Human immunodeficiency virus type 1 (HIV-1) diversity at time of infection is not restricted to certain risk groups or specific HIV-1 subtypes

African women frequently acquire several genetically distinct human immunodeficiency virus type 1 (HIV-1) variants from a heterosexual partner, whereas the acquisition of multiple variants appears to be rare in men. To determine whether newly infected individuals in other risk groups acquire genetically diverse viruses, we examined the viral envelope sequences in plasma samples from 13 women and 4 men from the United States infected with subtype B viruses and 10 men from Kenya infected with non-subtype B viruses. HIV-1 envelope sequences differed by more than 2% in three U.S. women, one U.S. man, and one Kenyan man near the time of seroconversion. These findings suggest that early HIV-1 genetic diversity is not exclusive to women from Africa or to infection with any particular HIV-1 subtype.     

Identification of three feline immunodeficiency virus (FIV) env gene subtypes and comparison of the FIV and human immunodeficiency virus type 1 evolutionary patterns.

Feline immunodeficiency virus (FIV) is a lentivirus associated with AIDS-like illnesses in cats. As such, FIV appears to be a feline analog of human immunodeficiency virus (HIV). A hallmark of HIV infection is the large degree of viral genetic diversity that can develop within an infected individual and the even greater and continually increasing level of diversity among virus isolates from different individuals. Our goal in this study was to determine patterns of FIV genetic diversity by focusing on a 684-nucleotide region encompassing variable regions V3, V4, and V5 of the FIV env gene in order to establish parallels and distinctions between FIV and HIV type 1 (HIV-1). Our data demonstrate that, like HIV-1, FIV can be separated into distinct envelope sequence subtypes (three are described here). Similar to that found for HIV-1, the pairwise sequence divergence within an FIV subtype ranged from 2.5 to 15.0%, whereas that between subtypes ranged from 17.8 to 26.2%. However, the high number of synonymous nucleotide changes among FIV V3 to V5 env sequences may also include a significant number of back mutations and suggests that the evolutionary distances among FIV subtypes are underestimated. Although only a few subtype B viruses were available for examination, the pattern of diversity between the FIV A and B subtypes was found to be significantly distinct; subtype B sequences had proportionally fewer mutations that changed amino acids, compared with silent changes, suggesting a more advanced state of adaptation to the host. No similar distinction was evident for HIV-1 subtypes. The diversity of FIV genomes within individual infected cats was found to be as high as 3.7% yet twofold lower than that within HIV-1-infected people over a comparable region of the env gene. Despite these differences, significant parallels between patterns of FIV evolution and HIV-1 evolution exist, indicating that a wide array of potentially divergent virus challenges need to be considered in FIV vaccine and pathogenesis studies.     

Subtypes of human immunodeficiency virus type 1 and disease stage among women in Nairobi, Kenya

In sub-Saharan Africa, where the effects of human immunodeficiency virus type 1 (HIV-1) have been most devastating, there are multiple subtypes of this virus. The distribution of different subtypes within African populations is generally not linked to particular risk behaviors. Thus, Africa is an ideal setting in which to examine the diversity and mixing of viruses from different subtypes on a population basis. In this setting, it is also possible to address whether infection with a particular subtype is associated with differences in disease stage. To address these questions, we analyzed the HIV-1 subtype, plasma viral loads, and CD4 lymphocyte levels in 320 women from Nairobi, Kenya. Subtype was determined by a combination of heteroduplex mobility assays and sequence analyses of envelope genes, using geographically diverse subtype reference sequences as well as envelope sequences of known subtype from Kenya. The distribution of subtypes in this population was as follows: subtype A, 225 (70.3%); subtype D, 65 (20.5%); subtype C, 22 (6.9%); and subtype G, 1 (0.3%). Intersubtype recombinant envelope genes were detected in 2.2% of the sequences analyzed. Given that the sequences analyzed represented only a small fraction of the proviral genome, this suggests that intersubtype recombinant viral genomes may be very common in Kenya and in other parts of Africa where there are multiple subtypes. The plasma viral RNA levels were highest in women infected with subtype C virus, and women infected with subtype C virus had significantly lower CD4 lymphocyte levels than women infected with the other subtypes. Together, these data suggest that women in Kenya who are infected with subtype C viruses are at more advanced stages of immunosuppression than women infected with subtype A or D. There are at least two models to explain the data from this cross-sectional study; one is that infection with subtype C is associated with a more rapid disease progression, and the second is that subtype C represents an older epidemic in Kenya. Discriminating between these possibilities in a longitudinal study will be important for increasing our understanding of the role of specific subtypes in the transmission and pathogenesis of HIV-1.     

Unprecedented degree of human immunodeficiency virus type 1 (HIV-1) group M genetic diversity in the Democratic Republic of Congo suggests that the HIV-1 pandemic originated in Central Africa

The purpose of this study was to document the genetic diversity of human immunodeficiency virus type 1 (HIV-1) in the Democratic Republic of Congo (DRC; formerly Zaire). A total of 247 HIV-1-positive samples, collected during an epidemiologic survey conducted in 1997 in three regions (Kinshasa [the capital], Bwamanda [in the north], and Mbuyi-Maya [in the south]), were genetically characterized in the env V3-V5 region. All known subtypes were found to cocirculate, and for 6% of the samples the subtype could not be identified. Subtype A is predominant, with prevalences decreasing from north to south (69% in the north, 53% in the capital city, and 46% in the south). Subtype C, D, G, and H prevalences range from 7 to 9%, whereas subtype F, J, K, and CRF01-AE strains represent 2 to 4% of the samples; only one subtype B strain was identified. The highest prevalence (25%) of subtype C was in the south, and CRF01-AE was seen mainly in the north. The high intersubtype variability among the V3-V5 sequences is the most probable reason for the low (45%) efficiency of subtype A-specific PCR and HMA (heteroduplex mobility assay). Eighteen (29%) of 62 samples had discordant subtype designations between env and gag. Sequence analysis of the entire envelope from 13 samples confirmed the high degree of diversity and complexity of HIV-1 strains in the DRC; 9 had a complex recombinant structure in gp160, involving fragments of known and unknown subtypes. Interestingly, the unknown fragments from the different strains did not cluster together. Overall, the high number of HIV-1 subtypes cocirculating, the high intrasubtype diversity, and the high numbers of possible recombinant viruses as well as different unclassified strains are all in agreement with an old and mature epidemic in the DRC, suggesting that this region is the epicenter of HIV-1 group M.     

Tat protein of human immunodeficiency virus type 1 subtype C strains is a defective chemokine

Human immunodeficiency virus type 1 (HIV-1)-associated dementia (HAD) is correlated with increased monocyte migration to the brain, and the incidence of HAD among otherwise asymptomatic subjects appears to be lower in India than in the United States and Europe (1 to 2% versus 15 to 30%). Because of the genetic differences between HIV-1 strains circulating in these regions, we sought to identify viral determinants associated with this difference. We targeted Tat protein for these studies in view of its association with monocyte chemotactic function. Analyses of Tat sequences representing nine subtypes revealed that at least six amino acid residues are differentially conserved in subtype C Tat (C-Tat). Of these, cysteine (at position 31) was highly (>99%) conserved in non-subtype C viruses and more than 90% of subtype C viruses encoded a serine. We hypothesized a compromised chemotactic function of C-Tat due to the disruption of CC motif and tested it with the wild type C-Tat (CS) and its two isogenic variants (CC and SC) derived by site-directed mutagenesis. We found that the CS natural variant was defective for monocyte chemotactic activity without a loss in the transactivation property. While the CC mutant is functionally competent for both the functions, in contrast, the SC mutant was defective in both. Therefore, the loss of the C-Tat chemotactic property may underlie the reduced incidence of HAD; although not presenting conclusive evidence, this study provides the first evidence for a potential epidemiologic phenomenon associated with biological differences in the subtype C viruses.     

Recombination in AIDS viruses

Recombination contributes to the generation of genetic diversity in human immunodeficiency viruses (HIV) but can only occur between viruses replicating within the same cell. Since individuals have not been found to be simultaneously coinfected with multiple divergent strains of HIV-1 or HIV-2, recombination events have been thought to be restricted to the rather closely related members of the quasispecies that evolves during the course of HIV infection. Here we describe examples of both HIV-1 and HIV-2 genomes that appear to be hybrids of genetically quite divergent viruses. Phylogenetic analyses were used to examine the evolutionary relationships among multiple HIV strains. Evolutionary trees derived from different genomic regions were consistent with respect to most of the viruses investigated. However, some strains of HIV-1 and HIV-2 exhibited significantly discordant branching orders indicative of genetic exchanges during their evolutionary histories. The crossover points of these putative recombination events were mapped by examining the distribution of phylogenetically informative sites supporting alternative tree topologies. A similar example of a recombinant simian immunodeficiency virus identified in West African green monkeys has also been described recently. These results indicate that coinfection with highly divergent viral strains can occur in HIV-infected humans and SIV-infected primates and could lead to the generation of hybrid genomes with significantly altered biological properties. Thus, future characterization of primate lentiviruses should include careful phylogenetic investigation of possible genomic mosaicism.Correspondence to: P.M. Sharp     

Variability of human immunodeficiency virus type 1 group O strains isolated from Cameroonian patients living in France.

Human immunodeficiency virus type 1 (HIV-1) nucleotide sequences encoding p24Gag and the Env C2V3 region were obtained from seven patients who were selected on the basis of having paradoxical seronegativity on a subset of HIV enzyme-linked immunosorbent assay detection kits and having atypical Western blot (immunoblot) reactivity. Sequence analyses showed that all of these strains were more closely related to the recently described Cameroonian HIV isolates of group O (HIV-1 outlier) than to group M (HIV-1 major). All seven patients had Cameroonian origins but were living in France at the time the blood samples were taken. Characterization of a large number of group M strains has to date revealed eight distinct genetic subtypes (A to H). Genetic distances between sequences from available group O isolates were generally comparable to those observed in M intersubtype sequence comparisons, showing that the group O viruses are genetically very diverse. Analysis of sequences from these seven new viral strains, combined with the three previously characterized group O strains, revealed few discernable phylogenetic clustering patterns among the 10 patients' viral sequences. The level of diversity among group O sequences suggests that they may have a comparable (or greater) age than the M group sequences, although for unknown reasons, the latter group dispersed first and is the dominant lineage in the pandemic.     

Inflammatory Genital Infections Mitigate a Severe Genetic Bottleneck in Heterosexual Transmission of Subtype A and C HIV-1

The HIV-1 epidemic in sub-Saharan Africa is driven largely by heterosexual transmission of non-subtype B viruses, of which subtypes C and A are predominant. Previous studies of subtype B and subtype C transmission pairs have suggested that a single variant from the chronically infected partner can establish infection in their newly infected partner. However, in subtype A infected individuals from a sex worker cohort and subtype B individuals from STD clinics, infection was frequently established by multiple variants. This study examined over 1750 single-genome amplified viral sequences derived from epidemiologically linked subtype C and subtype A transmission pairs very early after infection. In 90% (18/20) of the pairs, HIV-1 infection is initiated by a single viral variant that is derived from the quasispecies of the transmitting partner. In addition, the virus initiating infection in individuals who were infected by someone other than their spouse was characterized to determine if genital infections mitigated the severe genetic bottleneck observed in a majority of epidemiologically linked heterosexual HIV-1 transmission events. In nearly 50% (3/7) of individuals infected by someone other than their spouse, multiple genetic variants from a single individual established infection. A statistically significant association was observed between infection by multiple genetic variants and an inflammatory genital infection in the newly infected individual. Thus, in the vast majority of HIV-1 transmission events in cohabiting heterosexual couples, a single genetic variant establishes infection. Nevertheless, this severe genetic bottleneck can be mitigated by the presence of inflammatory genital infections in the at risk partner, suggesting that this restriction on genetic diversity is imposed in large part by the mucosal barrier.     

Human immunodeficiency virus type 1 variability and long-term non-progression.

A high heterogeneity is found in the HIV-1 genome in vivo, not only between individuals, but also within a single individual. Different types of genetic heterogeneity of HIV-1 can be analyzed: the extension and the evolution of the viral quasispecies in blood, the variation between the virus obtained from different body compartment, the differences between isolates from diverse individuals and between HIV-1 subtypes. The virus population during primary HIV-1 infection is generally homogeneous and the intrahost viral evolution is thought to be forced (in absence of antiviral therapy) by the immune system pressure and is generally related to the length of the immunocompetent period. A group of 12 Italian and Swedish well characterized HIV-1 infected long-term nonprogressors (LNTP) have been analyzed for the viral heterogeneity, calculated in the nef gene and in the long terminal repeat (LTR). The intra-sample variations in LTNP were found comparable with those from 8 progressor patients, while a lower inter-individual diversity was observed in the former. In one LTNP the viral evolution during a four-years period was extremely low suggesting that other factors than the host immune pressure may be involved in modulating the intra- and inter-sample HIV-1 diversity.     

Testing the hypothesis of a recombinant origin of human immunodeficiency virus type 1 subtype E

The human immunodeficiency virus type 1 (HIV-1) epidemic in Southeast Asia has been largely due to the emergence of clade E (HIV-1E). It has been suggested that HIV-1E is derived from a recombinant lineage of subtype A (HIV-1A) and subtype E, with multiple breakpoints along the E genome. We obtained complete genome sequences of clade E viruses from Thailand (93TH057 and 93TH065) and from the Central African Republic (90CF11697 and 90CF4071), increasing the total number of HIV-1E complete genome sequences available to seven. Phylogenetic analysis of complete genomes showed that subtypes A and E are themselves monophyletic, although together they also form a larger monophyletic group. The apparent phylogenetic incongruence at different regions of the genome that was previously taken as evidence of recombination is shown to be not statistically significant. Furthermore, simulations indicate that bootscanning and pairwise distance results, previously used as evidence for recombination, can be misleading, particularly when there are differences in substitution or evolutionary rates across the genomes of different subtypes. Taken jointly, our analyses suggest that there is inadequate support for the hypothesis that subtype E variants are derived from a recombinant lineage. In contrast, many other HIV strains claimed to have a recombinant origin, including viruses for which only a single parental strain was employed for analysis, do indeed satisfy the statistical criteria we propose. Thus, while intersubtype recombinant HIV strains are indeed circulating, the criteria for assigning a recombinant origin to viral structures should include statistical testing of alternative hypotheses to avoid inappropriate assignments that would obscure the true evolutionary properties of these viruses.     

Potent, broad-spectrum inhibition of human immunodeficiency virus type 1 by the CCR5 monoclonal antibody PRO 140

CCR5 serves as a requisite fusion coreceptor for clinically relevant strains of human immunodeficiency virus type 1 (HIV-1) and provides a promising target for antiviral therapy. However, no study to date has examined whether monoclonal antibodies, small molecules, or other nonchemokine agents possess broad-spectrum activity against the major genetic subtypes of HIV-1. PRO 140 (PA14) is an anti-CCR5 monoclonal antibody that potently inhibits HIV-1 entry at concentrations that do not affect CCR5's chemokine receptor activity. In this study, PRO 140 was tested against a panel of primary HIV-1 isolates selected for their genotypic and geographic diversity. In quantitative assays of viral infectivity, PRO 140 was compared with RANTES, a natural CCR5 ligand that can inhibit HIV-1 entry by receptor downregulation as well as receptor blockade. Despite their divergent mechanisms of action and binding epitopes on CCR5, low nanomolar concentrations of both PRO 140 and RANTES inhibited infection of primary peripheral blood mononuclear cells (PBMC) by all CCR5-using (R5) viruses tested. This is consistent with there being a highly restricted pattern of CCR5 usage by R5 viruses. In addition, a panel of 25 subtype C South African R5 viruses were broadly inhibited by PRO 140, RANTES, and TAK-779, although approximately 30-fold-higher concentrations of the last compound were required. Interestingly, significant inhibition of a dualtropic subtype C virus was also observed. Whereas PRO 140 potently inhibited HIV-1 replication in both PBMC and primary macrophages, RANTES exhibited limited antiviral activity in macrophage cultures. Thus CCR5-targeting agents such as PRO 140 can demonstrate potent and genetic-subtype-independent anti-HIV-1 activity.     

Hyperattenuated recombinant influenza A virus nonstructural-protein-encoding vectors induce human immunodeficiency virus type 1 Nef-specific systemic and mucosal immune responses in mice

We have generated recombinant influenza A viruses belonging to the H1N1 and H3N2 virus subtypes containing an insertion of the 137 C-terminal amino acid residues of the human immunodeficiency virus type 1 (HIV-1) Nef protein into the influenza A virus nonstructural-protein (NS1) reading frame. These viral vectors were found to be genetically stable and capable of growing efficiently in embryonated chicken eggs and tissue culture cells but did not replicate in the murine respiratory tract. Despite the hyperattenuated phenotype of influenza/NS-Nef viruses, a Nef and influenza virus (nucleoprotein)-specific CD8(+)-T-cell response was detected in spleens and the lymph nodes draining the respiratory tract after a single intranasal immunization of mice. Compared to the primary response, a marked enhancement of the CD8(+)-T-cell response was detected in the systemic and mucosal compartments, including mouse urogenital tracts, if mice were primed with the H1N1 subtype vector and subsequently boosted with the H3N2 subtype vector. In addition, Nef-specific serum IgG was detected in mice which were immunized twice with the recombinant H1N1 and then boosted with the recombinant H3N2 subtype virus. These findings may contribute to the development of alternative immunization strategies utilizing hyperattenuated live recombinant influenza virus vectors to prevent or control infectious diseases, e.g., HIV-1 infection.     

Molecular cloning and phylogenetic analysis of human immunodeficiency virus type 1 subtype C: a set of 23 full-length clones from Botswana

To better understand the virological aspect of the expanding AIDS epidemic in southern Africa, a set of 23 near-full-length clones of human immunodeficiency virus type 1 (HIV-1) representing eight AIDS patients from Botswana were sequenced and analyzed phylogenetically. All study viruses from Botswana belonged to HIV-1 subtype C. The interpatient diversity of the clones from Botswana was higher than among full-length isolates of subtype B or among a set of full-length HIV-1 genomes of subtype C from India (mean value of 9. 1% versus 6.5 and 4.3%, respectively; P < 0.0001 for both comparisons). Similar results were observed in all genes across the entire viral genome. We suggest that the high level of HIV-1 diversity might be a typical feature of the subtype C epidemic in southern Africa. The reason or reasons for this diversity are unclear, but may include an altered replication efficiency of HIV-1 subtype C and/or the multiple introduction of different subtype C viruses.     

Genetic characterization of new West African simian immunodeficiency virus SIVsm: geographic clustering of household-derived SIV strains with human immunodeficiency virus type 2 subtypes and genetically diverse viruses from a single feral sooty mangabey troop.

It has been proposed that human immunodeficiency virus type 2 (HIV-2) originated from simian immunodeficiency viruses (SIVs) that are natural infections of sooty mangabeys (Cercocebus torquatus atys). To test this hypothesis, SIVs from eight sooty mangabeys, including six new viruses from West Africa, were genetically characterized. gag and env sequences showed that while the viruses of all eight sooty mangabeys belonged to the SIVsm/HIV-2 family, each was widely divergent from SIVs found earlier in captive monkeys at American primate centers. In two SIVs from sooty mangabeys discovered about 100 miles (ca. 161 Km) from each other in rural West Africa, the amino acids of a conserved gag p17-p26 region differed by 19.3%, a divergence greater than that in four of five clades of HIV-2 and in SIVs found in other African monkey species. Analysis of gag region sequences showed that feral mangabeys in one small troop harbored four distinct SIVs. Three of the newly found viruses were genetically divergent, showing as much genetic distance from each other as from the entire SIVsm/HIV-2 family. Sequencing and heteroduplex analysis of one feral animal-derived SIV showed a mosaic genome containing an env gene that was homologous with other feral SIVsm env genes in the troop but having a gag gene from another, distinct SIV. Surprisingly a gag phylogenetic tree based on nucleotide sequences showed that the African relatives closest to all three household-derived SIVs were HIV-2 subtypes D and E from humans in the same West African areas. In one case, the SIV/HIV-2 cluster was from the same village. The findings support the hypothesis that each HIV-2 subtype in West Africans originated from widely divergent SIVsm strains, transmitted by independent cross-species events in the same geographic locations.     

Phenotypic and genotypic comparisons of CCR5- and CXCR4-tropic human immunodeficiency virus type 1 biological clones isolated from subtype C-infected individuals

Individuals infected with human immunodeficiency virus type 1 (HIV-1) subtype C infrequently harbour X4 viruses. We studied R5 and X4 biological clones generated from HIV-1 subtype C-infected individuals. All subtype C R5 viruses demonstrated slower profiles of replication on CD4⁺ lymphocytes in comparison to subtype B viruses, whereas subtype C X4 viruses replicated with comparable efficiency to subtype B X4 viruses. No differences were identified in CC or CXC chemokine inhibitions (RANTES and SDF-1α, respectively) between subtype C and subtype B viruses. Immature dendritic cells were shown in coculture experiments to similarly enhance the infection of subtype C and subtype B R5 as well as X4 viruses. By amino acid sequence analysis, we showed that the R5 and X4 subtype C gp120 envelope gene alterations were similar to those for a switching subtype B virus, specifically with respect to the V3 charge and envelope N-linked glycosylation patterns. By phylogenetic analysis, we showed that one patient was infected with HIV-1 C′ and the other was infected with HIV-1 C" and that one of the patients harbored a virus that was a recombinant in the gp120 env gene between an R5 and an X4 virus, with the resultant virus being R5. No differences were identified between the long terminal repeat regions of the subtype C R5 and X4 biological clones. These results indicate that even though R5 subtype C viruses are restrictive for virus replication, the R5-to-X4 phenotype switch can occur and does so in a manner similar to that of subtype B viruses.     

Genetic analysis of human immunodeficiency virus type 1 strains from patients in Cyprus: identification of a new subtype designated subtype I.

DNA sequences encoding the C2 to V3 region of envelope glycoprotein gp120 of human immunodeficiency virus type 1 (HIV-1) were amplified by PCR from uncultured peripheral blood mononuclear cells obtained from 24 of 25 HIV-1-seropositive patients from Cyprus. By using a heteroduplex mobility assay (HMA), all amplified products were studied genetically and compared with 16 previously characterized HIV-1 strains belonging to subtypes A through F. HMA results revealed that HIV-1 gp120 sequences from 15 of our patients were of subtype B of HIV-1, whereas one isolate was of subtype C. However, gp120 sequences from eight patients had no obvious similarities to the known subtypes as defined by HMA. DNA sequencing and phylogenetic analyses of molecular clones confirmed the HMA results and placed the eight undefined HIV-1 isolates into three distinct genetic clusters. On the basis of branch topology and lengths of the phylogenetic tree, we conclude that one group consisting of three clones from two patients represents a new HIV-1 env subtype, which we have termed subtype I. The remaining two sequence clusters, consisting of five sequences from four patients and two sequences from two other patients, are distally related to subtypes A and F. These data demonstrate the extensive heterogeneity of HIV-1 in Cyprus, including the presence of new subtype.     

Impact of natural polymorphism within the gp41 cytoplasmic tail of human immunodeficiency virus type 1 on the intracellular distribution of envelope glycoproteins and viral assembly

The motifs involved in the various functions of the human immunodeficiency virus type 1 (HIV-1) gp41 cytoplasmic tail (CT), particularly those related to the intracellular trafficking and assembly of envelope glycoproteins (Env) onto core particles, have generally been assessed with a restricted panel of T-cell laboratory-adapted virus strains. Here, we investigated gp41 CT sequences derived from individuals infected with HIV-1 viruses of various subtypes. We identified four patients harboring HIV variants with a natural polymorphism in the membrane-proximal tyrosine-based signal Y(712)SPL or the Y(802)W(803) diaromatic motif, which are two major determinants of Env intracellular trafficking. Confocal microscopy showed that the intracellular distribution of Env with a mutation in the tyrosine or diaromatic motif differed from that of Env with no mutation in these motifs. Surprisingly, the gp41 CTs of the primary viruses also had differential effects on the intracellular distribution of Env, independently of mutations in the tyrosine or diaromatic motifs, suggesting the involvement of additional determinants. Furthermore, analyses of virus replication kinetics indicated that the effects of mutations in the tyrosine or diaromatic motifs on viral replication depended on the gp41 CT context. These effects were at least partly due to differences in the efficiency of Env incorporation into virions. Thus, polymorphisms in primary HIV-1 gp41 CTs at the quasispecies or subtype level can influence the intracellular distribution of Env, its incorporation into virions, and viral replication capacity.     

Genetically Related Human Immunodeficiency Virus Type 1 in Three Adults of a Family with No Identified Risk Factor for Intrafamilial Transmission

A small number of cases of human immunodeficiency virus (HIV) infection have been reported in individuals with no identified risk factors for transmission. We report on the seroconversion of the 61-year-old mother and the subsequent finding of HIV seropositivity in the 66-year-old father of a 31-year-old AIDS patient. Extensive investigation failed to identify any risk factor for intrafamilial transmission. We conducted a genetic analysis and determined the amino acid signature patterns of the V3, V4, and V5 hypervariable domains and flanking regions in the HIV-1 gp120 env gene of 26 clones derived from proviral DNA in peripheral blood mononuclear cells of the members of the family. env sequences of the viruses isolated from the patients were compared with sequences of HIV-1 subtype B viruses from Europe and local field isolates. Phylogenetic analysis revealed that the sequences of the viruses isolated from the patients were genetically related and formed an intrafamilial cluster of HIV-1 distinct from other subtype B viruses. Interindividual nucleotide variability in the C2-V3 and V4-C4-V5 domains ranged between 1.2 and 5.0% and between 2.2 and 7.5%, respectively, whereas divergence between HIV strains from the patients and control viral strains ranged from 6.6 to 29.3%. The amino acid signature patterns of viral clones from the three patients were closely related. In the C2-V3 region, two minor clones derived from the son’s virus showed less nucleotide divergence (mean, 3.5 and 3.9%) than did the clones derived from the viruses of both parents or the seven other predominant clones derived from the virus from the son (mean, 5.4%). The top of the V3 loop of the last two clones and of all viral clones from the parents exhibited an unusual GPGG sequence. This is the first report of genotypic relatedness of HIV-1 in three adults of the same family in the absence of identified risk factor for transmission between the members of the family. Our findings suggest that atypical transmission of HIV may occur.