Human Immunodeficiency Virus Type 1 Population Research Articles

Metagenomic Sequencing of HIV-1 in the Blood and Female Genital Tract Reveals Little Quasispecies Diversity during Acute Infection.

Heterosexual transmission of human immunodeficiency virus type 1 (HIV-1) is associated with a significant bottleneck in the viral quasispecies population, yet the timing of that bottleneck is poorly understood. We characterized HIV-1 diversity in the blood and female genital tract (FGT) within 2 weeks after detection of infection in three women enrolled in a unique prospective cohort in South Africa. We assembled full-length HIV-1 genomes from matched cervicovaginal lavage (CVL) samples and plasma. Deep sequencing allowed us to identify intrahost single-nucleotide variants (iSNVs) and to characterize within-sample HIV-1 diversity. Our results demonstrated very little HIV-1 diversity in the FGT and plasma by the time viremia was detectable. Within each subject, the consensus HIV-1 sequences were identical in plasma and CVL fluid. No iSNV was present at >6% frequency. One subject had 77 low-frequency iSNVs across both CVL fluid and plasma, another subject had 14 iSNVs in only CVL fluid from the earliest time point, and the third subject had no iSNVs in CVL fluid or plasma. Overall, the small amount of diversity that we detected was greater in the FGT than in plasma and declined over the first 2 weeks after viremia was detectable, compatible with a very early HIV-1 transmission bottleneck. To our knowledge, our study represents the earliest genomic analysis of HIV-1 in the FGT after transmission. Further, the use of metagenomic sequencing allowed us to characterize other organisms in the FGT, including commensal bacteria and sexually transmitted infections, highlighting the utility of the method to sequence both HIV-1 and its metagenomic environment.IMPORTANCE Due to error-prone replication, HIV-1 generates a diverse population of viruses within a chronically infected individual. When HIV-1 is transmitted to a new individual, one or a few viruses establish the new infection, leading to a genetic bottleneck in the virus population. Understanding the timing and nature of this bottleneck may provide insight into HIV-1 vaccine design and other preventative strategies. We examined the HIV-1 population in three women enrolled in a unique prospective cohort in South Africa who were followed closely during the earliest stages of HIV-1 infection. We found very little HIV-1 diversity in the blood and female genital tract during the first 2 weeks after virus was detected in the bloodstream. These results are compatible with a very early HIV-1 population bottleneck, suggesting the need to study the HIV-1 population in the female genital tract before virus is detectable in the bloodstream.

Impact of the HIV-1 genetic background and HIV-1 population size on the evolution of raltegravir resistance

BackgroundEmergence of resistance against integrase inhibitor raltegravir in human immunodeficiency virus type 1 (HIV-1) patients is generally associated with selection of one of three signature mutations: Y143C/R, Q148K/H/R or N155H, representing three distinct resistance pathways. The mechanisms that drive selection of a specific pathway are still poorly understood. We investigated the impact of the HIV-1 genetic background and population dynamics on the emergence of raltegravir resistance. Using deep sequencing we analyzed the integrase coding sequence (CDS) in longitudinal samples from five patients who initiated raltegravir plus optimized background therapy at viral loads > 5000 copies/ml. To investigate the role of the HIV-1 genetic background we created recombinant viruses containing the viral integrase coding region from pre-raltegravir samples from two patients in whom raltegravir resistance developed through different pathways. The in vitro selections performed with these recombinant viruses were designed to mimic natural population bottlenecks.ResultsDeep sequencing analysis of the viral integrase CDS revealed that the virological response to raltegravir containing therapy inversely correlated with the relative amount of unique sequence variants that emerged suggesting diversifying selection during drug pressure. In 4/5 patients multiple signature mutations representing different resistance pathways were observed. Interestingly, the resistant population can consist of a single resistant variant that completely dominates the population but also of multiple variants from different resistance pathways that coexist in the viral population. We also found evidence for increased diversification after stronger bottlenecks. In vitro selections with low viral titers, mimicking population bottlenecks, revealed that both recombinant viruses and HXB2 reference virus were able to select mutations from different resistance pathways, although typically only one resistance pathway emerged in each individual culture.ConclusionsThe generation of a specific raltegravir resistant variant is not predisposed in the genetic background of the viral integrase CDS. Typically, in the early phases of therapy failure the sequence space is explored and multiple resistance pathways emerge and then compete for dominance which frequently results in a switch of the dominant population over time towards the fittest variant or even multiple variants of similar fitness that can coexist in the viral population.

HIV-1 molecular epidemiology among newly diagnosed HIV-1 individuals in Hebei, a low HIV prevalence province in China.

New human immunodeficiency virus type 1 (HIV-1) diagnoses are increasing rapidly in Hebei. The aim of this study presents the most extensive HIV-1 molecular epidemiology investigation in Hebei province in China thus far. We have carried out the most extensive systematic cross-sectional study based on newly diagnosed HIV-1 positive individuals in 2013, and characterized the molecular epidemiology of HIV-1 based on full length gag-partial pol gene sequences in the whole of Hebei. Nine HIV-1 genotypes based on full length gag-partial pol gene sequence were identified among 610 newly diagnosed naïve individuals. The four main genotypes were circulating recombinant form (CRF)01_AE (53.4%), CRF07_BC (23.4%), subtype B (15.9%), and unique recombinant forms URFs (4.9%). Within 1 year, three new genotypes (subtype A1, CRF55_01B, CRF65_cpx), unknown before in Hebei, were first found among men who have sex with men (MSM). All nine genotypes were identified in the sexually contracted HIV-1 population. Among 30 URFs, six recombinant patterns were revealed, including CRF01_AE/BC (40.0%), CRF01_AE/B (23.3%), B/C (16.7%), CRF01_AE/C (13.3%), CRF01_AE/B/A2 (3.3%) and CRF01_AE/BC/A2 (3.3%), plus two potential CRFs. This study elucidated the complicated characteristics of HIV-1 molecular epidemiology in a low HIV-1 prevalence northern province of China and revealed the high level of HIV-1 genetic diversity. All nine HIV-1 genotypes circulating in Hebei have spread out of their initial risk groups into the general population through sexual contact, especially through MSM. This highlights the urgency of HIV prevention and control in China.

Usefulness of calcaneal quantitative ultrasound stiffness for the evaluation of bone health in HIV-1-infected subjects: comparison with dual X-ray absorptiometry.

ObjectivesWith the development of effective treatments and the resulting increase in life expectancy, bone mineral density (BMD) alteration has emerged as an important comorbidity in human immunodeficiency virus type-1 (HIV-1)-infected individuals. The potential contributors to the pathogenesis of osteopenia/osteoporosis include a higher prevalence of risk factors, combined antiretroviral therapy (cART)-exposure, HIV-1 itself and chronic immune activation/inflammation. Dual-energy X-ray absorptiometry (DXA) is the “gold standard” technique for assessing bone status in HIV-1 population.MethodsWe conducted a cross-sectional study to investigate bone mineral status in a group of 158 HIV-1-infected subjects. The primary endpoint was the feasibility of calcaneal quantitative ultrasound (QUS) as a screening tool for BMD. All subjects were receiving stable cART and were virologically suppressed (HIV-RNA <37 copies/mL) from at least 12 months. Calcaneal QUS parameters were analyzed to obtain information on bone mass and microarchitecture. The results were compared with those obtained by DXA.ResultsNo correlations were found between DXA/QUS parameters and demographic or HIV-1-specific characteristics, also including cART strategies. In the univariate analyses BMD, QUS indexes, and Fracture Risk Assessment Tool scores conversely showed significant associations with one or more demographic or HIV-1-related variables. Moreover, a significant relationship between calcaneal quantitative ultrasound index/stiffness and femoral/lumbar BMD values from DXA was described. The multivariate analysis showed an independent association between calcaneal quantitative ultrasound index/stiffness and body mass index, higher CD4+ T-cell numbers and low 25-OH D2/D3 vitamin D levels <10 ng/mL (P-values: 0.004, 0.016, and 0.015, respectively).ConclusionAs an alternative and/or integrative examination to DXA, calcaneal QUS could be proposed as a useful screening in HIV-1-infected patients for assessing bone health impairment. In fact, the results obtained confirm that calcaneal QUS may be useful for monitoring bone status, being a noninvasive and inexpensive technique, especially in those subjects with the classical traditional risk factors for bone damage that were observed earlier in HIV-1 population.

Human APOBEC3 induced mutation of human immunodeficiency virus type-1 contributes to adaptation and evolution in natural infection.

Human APOBEC3 proteins are cytidine deaminases that contribute broadly to innate immunity through the control of exogenous retrovirus replication and endogenous retroelement retrotransposition. As an intrinsic antiretroviral defense mechanism, APOBEC3 proteins induce extensive guanosine-to-adenosine (G-to-A) mutagenesis and inhibit synthesis of nascent human immunodeficiency virus-type 1 (HIV-1) cDNA. Human APOBEC3 proteins have additionally been proposed to induce infrequent, potentially non-lethal G-to-A mutations that make subtle contributions to sequence diversification of the viral genome and adaptation though acquisition of beneficial mutations. Using single-cycle HIV-1 infections in culture and highly parallel DNA sequencing, we defined trinucleotide contexts of the edited sites for APOBEC3D, APOBEC3F, APOBEC3G, and APOBEC3H. We then compared these APOBEC3 editing contexts with the patterns of G-to-A mutations in HIV-1 DNA in cells obtained sequentially from ten patients with primary HIV-1 infection. Viral substitutions were highest in the preferred trinucleotide contexts of the edited sites for the APOBEC3 deaminases. Consistent with the effects of immune selection, amino acid changes accumulated at the APOBEC3 editing contexts located within human leukocyte antigen (HLA)-appropriate epitopes that are known or predicted to enable peptide binding. Thus, APOBEC3 activity may induce mutations that influence the genetic diversity and adaptation of the HIV-1 population in natural infection.

Selection of a Simian-Human Immunodeficiency Virus Strain Resistant to a Vaginal Microbicide in Macaques

PSC-RANTES binds to CCR5, inhibits human immunodeficiency virus type 1 (HIV-1) entry, and has been shown as a vaginal microbicide to protect rhesus macaques from a simian-human immunodeficiency virus chimera (SHIV(SF162-p3)) infection in a dose-dependent manner. In this study, env gene sequences from SHIV(SF162-p3)-infected rhesus macaques treated with PSC-RANTES were analyzed for possible drug escape variants. Two specific mutations located in the V3 region of gp120 (K315R) and C-helical domain of gp41 (N640D) were identified in a macaque (m584) pretreated with a 100 microM dose of PSC-RANTES. These two env mutations were found throughout infection (through week 77) but were found at only low frequencies in the inoculating SHIV(SF162-p3) stock and in the other SHIV(SF162-p3)-infected macaques. HIV-1 env genes from macaque m584 (env(m584)) and from inoculating SHIV(SF162-p3) (env(p3)) were cloned into an HIV-1 backbone. Increases in 50% inhibitory concentrations to PSC-RANTES with env(m584) were modest (sevenfold) and most pronounced in cells expressing rhesus macaque CCR5 as compared to human CCR5. Nonetheless, virus harboring env(m584), unlike inoculating virus env(p3), could replicate even at the highest tissue culture PSC-RANTES concentrations (100 nM). Dual-virus competitions revealed a dramatic increase in fitness of chimeric virus containing env(m584) (K315R/N640D) over that containing env(p3), but again, only in rhesus CCR5-expressing cells. This study is the first to describe the immediate selection and infection of a drug-resistant SHIV variant in the face of a protective vaginal microbicide, PSC-RANTES. This rhesus CCR5-specific/PSC- RANTES resistance selection is particularly alarming given the relative homogeneity of the SHIV(SF162-p3) stock compared to the potential exposure to a heterogeneous HIV-1 population in human transmission.

Major Coexisting Human Immunodeficiency Virus Type 1envGene Subpopulations in the Peripheral Blood Are Produced by Cells with Similar Turnover Rates and Show Little Evidence of Genetic Compartmentalization

A distinctive feature of chronic human immunodeficiency virus type 1 (HIV-1) infection is the presence of multiple coexisting genetic variants, or subpopulations, that comprise the HIV-1 population detected in the peripheral blood. Analysis of HIV-1 RNA decay dynamics during the initiation of highly active antiretroviral therapy (HAART) has been a valuable tool for modeling the life span of infected cells that produce the bulk HIV-1 population. However, different HIV-1 target cells may have different turnover rates, and it is not clear whether the bulk HIV-1 RNA decay rate actually represents a composite of the decay rates of viral subpopulations compartmentalized in different cellular subsets with different life spans. Using heteroduplex tracking assays targeting the highly variable V3 or V4-V5 regions of the HIV-1 env gene in eight subjects, we found that all detectable coexisting HIV-1 variants in the peripheral blood generally decayed at similar rates during the initiation of HAART, suggesting that all of the variants were produced by cells with similar life spans. Furthermore, single genome amplification and coreceptor phenotyping revealed that in two subjects coexisting HIV-1 variants with distinct CXCR4 or CCR5 coreceptor phenotypes decayed with similar rates. Also, in nine additional subjects, recombination and a lack of genetic compartmentalization between X4 and R5 variants were observed, suggesting an overlap in host cell range. Our results suggest that the HIV-1 env subpopulations detectable in the peripheral blood are produced by cells with similar life spans and are not genetically isolated within particular cell types.

Human Immunodeficiency Virus Type 1 Population Genetics and Adaptation in Newly Infected Individuals

Studies on human immunodeficiency virus type 1 (HIV-1) diversity are critical for understanding viral pathogenesis and the emergence of immune escape variants and for design of vaccine strategies. To investigate HIV-1 population genetics, we used single-genome sequencing to obtain pro-pol and env sequences from longitudinal samples (n = 93) from 14 acutely or recently infected patients. The first available sample after infection for 12/14 patients revealed HIV-1 populations with low genetic diversity, consistent with transmission or outgrowth of a single variant. In contrast, two patients showed high diversity and coexistence of distinct virus populations in samples collected days after a nonreactive enzyme-linked immunosorbent assay or indeterminate Western blot, consistent with transmission or outgrowth of multiple variants. Comparison of PR and RT sequences from the first sample for all patients with the consensus subgroup B sequence revealed that nearly all nonsynonymous differences were confined to identified cytotoxic T-lymphocyte (CTL) epitopes. For HLA-typed patients, mutations compared to the consensus in transmitted variants were found in epitopes that would not be recognized by the patient's major histocompatibility complex type. Reversion of transmitted mutations was rarely seen over the study interval (up to 5 years). These data indicate that acute subtype B HIV-1 infection usually results from transmission or outgrowth of single viral variants carrying mutations in CTL epitopes that were selected prior to transmission either in the donor or in a previous donor and that reversion of these mutations can be very slow. These results have important implications for vaccine strategies because they imply that some HLA alleles could be compromised in newly acquired HIV infections.

Sensitive Oligonucleotide Ligation Assay for Low-Level Detection of Nevirapine Resistance Mutations in Human Immunodeficiency Virus Type 1 Quasispecies

This study has adapted the oligonucleotide ligation assay (OLA) to probe for low-level nevirapine (NVP) resistance mutations K103N and Y181C in the human immunodeficiency virus type 1 (HIV-1) population of infected mother-infant pairs from Uganda. When NVP is used to prevent perinatal transmission, NVP-resistant HIV-1 clones may be rapidly selected due to a low barrier for mutation and a relatively high level of fitness (compared to that of other drug-resistant HIV-1 clones). Monitoring for even a low frequency of NVP resistance mutations may help predict the success of subsequent treatment or warrant the use of another regimen to prevent transmission in a subsequent pregnancy. The standard OLA was optimized by using nonstandard bases in oligonucleotides to allow promiscuous base pairing and accommodate significant HIV-1 heterogeneity. Radiolabeled as opposed to fluorescently tagged oligonucleotides increased the sensitivity, whereas alteration of the template, oligonucleotides, salt, and thermostable DNA ligase concentrations increased the specificity for the detection of minority codons. This modified OLA is now capable of detecting mutants with the K103N or the Y181C mutation present in an HIV-1 population at a frequency of approximately 0.4% and is at least 10- to 30-fold more sensitive than the original protocol. A cohort of 19 Ugandan mothers who received NVP treatment perinatally were sampled 6 weeks postdelivery. Ten of 19 HIV-1 DNA samples extracted from peripheral blood mononuclear cells had a detectable K103N (0.5 to 44%) or Y181C (0.8 to 92.5%) mutation, but only one plasma HIV-1 RNA sample had a viral population with the Y181C mutation. These findings suggest that OLA is a robust, sensitive, and specific method for the detection of low-frequency drug resistance mutations in an intrapatient HIV-1 population.

Increased Multinucleoside Drug Resistance and Decreased Replicative Capacity of a Human Immunodeficiency Virus Type 1 Variant with an 8-Amino-Acid Insert in the Reverse Transcriptase

Resistance to antiretroviral drugs is generally conferred by specific amino acid substitutions, rather than insertions or deletions, in reverse transcriptase (RT) of human immunodeficiency virus type 1 (HIV-1). The exception to these findings is the amino acid insertions found in the beta3-beta4 loop of the RT enzyme in response to treatment with nucleoside reverse transcriptase inhibitors. This insert consists most commonly of two amino acids, but we describe in detail the evolution of a variant with an 8-amino-acid (aa) insert in a patient treated with zidovudine (ZDV) and 2'-3'-dideoxycytidine (ddC). The 24-nucleotide insert is a partial duplication of local sequences but also contains a sequence segment of unknown origin. Extensive sequence analysis of longitudinal patient samples indicated that the HIV-1 population prior to the start of therapy contained not the wild-type amino acid 215T in RT but a mixture with 215D and 215C. Treatment with ZDV and subsequent ZDV-ddC combination therapy resulted in the evolution of an HIV-1 variant with a typical ZDV resistance genotype (41L, 44D, 67N, 69D, 210W, 215Y), which was slowly replaced by the insert-containing variant (41L, 44D, insert at position 69, 70R, 210W, 215Y). The latter variant demonstrated increased resistance to a wide range of drugs, indicating that the 8-aa insert augments nucleoside analogue resistance. The gain in drug resistance of the insert variant came at the expense of a reduction in replication capacity when assayed in the absence of drugs. We compared these data with the resistance and replication properties of 133 insert-containing sequences of different individuals present in the ViroLogic database and found that the size and actual sequence of the insert at position 69 influence the level of resistance to nucleoside analogues.

HIV-1 compartmentalization in diverse leukocyte populations during antiretroviral therapy.

CD4+ T lymphocytes are the primary target of human immunodeficiency virus type 1 (HIV-1), but there is increasing evidence that other immune cells in the blood, including CD8+ T lymphocytes and monocytes, are also productively infected. The extent to which these additional cellular reservoirs contribute to ongoing immunodeficiency and viral persistence during therapy remains unclear. In this study, we conducted a detailed investigation of HIV-1 diversity and genetic structure in CD4+ T cells, CD8+ T cells, and monocytes of 13 patients receiving highly active antiretroviral therapy (HAART). Analysis of molecular variance and nonparametric tests performed on HIV-1 envelope sequences provided statistically significant evidence of viral compartmentalization in different leukocyte populations. Signature pattern analysis and predictions of coreceptor use provided no evidence that selection arising from viral tropism was responsible for the genetic structure observed. Analysis of viral genetic variation in different leukocyte populations demonstrated the action of founder effects as well as significant variation in the extent of genetic differentiation between subpopulations among patients. In the absence of evidence for leukocyte-specific selection, these features were supportive of a metapopulation model of HIV-1 replication as described previously among HIV-1 populations in the spleen. Compartmentalization of the virus in different leukocytes may have significant implications for current models of HIV-1 population genetics and contribute to the highly variable way in which drug resistance evolves in different individuals during HAART.

Combination of drugs and drug-resistant reverse transcriptase results in a multiplicative increase of human immunodeficiency virus type 1 mutant frequencies.

Replication of drug-resistant human immunodeficiency virus type 1 (HIV-1) in the presence of drug can lead to the failure of antiretroviral drug treatment. Drug failure is associated with the accumulation of drug resistance mutations. Previous studies have shown that 3'-azido-3'-deoxythymidine (AZT), (-)2',3'-dideoxy-3'-thiacytidine (3TC), and AZT-resistant HIV-1 reverse transcriptase (RT) can increase the virus in vivo mutation rate. In this study, the combined effects of drug-resistant RT and antiretroviral drugs on the HIV-1 mutant frequency were determined. In most cases, a multiplicative effect was observed with AZT-resistant or AZT/3TC dually resistant RT and several drugs (i.e., AZT, 3TC, hydroxyurea, and thymidine) and led to increases in the odds of recovering virus mutants to over 20 times that of the HIV-1 mutant frequency in the absence of drug or drug-resistance mutations. This observation indicates that HIV-1 can mutate at a significantly higher rate when drug-resistant virus replicates in the presence of drug. These increased mutant frequencies could have important implications for HIV-1 population dynamics and drug therapy regimens.

Origin of human immunodeficiency virus type 1 quasispecies emerging after antiretroviral treatment interruption in patients with therapeutic failure.

The emergence of antiretroviral (ARV) drug-resistant human immunodeficiency virus type 1 (HIV-1) quasispecies is a major cause of treatment failure. These variants are usually replaced by drug-sensitive ones when the selective pressure of the drugs is removed, as the former have reduced fitness in a drug-free environment. This was the rationale for the design of structured ARV treatment interruption (STI) studies for the management of HIV-1 patients with treatment failure. We have studied the origin of drug-sensitive HIV-1 quasispecies emerging after STI in patients with treatment failure due to ARV drug resistance. Plasma and peripheral blood mononuclear cell samples were obtained the day of treatment interruption (day 0) and 30 and 60 days afterwards. HIV-1 pol and env were partially amplified, cloned, and sequenced. At day 60 drug-resistant variants were replaced by completely or partially sensitive quasispecies. Phylogenetic analyses of pol revealed that drug-sensitive variants emerging after STI were not related to their immediate temporal ancestors but formed a separate cluster, demonstrating that STI leads to the recrudescence and reemergence of a sequestrated viral population rather than leading to the back mutation of drug-resistant forms. No evidence for concomitant changes in viral tropism was seen, as deduced from env sequences. This study demonstrates the important role that the reemergence of quasispecies plays in HIV-1 population dynamics and points out the difficulties that may be found when recycling ARV therapies with patients with treatment failure.

A stochastic modeling of early HIV-1 population dynamics

In a recent paper, Tuckwell and Le Corfec [J. Theor. Biol. 195 (1998) 450–463] applied the multi-dimensional diffusion process to model early human immunodeficiency virus type-1 (HIV-1) population dynamics. The purpose of this paper is to assess certain features and consequences of their model in the context of Tan and Wu's stochastic approach [Math. Biosci. 147 (1998) 173–205].

A Stochastic Model for Early HIV-1 Population Dynamics

A simple stochastic mathematical model is developed and investigated for early human immunodeficiency virus type-1 (HIV-1) population dynamics. The model, which is a multi-dimensional diffusion process, includes activated uninfected CD4+T cells, latently and actively infected CD4+T cells and free virions occurring in plasma. Stochastic effects are assumed to arise in the process of infection of CD4+T cells and transitions may occur from uninfected to latently or actively infected cells by chance mechanisms. Using the best currently available parameter values, the intrinsic variability in response to a given initial infection is examined by solving the stochastic system numerically. We estimate the statistical distributions of the time of occurrence and the magnitude of the early peak in viral concentration. The maximum of the viral load has a value in the experimental range and its time of occurrence has a 95% confidence interval from 19.4 to 25.1 days. The stochastic nature of the growth of viral density is extremely pronounced in the first few days after initial infection. Threshold effects are noted at virion levels of about 3–5×10−5mm−3. In addition to modeling the intrinsic variability in HIV-1 growth, we have explored the effects of perturbations in the parameter values in order to assess the additional stochastic effects of between-patient variability. We found that changes in the initial number of virions or dose size, the rate at which latently infected CD4+T cells are converted to the actively infected form and the fraction of latent cells has only minor effects on the size, speed and variability of the response. In contrast, decreased speed and magnitude but greater variability in response were obtained when the death rate of uninfected CD4+T cells, the death rate of actively infected cells and the clearance rate of the virus were increased or when the appearance rate of uninfected CD4+T cells, the number of virions produced by infected cells, the infection rate of CD4+T cells and the initial number of uninfected activated CD4+T cells were decreased. We also determined the distribution of the time to reach a given virion density. From this distribution the probability of detection of the virus as a function of time can be estimated. The numerical results obtained are in the range of experimental values and are discussed in relation to recently proposed detection and testing procedures.

Human immunodeficiency virus type 1 RNA populations in faeces with higher homology to intestinal populations than to blood populations.

To determine whether human immunodeficiency virus type 1 (HIV-1) in faeces is representative of the HIV-1 population in intestinal tissue, we studied HIV-1 V3 variation in faeces, intestinal biopsies and serum from two individuals. Phylogenic analysis of HIV-1 V3-coding RNA in faeces from one individual showed three distinct genotypes. Viruses belonging to all three genotypes were also present in sigmoidal tissue and in serum. Jejunal tissue contained two of these three genotypes. Analysis of the V3-coding RNA in faeces of the other individual showed five distinct genotypes. One of these genotypes was present in all specimens from this individual. Besides this shared genotype, jejunal tissue and serum contained sequences belonging to one other genotype. In addition, one of the other three V3 variants was detected in sigmoidal tissue. For both persons the shared HIV-1 RNA genotypes in faeces and serum displayed a distinctly different frequency distribution. In one individual, the genotype which was detected in a majority of the clones in faeces (59%) and as a minority in serum (11%), was the most abundant genotype in jejunal and sigmoidal tissue (61% and 80%, respectively). For the other individual the genotype that was present in faeces in a significant number of clones (43%) was detected in serum as a minority (8%), whereas this genotype composed 47% of the clones isolated from jejunal tissue. Taken together these data suggest that faeces contain HIV-1 sequences that are derived from local HIV-1 replication in intestinal tissue.