INTRODUCTION

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The genetic diversity and rapid variation of human immunodeficiency virus type 1 (HIV-1) continues to complicate the development of effective vaccines to limit the AIDS pandemic. While there are regional clusters of more closely related HIV-1 variants, viral strains that originate from different continents show more significant genetic differences. Group M represents the main group of HIV-1 strains; in addition, there is an outgroup of HIV-1 variants (group O) whose envelope sequences have diverged from those of the group M viruses by approximately 50% (52). The group M viruses have been further subdivided into subtypes, or clades, A through J, whose envelope gene sequences differ from each other by as much as 30 to 35% (52). Intersubtype recombinant genomes have also been identified (3, 4, 11, 12, 23, 28, 47, 48, 50), and this has complicated the analysis of HIV-1 genetic diversity.

Many of the first HIV-1 genomes to be analyzed at both the molecular and phenotypic levels were subtype B variants from the United States and Europe. Thus, much of our understanding of the biology and pathogenesis of HIV-1 comes from analyses of clade B viruses (2). It remains unclear whether the subtypes, which are defined purely on the basis of sequence similarity, also define groups with any biological or immunological differences. As a result, it is also unclear whether the development of effective vaccines will require the use of geographically and/or subtype-specific vaccine strains. Over the past few years, more and more viral strains from throughout the world have been characterized (35). However, the majority of these studies have focused on a relatively small number of viral isolates, in some cases repetitive isolates from a single individual. Thus, the true extent of viral diversity within a particular HIV-1-infected endemic population has rarely been examined in detail.

Representatives of most of the group M HIV-1 subtypes have been found in sub-Saharan Africa (2, 15), where the global impact of HIV-1 infection and disease has been most apparent (55). It is estimated that 70% of individuals infected with HIV-1 reside in sub-Saharan Africa and that new infections are occurring there at a rate of 4 million per year, including 0.5 million new infections per year in infants (55). The distribution of different subtypes within African populations is usually not linked to particular risk behaviors. In contrast, different subtypes are clustered within distinct risk groups in some Asian countries (8, 22, 42, 56). Thus, Africa is an ideal setting in which to examine in more detail the diversity and mixing of viruses of different subtypes on a population basis.

In Kenya, HIV-1 infection was first reported in the mid-1980s in a group of Nairobi sex workers (21, 38). Of the African countries, Kenya has one of the highest prevalences of HIV-1 (32), and the epidemic has included the spread of at least two group M HIV-1 subtypes (17, 26, 45, 57). To gain a comprehensive picture of HIV-1 diversity in Kenya, we have determined HIV-1 subtypes by using proviral envelope gene sequences from 320 seropositive Nairobi women who were participants in a breast-feeding transmission study of HIV-1 (19, 36). Subtype A, C, D, and G genomes were identified, as well as several recombinant envelope gene sequences that are distinct from previously described HIV-1 group M or O genomes. Among these subjects, plasma RNA levels and CD4 lymphocyte counts were measured at the same time to determine whether there were differences in disease stage among persons infected with different subtypes.

	MATERIALS AND METHODS

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Study population. The 347 women who provided blood samples for this study were participants in a randomized clinical trial of breast and formula feeding to determine the frequency of breast milk transmission of HIV-1 in Nairobi, Kenya (19, 36). After giving informed consent, the women were tested for HIV-1-specific antibodies, typically during their third trimester. Sera were tested by the use of a peptide enzyme-linked immunosorbent assay (Behring, Ausgabe, Germany), and samples that were reactive were confirmed by using a second enzyme-linked immunosorbent assay (Cambridge Biotech, Rockville, Md.). Each HIV-1-seropositive woman who enrolled in the trial underwent a baseline clinical evaluation, including determination of CD4 and CD8 lymphocyte counts by flow-cytometric analysis using specific monoclonal antibodies (Becton Dickinson). The blood samples were separated into cellular and plasma fractions by standard methods, and the samples were stored in liquid nitrogen and a 70°C freezer, respectively, for subsequent virological analyses.

PCR amplification. Subjects' peripheral blood mononuclear cell (PBMC) samples, which were isolated from approximately 5 to 10 ml of blood, were lysed in the presence of proteinase K as described previously (34). Typically, 2 µl of the total 100-µl volume of lysate was used in a PCR. Primers and reaction conditions that have been used extensively in our previous studies to amplify envelope sequences from Kenyan subjects were used in the first-round PCR (44, 46). These primers (env13 [5'-CCA CTC TAT TTT GTG CAT CAG A-3'] and env12 [5'-CCT GGT GGG TGC TAC TCC TA-3']) amplify a 1.2-kb fragment encoding V1 through V5 of the extracellular envelope glycoprotein. This first-round product was used for all subsequent second-round PCRs.

HMA. The protocol for subtype analysis was based on an HIV-1 envelope gene HMA method described previously (5-7). However, in order to maximize and streamline our ability to determine Kenyan HIV-1 subtypes, modifications were made to this protocol as we accumulated subtype information from our cohort. Specifically, envelope sequences from known Kenyan clade A and D sequences were added to the reference strain panel when they became available (45), as described below.

Cloning and sequence analyses. The 1.2-kb envelope fragment spanning V1 through V5 was amplified and cloned into M13 by methods described previously (44, 46). For each subject, two to four clones were obtained from at least two independent first-round PCRs. The sequences of both strands were determined by using an ABI 377 automated sequencer (Applied Biosystems).

Phylogenetic and recombinant analyses. A multiple alignment of the 16 new envelope sequences from Kenya with several reference HIV-1 sequences of each subtype was generated. For 15 of the 16 Kenyan sequences, sequences encompassing C2 through V5 were analyzed. For one envelope gene (MM2227), the clone included only V1 through V3 sequences, and so this sequence was analyzed in a separate phylogenetic analysis. Gaps which had to be introduced to create the alignment were eliminated in the analysis. Reference isolates of subtype A (Q23 from Kenya, UG037 from Uganda, DJ263 from Djibouti, and IbNG from Nigeria), subtype B (MN and SF2 from the United States), subtype C (C2220 from Ethiopia, BR025 from Brazil, UG268 from Uganda, and SM145 from Somalia), subtype D (MB2059 and TK1316 from Kenya, UG274 and UG114 from Uganda, and NDK and ELI from Zaire), subtype E (CM240 from Thailand and CAR402 from Central African Republic), subtype F (F9363 from Zaire and BZ163 from Brazil), G (HH8793 from Kenya, NG083 from Nigeria, and SE6165 from Congo), H (V1991 from Zaire and CF056 from Central African Republic), and J (SE9280 and SE9173 from Zaire) were used (35). The multiple alignment was then broken into overlapping segments of equal length, and each segment was analyzed separately. Briefly, phylogenetic trees were constructed and the consistency of branching order was evaluated by using the SEQBOOT, DNADIST, NEIGHBOR, CONSENSE, and DNAPARS modules of the Phylip package (V3.52c) (10) and TREETOOL (27). Bootscanning, an analytical approach that tracks the bootstrap value of the node joining an unknown sequence with known sequences progressively across the genome, was used to identify putative recombination breakpoints (51). A bootstrap value equal to or greater than 70% was considered definitive (14). In addition, distance scanning (3) was performed by computing the genetic distances between isolates, using the technique of maximum likelihood with a transition-transversion ratio of 2.0, and the distances between the unknown isolate and isolates of known subtypes were evaluated for each segment. These distances were normalized within each fragment in order to aid comparability across the genome. Using these two scanning techniques, breakpoints were identified, and the segments were then used in separate phylogenetic analyses to confirm the subtype origin of the segment. Each segment was analyzed by building a phylogenetic tree via the neighbor-joining method (49), and the stability of the nodes was assessed by using maximum parsimony (10, 53) with the bootstrap value (9).

Plasma RNA methods. To determine the viral loads of plasma samples, we used a quantitative assay for HIV-1 RNA which is under development at Gen-Probe Incorporated. This method utilizes an integrated approach in which sample preparation, amplification, and detection are performed in a single tube. The assay protocol includes three hybridization-based procedures: (i) target capture and magnetic microparticle-based sample preparation, (ii) amplification of viral sequences by transcription-mediated amplification (TMA), and (iii) detection of the amplicon by use of the hybridization protection assay. This integrated approach is highly effective and allows processing of 200 samples in less than 6 h (13). The specimen processing method releases and stabilizes the viral RNA, which is then captured with oligonucleotides that contain sequences complementary to the viral RNA and poly(dA) tails complementary to poly(T) tails on a magnetic particle. The use of magnetic racks allows washing of the magnetic particles and elimination of unwanted clinical specimen. Amplification of HIV-1-specific viral sequences is performed by TMA (29). TMA is an exponential isothermal reaction that utilizes reverse transcriptase and T7 RNA polymerase. Briefly, the reaction is initiated by the annealing of a chimeric primer that contains a T7 polymerase promoter coupled to an HIV-1-specific primer, which serves to prime DNA synthesis via reverse transcriptase. The T7 primer is extended by reverse transcriptase to form an RNA-DNA duplex, and the RNase H activity of the reverse transcriptase degrades the RNA in this duplex. A second HIV-1-specific primer is used to create a double-stranded DNA copy of the target RNA. The DNA, which is engineered to include 5' promoter sequences, then serves as a template for RNA synthesis by T7 polymerase. The RNA product is then subjected to the same cycle of DNA synthesis and RNA amplification, leading to a greater than 10⁹-fold amplification of the specific target nucleic acid. The amplified product is detected by using chemiluminescence-labeled oligonucleotide probes in a homogeneous hybridization assay, the hybridization protection assay (37). Results (in relative light units) from each sample are converted to copies per milliliter of HIV-1 virus by interpolation against an external standard curve run at the same time as the samples. The current Gen-Probe quantitative assay has a dynamic range of 50 to 100,000 copies/ml and between-run coefficients of variation of less than 20% down to 500 copies/ml. The use of capture oligonucleotides, primers, and detection probes targeted to conserved regions of the pol gene of HIV-1 allows detection and quantification of all HIV-1 viral subtypes, including group O strains, with similar efficiencies.

Statistical methods. Median CD4 cell counts and plasma viral loads of subtypes were compared by nonparametric statistical tests. Comparisons were conducted between each subtype and other subtypes, with the latter being considered both as a grouped and as a separate variable (e.g., for subtype A, A versus non-A, A versus D, and A versus C). Plasma viral load was divided into quartiles (<10,000, 10,000 to 42,000, 42,000 to 162,000, and >162,000). Women with viral loads in the highest and lowest quartiles were compared with the remainder of the cohort by the use of nonparametric statistical tests for continuous variables (CD4 cell counts) and chi-square tests for dichotomous variables (subtypes).

Nucleotide sequence accession numbers. The 16 Kenyan envelope sequences described here are available under GenBank accession no. AF101456 to AF101471. The GenBank accession numbers for Q23, TK1316, and 2059 are AF004885, AF133822, and AF133821, respectively.

	RESULTS

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PCR amplification of sequences from PBMC samples. A total of 347 PBMC samples from pregnant women participating in a breast feeding transmission study of HIV-1 in Nairobi were analyzed. The envelope sequences of 23 samples from subjects identified serologically as being HIV-1 positive could not be amplified. In 19 of these cases, a 142-bp HIV-1 gag sequence was amplified, which suggested that the sample contained HIV-1 proviruses. We were unable to determine why envelope sequences, from these samples could not be amplified, but possible explanations include partially degraded or very small amounts of PBMC sample, a very low proviral copy number, and inefficient amplification due to extensive sequence divergence from the primers. For four additional samples, the amount of envelope PCR product was not sufficient for HMA analysis. For the purposes of this report, these 4 samples, along with the 23 amplification-negative samples, were not considered further, and the analyses include only the 320 samples whose subtypes were determined.

Characterization of viral subtype by HMA of envelope sequences. The envelope sequences amplified from the 320 PBMC samples were analyzed by HMA against reference strains of known subtype, using a sequential approach described in Materials and Methods. In the primary screen, a fragment encompassing V1 through C3 was analyzed against clade A, D, and C subtype reference strains, and the subtypes of 167 (64%) of 261 samples, including 123 subtype A, 32 subtype D, and 12 subtype C, were determined by this approach. However, the subtypes of the remaining 94 samples could not be assigned with these six reference strains by using V1 through C3 sequences.

View larger version (21K): [in this window] [in a new window]   FIG. 1.   Summary flowchart of subtype analysis based on the eventual subtype identified in the sample. The different steps in this analyses included a primary screen using V1 through V3 sequences versus standard reference strains from the AIDS Research and Reference Reagent Program, a secondary screen using V1 through V3 sequences against Kenyan reference strains, and a tertiary screen using a C2-C3 fragment against the standard reference strains, as described in Materials and Methods. The envelope sequences used for HMA are indicated for each subtype, and the number of samples tested against these reference sequences is shown in the flowchart. Samples that were successfully typed at each stage are indicated at the base of a closed arrow, and samples that could not be typed with the indicated sequences are indicated following a dotted line at the base of an open arrowhead. The latter were then tested with the next screen, and the results of this screen are similarly displayed. In cases in which samples were not analyzed with the primary screen, the flowchart begins at the secondary screen, and these samples are shown separately to the right. The percentages that were typed or not typed at each screen are indicated in parentheses. Viral envelope genes that could not be assigned subtypes based on the HMA sequential screening method and required sequence analysis are indicated with asterisks. The results are for subtypes A (A), D (B), and C (C) and recombinants or subtype G (D).

Sequence analysis of unusual envelope genes. The envelope gene fragments of 16 proviral genomes that could not be assigned a subtype by the HMA approach were amplified and cloned. Two to four envelope clones from at least two PCRs were obtained from the PBMCs from each subject. The sequences spanning V1 through V5 from each of the 16 subjects were compared to a panel of HIV-1 subtype sequences by phylogenetic methods (Fig. 2). The sequences from an individual clustered closely together in the analysis (data not shown), indicating that the ambiguity in the HMA was not likely to be the result of extensive intrapatient viral sequence heterogeneity. For convenience, only one subject's sequence is represented in Fig. 2. The known-subtype reference sequences included in this analysis are representative of subtypes A through J (sequences for subtype I were not available). Seven of the Kenyan envelope genes (MM13376, MM9846, MM14999, MM1324, MM1480, MM9885, and MM2227 grouped with the clade C viruses (Fig. 2 and data not shown). Interestingly, one envelope gene (MM4089) clearly grouped with type G HIV-1; this is the only example of a subtype G variant in our cohort. One sequence (MM5035) grouped with clade D. MM5035 was unusual relative to other subtype D viruses from Kenya that we have examined because it grouped midway between the Zairian variants and the East African viruses. A more detailed analysis of this envelope sequence was performed, but the precise structure could not be resolved (data not shown). Seven sequences were outliers that showed weak associations with either subtype A, C, D, or G but were clearly distinct (data not shown).

View larger version (24K): [in this window] [in a new window]   FIG. 2.   Phylogenetic tree of HIV-1 envelope sequences. Phylogenetic analysis was performed as described in Materials and Methods, using C2 through V5 sequences and the subtype references sequences indicated. The number at each node indicates the percentage of bootstrap support as determined from 100 bootstrap resamplings and maximum-parsimony analysis. The phylogenetic tree was built by neighbor-joining analysis. The sequences from this cohort are shown in boldface.

Detection of subtype recombinants. The sequences of the seven outlier envelope genes were subjected to additional analyses, using a bootscan method designed to characterize subtype recombinants of HIV-1 (51). By using this method, segments of sequence that were highly related to known subtypes (A, C, D, and G) were defined, although other sequences were not clearly derived from any HIV-1 subtype A to J group. The bootscan analysis results for the four sequences derived from known subtypes are presented in Fig. 3. All four of these recombinants had subtype A-derived V4-V5 sequences, and two of these also had subtype A V3 sequences. Each viral envelope gene is highly distinct, with different apparent sites of recombination. In particular, there are examples among these clones of 5' sequences that originated from each of the other subtypes found in the Nairobi cohort (clades C, D, and G). Thus, each of these recombinants has a unique origin.

View larger version (43K): [in this window] [in a new window]   FIG. 3.   Recombination analysis: bootscans. Bootscans were performed as described in Materials and Methods, using subtype A (Q23), subtype C (SM145), subtype D (UG274), and subtype G (HH8793) HIV-1. The analysis included C2 through V5 sequences. Analysis of MM13898 included A (red), C (gold), and D (black). Analysis of MM2760 used A (red), G (green), and D (black). Analyses of MM13324 and MM6535 used A (red), D (blue), and C (black). A black triangle marks the spot where the sequences were separated. The phylogenetic trees are neighbor-joining trees with parsimony bootstrap values at the nodes containing the unknown. Schematics showing the deduced subtype structures are shown below the bootscans. V3, the location of the V3 loop (shown for orientation).

View larger version (50K): [in this window] [in a new window]   FIG. 4.   Recombination analysis: distance scans. Distance scanning was performed as described in Materials and Methods. The analysis included C2 through V5 sequences. Distances between the unknown and subtypes A (red), B (light blue), C (gold), D (dark blue), E (brown), F (magenta), G (green), H (black), and J (purple) were plotted. The black triangles represent locations where the sequences were broken to run phylogenetic trees. The phylogenetic trees were computed by the neighbor-joining method with parsimony bootstrap values at the nodes with the unknown. The deduced subtype structure of the each virus is shown below the distance scan. Regions in white could not be assigned to any known subtype. V3, the location of the V3 loop (shown for orientation).

View larger version (39K): [in this window] [in a new window]   FIG. 5.   Summary of the subtype distribution of viruses from the 320 women analyzed. (A) Pie chart of subtype distribution in the overall cohort. The percentage of viruses that belong to each subtype is indicated in the relevant pie section or above it. Rec, recombinant. (B) Geographic distribution of subtypes. The regions of origin were defined on the basis of the ethnicities of the women. This cohort represented ethnic groups from throughout Kenya.

Analysis of markers for disease in relation to viral subtype. Both CD4 lymphocyte counts and plasma viral loads are markers for the stage of disease and the level of immunosuppression (30, 31, 39, 40). To address whether infection with a particular subtype was associated with a more advanced disease stage, plasma RNA levels were measured by a quantitative HIV-1 viral load assay (Gen-Probe) that was designed to detect all HIV-1 subtypes with the same efficiency (1, 13). Additional pilot studies were performed with Kenyan viral isolates and plasma samples, using the Gen-Probe quantitative assay versus a quantitative RNA PCR method (24), and these studies further validated the use of this approach for quantitative analysis of subtype A, C, and D HIV-1 (data not shown). The median viral load for this cohort was 41,400 copies per ml (range, 112 to 1,228,480). The highest median viral load was seen in women infected with subtype C HIV-1 (71,800 copies/ml), although this was not significantly different from the median plasma RNA levels in women infected with clade A or D virus (39,040 and 36,400 copies/ml, respectively) (Table 1). Women infected with clade C viruses were the most likely to have viral loads in the upper quartile and the least likely to have viral loads in the lowest quartile. Most striking was the fact that women infected with subtype C had significantly lower CD4 cell counts than women infected with subtype A and D (P = 0.01 for C versus non-C). Although women infected with subtype D had only a very slightly lower median viral load than the population as a whole, they were significantly less likely to have a viral load in the upper quartile.

	DISCUSSION

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This study presents a detailed analysis of the envelope subtypes that are currently circulating in Kenya, which has one of the highest prevalences of HIV-1 infection among countries in sub-Saharan Africa (33). In that country, particular subtypes are not preferentially circulating within particular risk groups, allowing us to generate a representative picture of both the pure subtypes and the intersubtype envelope recombinants in Kenya. The majority of women were infected with clade A virus (70.3%); a significant fraction were infected with clade D (20.3%) or clade C (6.9%) HIV-1. One (0.3%) subtype G virus and seven (2.2%) intersubtype recombinants were also detected. The cohort analyzed in this study included women from western, central, and eastern Kenya, suggesting a very similar distribution of subtypes throughout that country.

HMA provides a relatively rapid tool for determining HIV-1 subtypes (7). While the existing reference strains provide a reasonable starting point for subtype assessment in a new population, our analyses suggest that when using these strains it is difficult to assign a significant fraction of Kenyan viruses (about 30%) by HMA of envelope sequences. This is similar to what was seen in a French study with participants from various parts of the world, in which HMA did not predict all viral subtypes (25). The use of geographically specific viruses significantly enhanced our power to determine subtypes in this cohort. In fact, we were successful at assigning the subtype for each of the subtype A and all but one of the subtype D viruses by using a combination of Kenyan and standard reference strains. Because we lacked Kenyan subtype C reference strains, we were not able to achieve the same success in typing subtype C envelope sequences. Conversely, we were not able to assign a subtype to all of the envelope genes examined by using only Kenyan reference sequences. Thus, although local reference strains perform better than subtype reference strains from different geographic locations, a combination of both appears to be optimal when using HMA to define HIV-1 subtypes.

By surveying such a large cohort, intersubtype recombinants representing viruses that had exchanged sequences within the coding region for the extracellular envelope glycoprotein were also identified. Proviral genomes that are mosaic over the length of the genome as well as recombinant forms of individual HIV-1 genes have been described (3, 4, 11, 12, 23, 28, 47, 48, 50). Although some of these recombinants were characterized directly from patients' samples (4, 50), many were identified in viruses amplified in culture. In the latter case, it is unclear whether the recombinant was present in the individual or it arose in culture from two pure subtypes in the individual. Moreover, there have been limited population-based studies of the prevalence of recombinant viruses. A survey of 320 subjects who came from throughout Kenya to work and live in Nairobi provided us with an opportunity to estimate the frequency of intersubtype recombinant genomes in the population. We detected seven (2.2%) intersubtype recombinants whose exchanges occurred within a segment of the gene coding for the extracellular envelope glycoprotein. This is likely to be a minimal estimate of the envelope recombinants in this population, since it is certainly possible that additional envelope recombinants were not identified in the HMA screen because either the majority of the sequence represented one subtype, leading it to behave like a pure subtype in the HMA, or recombination occurred outside of the V1 through C3 region analyzed in the HMA. Approximately 10% of the viral genome was analyzed. If we extrapolate from the frequency of recombinant envelope genes observed here and assume that there is not a strong bias for or against protein function of a recombinant envelope gene in comparison to other viral genes, then >20% of Kenyan HIV-1 proviral genomes would be predicted to be intersubtype recombinants. These estimates are likely to be relevant for most of sub-Saharan Africa because there are similar mixtures of multiple subtypes in many countries in this region (2).

Among the recombinant envelope genes analyzed here, sequences that did not closely associate with any known subtype were identified. Distance scan analyses demonstrated that these sequences originated from viruses that are within the group M cluster rather than from a more divergent HIV-1 group. These sequences may represent multiple small fragments of known subtype or novel group M subtypes not yet described.

Recombination between the diploid RNA genomes during DNA synthesis is a common mechanisms for generating diversity in retroviruses (20). To generate a recombinant during reverse transcription, a single cell must be coinfected with two different viruses so that copackaging of heterologous RNAs can occur. Thus, detection of intersubtype recombinants implies that at some previous time a person was coinfected with HIV-1 of two different subtypes. This may have occurred as a result of simultaneous transmission of multiple variants, a common occurrence in women (43, 46), or by a second infection of the individual, with a different subtype, at a later time. At some point, dual infection must have occurred to initiate the cascade of events leading to the detection of recombinant genomes. Because recombination is predicted to occur quite commonly when there is coinfection of a cell by two highly related retroviruses (16, 20), the limiting event in the generation of intersubtype recombinants is likely to be dual infection. At this point, little is known about the factors that determine the frequency of dual infection in a population. As discussed herein, the predicted frequency of intersubtype recombinant genomes in this cohort is estimated to be approximately 20%, using fairly conservative assumptions. This implies either that reinfection occurs very commonly in HIV-1-infected individuals or that recombinant viruses are favored for transmission. In the latter case, the viruses could originate from a true genetic recombinant provirus, or they could represent particles assembled from viral proteins expressed from distinct viral genomes in a dually infected individual. Certainly it will be important to distinguish between these possibilities in considering the design of vaccines for African populations.

The subtypes define broad categories of viruses with similar sequence characteristics. In the viruses examined here, subtype was defined on the basis of sequences coding for the extracellular envelope protein, which is the protein that determines cell tropism as well as the replication patterns of the virus. Thus, it is possible that viruses that have different envelope subtypes will replicate with different efficiencies in the host, leading to different clinical outcomes. To address this possibility, we analyzed plasma viral loads and CD4 lymphocyte counts as markers for disease stage in this cohort. Women infected with clade C viruses tended to have higher viral loads, and they had significantly lower CD4 cell counts than women infected with clade A or D HIV-1. From these data, we cannot discriminate between the possibility that women infected with clade C virus have been infected for a longer period of time and the possibility that clade C infection is associated with a more rapid disease progression and, as a consequence, a more rapid CD4 cell count decline and higher viral loads. Recent studies suggest that viruses which can use the CXCR4 coreceptor are underrepresented among subtype C HIV-1, including viruses that were derived from individuals at later stages of infection and disease (54). This finding may suggest that subtype C variants use novel coreceptor proteins for entry into the cell. Interestingly, women infected with clade C viruses in the cohort described here were also significantly more likely to shed HIV-1-infected cells in their vaginal secretions (18). Together, these data suggest that viruses with clade C-derived envelope sequences may have unique replication properties which may affect transmission and/or progression. This can only be directly addressed by longitudinal studies of disease progression in relation to viral subtype, particularly in cohorts in which clade C virus infection represents a more significant fraction of all HIV-1 infections. Certainly, such information on differences in the biological properties or pathogenesis of different subtypes will be important in designing strategies to limit the spread of the most virulent HIV-1 variants.