Infection of macaques with chimeric simian and human immunodeficiency viruses containing Env from subtype F.

Chimeric simian and human immunodeficiency viruses (SHIVs) are useful for investigating the pathogenicity of human immunodeficiency virus (HIV-1) and to develop an anti-HIV-1 vaccine. We attempted to construct SHIVs containing Env from various subtypes, because almost all SHIVs which have been reported so far have Env from HIV-1 that belongs to subtype B. Two infectious SHIVs containing Env from two strains of HIV-1, CMR304 and CMR306, which belong to subtype F and A, respectively, were newly obtained. These SHIVs essentially showed a coreceptor usage and a neutralization pattern that were similar to those of the parental HIV-1s. In macaque PBMC, SHIVcmr304 replicated with kinetics similar to that of prototypic SHIV-NM-3rN with HIV-1 NL432 Env, but SHIVcmr306 replicated poorly. Inoculation of four rhesus macaques with SHIVcmr304 resulted in an increase of plasma viral load in all the macaques, though viral RNA copies were 100-fold lower than that in the infection with NM-3rN. This SHIV containing Env from HIV-1 subtype F will be a valuable source for the analysis of HIV-1 subtype F and the evaluation of vaccine candidates as a genetically divergent challenge virus.

Natural infection of wild-born mandrills (Mandrillus sphinx) with two different types of simian immunodeficiency virus.

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.

Genetic subtypes of HIV type 1 in Republic of Congo.

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.

Natural infection of chimpanzees with new lentiviruses related to HIV-1/SIVcpz.

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.

Molecular epidemiology of human T-cell leukemia virus type I (HTLV-1) Brazil: the predominant HTLV-1s in South America differ from HTLV-ls of Japan and Africa, as well as those of Japanese immigrants and their relatives in Brazil.

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.

HIV type 1 genetic variability in the northern part of Cameroon.

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.

Human immunodeficiency virus type 1 intergroup (M/O) recombination in cameroon.

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.

Genetic diversity of HIV-1 group M from Cameroon and Republic of Congo.

We analyzed 57 HIV-1 isolates from Cameroon and the Republic of Congo, with respect to the env C2V3 and/or the pol integrase regions. The results indicated that the topology of the pol tree correlated well with that of the env tree for four clusters of subtype D, F G and H, suggesting that these trees reflect the true evolution of the overall genome structures of these subtypes. However, of 22 Cameroonian isolates that were classified as subtype A based on env, 20 of them diverged in their pol sequence into two lineages that were completely different from the prototypical subtype A, tentatively designated as subtypes A1 and A2. The subtype A1 isolates (6 out of 22) were related in their env C2V3 regions with prototypical subtype A strain, but in their pol regions, they formed an independent cluster that diverged from known HIV-1 subtypes so far reported (except for subtypes I and J). The subtype A2 isolates (14 out of 22), which represent the major epidemic type of HIV-1 in Cameroon, clustered distinctly in both the env and pol trees with the recently described A/G mosaic strains from Nigeria and Djibouti. These two lineages were not spreading in the neighboring Republic of Congo.

Various types of HIV mixed infections in Cameroon.

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.

African origin of GB virus C/hepatitis G virus.

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.

The rapid spread of recombinants during a natural in vitro infection with two human immunodeficiency virus type 1 strains.

We quantified a population of recombinants in a natural in vitro infection, using wild-type viruses without any pressure. It was found that recombinants emerged early after infection and constituted more than 20% of the whole proviral population 15 days after infection. Furthermore, recombinants were isolated as infectious viruses by simple limiting dilution. These results imply that, in addition to the high mutation rate of human immunodeficiency virus type 1 (HIV-1), recombination among HIV-1 strains plays a significant part in the development of the high diversity of HIV-1.

Prevalence and phylogenetic analysis of HTLV-I isolates in Cameroon, including those of the Baka Pygmy.

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.

Phylogenetic relationships of HTLV-I/STLV-I in the world.

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.

Phylogenetic analysis of human immunodeficiency virus 1 in Ghana.

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.

PIP: The molecular epidemiology of HIV-1 in Ghana was investigated through genomic and phylogenetic analysis of isolates from 11 AIDS or AIDS-related complex patients obtained in 1986-94. A phylogenetic tree constructed by analyzing the env region indicated that heterogeneous HIV-1 strains are circulating in Ghana. 9 of the isolates belonged to clade A, 1 to subtype D, and 1 to "clade G"--an A/G intersubtype recombinant. The V3 loops of all isolates were composed of 35 amino acid residues--a characteristic not previously described. These molecular data on the genetic variability of the envelope glycoprotein of HIV-1 should be useful for future vaccine studies in West Africa.

Isolation of STLV-I from orangutan, a great ape species in Southeast Asia, and its relation to other HTLV-Is/STLV-Is.

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.

Molecular phylogeny of human T-cell leukemia virus type I and II of Amerindians in Colombia and Chile.

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.

Genomic and biological alteration of a human immunodeficiency virus type 1 (HIV-1)-simian immunodeficiency virus strain mac chimera, with HIV-1 Env, recovered from a long-term carrier monkey.

A macaque monkey infected with NM-3, a human immunodeficiency virus type 1 (HIV-1)-simian immunodeficiency virus strain mac (SIVmac) chimeric virus with env, rev, tat and vpu derived from HIV-1 and LTR, gag, pol, vif and vpx derived from SIVmac, became a long-term carrier (more than 2.8 years). This monkey produced neutralizing antibodies to the original NM-3 as well as to the parental HIV-1. The virus recovered at 116 weeks replicated more rapidly and productively in macaque peripheral blood mononuclear cells than the original virus. The recovered virus was not neutralized either by antibodies raised early in the monkey or by a neutralizing monoclonal antibody that recognizes the V3 loop of HIV-1 Env, whereas both the early antibodies and the monoclonal antibody neutralized the original NM-3. Analysis of the virus genomic population revealed a few common mutations in the V3 region that caused amino acid changes. These data are consistent with the hypothesis that the virus escaped from the early antibodies and that the observed mutations contributed to this, as with HIV-1-infected humans. The observed mutations could equally well be the result of adaptation to simian cells. These results suggest that the HIV-1-SIVmac chimeric virus will be useful for investigating genetic variation of HIV-1 env and alteration of biological properties in vivo in relation to the host immune response.

Geographical distribution of the human polyomavirus JC virus type A and B and isolation of a new type from Ghana.

The JC polyomavirus (JCV) is ubiquitous in humans infecting children asymptomatically, then persisting in renal tissue. Since JCV DNA can be readily isolated from urine, it should be a useful tool with which to study the evolution of DNA viruses in humans. We showed that JCV DNA from the urine of Japanese, Taiwanese, Dutch and German patients can be classified into A and B types, based upon restriction fragment length polymorphisms (RFLPs). This work was extended in the present study. We established multiple JCV DNA clones from the UK, Spain, Italy, Sweden, South Korea, People's Republic of China, Malaysia, Indonesia, Mongolia, India, Sri Lanka, Saudi Arabia, Ethiopia, Kenya, Zambia, South Africa and Ghana. Using type-specific RFLPs, most clones except the four clones from Ghana were classified as either type A or B. We constructed a molecular phylogenetic tree for the Ghanaian clones and several representative type A and B clones. According to the phylogenetic tree, the Ghanaian clones constituted a major new group, tentatively named type C. From the findings presented here and elsewhere, the following conclusions were drawn: (i) type A is prevalent only in Europe; (ii) type B is found mainly in Asia and Africa; and (iii) type C is localized to part of Africa. Our findings should help to clarify how JCV evolved in humans.