. In addition, the results do not support the prevailing hypothesis that HTLV-ls in South America were introduced by blacks who were brought from Africa as slaves. Rather, these results suggest that the majority of HTLV-1s prevailing in South America have spread from Amerindians, some of whom are likely to have possessed this human retrovirus from the beginning of their settlement in South America.

To determine newly identified lentiviruses, termed simian immunodeficiency virus (SIV)cpz97CG4 and SIVcpz97CG6, from two wild-captured juvenile brother chimpanzees in the Republic of Congo, subgenomic pol (integrase, 288 bp), 5'tat/rev-env Cl (including vpu, 354 bp) and env (C2-C4, 544 bp) gene fragments were amplified and sequenced. The analysis revealed significantly discordant phylogenetic positions of SIVcpz97CG in each genomic region. In the trees derived from partial env sequences (V3), both SIVcpz strains clustered in human immunodeficiency virus type 1 (HIV-1) subtype A. However, in the trees derived from partial pol (integrase) and 5'tat/rev-env C1 (including vpu) sequences, they clustered independently from any of the known HIV-1 subtypes. Especially, in the 5'tat/rev-vpu tree, they branched before the root of HIV-1 group M. These findings suggest that these Congolese SIVcpz genomes are mosaic, probably due to a recombinational event in the recent past, and it provides evidence for a rather recently occurring cross-species transmission between humans and chimpanzees.

Here we describe, for the first time, recombinants between two highly divergent major groups of human immunodeficiency virus type 1 (HIV-1), M and O, within a Cameroonian woman infected with three different HIV-1 strains, a group O virus, a subtype D virus, and a recently reported IBNG (A/G)-like recombinant virus. Using nested extra-long PCR amplification, we sequenced from the pol region to the env region including accessory genes of the viral genome obtained from the patient's uncultured peripheral blood mononuclear cells and examined the phylogenetic position of each gene. Compared with sequential blood samples obtained in 1995 and 1996, there were multiple segmental exchanges between three HIV-1 strains (O, D, and IBNG) and all the recombinants appeared to be derived from a common M/O ancestor. Importantly, recombination between groups M and O occurred, even though the homology between these two groups is 69, 76, 68, and 55% in the gag, pol, vif-vpr, and env regions, respectively. Recombination between strains with such distant lineages may contribute substantially to generating new HIV-1 variants.

In 1995, 53 blood samples from Muslim patients with AIDS, or who were thought to have AIDS, were collected in the main hospitals of Adamaoua Province, in the northern part of Cameroon. The variable env C2V3 region of HIV-1 was amplified by nested PCR and phylogenetically analyzed. The results indicated that of 15 amplified samples, 1 belonged to HIV-1 group O, 1 to HIV-1 subtype D, 1 to subtype G, 2 to subtype H, and 10 to subtype A. Furthermore, the northern Cameroonian subtype A could be divided into at least two subclusters as shown by the env tree as well as by two remarkably conserved hexameric amino acid sequences in the apex of V3 (GPGQAF in one subcluster and GPGQTF in the other). This distinction suggests that the HIV-1 subtype A circulating in northern Cameroon evolved from two main sources. More recently, three HIV-1 strains from Nigeria (IBNG) and Djibouti (DJ263 and DJ264), previously reported on the basis of their env C2V3 sequences as subtype A, were found to have a similar A/G mosaic structure alongside their full-length sequence and were tentatively designated as members of a new subtype called "IBNG." Interestingly, within the northern Cameroonian subtype A described, the isolates of the second subcluster clustered distinctly with these A/G mosaic strains, strongly suggesting that they may be members of the IBNG subtype.

In order to assess the incidence of HIV mixed infection as well as to clarify the molecular epidemiology of HIV in central Africa, we investigated 43 HIVs obtained from 211 Cameroonian AC, ARC, and AIDS patients in 1994 and 1995. Part of the pol region and part of the env region were phylogenetically analyzed. The genotypes observed were varied: of 43 specimens, 28 (65%) were subtype A, 1 (2%) was subtype B, 2 (5%) were subtype D, 3 (7%) were subtype F, and 2 (5%) were group O. Of the remaining 7 specimens, 3 were mixed infections with HIV-1 subtypes A and C, HIV-1 subtypes C and F, and HIV-2 subtype A and HIV-1 subtype A; 1 was a mixed infection with HIV-1 subtypes A and D and the highly divergent group O (triple infection); another 3 appeared to consist of mosaic genomes (A/G, A/E, and B/A recombinant). These data show that various types of mixed infection, such as between different subtypes of HIV-1 group M, between HIV-1 and HIV-2, and even between HIV-1 groups O and M, were confirmed at a rather high frequency (approximately 10%). The mixed infection is particularly significant where there is a greater variety of HIV-1 subtypes circulating, since it results in new genetic diversity generated by intersubtype recombination.

Ninety-four GB virus C/hepatitis G virus (GBV-C/ HGV) RNA-positive serum samples were obtained from all over the world. We found that all 15 GBV-C/HGV isolates from the Pygmies and the Bantu in the Central African region had a 12-amino acid indel (i.e. insertion or deletion) in the non-structural protein (NS) 5A region. Phylogenetic analyses of the NS5A region, using GBV-A as an outgroup, showed that these 15 isolates had diverged from the common ancestor much earlier than the remaining isolates, indicating an African origin of GBV-C/HGV.

Our previous analysis of an HTLV-I isolate (CMR229) from a Cameroonian Pygmy demonstrated that the isolate is distinct from typical HTLV-Is of the "Central African group," which has a close similarity to HTLV-I-related simian viruses (STLV-I) in Africa. In this study, we analyzed six new HTLV-Is from Cameroon consisting of three isolates from the Pygmy and three from the Bantu to examine further the genetic features of HTLV-I in Cameroon, especially in the Pygmy. A phylogenetic tree based on the long terminal repeats (LTR) region showed that all the new HTLV-Is belong to the Central African group. On the other hand, an env-based analysis of CMR229 confirmed the previous finding derived from LTR-based analysis that CMR229 has a similarity to African STLV-Is, but is distinct from the typical Central African group of HTLV-I. This suggests that multiple interspecies transmissions from non-human primates to humans have occurred in Central Africa, resulting in the presence of two distinct HTLV-I strains in this area. In addition, it seems likely that the Pygmy harbors the heterogeneous HTLV-I strains from which the main HTLV-I population spread into the Bantu.

In an effort to delineate the origin and evolution of HTLV-I/STLV-I, we have been conducting phylogenetic analyses on LTR sequences of this virus group. HTLV-I isolates newly analyzed in the present study were from Iran, South Africa, Cameroon, Sakhalin and Brazil where little is known concerning the genetic features of HTLV-I. In addition, STLV-I isolates were obtained from non-human primates in Africa and Asia including an isolate from orangutans in Indonesia. Proviral LTR sequences were amplified by nested PCR, and then sequenced. Phylogenetic trees were constructed by the neighbor joining method. The results obtained are: 1) African STLV-I isolates formed one large cluster together with the Central African group of HTLV-I in the tree; 2) Asian STLV-I isolates including that of an orangutan in Indonesia were highly divergent from African STLV-I and the Cosmopolitan group of HTLV-I, but not so closely related to each other and to the Melanesian group of HTLV-I; 3) An HTLV-I isolate of Cameroon Pygmy was related to African STLV-I isolates, but distinct from the Central African group of HTLV-I; 4) The majority of HTLV-I isolates belonged to subgroup A which is the most widespread subgroup of the Cosmopolitan group of HTLV-I, while some Brazilian isolates from descendants of Japanese immigrants belonged to subgroup B which mainly consists of HTLV-I isolates from Japan. 5) In the phylogenetic tree, several HTLV-I isolates of subgroup A from the same areas appear to form monophyletic clusters such as a subcluster of Brazilian and Colombian isolates and that of Iranian isolates.

Eleven human immunodeficiency virus 1 (HIV-1) isolates from Ghanaian acquired immunodeficiency syndrome (AIDS) or AIDS-related complex (ARC) patients obtained by our serosurvey in 1986-1994 were genomically analyzed and phylogenetically compared with other known strains. A phylogenetic tree constructed by analyzing the env region indicated that heterogeneous HIV-1 strains were circulating in Ghana and the majority of them (9 of 11 isolates) belonged to clade (subtype) A which is now furiously epidemic in Africa. Another isolate (1 of 11) belonged to clade D, and the remaining one (1 of 11) belonged to "clade G". This "clade G" virus grouped by the env analysis belonged to clade A by its pol sequence, suggesting an A/G intersubtype recombinant. The characteristic sequences in the V3 tip which have not yet been reported were observed in these Ghanaian isolates, which should be taken into account for future vaccine programs.

To study the evolutionary origin of human T-lymphotropic virus type I/simian T-lymphotropic virus type I (HTLV-I/STLV-I), we isolated and characterized STLV-I from orangutans (Pongo pygmaeus). Plasma samples from 3 out of 41 animals examined were reactive by particle agglutination and immunofluorescence, and one of these three was confirmed to be anti-HTLV-I antibody-positive by western blotting (WB). Cultured peripheral blood mononuclear cells from the WB-positive orangutan were reactive to anti-STLV-I-positive rhesus monkey plasma. The proviral long terminal repeat region was amplified by polymerase chain reaction and sequenced. A phylogenetic analysis indicated that orangutan STLV-I is related to the Melanesian group of HTLV-Is and other Asian STLV-Is, but the degree of divergence is considerable.

Six human T-cell leukemia virus type I (HTLV-I) and eight human T-cell leukemia virus type II (HTLV-II) cases newly isolated from the South American countries of Colombia and Chile were analyzed together with the two Amerindian HTLV-I isolates previously reported. All of the HTLV-I isolates belonged to the transcontinental subgroup of the "cosmopolitan" group, and Colombian isolates, including those from native Amerindians and Negroes, formed a single tight cluster within this subgroup. The transcontinental subgroup consisted of isolates from various regions such as the Caribbean basin, India, Iran, South Africa, Sakhalin, and Japan, and included isolates from the "Ainu" and "Okinawa" people, regarded as relatively pure Japanese descended from the prehistoric "Jomon" period which began more than 10,000 years ago. This implied a dissemination of the subgroup associated with the movement of human beings in ancient times. On the other hand, all of the HTLV-II isolates from native Amerindians in Colombia and Chile belonged to the HTLV-IIb subtype which has previously been reported to be mainly endemic in certain populations of native Amerindians. The southernmost isolate from Chile, showing wide distribution of the IIb subtype in native South Amerindians and largest heterogeneity of the subtype in Colombian isolates, supported the idea that the HTLV-IIb subtype has been endemic for a long time in native Indians of South America.

A new endemic focus of human T-lymphotropic virus type I (HTLV-I) was recently reported among Mashhadi Jews, a group of immigrants from northeastern Iran to Israel. We extracted DNAs from fresh peripheral blood mononuclear cells (PBMCs) and/or gargle mouthwash from 10 HTLV-I carriers, who consisted of members of one family, and HTLV-I-associated myelopathy (HAM) and adult T-cell leukemia (ATL) patients. Long terminal repeat (LTR) regions of proviral DNAs were sequenced and analyzed phylogenetically. In a phylogenetic tree, all the Mashhadi HTLV-I isolates belonged to subtype A, one of the three subtypes of the cosmopolitan type of HTLV-I, and made a tight cluster distinct from the other isolates of subtype A from Japan, India, the Caribbean Basin, and South America. Although a few nucleotide substitutions were observed among the clones sequenced, no characteristic sequence variation was found in different disease manifestations, even in one family or different sources of DNA preparation.

Objective: To determine seroprevalence among suspected AIDS patients in Ghana in relation to clinical manifestations., Materials and Methods: Blood samples and medical records were collected from 290 Ghanaian patients with suspected AIDS in 1990 and 1992. Seroprevalence of HIV-1, HIV-2 and human T-cell leukemia virus (HTLV-1) were investigated by the particle agglutination method, indirect immunofluorescence assay, the monoepitope enzyme-linked immunosorbent assay and Western blot., Results: The specimens were classified into five serologic categories: 78 were HIV-1-positive (26.9%), 25 were HIV-2-positive (8.6%), 17 dual-positive (5.9%), 16 indeterminate (5.5%) and 154 seronegative (53.1%). No significant difference was found between the clinical symptoms of patients with HIV-1 and HIV-2 infection. Of the patients, 14 (4.8%) were HTLV-1-seropositive, of whom 11 were also HIV-positive, indicating a significant correlation between the two groups of viral infections (P < 0.01). However, there was no evidence of an increase in severity of symptoms in cases of dual infection with HTLV-1 and HIV., Conclusions: HIV-1 infection is now dominant in Ghana in contrast to our previous survey in 1986 which showed the dominance of HIV-2. The change in seroprevalence suggests that an HIV-1 epidemic has been developing in recent years in this country, where HIV-2 was originally endemic. A relatively high prevalence of dual-reactive specimens implies the existence of highly cross-reactive strains of HIV or frequent coinfection with HIV-1 and HIV-2 in the region. The large number of seronegative patients with clinically diagnosed AIDS raises the question of the inadequacy of AIDS definitions based on clinical manifestations only.