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Objective: Recent surveillance data suggests the United States (U.S.) Army HIV epidemic is concentrated among men who have sex with men. To identify potential targets for HIV prevention strategies, the relationship between demographic and clinical factors and membership within transmission clusters based on baseline pol sequences of HIV-infected Soldiers from 2001 through 2012 were analyzed., Methods: We conducted a retrospective analysis of baseline partial pol sequences, demographic and clinical characteristics available for all Soldiers in active service and newly-diagnosed with HIV-1 infection from January 1, 2001 through December 31, 2012. HIV-1 subtype designations and transmission clusters were identified from phylogenetic analysis of sequences. Univariate and multivariate logistic regression models were used to evaluate and adjust for the association between characteristics and cluster membership., Results: Among 518 of 995 HIV-infected Soldiers with available partial pol sequences, 29% were members of a transmission cluster. Assignment to a southern U.S. region at diagnosis and year of diagnosis were independently associated with cluster membership after adjustment for other significant characteristics (p<0.10) of age, race, year of diagnosis, region of duty assignment, sexually transmitted infections, last negative HIV test, antiretroviral therapy, and transmitted drug resistance. Subtyping of the pol fragment indicated HIV-1 subtype B infection predominated (94%) among HIV-infected Soldiers., Conclusion: These findings identify areas to explore as HIV prevention targets in the U.S. Army. An increased frequency of current force testing may be justified, especially among Soldiers assigned to duty in installations with high local HIV prevalence such as southern U.S. states.

Characterization of HIV-1 subtype diversity in regions where vaccine trials are conducted is critical for vaccine development and testing. This study describes the molecular epidemiology of HIV-1 within a tea-plantation community cohort in Kericho, Kenya. Sixty-three incident infections were ascertained in the HIV and Malaria Cohort Study conducted in Kericho from 2003 to 2006. HIV-1 strains from 58 of those individuals were full genome characterized and compared to two previous Kenyan studies describing 41 prevalent infections from a blood bank survey (1999-2000) and 21 infections from a higher-risk cohort containing a mix of incident and prevalent infections (2006). Among the 58 strains from the community cohort, 43.1% were pure subtypes (36.2% A1, 5.2% C, and 1.7% G) and 56.9% were inter-subtype recombinants (29.3% A1D, 8.6% A1CD, 6.9% A1A2D, 5.2% A1C, 3.4% A1A2CD, and 3.4% A2D). This diversity and the resulting genetic distance between the observed strains will need to be addressed when vaccine immunogens are chosen. In consideration of current vaccine development efforts, the strains from these three studies were compared to five candidate vaccines (each of which are viral vectored, carrying inserts corresponding to parts of gag, pol, and envelope), which have been developed for possible use in sub-Saharan Africa. The sequence comparison between the observed strains and the candidate vaccines indicates that in the presence of diverse recombinants, a bivalent vaccine is more likely to provide T-cell epitope coverage than monovalent vaccines even when the inserts of the bivalent vaccine are not subtype-matched to the local epidemic.

The RV144 Thai vaccine trial has been the only vaccine study to show efficacy in preventing HIV infection. Ongoing molecular surveillance of HIV-1 in Southeast Asia is vital for vaccine development and evaluation. In this study a novel tool, the multi-region subtype specific PCR (MSSP) assay, that was able to identify subtypes B, C, CRF01&#95;AE for Thailand, other Southeast Asian countries, India and China is described. The MSSP assay is based on a nested PCR strategy and amplifies eight short regions distributed along the HIV-1 genome using subtype-specific primers. A panel of 41 clinical DNA samples obtained primarily from opiate users in northern Thailand was used to test the assay performance. The MSSP assay provided 73-100% sensitivity and 100% specificity for the three subtypes in each genome region. The assay was then field-tested on 337 sera from HIV infected northern Thai drug users collected between 1999 and 2002. Subtype distribution was CRF01&#95;AE 77.4% (n=261), subtype B 3.3% (n=11), CRF01&#95;AE/B recombinant 12.2% (n=41), CRF01&#95;AE/C recombinant 0.6% (n=2), and non-typeable 6.5% (n=22). The MSSP assay is a simple, cost-effective, and accurate genotyping tool for laboratory settings with limited resources and is sensitive enough to capture the recombinant genomes and dual infections., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Standardized assays to assess vaccine and antiviral drug efficacy are critical for the development of protective HIV-1 vaccines and drugs. These immune assays will be advanced by the development of standardized viral stocks, such as HIV-1 infectious molecular clones (IMC), that i) express a reporter gene, ii) are representative of globally diverse subtypes and iii) are engineered to easily exchange envelope (env) genes for expression of sequences of interest. Thus far, a subtype B IMC backbone expressing Renilla luciferase (LucR), and into which the ectodomain of heterologous env coding sequences can be expressed has been successfully developed but as execution of HIV-1 vaccine efficacy trials shifts increasingly to non-subtype B epidemics (Southern African and Southeast Asia), non-subtype B HIV-1 reagents are needed to support vaccine development. Here we describe two IMCs derived from subtypes C and CRF01&#95;AE HIV-1 primary isolates expressing LucR (IMC.LucR) that were engineered to express heterologous gp160 Envs. 18 constructs expressing various subtypes C and CRF01&#95;AE Envs, mostly acute, in subtype-matched and -unmatched HIV backbones were tested for functionality and neutralization sensitivity. Our results suggest a possible effect of non-env HIV-1 genes on the interaction of Env and neutralizing antibodies and highlight the need to generate a library of IMCs representative of the HIV-1 subtype spectrum to be used as standardized neutralization assay reagents for assessing HIV-1 vaccine efficacy.

Here we explore the association between killer cell immunoglobulin-like receptor (KIR)/HLA and human immunodeficiency virus type 1 (HIV-1) acquisition with different viral subtypes circulating in East Africa. In the prospective Cohort Development (CODE) cohort (Mbeya, Tanzania), carriers of KIR3DS1 and its putative ligand (HLA-A or HLA-B Bw4-80Ile alleles) showed increased HIV-1 acquisition risk (odds ratio [OR] = 3.46; 95% confidence interval [CI], 1.12-10.63; P = .04) and a trend for enrichment for subtype A and A-containing recombinants (78% vs. 46%; OR = 4.05; 95% CI, .91-28.30; P = .09) at the expense of subtype C (11% vs. 43%; OR = 0.17; 95% CI, .01-.97; P = .08). In vitro, only natural killer cells from KIR3DS1(+)/HLA-Bw4-80Ile(+) healthy donors showed a 2-fold increased capacity to inhibit replication of subtype C vs subtype A viruses (P = .01). These findings suggest the presence of an innate sieve effect and may inform HIV-1 vaccine development.

The RV144 HIV-1 vaccine trial (Thailand, 2003 to 2009), using immunogens genetically matched to the regional epidemic, demonstrated the first evidence of efficacy for an HIV-1 vaccine. Here we studied the molecular evolution of the HIV-1 epidemic from the time of immunogen selection to the execution of the efficacy trial. We studied HIV-1 genetic diversity among 390 volunteers who were deferred from enrollment in RV144 due to preexisting HIV-1 infection using a multiregion hybridization assay, full-genome sequencing, and phylogenetic analyses. The subtype distribution was 91.7% CRF01&#95;AE, 3.5% subtype B, 4.3% B/CRF01&#95;AE recombinants, and 0.5% dual infections. CRF01&#95;AE strains were 31% more diverse than the ones from the 1990s Thai epidemic. Sixty-nine percent of subtype B strains clustered with the cosmopolitan Western B strains. Ninety-three percent of B/CRF01&#95;AE recombinants were unique; recombination breakpoint analysis showed that these strains were highly embedded within the larger network that integrates recombinants from East/Southeast Asia. Compared to Thai sequences from the early 1990s, the distance to the RV144 immunogens increased 52% to 68% for CRF01&#95;AE Env immunogens and 12% to 29% for subtype B immunogens. Forty-three percent to 48% of CRF01&#95;AE sequences differed from the sequence of the vaccine insert in Env variable region 2 positions 169 and 181, which were implicated in vaccine sieve effects in RV144. In conclusion, compared to the molecular picture at the early stages of vaccine development, our results show an overall increase in the genetic complexity of viruses in the Thai epidemic and in the distance to vaccine immunogens, which should be considered at the time of the analysis of the trial results.

Background: Dual infection with diverse HIV strains can foster the emergence of recombinants. The resulting increase in viral genetic diversity is a major challenge for vaccine development HIV treatment. In this study we aim to investigate the socio demographic factors associated with an increasing level of genetic diversity among HIV strains in a population of drug-users in Northern Thailand., Methods: From 1999 through 2000, 2231 volunteers were enrolled in the Opiate-Users Research in Chiang Mai, Thailand. HIV subtype analysis was conducted among those HIV-1 seropositive (n=347) using a multi-region hybridization assay. Social and demographic variables were assessed using a structured questionnaire., Results: Overall, 336/347 (96.8%) of the samples could be typed. 81.8% were CRF01&#95;AE, 3.9% were subtype B, 9.2% were recombinants (mostly between CRF01&#95;AE and B) and 5.1% were dual infections. Dual infections were more frequent among those with a lower education level (AOR: 5.2; 95% CI 1.4-20.3), those who have initiated injecting in the last 3 years (AOR: 3.9; 95% CI 1.1-14.6), and those reporting frequent needle sharing in the last 3 months (AOR: 7.0; 95% CI 1.5-34.1). Both recombinant strains and dual infection were more frequent among those reporting frequent needle sharing in the last 3 months (AOR: 5.3; 95% CI 1.6-17.1)., Conclusion: To limit the expanding complexity of HIV-1 strains, early intervention should be aimed at reduction in needle sharing, especially among new intravenous drug users., (Copyright © 2010 Elsevier Ireland Ltd. All rights reserved.)

The crucial role of recombination in HIV-1 biology is being increasingly recognized. In vitro studies have shown that up to 30 strand-transfer events may occur per viral replication cycle. Thus, recombination may surpass mutation as a major mechanism driving HIV-1 evolution. Currently, recombinant strains comprise 37% of the full-genome HIV-1 sequence database, including sequences representing 47 Circulating Recombinant Forms (CRFs) and more than 250 different Unique Recombinant Forms (URFs). Mapping of recombination breakpoints helps establish relationships among strains that are related by descent, such as CRF07&#95;BC and CRF08&#95;BC in China, and sheds light on their origin and epidemic spread. Additionally, unrelated recombinants sharing common breakpoints may reflect recombination hotspots within the viral genome. Here we present a software tool, RecDraw, for the graphical representation and efficient comparison of recombinant HIV-1 structures and breakpoints. RecDraw is a platform-flexible, Java stand-alone application available through http://www.hivresearch.org/research.php?ServiceID = 5&SubServiceID = 6 .

Here we explore associations between HLA variation and human immunodeficiency virus type 1 (HIV-1) acquisition and disease progression in a community cohort in Mbeya, Tanzania, a region that, despite harboring high rates of HIV-1 infection, remains understudied. African-specific allele HLA-A*74:01 was associated with decreased risk of infection (odds ratio [OR], 0.37; 95% confidence interval [CI], 0.14-0.80; P = .011) and with protection from CD4(+) cell counts <200 cells/uL in women (OR, 0.31; 95% CI, 0.07-0.91; P = .032) and men (OR, 0.15; 95% CI, 0.01-0.78; P = .020). These associations remained significant after adjustment for linkage disequilibrium with HLA-B and HLA-C alleles. This observation calls for additional investigation of mechanisms by which HLA-A*74:01 may influence HIV-1 acquisition and control of the infection.

Since 1985, the U.S. Department of Defense has periodically screened all military personnel for HIV allowing for the monitoring of the infection in this dynamic cohort population. A nested case-control study was performed to study sociodemographics, overseas assignment, and molecular analysis of HIV. Cases were newly identified HIV infections among U.S. Army and Air Force military personnel from 2000 to 2004. Controls were frequency matched to cases by gender and date of case first positive HIV screening test. Genotyping analysis was performed using high-throughput screening assays and partial genome sequencing. HIV was significantly associated with black race [odds ratio (OR) = 6.65], single marital status (OR = 4.45), and age (OR per year = 1.07). Ninety-seven percent were subtype B and 3% were non-B subtypes (A3, CRF01&#95;AE, A/C recombinant, G, CRF02&#95;AG). Among cases, overseas assignment in the period at risk prior to their first HIV-positive test was associated with non-B HIV subtype infection (OR = 8.44). Black and single military personnel remain disproportionately affected by HIV infection. Most non-B HIV subtypes were associated with overseas assignment. Given the increased frequency and length of assignments, and the expanding HIV genetic diversity observed in this population, there is a need for active HIV genotyping surveillance and a need to reinforce primary HIV prevention efforts.

Background: The viral load setpoint (VLS) is an important predictor of HIV disease progression, but there is a lack of information regarding the VLS and its possible determinants in African populations., Methods: Initially HIV-negative adults from 3 distinct groups(female bar workers, females, and males from the general population)were followed for up to 4 years. The VLS was calculated for 108 seroconverters and associations of the VLS with possible risk factors were analyzed using univariate and multivariate regression., Results: The median VLS for female bar workers, females, and males from the general population were 69,850, 28,600, and 158,000 RNA copies per milliliter, respectively. Significant associations with an elevated viral load were observed for male gender [risk ratio(RR) = 1.83, 95% confidence interval (95% CI) = 1.14 to 2.93], the expression of harmful HLA I alleles (RR = 1.73, 95% CI = 1.13 to 2.66) and multiple infection with different HIV-1 subtypes (RR =1.65, 95% CI = 1.03 to 2.66). Bar workers were considerably more often infected with different HIV-1 subtypes than participants from the general population., Conclusions: Our study confirms that gender and the expression of different HLA class I alleles are important determinants of the viremia at VLS, and it also corroborates an earlier finding that multiple infection with different HIV-1 subtypes is associated with a higher VLS.

Little information is available regarding a circulating HIV genotype among high-risk groups in Afghanistan; we describe HIV genotypes among injecting drug users (IDUs) and sex workers (SWs) in four Afghan cities. Participants completed behavioral questionnaires and HIV testing. Western blot-confirmed specimens had peripheral mononuclear blood cells isolated for genotyping. Analysis of recombinants was done by bootscanning and manual sequence alignment. The single SW sample harbored a CRF01&#95;AE strain. Of 10 IDUs available for analysis, all were CRF35&#95;AD and from Hirat. Analyzed subregions (gag p17 and env C1-C5) revealed close homology between the Hirat specimens. Three distinct subclusters comprising two or three strains were identified, whereas two other strains were generally equidistant from previously identified Kabul strains. Results suggest that the nascent HIV epidemic among IDUs in Hirat is largely, if not entirely, subtype CRF35&#95;AD, and the close homology suggests recent infection; harm reduction should be supported to avert further transmission.

Background: The HIV pandemic disseminated globally from Central West Africa, beginning in the second half of the twentieth century. To elucidate the virologic origins of the pandemic, a cross-sectional study was conducted of the genetic diversity of HIV-1 strains in villagers in 14 remote locations in Cameroon and in hospitalized and STI patients. DNA extracted from PBMC was PCR amplified from HIV(+) subjects. Partial pol amplicons (N = 164) and nearly full virus genomes (N = 78) were sequenced. Among the 3956 rural villagers studied, the prevalence of HIV infection was 4.9%; among the hospitalized and clinic patients, it was 8.6%., Results: Virus genotypes fell into two distinctive groups. A majority of the genotyped strains (109/164) were the circulating recombinant form (CRF) known to be endemic in West Africa and Central West Africa, CRF02&#95;AG. The second most common genetic form (9/164) was the recently described CRF22&#95;01A1, and the rest were a collection of 4 different subtypes (A2, D, F2, G) and 6 different CRFs (-01, -11, -13, -18, -25, -37). Remarkably, 10.4% of HIV-1 genomes detected (17/164) were heretofore undescribed unique recombinant forms (URF) present in only a single person. Nearly full genome sequencing was completed for 78 of the viruses of interest. HIV genetic diversity was commonplace in rural villages: 12 villages each had at least one newly detected URF, and 9 villages had two or more., Conclusions: These results show that while CRF02&#95;AG dominated the HIV strains in the rural villages, the remainder of the viruses had tremendous genetic diversity. Between the trans-species transmission of SIVcpz and the dispersal of pandemic HIV-1, there was a time when we hypothesize that nascent HIV-1 was spreading, but only to a limited extent, recombining with other local HIV-1, creating a large variety of recombinants. When one of those recombinants began to spread widely (i.e. became epidemic), it was recognized as a subtype. We hypothesize that the viruses in these remote Cameroon villages may represent that pre-epidemic stage of viral evolution.

Several studies have reported an increasing number of non-CRF01&#95;AE infections in high-risk groups in Thailand suggesting a more complex HIV-1 epidemic. This study assessed the complexity of the HIV epidemic among high-risk clients tested for HIV-1 at the Thai Red Cross Anonymous Clinic (TRCAC) between July 1, 2006 and February 28, 2007. HIV-1 genotypes were determined from plasma of infected subjects (n = 401) by the multiregion hybridization assay (MHAbce, v.2). Univariate and multivariate logistic regression analyses were used to determine risk factors associated with HIV prevalence and non-CRF01&#95;AE infection. The estimated overall HIV prevalence was 14.1%: 25.3% among men who have sex with men (MSM), 18.4% among heterosexual women, and 9.6% among heterosexual men. Among the risk factors found to be associated with HIV prevalence were age (25-29 years), risk behavior (MSM), marital status (not single), education (less than high school), and inconsistent condom use. Overall, non-CRF01&#95;AE strains accounted for 18.9% of the infections: 25.3% among MSM and 14.8% and 20.4% among heterosexual women and men, respectively. Our results indicate a concentrated and genetically complex HIV epidemic among Thai MSM. These findings advocate for targeted intervention and prevention measures among high-risk populations in Thailand.

In preparation for HIV-1 vaccine trials in Kenya, 2801 study volunteers, from a tea plantation in Kericho, were recruited as part of a prospective vaccine cohort development study. Cryopreserved plasma was available from 401 HIV-positive volunteers, and was the source of viral RNA for genotyping by the multiregion hybridization assay (MHA). Logistic regression was performed to determine association of risk factors and HIV-1 recombinant and dual infections. At baseline, HIV-1 subtype A was the dominant circulating pure subtype (56%), followed by subtype D (10%) and C (5%). Recombinant HIV-1 strains accounted for almost one-third of all infections (29%), with 7% infected with a dual strain of the HIV-1 variants described. A higher number of HIV-1 recombinant and dual infections was observed among volunteers who were 18-24 and 25-29 years of age, affiliated with the Luo tribe, had been married two or more times, reported not being circumcised, and had STI symptoms in the past 6 months. Adjusted odds ratios (AOR) significantly associated with HIV-1 recombinant and dual infection were age difference from current spouse (5-9 years; AOR = 2.5, 95% CI = 1.2-5.3 and > or = 10 years; AOR = 3.1, 95% CI = 1.5-6.4) and reported STI symptoms in the past 6 months (AOR = 4.8, 95% CI = 2.0-11.6), respectively. In conclusion, our results suggest that there is considerable heterogeneity with respect to HIV-1 subtype diversity in this population that should be considered in the planning for future vaccine trials in the region.

Little is known about the neutralization properties of HIV-1 in India to optimally design and test vaccines. For this reason, a functional Env clone was obtained from each of ten newly acquired, heterosexually transmitted HIV-1 infections in Pune, Maharashtra. These clones formed a phylogenetically distinct genetic lineage within subtype C. As Env-pseudotyped viruses the clones were mostly resistant to IgG1b12, 2G12 and 2F5 but all were sensitive to 4E10. When compared to a large multi-subtype panel of Env-pseudotyped viruses (subtypes B, C and CRF02&#95;AG) in neutralization assays with a multi-subtype panel of HIV-1-positive plasma samples, the Indian Envs were remarkably complex. With the exception of the Indian Envs, results of a hierarchical clustering analysis showed a strong subtype association with the patterns of neutralization susceptibility. From these patterns we were able to identify 19 neutralization cluster-associated amino acid signatures in gp120 and 14 signatures in the ectodomain and cytoplasmic tail of gp41. We conclude that newly transmitted Indian Envs are antigenically complex in spite of close genetic similarity. Delineation of neutralization-associated amino acid signatures provides a deeper understanding of the antigenic structure of HIV-1 Env.

APOBEC-mediated cytidine deamination of HIV-1 genomes during reverse transcription has been shown to be a potent mechanism of host restriction for HIV-1 infection ex vivo and in vitro. However, this defense system can be overcome by the viral protein Vif. Unlike other mechanisms of host restriction, the APOCEC-Vif interaction leaves an imprint on integrated proviruses in the form of G-->A hypermutation. In the current work we systematically studied levels, contexts, and patterns of HIV-1 hypermutation in vivo. The analysis of 24 full-genome HIV-1 sequences retrieved from primary PBMCs, representing infections with several HIV-1 clades, and the inclusion of 7 cognate pairs of hypermutated/non-hypermutated sequences derived from the same patient sample, provided a comprehensive view of the characteristics of APOBEC-mediated restriction in vivo. Levels of hypermutation varied nearly 5-fold among the studied proviruses. GpG motifs were most frequently affected (22/24 proviruses). Levels of hypermutation varied across the genome. The reported "twin peak" pattern of hypermutation was observed in 18/24 hypermutants, but the remainder exhibited singular non-conforming patterns. These data suggest considerable complexity in the interplay of host restriction and viral defense during HIV-1 infection.

In AIDS vaccine development the pendulum has swung towards a renewed emphasis on the potential role for neutralizing antibodies in a successful global vaccine. It is recognized that vaccine-induced antibody performance, as assessed in the available neutralization assays, may well serve as a "gatekeeper" for HIV-1 subunit vaccine prioritization and advancement. As a result, development of a standardized platform for reproducible measurement of neutralizing antibodies has received considerable attention. Here we review current advancements in our knowledge of the performance of different types of antibodies in a traditional primary cell neutralization assay and the newer, more standardized TZM-bl reporter cell line assay. In light of recently revealed differences (see accompanying article) in the results obtained in these two neutralization formats, parallel evaluation with both platforms should be contemplated as an interim solution until a better understanding of immune correlates of protection is achieved.

A panel of paired primary virus isolates and envelope pseudoviruses from sixty strains representing six HIV-1 clades was tested for neutralization using pooled, clade-specific plasma in two prominently utilized neutralization platforms: a primary isolate assay using peripheral blood mononuclear cells (PBMC) and a pseudovirus assay using a reporter epithelial cell line. Using the PMBC assay, pairing of the antibody pool against homologous clade viruses generated the highest geometric mean neutralizing antibody titer in 4 out of 6 clades tested, and neutralization patterns showed numerous examples of reciprocal cross-recognition between antibody and viruses of specific clade pairs. In the pseudovirus assay, cross-clade neutralization was more limited, with fewer distinct cross-clade relationships evident. The clade C antibody pool was broadly cross-reactive, neutralizing the greatest number of viruses in both assays. These data highlight the importance of the neutralization assay format employed and suggest that clade C envelopes merit further evaluation for the elicitation of broadly neutralizing antibodies.

In Thailand, the circulating HIV-1 strains include CRF01&#95;AE, subtype B, and their recombinants. Genotyping and full-genome sequencing had previously identified circulating recombinant form CRF15&#95;01B within a cohort of 347 HIV-1-infected individuals enrolled in the Opiate Users Research (OUR) study in northern Thailand. Using an improved MHAbce in six to eight genome regions and archived OUR serum samples, seven strains were identified with a new and complex 01/B recombinant pattern in common, different from that of CRF15&#95;01B. Complete sequencing of three strains, amplified from serum as overlapping half-genomes, confirmed their common recombinant structure, mostly CRF01&#95;AE, but with segments of subtype B in pol and gp41, plus a region of frequent 01/B crossovers in pol. OUR strains 1969P, 2275P, and 2478P were from individuals without direct epidemiological linkage and thus establish CRF34&#95;01B. More comprehensive HIV-1 prevention and treatment programs in IDU can help to limit the growing complexity of HIV-1 strains in Thailand.

Injecting drug use (IDU), common in global centers of heroin production, confers significant risk for HIV-1 infection. Once introduced into IDU networks, an explosive rise in HIV-1 infection typically occurs, fueled principally by needle sharing. New HIV-1 epidemics in IDUs have occurred in Russia, China, Thailand, Spain, Iran, and in other countries, and some have spread into other risk groups in their respective countries. In Afghanistan, the introduction of HIV-1 into IDU networks has begun, but a recent report of 3% HIV-1 prevalence suggests that the epidemic is still at an early stage. Here we establish, by complete genome sequencing and phylogenetic analysis of four viral strains from Afghan IDUs, that all are the same complex recombinant strain, combining HIV-1 subtypes A and D and herein termed CRF35&#95;AD. Published partial HIV-1 sequences from an HIV-1 epidemic among IDUs in Iran, already at 23.2% HIV-1 prevalence, are either CRF35&#95;AD or a related recombinant. Voluntary HIV-1 screening and harm reduction programs in Afghanistan, applied now, could limit the spread of HIV-1, both in IDUs and in other social networks.

Molecular characterization of C2-V5 envelope sequences from maternal plasma, peripheral blood mononuclear cells (PBMC), cervical secretions and infant PBMC was performed in eight CRF01&#95;AE-infected mother/infant pairs. Maternal viruses were relatively homogeneous within a compartment but distinct in different compartments in mothers with high CD4 cell counts. Infant viruses were almost distinct, but phylogenetically related, to maternal viruses, mostly from the maternal PBMC compartment, reflecting the frequent transmission of HIV-1 from maternal cells rather than free viruses.

G-to-A hypermutation, a massive substitution of G for A, was first observed in HIV genomes 15 years ago. Initially ascribed to fluctuating nucleotide pools or PCR errors, it has now been recognized as the result of an important host-defense mechanism mediated by APOBECs, a family of sequence-specific cytidine deaminases. Levels of hypermutation vary, and APOBECs have different preferences for the sequence context of cytidines targeted for deamination; analysis of hypermutation provides important information about the host-virus interaction during viral replication. Here we present HyperPack, a software package to support systematic characterization of hypermutated sequences. Users can define the context of substitution for independent study of the effects of different APOBECs. The SubstrateScan and HyperScan modules are overlapping sliding-window strategies to analyze the distribution of targeted motifs and their substitution levels, respectively, across the viral genome. HyperPack is a platform-flexible, Java stand-alone application available through http://www.hivresearch.org/hyperpack/.

High-resolution HIV-1 genotyping of large sample sets is crucial to define the evolving and dynamic epidemics in Asia. Here we present MHAbce v.2, a multi-region hybridization assay that individually discriminates subtypes B, C, CRF01&#95;AE, and virtually all of their described recombinants, based on real-time PCR using subtype-specific TaqMan probes in 8 regions throughout the viral genome. In a validation panel (n=70), the assay performed with a sensitivity of 95.7% and specificity of 99.8%. The assay was field-tested on samples from a retrospective MTCT cohort (n=180; Lampang Province, Northern Thailand; 1996-1998). 177/180 of the samples were typeable, and 94.4% were typed as CRF01&#95;AE. The remaining strains represented even proportions of subtype B and B/CRF01&#95;AE recombinants and were confirmed by sequencing, revealing early links between the heterosexual and IDU HIV-1 epidemics in Thailand. MHAbce v.2, with an area of application including China, India, Southeast Asia, and the Pacific Rim, can be used to develop a comprehensive and detailed picture of this important component of the HIV/AIDS pandemic.

Objective: To determine the association between the incidence of HIV-1 infection and the genetic complexity of HIV-1 strains in 2 geographic strata within Rakai District, Uganda., Methods: Study volunteers with recent HIV-1 infections during the period 1997 through 2003 were recruited from 10 communities that were geographically stratified as a main road trading center (n = 5) or a secondary road trading village (n = 5). Cryopreserved plasma was available from 384 volunteers and was the source of viral RNA for genotyping by the multiregion hybridization assay. Hazard ratios (HRs) for a single HIV subtype, a recombinant form, or dual infection for gender and geographic strata were obtained using Cox proportional hazards analysis., Results: The HIV-1 incidence rate during the period 1999 through 2002 was 1.3 per 100 person-years (PYs) in the trading centers and 1.1 per 100 PYs in the trading villages. The HR for infection with an HIV-1 recombinant strain in trading centers relative to trading villages was 2.3 (95% confidence interval [CI]: 1.0 to 6.7). Among those who changed residence between village strata, the HR for a recombinant HIV-1 infection was 8.1 (95% CI: 0.4 to 47.7)., Conclusions: HIV-1 incidence and genetic complexity are associated with geographic strata and population mobility in Rakai District and are important variables to be considered in planning and recruitment for vaccine trials.

To characterize HIV-1 genotypes in candidate populations for a prime-boost phase III vaccine trial in Thailand, specimens from prevalent and incident HIV-1 infections from a family planning clinic population in Rayong Province and a community cohort in Chon Buri Province, collected from 1998 to 2001, were genotyped. A new multiregion hybridization assay, MHAbce, capable of distinguishing HIV-1 CRF01&#95;AE, subtype B, and subtype C and their recombinants, was developed and applied to prevalent infections. Most incident and selected prevalent infections were studied by complete genome sequencing. By MHAbce, 168 of 194 prevalent infections were genotyped. Of these, 90.5% were CRF01&#95;AE, 2.4% were subtype B, and 7.2% showed discordant or dual probe reactivity, indicative of recombination or dual infection, respectively. Among 23 incident infections, 20 were sequenced. Eighteen CRF01&#95;AE, one subtype B, and one CRF01/B recombinant strains were seen. Two CRF01/B and one CRF01/C recombinant were identified among selected prevalent infections. These results indicate that incident and prevalent HIV-1 infections in Rayong and Chon Buri during 1998-2001 were 90% CRF01&#95;AE, 3% subtype B, and 7% either recombinant or dual. This study frames the genetic diversity of HIV-1 in these cohorts in their preparatory phase for the ongoing ALVACHIV (vCP1521) prime, AIDSVAX B/E boost, phase III vaccine trial and will provide a benchmark for interpretation and analysis.

HIV-1 diversity, frequency of recombinants, and dual infection were determined in two populations with different HIV risk behavior. A high-risk cohort of 600 female bar workers and a normal-risk population of 1,108 antenatal clinic attendees and blood donors were recruited. Behavioral data were assessed and blood for HIV- 1 diagnosis and genotyping was sampled. HIV-1 subtypes were defined through the multiregion hybridization assay (MHA(acd)). HIV-1 prevalence differed significantly among the two populations. The prevalence was 67.8% in the population of bar workers and 17% in the normal-risk population (antenatal care attendees and blood donors). Within the normal-risk population the HIV-1 prevalence was lowest in the group of volunteer blood donors. The frequency of HIV-1 infection in women was 1.7 times higher than in men. The overall subtype distribution was A (8.5%), C (40.8%), D (3.8%), AC (25.4%), AD (5.4%), CD (8.8%), and ACD (7.3%). In the high-risk population there was a higher percentage of HIV-1 recombinant strains (54% vs. 40%, p < 0.05) and a higher frequency of dual infections (19% vs. 9%, p < 0.02) compared to the normal-risk population. High-risk populations may play an important role in the evolution of HIV, as they can provide an opportunity for the virus to coinfect, recombine, and adapt to the host-specific genetic background.

Background: Subtype A is a major strain in the HIV-1 pandemic in eastern Europe, central Asia and in certain regions of east Africa, notably in rural Kenya. While considerable effort has been focused upon mapping and defining immunodominant CTL epitopes in HIV-1 subtype B and subtype C infections, few epitope mapping studies have focused upon subtype A., Results: We have used the IFN-gamma ELIspot assay and overlapping peptide pools to show that the pattern of CTL recognition of the Gag and Nef proteins in subtype A infection is similar to that seen in subtypes B and C. The p17 and p24 proteins of Gag and the central conserved region of Nef were targeted by CTL from HIV-1-infected Kenyans. Several epitope/HLA associations commonly seen in subtype B and C infection were also observed in subtype A infections. Notably, an immunodominant HLA-C restricted epitope (Gag 296-304; YL9) was observed, with 8/9 HLA-CW0304 subjects responding to this epitope. Screening the cohort with peptide sets representing subtypes A, C and D (the three most prevalent HIV-1 subtypes in east Africa), revealed that peptide sets based upon an homologous subtype (either isolate or consensus) only marginally improved the capacity to detect CTL responses. While the different peptide sets detected a similar number of responses (particularly in the Gag protein), each set was capable of detecting unique responses not identified with the other peptide sets., Conclusion: Hence, screening with multiple peptide sets representing different sequences, and by extension different epitope variants, can increase the detectable breadth of the HIV-1-specific CTL response. Interpreting the true extent of cross-reactivity may be hampered by the use of 15-mer peptides at a single concentration and a lack of knowledge of the sequence that primed any given CTL response. Therefore, reagent choice and knowledge of the exact sequences that prime CTL responses will be important factors in experimentally defining cross-reactive CTL responses and their role in HIV-1 disease pathogenesis and validating vaccines aimed at generating broadly cross-reactive CTL responses.

HIV is among the most generically variable of human pathogens. A comprehensive and detailed description of HIV strains in the pandemic is an important foundation for diagnosis, treatment, and prevention. The current sequence database for HIV includes almost 800 complete genome sequences, documenting HIV-1 groups M, O, and N, and HIV-2. Among HIV-1 group M strains, responsible for the vast majority of HIV infections worldwide, 743 sequences represent 9 genetic subtypes, 16 circulating recombinant forms (CRF) that are spreading in populations, and a variety of unique recombinant forms (URF), identified so far only from a single individual. The global distribution of HIV is complex and dynamic with regional epidemics harboring only a subset of the global diversity. HIV strains differ enormously in terms of global prevalence. Six strains account for the majority of HIV infections: HIV-1 subtypes A, B, C, D, and two of the CRF, CRF01-AE and CRF02&#95;AG, respectively. Many of the known subtypes and recombinant forms are currently rare in the epidemic, but could spread more widely if favorable conditions arise. HIV-2 is largely restricted to West Africa at relatively low prevalence there. Groups O and N of HIV-1 are very rare in the pandemic. The goal of universal coverage of HIV-1 strains by diagnostic tests can be met by minimizing false negative test rates for the six globally prevalent HIV-1 group M strains and HIV-2, and by evaluating systematically coverage of rare subtypes and recombinant forms.

Objective: To characterize HIV-1 strains in a potential vaccine trial cohort (CODE) in the Mbeya region of southwest Tanzania., Design: Study volunteers (n = 3096) were recruited from urban areas in Mbeya Town, using two different recruitment strategies, and in a nearby rural village., Methods: Cryopreserved plasma from 507 HIV-1 prevalent cases was the source of viral RNA for HIV-1 genotyping by the Multi-region Hybridization Assay, the MHA(acd), and selected strains were confirmed by complete genome sequencing., Results: The overall HIV-1 prevalence was 16.6% [95% confidence interval (CI), 15.3-17.9] within the cohort. HIV-1 prevalence was higher among women, and in urban areas. Recruitment through advertisement targeted a high-risk urban male population for HIV-1 infection [adjusted odds ratio (adj. OR), 1.68; 95% CI, 1.13-2.51] when compared with men recruited door-to-door. The complexity of the HIV-1 epidemic was also higher in urban areas evidenced by the high-risk of HIV-1 infection with a recombinant strain (adj. OR, 2.69; 95% CI, 1.08-6.69) and HIV-1 dual infection (adj. OR, 5.16; 95% CI, 1.07-24.9), mainly driven by urban men recruited through advertisement., Conclusions: Overall the urban epidemic was more genetically complex, with higher prevalence and more recombinants and dual infections. Vaccine trials in Mbeya region can assess a complex HIV-1 population dynamic and determine vaccine efficacy in relationship to the genetic diversity of HIV-1 strains that challenge vaccines.

Human immunodeficiency virus type 1 (HIV-1) superinfection refers to the acquisition of another strain by an already infected individual. Here we report a comprehensive genetic analysis of an HIV-1 superinfection acquired heterosexually. The infected individual was in a high-risk cohort in Tanzania, was exposed to multiple subtypes, and was systematically evaluated every 3 months with a fluorescent multi-region genotyping assay. The subject was identified in the window period and was first infected with a complex ACD recombinant strain, became superinfected 6 to 9 months later with an AC recombinant, and was monitored for >2.5 years. The plasma viral load exceeded 400,000 copies/ml during the first 9 months of infection but resolved to the set point of 67,000 copies/ml by 3 months after superinfection; the CD4 cell count was 377 cells/mul at 30 months. Viral diversity was evaluated with techniques designed to fully sample the quasi-species, permitting direct observation of the evolution, temporal fluctuation, and intercompartment dynamics of the initial and superinfecting strains and recombinants derived from them. Within 3 months of superinfection, seven different molecular forms were detected in gag and six were detected in env. The proportions of forms fluctuated widely over time in plasma and peripheral blood mononuclear cells, illustrating how challenging the detection of dually infected individuals can be. Strain-specific nested PCR confirmed that the superinfecting strain was not present until the 9 month follow-up. This study further defines the parameters and dynamics of superinfection and will foster appropriate studies and approaches to gain a more complete understanding of risk factors for superinfection and its impact on clinical progression, epidemiology, and vaccine design.

A critical priority for human immunodeficiency virus type 1 (HIV-1) vaccine development is standardization of reagents and assays for evaluation of immune responses elicited by candidate vaccines. To provide a panel of viral reagents from multiple vaccine trial sites, 60 international HIV-1 isolates were expanded in peripheral blood mononuclear cells and characterized both genetically and biologically. Ten isolates each from clades A, B, C, and D and 10 isolates each from CRF01&#95;AE and CRF02&#95;AG were prepared from individuals whose HIV-1 infection was evaluated by complete genome sequencing. The main criterion for selection was that the candidate isolate was pure clade or pure circulating recombinant. After expansion in culture, the complete envelope (gp160) of each isolate was verified by sequencing. The 50% tissue culture infectious dose and p24 antigen concentration for each viral stock were determined; no correlation between these two biologic parameters was found. Syncytium formation in MT-2 cells and CCR5 or CXCR4 coreceptor usage were determined for all isolates. Isolates were also screened for neutralization by soluble CD4, a cocktail of monoclonal antibodies, and a pool of HIV-1-positive patient sera. The panel consists of 49 nonsyncytium-inducing isolates that use CCR5 as a major coreceptor and 11 syncytium-inducing isolates that use only CXCR4 or both coreceptors. Neutralization profiles suggest that the panel contains both neutralization-sensitive and -resistant isolates. This collection of HIV-1 isolates represents the six major globally prevalent strains, is exceptionally large and well characterized, and provides an important resource for standardization of immunogenicity assessment in HIV-1 vaccine trials.

Infections with non-B HIV-1 subtypes are rare in the United States, but comprise a significant percentage of infections among U.S. military personnel. Risk behavior while on overseas deployment correlates with non-B infection in this population. Extensive genetic characterization will be required to define HIV-1 diversity, and to effectively evaluate requirements for HIV-1 vaccines and other prevention strategies in this group. From 1997 to 2000, 520 recent seroconverters, identified through routine HIV-1 testing in the U.S. active military force, volunteered for a prospective study. V3 loop serology or partial genome sequencing identified 28 non- B subtype infections; 14 were studied by full genome sequencing and phylogenetic analysis. Five strains were CRF01&#95;AE. Four of these clustered with CM240 from Thailand, and one clustered with African CRF01&#95;AE. Four strains were CRF02&#95;AG, prevalent in West and West Central Africa. Two strains were subtype C. One strain was a unique recombinant between CRF01&#95;AE and subtype B, and another was a complex unique recombinant between subtype A and D. The final strain was a member of a complex circulating recombinant first identified in Senegal, CRF09&#95;cpx, incorporating subtypes A, F, G, and an unclassified genome. This diversity of non-B subtype HIV-1 strains, encompassing three globally prevalent non-B strains and including rare or even possibly unique strains, illustrates the breadth of U.S. military exposure while deployed and sets the bar higher for breadth of cross-subtype protection to be afforded by an HIV-1 vaccine.

HIV-1 is endemic in Tanzania where three different subtypes, A, C, and D, have been identified. Information on HIV-1 genetic diversity is crucial to define requirements for an effective vaccine, in regions where HIV-1 vaccine trials are planned. To define the subtype distribution of HIV-1 in the Mbeya region of southwest Tanzania, peripheral blood mononuclear cells (PBMC) and plasma were obtained from 36 discarded HIV seropositive blood units. Multiregion hybridization assay (MHA) was performed on both PBMC DNA and plasma RNA to determine the subtype distribution. Twenty virtually full-length HIV-1 sequences were amplified from the extracted DNA, sequenced, and phylogenetically analyzed. Subtype distribution determined by all three assays was comparable. More than 50% of the samples analyzed were subtype C, followed by a high proportion of subtype C-containing intersubtype recombinants. Based on this work, subtype C appears to be the prevalent subtype in southwest Tanzania, followed by a high proportion of intersubtype recombinants.

Two HIV-1 intersubtype recombinant forms are circulating widely in populations and have become important strains in the pandemic: CRF01&#95;AE in Southeast Asia and CRF02&#95;AG in West and West Central Africa, respectively. Several other circulating recombinant forms (CRF) have also been identified, but with fewer numbers of infections and/or more limited geographic spread. Here we expand knowledge of HIV-1 CRF using clinical samples, principally from West Africa, that were difficult to classify by partial genome sequencing. DNA was extracted from primary patient peripheral blood mononuclear cells (PBMC). The virtually complete HIV-1 genome was amplified by polymerase chain reaction (PCR) and directly sequenced. Additional strains were characterized by partial envelope sequencing. Phylogenetic analysis was used to identify and map intersubtype recombination breakpoints. Four virtually complete genome sequences and two partial envelope sequences represent CRF09&#95;cpx, a newly identified complex recombinant HIV-1 whose principal focus seems to be in West Africa. This recombinant includes segments of subtypes A, F, G, and unclassified genetic material. It shares unique unclassified regions with the early Zaire strain Z321. There are similarities in structure, but considerable genetic distances, between CRF09&#95;cpx and CRF02&#95;AG IbNG. In conclusion, it is possible that this CRF shared common ancestors with both Z321 and CRF02&#95;AG in the course of the pandemic, perhaps arising by recombination between earlier forms of these strains. Although newly identified, at least one infection with CRF09&#95;cpx has already occurred outside of Africa.

CRF01&#95;AE and subtype B have dominated the HIV-1 epidemic in Thailand since 1989. We reported a new circulating recombinant form of HIV-1, CRF15&#95;01B, as well as other unique CRF01&#95;AE/B recombinants among prevalent HIV infections in Thailand. We sought to study this challenging molecular picture through assessment of subtypes among recent HIV-1 seroconverters in northern Thai drug users. A total of 847 HIV-1 seronegative drug users (342 IDU and 505 non-IDU) were enrolled, from 1999 to 2002, in a prospective study; 39 HIV-1 incident cases were identified and characteristics were collected. The overall HIV-1 incidence rate was 2.54/100PY, but it was 10.0/100PY among male IDU. HIV was strongly associated with injection history; 38 of 39 seroconverters gave a history of IDU. A near full-length genome of HIV-1 was recovered by PCR amplification and sequenced from peripheral mononuclear cell extracted DNA of 38 seroconverters. Phylogenetic analysis revealed that 33 (86.8%) were CRF01&#95;AE and 5 (13.2%) were CRF01&#95;AE/B recombinants. These recombinants had different structure but shared some common breakpoints, indicating an ongoing recombination process. Recombinant infection increased with year of sampling (0 to 57.1%). The molecular epidemiology of HIV-1 among drug users in northern Thailand has thus entered a new era. CRF01&#95;AE remains predominant while pure subtype B is becoming rare, and now a substantial component of the epidemic. These findings support the need for CRF01&#95;AE and subtype B components in clade-matched vaccine strategies for Thai phase III trials. Ongoing molecular surveillance of circulating HIV-1 strains is imperative for the evaluation of HIV vaccine efficacy.

Synonymous nucleotide substitutions in protein-coding sequences are often regarded as evolutionarily neutral and not subject to selective pressure. However, synonymous codons can sometimes lead to different patterns of amino acid substitution by single nucleotide changes. Based on the deconstruction of the standard genetic code, we propose the term 'quasi-synonymous' to describe codons that specify the same amino acid, but lie on different mutational pathways, and we show that in at least one rapidly evolving organism, HIV-1, quasi-synonymy plays a role in its evolution. We present concrete examples that demonstrate the relevance of codon usage in the development of antiretroviral-drug resistance. In the case of the host immune response, the data indicates that viral evasion is achieved through use of codons that lie on the direct path to escape mutants, and equally, permit rapid reversion to wild-type in the absence of these selective pressures. Quasi-synonymy conditions HIV-1 and, potentially, other rapidly evolving organisms in their exploration of the mutational space.

The impact of HIV-1 genetic diversity on candidate vaccines is uncertain. One approach to minimize genetic diversity in the evaluation of HIV-1 vaccines is to match the vaccine sequence to the predominant subtype in a vaccine cohort. Over two million Ethiopians are infected with HIV-1, and the predominant subtype is thought to be subtype C. Understanding the phylogenetic relationships between sequences from Ethiopia and within subtype C can help decide what sequence(s) should comprise a candidate vaccine. To that end, nearly full genome sequencing was used to characterize HIV-1 from volunteers who emigrated from Ethiopia. DNA extracted from peripheral blood mononuclear cells (PMBC) was amplified using primers in the long terminal repeats to generate nearly full-length genomes. Amplicons were directly sequenced with dye terminators and automated sequencers. Sequences were phylogenetically analyzed by neighbor joining. The six new Ethiopian sequences were all subtype C, consistent with previous partial and full genome analysis. Together with two other Ethiopian sequences, the new sequences formed a geographic cluster when the complete genome was analyzed. However, subgenomic trees showed only a weak geographic cluster, or none, with respect to Ethiopian strains. Although immunological responses must be considered, from a phylogenetic perspective, there is no compelling support for use of Ethiopian subtype C sequences, compared to other subtype C, as vaccine prototype strains.

Objective: To describe the demographics, risk behaviors, and HIV-1 subtypes in a large cohort of recently HIV-infected military personnel., Design: Descriptive, cross-sectional study., Methods: US military personnel with recent HIV seroconversion from six medical referral centers were enrolled with a self-administered questionnaire, CD4 cell counts, syphilis and hepatitis B serologies, plasma viral RNA levels, and HIV-1 subtype nucleic acid sequencing., Results: Between February 1997 and May 2000, 520 patients were enrolled. Most [488 (94.3%)] were infected with HIV-1 subtype B. The most prevalent non-B subtype was a circulating recombinant form (CRF01&#95;AE) [17 (61%)]; however, two pure subtypes (C and D), as well as CRF02&#95;AG, CRF09&#95;cpx and a BE recombinant were identified. The likely area of HIV-1 acquisition was the United States for 70% of the volunteers. At least three non-B subtype infections (two subtype C, one subtype CRF01&#95;AE) were apparently acquired domestically. Risk behaviors and comorbid sexually transmitted diseases were reported during the seroconversion period. Volunteers with non-B subtype HIV infection were more likely to report heterosexual contacts [92% vs. 39%; odds ratio (OR), 10.0], including contacts with commercial sex workers (41% vs. 13%; OR, 4.9). The Roche Amplicor version 1.0 assay was less sensitive for non-B subtype infections than the Roche Amplicor version 1.5 assay., Conclusion: There is a high prevalence and diversity of non-B HIV subtypes in this large cohort. Efficient diagnosis of acute primary HIV-1 infection was identified as a goal for prevention programs. Modifiable risk behaviors and target populations for intervention were identified.

Isolation and characterization of HIV-1 from asymptomatic, slow-progressing individuals are important in studying viral pathogenesis and facilitate the development of vaccines and antivirals. In this study we identified two slow-progressing HIV-1-infected siblings, isolated viruses, and sequenced the full-length genome, to identify virus attenuations that may contribute to their altered rate of disease progression. Proviral DNA from strains 99ZATM10 and 01ZATM45 was isolated from peripheral blood mononuclear cells (PBMC) coculture.Virtually full-length genomes and long terminal repeat (LTR) regions were polymerase chain reaction (PCR) amplified, sequenced, and assembled to generate the complete genomes. Phylogenetic analysis confirmed that both isolates were subtype C throughout their genome. Predicted amino acid sequence analysis for all the HIV-1 proteins showed that both viruses had open reading frames for all genes, and encoded proteins of the expected length, except for the rev gene. The 3' end of rev exon 2 did not have the 16-amino acid (aa) truncation characteristic of subtype C viruses, and in addition, had a three-aa extension (GlyCysCys). Rev is a necessary regulatory factor for HIV expression, and changes in the protein may affect viral replication. These results suggest that slower HIV disease progression in these children may be attributed, at least in part, to an altered Rev protein.

Objectives: To quantitate rapidly the frequency of HIV-1 subtype-specific and broadly HIV-1 cross-subtype-reactive CD8 T cells in the peripheral blood of HIV-1-infected individuals from a geographical region of multiple subtype endemicity., Methods: Autologous B-lymphoblastoid cell lines infected with recombinant vaccinia-viruses expressing gag, env and nef gene products from HIV-1 subtypes A-H were used as antigen-presenting cells to stimulate CD8 T cells from cryopreserved peripheral blood mononuclear cells. Cross-subtype and subtype-specific CD8 cell responses were assessed by flow cytometry for the upregulation of IFN-gamma gene expression in specifically activated CD8 T cells., Results: Strikingly high frequencies of circulating CD8 T cells (up to 11.3% of peripheral CD8 T cells) with specificity for HIV-1 were detectable using this methodology. Both subtype-specific and broadly cross-subtype-reactive CD8 T cells were detected as assessed by IFN-gamma production after stimulation. The pattern of cross-subtype reactivity appeared to be random when the results were assessed as a population, but analysis of the full-length sequence of the infecting virus for each individual showed some skewing of the CD8 cell response towards the infecting subtype., Conclusion: High frequencies of HIV-1 cross-subtype-reactive peripheral CD8 T cells can be detected in individuals from a multiple subtype endemic region, providing an incentive for vaccine advancement in such locations. The future assessment of the subtype specificity of cellular immune responses requires full-length sequencing of the infecting virus in conjunction with a comprehensive analysis of phenotypic and functional parameters.

HIV-1 subtype B and CRF01&#95;AE have been in circulation in Thailand and Southeast Asia for more than a decade. Initially separated by risk group, the two strains are increasingly intermixed, and two recombinant strains of essentially reciprocal structure have been recently reported. Here we identify additional CRF&#95;01B recombinants and provide the evidence that HIV-1 strains now pass freely between the two high-risk populations. HIV isolates that showed discordance between CRF01&#95;AE and subtype B in multi-region genotyping assays were selected for the study. They were drawn from 3 different cohorts in Thailand representing different risk behaviors and demographic characteristics: a drug user cohort in the north, a family planning clinic attendee cohort in the southeast, and a cohort study of the mucosal virology and immunology of HIV-1 infection in Thailand. The DNA from these isolates was PCR amplified to recover the full HIV-1 genome and subjected to sequencing and phylogenetic analysis. We establish that one particular CRF&#95;01B recombinant, with the external envelope of subtype B and the rest of the genome from CRF01&#95;AE, is circulating widely in Thailand. Termed CRF15&#95;01B (also referred to as CRF15), the strain was primarily heterosexually transmitted, although injecting drug use (IDU) also played a role. In aggregate data from the studies, CRF15 constituted 1.7% of all HIV-1 infections (95% confidence interval 0.5-4.4%) and was dispersed widely in the country. The previously separate heterosexual and IDU epidemics have apparently been bridged by a new CRF. The entry of CRF15 into the mainstream of the epidemic signals new complexity in the long stable molecular picture in Thailand. These recombinants must be considered in ongoing or projected efficacy evaluations of HIV-1 vaccines and antiviral therapies.

CRF01&#95;AE and subtype B are circulating in Thailand and the strains have become intermixed in some high-risk groups, establishing the possibility of intersubtype recombination. The first such recombinant, mostly B with gp120 from CRF01&#95;AE, was recently identified. Here we report a heterosexually acquired recombinant of different structure, with most of the genome from CRF01&#95;AE but almost the entire envelope from subtype B. Surveillance by V3 serotype and genotype in multiple regions, followed by full-genome sequencing, was used to identify this strain. Pending vaccine trials in Thailand require knowledge of the presence of such strains in the population, and these recombinants provide valuable reagents for the laboratory evaluation of cross-subtype immunity. Studies are underway to determine whether either recombinant is circulating widely.

Objective: To enable more rapid and efficient genotyping of HIV-1 in East Africa, where subtypes A, C, and D and their recombinants are co-circulating., Design: Full-genome sequencing of HIV-1 provides complete discrimination of subtypes and recombinant forms but is costly and low-throughput compared to other genotyping approaches. Here we describe the development and evaluation of a Multi-region Hybridization Assay (MHA) for the efficient determination of HIV-1 subtypes A, C, D, recombinants, and dual infections., Methods: Five genome regions containing clustered mutations distinguishing subtypes A, C, and D were identified and used to design subtype-specific probes. DNA from primary peripheral blood mononuclear cells was used as template for real-time PCR using the fluorescent, subtype-specific probes., Results: A panel of 45 clinical samples from Uganda, Kenya, and Tanzania, previously characterized by full-genome sequencing and including 26 pure subtypes and 19 recombinant strains, was evaluated by MHA. The MHA provided 90% sensitivity and 98% specificity for the three subtypes, efficiently discriminated subtypes from recombinant forms, and detected several dual infections., Conclusions: Accurate and efficient genotyping of HIV-1 strains in vaccine trial populations in East Africa, ascertainment of dual infections, and elucidation of the genesis of recombinant forms in individuals can be facilitated by the application of MHA., (Copyright 2002 Lippincott Williams & Wilkins)

Objective: To further define the genetic diversity of HIV-1 in Kenya using approaches that clearly distinguish subtypes from inter-subtype recombinants., Design: Near full genome sequencing and analysis were used, including sensitive new tools for detection and mapping of recombinants., Methods: Purified peripheral blood mononuclear cell DNA from 41 HIV-1 positive blood donations collected from six hospitals across southern Kenya was used to amplify near full-length genomes by nested PCR. These were sequenced on an ABI 3100 automated sequencer and analyzed phylogenetically., Results: Among 41 near full-length genomes, 25 were non-recombinant (61%) and 16 were recombinant (39%). Of the 25 pure subtypes, 23 were subtype A, one was subtype C and one was subtype D. Most recombinants consisted of subtype A and either subtype C or subtype D; a few contained A2, a recently identified sub-subtype. Two A2/D recombinants had identical breakpoints and may represent a circulating recombinant form. A third A2/D recombinant had the same structure as a previously described Korean isolate, and these may constitute a second A2-containing circulating recombinant form., Conclusions: In Kenya, 93% of HIV-1 genomes were subtype A or A-containing recombinant strains. Almost 40% of all strains were recombinant. Vaccine candidates tested in Kenya should be based on subtype A strains, but the methods used for evaluation of breakthrough infections during future vaccine trials should be capable of identifying non-A subtypes, the A2 sub-subtype, and recombinants., (Copyright 2002 Lippincott Williams & Wilkins)

The HIV-1 epidemic in South Africa is largely due to subtype C viruses, which preferentially use CCR5 as a coreceptor for infection. We describe full-length genome sequences of two CXCR4-utilizing HIV-1 subtype C viruses and two intersubtype recombinants from South Africa. Three of the viruses (99ZACM4, 99ZACM9, and 99ZASW7) were isolated in 1999 from AIDS patients in Johannesburg, and a fourth virus (98ZADu178) was isolated in Durban in 1998 from an asymptomatic female sex worker. Isolates 99ZASW7 and 99ZACM9 from Johannesburg were subtype C throughout the genome, 99ZASW7 used the CXCR4 coreceptor, and 99ZACM9 used both CCR5 and CXCR4. Isolate 98ZADu178 from Durban was a novel recombinant between subsubtype A2 and subtype C. The third isolate from Johannesburg, 99ZACM4, was a complex, novel recombinant with multiple breakpoints and contained segments of subtypes A, C, D, G, and K. These results establish the presence of intersubtype recombinants in South Africa, indicating that ongoing surveillance for other subtypes and recombinants is necessary.

The molecular epidemiology of HIV-1 in Argentina is more complex than was previously appreciated. One circulating recombinant form, CRF12&#95;BF, and many related BF recombinant forms predominate in the capital city, Buenos Aires. This study of HIV-1 subtypes acquired perinatally between 1984 and 2000 has permitted, for the first time, a reconstruction of the history of BF recombination in Argentina. Sequencing of a partial genome region from the beginning of vpu to the beginning of env(gp120), which spans a breakpoint common in most contemporary Argentine BF recombinants, enabled samples to be rapidly screened. Among 23 children born between 1984 and 2000, 15 including 1 child born in 1986, harbored a BF recombinant. Thirteen of the 15 recombinants shared a common breakpoint at the 5' end of env(gp120). Full genome sequencing of two viruses, from 1986 and 1987, respectively, revealed them to be genetically related but not identical to CRF12&#95;BF. Both contained more subtype B sequence than did CRF12&#95;BF. BF recombinants related to CRF12&#95;BF have been in circulation in Buenos Aires since 1986 and continue to predominate in perinatal transmissions.

Objective: To describe the genetic diversity of HIV-1 in South America by full genome sequencing and analysis., Methods: Purified peripheral blood mononuclear cell DNA from HIV-infected individuals in Argentina, Uruguay and Bolivia was used to amplify full HIV-1 genomes. These were sequenced using the ABI 3100 automated sequencer and phylogenetically analysed., Results: Twenty-one HIV-1 strains from three South American countries, 17 of which were pre-screened by envelope heteroduplex mobility assay (HMA), were studied. Ten out of 10 HMA subtype F and four out of seven HMA subtype B strains were actually BF recombinants upon full genome analysis. Two BF recombinants from Argentina and two from Uruguay had the same structure, representing a new circulating recombinant form termed CRF12&#95;BF(ARMA159). Twelve other BF recombinants had structures related to CRF12 but with additional segments of subtype B; each was unique. BF recombinants were temporally and geographically widespread, found as early as 1986-1987 in vertically infected Argentinian children and in Argentina, Uruguay, and Bolivia.