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International audience; Background: Global HIV-1 genetic diversity and evolution form a major challenge to treatment and prevention efforts. An increasing number of distinct HIV-1 recombinants have been identified worldwide, but their contribution to the global epidemic is unknown. We aimed to estimate the global and regional distribution of HIV-1 recombinant forms during 1990-2015.Methods: We assembled a global HIV-1 molecular epidemiology database through a systematic literature review and a global survey. We searched the PubMed, Embase (Ovid), CINAHL (Ebscohost), and Global Health (Ovid) databases for HIV-1 subtyping studies published from Jan 1, 1990, to Dec 31, 2015. Unpublished original HIV-1 subtyping data were collected through a survey among experts in the field who were members of the WHO-UNAIDS Network for HIV Isolation and Characterisation. We included prevalence studies with HIV-1 subtyping data collected during 1990-2015. Countries were grouped into 14 regions and analyses were done for four time periods (1990-99, 2000-04, 2005-09, and 2010-15). The distribution of circulating recombinant forms (CRFs) and unique recombinant forms (URFs) in individual countries was weighted according to the UNAIDS estimates of the number of people living with HIV in each country to generate regional and global estimates of numbers and proportions of HIV-1 recombinants in each time period. The systematic review is registered with PROSPERO, CRD42017067164.Findings: Our global data collection yielded an HIV-1 molecular epidemiology database of 383 519 samples from 116 countries in 1990-2015. We found that the proportion of recombinants increased over time, both globally and in most regions, reaching 22·8% (7 978 517 of 34 921 639) of global HIV-1 infections in 2010-15. Both the proportion and the number of distinct CRFs detected increased over time to 16·7% and 57 CRFs in 2010-15. The global and regional distribution of HIV-1 recombinants was diverse and evolved over time, and we found large regional variation in the numbers (0-44 CRFs), types (58 distinct CRFs), and proportions (0-80·5%) of HIV-1 recombinants. Globally, CRF02_AG was the most prevalent recombinant, accounting for 33·9% (2 701 364 of 7 978 517) of all recombinant infections in 2010-15. URFs accounted for 26·7% (2 131 450 of 7 978 517), CRF01_AE for 23·0% (1 838 433), and other CRFs for 16·4% (1 307 270) of all recombinant infections in 2010-15. Although other CRFs accounted for small proportions of infections globally (

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_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_AE 77.4% (n = 261), subtype B 3.3% (n = 11), CRF01_AE/B recombinant 12.2% (n = 41), CRF01_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.

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 U.S. Army initiated an investigation in response to observations of a possible increase in HIV incidence among soldiers deployed to combat. Human immunodeficiency virus (HIV)-infected U.S. Army soldiers are not eligible to deploy. Combat presents a health hazard to HIV-infected soldiers and they pose a threat to the safety of the battlefield blood supply and their contacts. All soldiers are routinely screened for HIV every 2 years and those who deploy are also screened both prior to and after deployment. Seroconversion rates were estimated for all soldiers who deployed to Afghanistan or Iraq in the period 2001-2007 and all active duty soldiers who did not. Seroconverters with an estimated date of infection, based on calculation of the midpoint between the last seronegative and first seropositive test date, that was either before or during deployment were eligible for inclusion. Confidential interviews and medical record reviews were conducted to determine the most likely time, geographic location, and mode of infection. Reposed predeployment samples were tested for HIV ribonucleic acid. The HIV seroconversion rate among all soldiers who deployed was less than the rate among those who did not deploy: 1.04 and 1.42 per 10,000 person-years, respectively. Among 48 cases, most were determined to have been infected in the United States or Germany and prior to deployment (n=20, 42%) or during rest and relaxation leave (n=13, 27%). Seven seronegative acute infections were identified in the predeployment period. Subtype was determined for 40 individuals; all were subtype B infections. All were acquired through sexual contact. These findings can inform development of preventive interventions and refinement of existing screening policy to further reduce HIV-infected deployed soldier person time.

We analyzed HIV-1 genome sequences from 68 newly infected volunteers in the STEP HIV-1 vaccine trial. To determine whether the vaccine exerted selective T cell pressure on breakthrough viruses, we identified potential T cell epitopes in the founder sequences and compared them to epitopes in the vaccine. We found greater distances to the vaccine sequence for sequences from vaccine recipients than from placebo recipients. The most significant signature site distinguishing vaccine from placebo recipients was Gag amino acid 84, a site encompassed by several epitopes contained in the vaccine and restricted by human leukocyte antigen (HLA) alleles common in the study cohort. Moreover, the extended divergence was confined to the vaccine components of the virus (HIV-1 Gag, Pol and Nef) and not found in other HIV-1 proteins. These results represent what is to our knowledge the first evidence of selective pressure from vaccine-induced T cell responses on HIV-1 infection in humans.

Specific glycosphingolipids (GSL), found on the surface of target immune cells, are recognized as alternate cell surface receptors by the human immunodeficiency virus type 1 (HIV-1) external envelope glycoprotein. In this study, the globotriose and 3'-sialyllactose carbohydrate head groups found on two GSL were covalently attached to a dendrimer core to produce two types of unique multivalent carbohydrates (MVC). These MVC inhibited HIV-1 infection of T cell lines and primary peripheral blood mononuclear cells (PBMC) by T cell line-adapted viruses or primary isolates, with IC50s ranging from 0.1 to 7.4μg/ml. Inhibition of Env-mediated membrane fusion by MVC was also observed using a dye-transfer assay. These carbohydrate compounds warrant further investigation as a potential new class of HIV-1 entry inhibitors. The data presented also shed light on the role of carbohydrate moieties in HIV-1 virus–host cell interactions.

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_BC and CRF08_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 = 5SubServiceID = 6 .

BACKGROUND: Current US military clinical practice guidelines permit emergency transfusions of non–Food and Drug Administration (FDA)-compliant freshly collected blood products in theaters of war. This investigation aimed to characterize the risks of transfusion-transmitted infections (TTIs) associated with battlefield transfusions of non–FDA-compliant blood products. STUDY DESIGN AND METHODS: US Service members who received emergency transfusion products in Iraq and Afghanistan (March 1, 2002-September 30, 2007) were tested for hepatitis C virus (HCV), human immunodeficiency virus (HIV), and hepatitis B virus (HBV) infections using reposed pre- and posttransfusion sera. Selected regions of viral genomes from epidemiologically linked infected recipients and their donors were sequenced and compared. RESULTS: Of 761 US Service members who received emergency transfusion products, 475 were tested for HCV, 472 for HIV, and 469 for HBV. One transfusion-transmitted HCV infection (incidence rate of 2.1/1000 persons) was identified. The pretransfusion numbers (prevalence per 1000 persons) were HCV—four (8/1000), HIV—zero (0/1000), chronic HBV—two (4 /1000), and naturally immune (antibody to HBV core antigen)—nine (19/1000). CONCLUSION: One HCV TTI was determined to be associated with emergency blood product use. The pretransfusion HCV and HBV prevalence in transfusion recipients, themselves members of the potential donor population, indicates better characterization of the deployed force's actual donor population, and further investigations of the TTI prevalence in these donors are needed. These data will inform countermeasure development and clinical decision making.

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_AE, A/C recombinant, G, CRF02_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.

Several studies have reported an increasing number of non-CRF01_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_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_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 andor = 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.

Background and Objective: Natural killer (NK) cells most likely contribute toward limiting HIV-1 replication, and investigation into their function throughout the course of infection is therefore important. We here aimed to determine the state of the NK cell compartment in Ugandans with untreated HIV-1 clade A or D infection in comparison with matched uninfected controls. Methods and Results: The function and phenotype of NK cells were investigated using 10-color flow cytometry. Surprisingly, NK cells displayed elevated production of interferon-g and macrophage inflammatory protein 1b, as well as CD107a degranulation in infected subjects. This included unexpected levels of degranulation in the CD56bright subset of NK cells and high levels of macrophage inflammatory protein 1b in CD56negative NK cells. HIV-1 infection was associated with reduced expression of KIR2DL1, NKG2A, CD161, and NKp30 in CD56dim and CD56negative NK cells, whereas lowered CD161 expression was the only alteration in the CD56bright subset. Interestingly, low CD4 counts were associated with increased levels of interferon-g and degranulation in CD56bright NK cells, as well as increased NKp44 expression in the CD56dim cells. Conclusions: NK cells in HIV-1‐infected Ugandans display elevated activity, despite an altered functional and phenotypic profile. Furthermore, specific alterations in the CD56bright and CD56dim subsets occur in patients with severe CD4 loss.

Killer immunoglobulin-like receptors (KIRs) expressed on natural killer cells are critical components of innate immunity. Interactions between KIRs and their human leukocyte antigen (HLA) ligands have been shown to influence autoimmune and infectious disease course in defined populations. However, the low throughput and high cost of current methods impede confirmation of the universality of these findings. To support large epidemiology surveys, we developed a high-throughput real-time polymerase chain reaction-based assay to identify carriers of KIR3DL1, KIR3DS1, KIR2DL2, and KIR2DL3 and their HLA ligands. The platform performed with 100% sensitivity and specificity in detection of carrier and non-carrier on reference samples. The application of this platform will further clarify the nature and impact of the KIR-HLA epistatic interaction on disease course in large global population-based studies.

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_AG) in neutralization assays with a multi-subtype panel of HIV-1-positive plasma samples, the Indian Envs were remarkably complex antigenically. 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.

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.

In Thailand, the circulating HIV-1 strains include CRF01_AE, subtype B, and their recombinants. Genotyping and full-genome sequencing had previously identified circulating recombinant form CRF15_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_01B. Complete sequencing of three strains, amplified from serum as overlapping half-genomes, confirmed their common recombinant structure, mostly CRF01_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_01B. More comprehensive HIV-1 prevention and treatment programs in IDU can help to limit the growing complexity of HIV-1 strains in Thailand.

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/.

The importance of HLA class I-restricted CD8 T-cell responses in the control of human immunodeficiency virus (HIV) infection is generally accepted. While several studies have shown an association of certain HLA class I alleles with slower disease progression, it is not fully established whether this effect is mediated by HIV-specific CD8 T-cell responses restricted by these alleles. In order to study the influence of the HLA class I alleles on the HIV-specific CD8 T-cell response and on viral control, we have assessed HIV-specific epitope recognition, plasma viral load, and expression of HLA class I alleles in a cohort of HIV-seropositive bar workers. Possession of the HLA class I alleles B5801, B8101, and B0702 was associated with a low median viral load and simultaneously with a broader median recognition of Gag epitopes compared to all other HLA alleles (twofold increase) (P= 0.0035). We further found an inverse linear relationship between the number of Gag epitopes recognized and the plasma viral load (R= −0.36;P= 0.0016). Particularly, recognition of multiple epitopes within two regions of Gag (amino acids [aa] 1 to 75 and aa 248 to 500) was associated with the maintenance of a low steady-state viremia, even years after acute infection.

Eliciting broadly reactive functional antibodies remains a challenge in human immunodeficiency virus type 1 (HIV-1) vaccine development that is complicated by variations in envelope (Env) subtype and structure. The majority of new global HIV-1 infections are subtype C, and novel antigenic properties have been described for subtype C Env proteins. Thus, an HIV-1 subtype C Env protein (CO6980v0c22) from an infected person in the acute phase (Fiebig stage I/II) was developed as a research reagent and candidate immunogen. The gp145 envelope is a novel immunogen with a fully intact membrane-proximal external region (MPER), extended by a polylysine tail. Soluble gp145 was enriched for trimers that yielded the expected “fan blade” motifs when visualized by cryoelectron microscopy. CO6980v0c22 gp145 reacts with the 4E10, PG9, PG16, and VRC01 HIV-1 neutralizing monoclonal antibodies (MAbs), as well as the V1/V2-specific PGT121, 697, 2158, and 2297 MAbs. Different gp145 oligomers were tested for immunogenicity in rabbits, and purified dimers, trimers, and larger multimers elicited similar levels of cross-subtype binding and neutralizing antibodies to tier 1 and some tier 2 viruses. Immunized rabbit sera did not neutralize the highly resistant CO6980v0c22 pseudovirus but did inhibit the homologous infectious molecular clone in a peripheral blood mononuclear cell (PBMC) assay. This Env is currently in good manufacturing practice (GMP) production to be made available for use as a clinical research tool and further evaluation as a candidate vaccine. IMPORTANCE At present, the product pipeline for HIV vaccines is insufficient and is limited by inadequate capacity to produce large quantities of vaccine to standards required for human clinical trials. Such products are required to evaluate critical questions of vaccine formulation, route, dosing, and schedule, as well as to establish vaccine efficacy. The gp145 Env protein presented in this study forms physical trimers, binds to many of the well-characterized broad neutralizing MAbs that target conserved Env epitopes, and induce cross-subtype neutralizing antibodies as measured in both cell line and primary cell assays. This subtype C Env gp145 protein is currently undergoing good manufacturing practice production for use as a reagent for preclinical studies and for human clinical research. This product will serve as a reagent for comparative studies and may represent a next-generation candidate HIV immunogen.

HIV-1 strains containing subsubtype A2 are relatively rare in the pandemic but have been repeatedly identified in Kenya, where candidate vaccines based in part on subtype A, but not A2 strains, may be evaluated. Among the most recent is CRF16_A2D, a circulating recombinant form (CRF) whose prototypes are complete or partial HIV-1 sequences from Kenya, Korea, and Argentina. Using samples from blood bank discards in Kenya and complete genome sequencing, this report further documents CRF16_A2D and related recombinants and identifies a second CRF, CRF21_A2D. The two A2-containing CRFs, and two recombinants related to CRF16_A2D, share common structural elements but appear to have been independently derived. Concerted selection may have influenced the emergence and spread of certain A2-containing strains in Kenya. The second complete subtype C sequence from Kenya is also reported here. Monitoring of A2-containing recombinants and subtype C strains, both relatively rare in Kenya, may be informative in the course of cohort development and evaluation of candidate vaccines.

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_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.

The magnitude of the HIV pandemic and its extensive genetic variation may earn it a unique place among infectious agents. A high mutation rate and a rampant recombination are driving HIV's evolution. Nine subtypes and a variety of recombinant forms of HIV now exist. The source of recombinant forms is the multiple infection of target cells, which becomes highly significant when individuals become infected with two or more divergent strains. In the current paper, we re-examine the role of dual infection and recombination in the generation of HIV-1 diversity, both in individuals and on a global scale. The current molecular epidemiology of HIV-1 is reviewed, emphasizing the latest reports from regional epidemics.

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_AE. Four of these clustered with CM240 from Thailand, and one clustered with African CRF01_AE. Four strains were CRF02_AG, prevalent in West and West Central Africa. Two strains were subtype C. One strain was a unique recombinant between CRF01_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_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.

The HLA-B57 allele family is associated with slow progression to disease in HIV-1-infected individuals and restricts a potent CD8 response against the p24 protein. This study was designed to assess the sequence variation and the CD8 response against B57-restricted epitopes of the p24 protein in a cohort of HIV-1 subtype C-infected individuals possessing a high frequency of the B5703 allele. Gag sequences were amplified by PCR, cloned, and sequenced from 19 individuals including 8 B57-negative individuals. CD8 responses were assessed by interferon-gamma ELISPOT assay directly from PBMC using synthetic peptides matching the autologous virus as well as the peptides representing the sequence variants circulating within the B5703 individuals. The KF11 epitope (p24 amino acids 162-172) and variants of this epitope were immunodominant in subjects possessing the B5703 allele. Three variants were observed only in B5703 individuals. Differing patterns of cross-reactivity against variant peptides were observed and were dependent upon the sequence of the autologous virus. Subjects infected with the A2G, S4N variant of KF11 demonstrated poor cross-reactivity against all other variant peptides. Determination of the breadth of viral quasispecies circulating in a population provided crucial information for studying potential escape variants of an immunodominant epitope. The presented data show that the sequence of autologous virus is critical in determining the extent of cross-reactivity of a CD8 T cell response against heterologous virus variants. Efforts to optimize the cross-reactivity of vaccine-induced CD8 T cells may need to focus on the relative immunogenicity of minor sequence variation.

A high percentage of rural villagers are exposed to blood of nonhuman primates and risk acquiring infectious diseases., Exposure to nonhuman primates has led to the emergence of important diseases, including Ebola hemorrhagic fever, AIDS, and adult T-cell leukemia. To determine the extent of exposure to nonhuman primates, persons were examined in 17 remote villages in Cameroon that represented three habitats (savanna, gallery forest, and lowland forest). Questionnaire data were collected to assess whether persons kept wild animal pets; hunted and butchered wild game; had experienced bites, scratches, or injuries from live animals; or had been injured during hunting or butchering. While all villages had substantial exposure to nonhuman primates, higher rates of exposure were seen in lowland forest sites. The study demonstrates that exposure is not limited to small groups of hunters. A high percentage of rural villagers report exposure to nonhuman primate blood and body fluids and risk acquiring infectious diseases.

Phylogenetic analyses demonstrate significant diversity in worldwide circulating strains of human immunodeficiency virus type 1 (HIV-1). Detailed studies have revealed a complex pattern of intersubtype recombinations, as well as evidence of sub-subtypes circulating in various populations. In this study, we characterized an HIV-1 strain that had previously been identified as a distinct subcluster within the subtype A radiation based on partial sequence data. These viruses were of particular interest given that we recently found that their prevalence was significantly higher in dually infected individuals compared to women who were singly infected with HIV-1. Five viruses isolated from commercial sex workers in Dakar, Senegal, were full-length PCR amplified and sequenced. Phylogenetic analyses indicated that, whereas three of these viruses were closely related and clustered overall within the HIV-1 subtype A radiation, they were distinct from previously characterized sub-subtype A1 and A2 viruses. The clustering pattern was maintained in the individual gag , pol , and env regions of the genome. Distance calculations between these viruses, which we termed A3, and other reference sub-subtype A1 and A2 viruses fell in the range of distances between previously characterized sub-subtype groups. In addition, we found evidence of two A3-containing recombinants in our cohort. These recombinants are mosaics composed of sequence from both sub-subtype A3 and CRF02_AG, the major circulating recombinant form in this West African population. Based on phylogenetic analyses, we propose that the group of viruses found in the Dakar sex worker cohort, previously referred to as HIV-1 A subcluster 2, be referred to as HIV-1 sub-subtype A3.

Two HIV-1 intersubtype recombinant forms are circulating widely in populations and have become important strains in the pandemic: CRF01_AE in Southeast Asia and CRF02_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_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_cpx and CRF02_AG IbNG. In conclusion, it is possible that this CRF shared common ancestors with both Z321 and CRF02_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_cpx has already occurred outside of Africa.

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.

In West/West Central Africa, CRF02_AG is the most prevalent HIV-1 strain and circulates in the milieu of rare subtypes, circulating recombinant forms (CRFs), and unique recombinant forms (URFs). The molecular complexity of HIV-1 epidemics in this region and the need to extensively sample large populations, such as in the case of vaccine trials, pose seemingly conflicting requirements between full-genome sequencing and high-throughput low-resolution assays. Here we describe the development and evaluation of a multiregion hybridization assay (MHAcrf02) for the efficient genotyping of CRF02_AG in West/West Central Africa. Subtype A, G, and CRF02_AG-specific fluorescent probes were designed flanking five recombination breakpoints in CRF02_AG and were used in real-time PCRs. A panel representing West/West Central African HIV-1 genetic diversity was evaluated by MHAcrf02. The sample set, previously characterized by full-genome sequencing, included CRF02_AG and CRF02_AG-containing recombinants (n = 28), other subtypes, CRFs, and URFs (n = 34). DNA from peripheral blood mononuclear cells, cocultures, and plasmids was used as template. When the patterns of probe reactivity were evaluated. CRF02_AG was identified with a 100% specificity and sensitivity. In conclusion, MHAcrf02 will permit more efficient characterization of HIV-1 in West/West Central Africa, where CRF02_AG is an important strain. Together with other regional genotyping assays MHAcrf02 will contribute to the development of a global picture of HIV-1 diversity and geographic distribution, providing a strong foundation for intervention, including vaccine development.

Summary Background Hunting and butchering of wild non-human primates infected with simian immunodeficiency virus (SIV) is thought to have sparked the HIV pandemic. Although SIV and other primate retroviruses infect laboratory workers and zoo workers, zoonotic retrovirus transmission has not been documented in natural settings. We investigated zoonotic infection in individuals living in central Africa. Methods We obtained behavioural data, plasma samples, and peripheral blood lymphocytes from individuals living in rural villages in Cameroon. We did serological testing, PCR, and sequence analysis to obtain evidence of retrovirus infection. Findings Zoonotic infections with simian foamy virus (SFV), a retrovirus endemic in most Old World primates, were identified in people living in central African forests who reported direct contact with blood and body fluids of wild non-human primates. Ten (1%) of 1099 individuals had antibodies to SFV. Sequence analysis from these individuals revealed three geographicallyindependent human SFV infections, each of which was acquired from a distinct non-human primate lineage: De Brazza’s guenon (Cercopithecus neglectus), mandrill (Mandrillus sphinx), and gorilla (Gorilla gorilla), two of which (De Brazza’s guenon and mandrill) are naturally infected with SIV. Interpretation Our findings show that retroviruses are actively crossing into human populations, and demonstrate that people in central Africa are currently infected with SFV. Contact with non-human primates, such as happens during hunting and butchering, can play a part in the emergence of human retroviruses and the reduction of primate bushmeat hunting has the potential to decrease the frequency of disease emergence.

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.

We have characterized six novel genomes of human immunodeficiency virus type 1 (HIV-1) sampled from individuals infected in Uganda and former Zaire. Four isolates (SE6954, SE8603, UG035, and UG266) had clear recombination patterns that included subtypes A1, D and C. The two remaining strains (SE8646 and SE9010) also appeared to be recombinant but had a more complex pattern. To facilitate the classification of these two genomes we developed a metric, the branching index, for characterization of “problematic” sequence fragments that associate to a subtype cluster with a high bootstrap value but are only distantly related to the reference sequences. The branching index is able to signal when parental representatives may be missing and a subtype classification thus is not meaningful. Several fragments of SE8646 and SE9010 had a branching index below the subtype defining cutoff value (0.55) and, therefore, these genomes could not be unequivocally classified. The branching index, with a cutoff value defined from analyses of HIV-1 reference sequences, may be a useful approach not only for more conservative classifications of HIV-1 subtypes but also for analyzing relationships among other types of sequences.

HIV-1 subtype B and CRF01_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_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_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_01B recombinant, with the external envelope of subtype B and the rest of the genome from CRF01_AE, is circulating widely in Thailand. Termed CRF15_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.

The impact of HIV-1 genetic diversity on candidate vaccines is uncertain. To minimize genetic diversity in the evaluation of HIV-1 vaccines, vaccine products must be matched to the predominant subtype in a vaccine cohort. To that end, full genome sequencing was used to detect and characterize HIV-1 subtypes and recombinant strains from individuals in Rakai District, Uganda. DNA extracted from peripheral blood mononuclear cells (PMBC) was PCR amplified using primers in the long terminal repeats (LTRs) to generate nearly full length genomes. Amplicons were directly sequenced with dye terminators and automated sequencers. Sequences were phylogenetically analyzed and recombinants were detected and mapped with distance scan and bootscan. Among 46 sequences, 54% were subtype D, 15% were subtype A, and 30% were recombinant. All recombinants were individually unique, and most combined subtypes A and D. Subtype D comprised more than 70% of all the HIV-1 genomes in Rakai when both pure subtypes and recombinants were considered. Candidate vaccines based on HIV-1 subtype D would be appropriate for evaluation in Rakai District, Uganda.

Two different BC recombinant HIV-1 strains have arisen and begun to circulate among intravenous drug users in China. The recombinants are mostly subtype C with a few small subtype B segments. Additional full-genome sequences of the two recombinants, termed CRF07_BC and CRFO&_BC, are now available for analysis. Four CRF07_BC strains, including c54, 97CNU01, 98CN009, and a new strain CNGL-179, described here, and four CRF08_BC strains, including 97CNGX-6, 97CNGX-7, 97CNGX-9, and 98CN006, were compared for their recombination breakpoints by bootscanning and software for fine mapping of recombinants. The four CRF07_BC strains shared an identical recombination structure and the four CRF08_BC strains shared an identical, but different, recombination structure. The two CRFs share five precise subtype B/C boundaries, although although other segments differ between them, suggesting that they shared a common ancestor, itself a BC recombinant that separately "back-crossed" onto different subtype C strains. Both CRFs are broadly distributed from north to south in western China and have maintained low interpatient diversity.

Objective: To enable more rapid and effificient genotyping of HIV-1 in East Africa where subtypes A C and Dand 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 Multiregion 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 Dwere 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. (author’s)

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. (author’s)

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 high genetic heterogeneity of HIV-1 in the Democratic Republic of Congo (DRC) constitutes a real challenge for the development of vaccines to counter the spread of the HIV-1 epidemic. It is important to continue to monitor the epidemic by studying the circulating strains and their impact on the overall spread. As part of the ongoing effort to study the global distribution of HIV-1 subtypes and circulating recombinant forms (CRFs), here we describe a new phylogenetic clade of HIV-1 by the analysis of two full-length sequences (83CD003 and 90CD121E12) collected from two individuals at a 7-year interval (1983 and 1990, respectively). One of the two sequences (90CD121E12) was obtained from a vertically infected, 12-month-old baby in Kimpese, rural DRC, an area with low and stable seroprevalence of HIV-1 in women attending antenatal clinics. The two sequences are genetically similar by 95% of their full genome and topologically form a distinct cluster that is equidistant from the existing subtypes (A through K) by the analysis of both the full genome and subgenomic regions. Furthermore, they share several other genetic features, including an additional pair of cysteine residues, predictive of an extra disulfide bridge, in the V4 loop of gp120. This new clade is currently rare, spreading at a slower pace than the other subtypes found in the DRC region. Pending the identification of at least one partial length sequence of the same lineage from another patient who is epidemiologically unlinked to those described here, this clade is not yet named as a subtype as per the recommendation of the nomenclature committee.

HIV-1 outbreaks in Guangxi Province, southern China were initiated from two separate border cities in 1996 and 1997. Drug users in Pingxiang City, which borders Vietnam, were infected with CRF01_AE HIV-1, and drug users in Baise City, which borders Yunan Province, were infected with a novel B/C recombinant HIV-1. Since 1997, HIV-1 has been rapidly spreading in Guangxi, including its capital city Nanning. Survey data indicated that HIV-1 prevalence among IDUs in new outbreak regions increased 8 to 42% within 1 year. The B/C recombinants obtained from five separate regions in Guangxi, which span a 4-year time frame, were remarkable for their low intersubject env V3 diversity, less than 0.2%. Similarly, the CRF01_AE from IDUs over a 3-year time frame had low intersubject env V3 diversity of less than 1.6%. Different patterns of sequence variations in the V3 and V4 regions were observed for the B/C recombinant and the CRF01_AE HIV1. The rapid spreading of homogeneous HIV-1 strains in Guangxi may have important implications for HIV transmission as well as vaccine development and evaluation.

Neutralizing antibodies (nAbs) are a high priority for vaccines that aim to prevent the acquisition of HIV-1 infection. Vaccine effectiveness will depend on the extent to which induced antibodies neutralize the global diversity of circulating HIV-1 variants. Using large panels of genetically and geographically diverse HIV-1 Env-pseudotyped viruses and chronic infection plasma samples, we unambiguously show that cross-clade nAb responses are commonly induced in response to infection by any virus clade. Nonetheless, neutralization was significantly greater when the plasma clade matched the clade of the virus being tested. This within-clade advantage was diminished in older, more-diverse epidemics in southern Africa, the United States, and Europe compared to more recent epidemics in Asia. It was most pronounced for circulating recombinant form (CRF) 07_BC, which is common in China and is the least-divergent lineage studied; this was followed by the slightly more diverse Asian CRF01_AE. We found no evidence that transmitted/founder viruses are generally more susceptible to neutralization and are therefore easier targets for vaccination than chronic viruses. Features of the gp120 V1V2 loop, in particular, length, net charge, and number of N-linked glycans, were associated with Env susceptibility and plasma neutralization potency in a manner consistent with neutralization escape being a force that drives viral diversification and plasma neutralization breadth. The overall susceptibility of Envs and potencies of plasma samples were highly predictive of the neutralization outcome of any single virus-plasma combination. These findings highlight important considerations for the design and testing of candidate HIV-1 vaccines that aim to elicit effective nAbs. IMPORTANCE An effective HIV-1 vaccine will need to overcome the extraordinary variability of the virus, which is most pronounced in the envelope glycoproteins (Env), which are the sole targets for neutralizing antibodies (nAbs). Distinct genetic lineages, or clades, of HIV-1 occur in different locales that may require special consideration when designing and testing vaccines candidates. We show that nAb responses to HIV-1 infection are generally active across clades but are most potent within clades. Because effective vaccine-induced nAbs are likely to share these properties, optimal coverage of a particular clade or combination of clades may require clade-matched immunogens. Optimal within-clade coverage might be easier to achieve in regions such as China and Thailand, where the epidemic is more recent and the virus less diverse than in southern Africa, the United States, and Europe. Finally, features of the first and second hypervariable regions of gp120 (V1V2) may be critical for optimal vaccine design.

New outbreaks of human immunodeficiency virus type 1 (HIV-1) among injecting drug users (IDUs) are spreading in China along heroin trafficking routes. Recently, two separate HIV-1 epidemics among IDUs were reported in Guangxi, Southern China, where partial sequencing of the env gene showed subtype C and circulating recombinant form (CRF) AE. We evaluated five virtually full-length HIV-1 genome sequences from IDUs in Guangxi to determine the genetic diversity and the presence of intersubtype recombinants. Sequence analysis showed two geographically separated, highly homogeneous HIV-1 strains. B/C intersubtype recombinants were found in three IDUs from Baise City, in a mountainous region near the Yunnan-Guangxi border. These were mostly subtype C, with portions of the capsid and reverse transcriptase (RT) genes from subtype B. The subtype B portion of the capsid was located in the N-terminal domain, which has been shown to influence virus core maturation, virus infectivity, and binding to cyclophilin A, whereas the subtype B portion of RT was located in the palm subdomain, which is the active site of the enzyme. These BC recombinants differed from a BC recombinant found in Xinjiang Province in northwestern China. CRF AE strains were found in IDUs from Nanning, the capital of Guangxi, and in IDUs from Pingxiang City near the China-Vietnam border. The AE and BC recombinants were both remarkable for their low interpatient diversity, less than 1% for the full genome. Rapid spread of HIV-1 among IDUs may foster the emergence of highly homogeneous strains, including novel recombinants in regions with multiple subtypes.

Objective To characterize near-full-length genomes of two HIV-1 subtype H strains. To extend sequence data to include full env and gag, and analyse and redefine, previously documented subtype H strains. Design Near-full-length genomes of HIV-1 env subtype H strains VI991 and VI997 were amplified, cloned, sequenced, phylogenetically analysed and compared with a panel of 23 HIV-1 group M reference isolates. The mosaic nature of previously published subtype H strains VI557 and CA13 was reanalysed. Materials and methods Peripheral blood mononuclear cells (PBMC) from individuals harbouring strains VI991 and VI997 were co-cultivated with PHA stimulated donor PBMC. Near-full-length genomes of VI991 and VI997, and gag and env genes of CA13 and VI557, were amplified by polymerase chain reaction, cloned and sequenced. Intersubtype recombination analyses were performed by similarity plot, bootscanning and phylogenetic analysis. Results Near-full-length clones of HIV-1 VI991 and VI997 are representative of subtype H. They form a phylogenetic cluster with the only previously described subtype H representative HIV-1 90CF056.1, regardless of the genome region analysed. VI557 is redefined as a gag and env subtype H mosaic virus containing unclassified fragments. CA13 is a complex intersubtype recombinant between subtypes A, H and unclassified strains Conclusion Near-full-length genome analysis identified HIV-1 VI991 and VI997 as two new subtype H representatives. These reagents will allow defining and classifying non-recombinant as well as recombinant HIV-1, eventually helping to solve the puzzle of HIV-1 subtypes.

The envelope-coding sequence of human immunodeficiency virus type 1 (HIV-1) was determined for 11 Thai seroconverters between 1995 and 1996. On the basis of the env sequences, all subjects were infected with HIV subtype E. Compared with the interpatient protein diversity among HIV-1 Thai reference sequences from 1990 to 1992 (4.4%), the diversity among the 1995–1996 seroconverters was approximately double (9.5%). The tetrapeptide tip of the V3 loop was invariant for 10 of the 11 seroconverters, and identical to that observed in sequences derived from the 1990–1992 group. However, in the V3 region, sequences from 2 of the 11 subjects demonstrated more than 5 amino acid changes relative to the reference strains. This may represent the "aging" of the HIV epidemic seen in other endemic regions. These findings may have substantial implications for vaccine development and evaluation for both HIV antibody and cytotoxic T lymphocyte repertoire recognition.

The objective was to improve our understanding of the genetic complexity of HIV-1 subtype A by increasing the number of subtype A isolates that have been sequenced in their entirety. Nine HIV- 1-seropositive patients from Africa living in Sweden contributed peripheral blood mononuclear cells for this study. Sequencing of the C2-V3 region of env had shown them to be subtype A. Deoxyribonucleic acid from virus cultures was used for the amplification of virtually full-length proviral sequences and the resulting fragment was sequenced. 6 of the 9 viral isolates were subtype A throughout the genome or non-recombinant and all of these were from East Africa. One virus from the Ivory Coast had the AG (IbNG) genetic form a recombinant form common in West Africa. Two of the isolates were novel recombinants: one was an A/C recombinant and the other was A/D. Analysis of gag reveals three subclusters within the A subtype: one containing the AG (IbNG) subtype viruses one containing the AE (CM240) viruses and one containing the non-recombinant A viruses. These genetic clusters have different geographical distributions in Africa. The prevailing view of HIV-1 subtype A forming a uniform band across the center of sub-Saharan Africa needs revision. In all probability the most common subtype in West Africa and West Central Africa is the AG recombinant AG (IbNG) whereas in East Central Africa it is the non-recombinant subtype A. (authors)

Accurate determination of plasma human immunodeficiency virus type 1 (HIV-1) RNA levels is critical for the effective management of HIV-1 disease. The AMPLICOR HIV-1 MONITOR Test, a reverse transcription-PCR-based test for quantification of HIV-1 RNA in plasma, was developed when little sequence information on HIV-1 isolates from outside North America was available. It has since become apparent that many non-subtype B isolates, particularly subtypes A and E, are detected inefficiently by the test. We describe here the AMPLICOR HIV-1 MONITOR Test, version 1.5, an upgraded test developed to minimize subtype-related variation. We also developed a panel of HIV-1 standards containing 30 HIV-1 isolates of subtypes A through G. The virus particle concentration of each cultured viral stock was standardized by electron microscopic virus particle counting. We used this panel to determine the performance of the original AMPLICOR HIV-1 MONITOR Test and version 1.5 of the test with HIV-1 subtypes A through G. The original test underestimated the concentration of HIV-1 subtype A, E, F, and G RNA by 10-fold or more, whereas version of the 1.5 test yielded equivalent quantification of HIV-1 RNA regardless of the subtype. In light of the increasing intermixing of HIV-1 subtypes worldwide, standardization of PCR-based tests against well-characterized viral isolates representing the full range of HIV-1 diversity will be essential for the continued utility of these important clinical management tools.

293 THE RAPID VIRAL TURNOVER and high mutation rate of HIV1 result in remarkable genetic variation among strains obtained from different parts of the world. This has led to the development of evolutionary lineages (genetic subtypes) that can be identified by phylogenetic analysis of gene sequences.2,3 Eight genetic subtypes, named A through H in group M (major) and a group of other strains, group O (outlier), have previously been determined by phylogenetic analysis, mainly of gag and env genes. The genetic subtype classification is based on consistent clustering of the sequences in different parts of the genome, and approximate equidistance to strains belonging to other subtypes. Two additional subtypes, I and J, have been proposed, on the basis of analysis of gene subfragments. Sequences from these strains were shown to group as independent, monophyletic lineages in phylogenetic comparisons to representative reference strains of previously described subtypes A through H.2,3 However, the definition of a genetic subtype is dependent on consistent clustering of sequences independent of the genome fragment analyzed (provided that enough informative variation is present in the region). This means that the classification results must be similar regardless of the genome region examined. Since HIV-1 strains of different subtypes have been shown to recombine, a classification based on a single subgenomic fragment does not necessarily indicate the genetic subtype of the complete genome. When a putative new genetic subtype is found the complete genome should be analyzed to verify that all regions of the virus cluster independently. In this study we have cloned and sequenced the first virtually complete proviruses of two independently obtained, epidemiologically unlinked primary isolates of HIV-1 subtype J. They form an independent cluster, approximately equidistant to the other subtypes over all of the genome, verifying their classification as a new subtype. Blood samples SE9173 and SE9280 for virus isolation were collected in Sweden from two previously identified HIV-1-infected individuals carrying the putative new subtype J viruses.8 Both individuals were immigrants from the Democratic Republic of Congo (formerly Zaire). A thorough epidemiological investigation, including contact tracing, did not reveal any direct or indirect contact between the two individuals. The initial identification of subtype J was based on direct sequencing and phylogenetic analysis of the gag p17 and env V3 regions amplified from uncultured patient lymphocytes. Samples SE9173 and SE9280 were obtained from the same individuals as the previously described samples SE7022 and SE7887, respectively. A seminested extended polymerase chain reaction (PCR) was used to amplify the virtually complete provirus from peripheral blood mononuclear cell (PBMC) cocultures essentially as has been previously described. Briefly, primers MSF12, binding to the tRNA-binding site located in the U5-gag leader junction of the 59 long terminal repeat (LTR), and MSR5, binding to the mRNA-polyadenylation signal site located in the R-U5 junction of the 3 9 LTR10 (Fig. 1), were used for first-round amplification. For second-round amplification one-tenth of the reaction was reamplified using primers MSF14 and MSR5. Second-round amplicons were cloned with a TA-cloning kit (pCR2.1 cloning kit; Invitrogen, San Diego, CA). Clones were sequenced on both strands by primer walking, using an ABI 377 automated sequencer and dye-terminator chemistry (Perkin-Elmer Applied Biosystems Division, Foster City, CA). The sequences have been submitted to GenBank with accession numbers AF082394 and AF082395. The lengths of the clones from strains SE9173 and SE9280 were 8953 and 8943 bp, respectively. The genome structure was similar to all other subtypes of HIV-1, with the gag, pol, and env structural genes, and reading frames for vif, vpr, vpu, and nef single exon regulatory/accessory genes (Table 1). The tat and rev exons 1 and 2 were also discernible at their usual positions. All reading frames appeared open and of complete length. The newly obtained SE9173 and SE9280 sequences were 94.2 and 97.6% similar to the SE7022 and SE7887 sequences in the V3 region, respectively.

Multiple human immunodeficiency virus type 1 (HIV-1) genetic subtypes, intersubtype recombinants, and group O have been found in west central Africa. In Nigeria, where HIV-1 prevalence is rising rapidly, characterization of HIV-1 strains has been limited. Each of three full-length genome sequences acquired to date shows evidence of recombination: two are largely subtype G with subtype A segments in the midgenome accessory region; the third, IbNG, is subtype G with the long terminal repeats and two segments of pol from subtype A. In this study, peripheral blood mononuclear cells obtained in 1994–1995 from 10 patients hospitalized in northeastern Nigeria were evaluated by sequencing of the complete envelope and, from 7 patients, a portion ofgag.Four patients harbored subtype G viruses and six patients had recombinant viruses. Two had strains sharing the A/G recombinant structure of IbNG. Two had a previously undescribed recombinant, mostly subtype A, whose carboxyl-terminal gp41 could not be classified. An A/G recombinant different from IbNG but similar to CA1, a Cameroonian strain, was found in one patient. The remaining patient had a strain that was otherwise subtype G but shared an unclassified carboxyl-terminal gp41 segment with the CA1-like strains. Other subtypes and group O were not found.