Molecular characterization of HIV type 1 among HIV-infected respondents in a cohort being prepared for HIV Phase III vaccine clinical trials, Western Kenya.

Kenya is one of the sub-Saharan African countries affected by HIV-1 infection and AIDS. We investigated HIV-1 genetic diversity in 130 individuals from Busia, Bungoma, and Kakamega in western Kenya as part of an HIV-1 vaccine feasibility study in preparation for Phase III efficacy clinical trials. After RNA extraction the partial gag (484 bp) and env (1297 bp) regions were amplified and directly sequenced. Phylogenetic analysis was done using MEGA version 4 and recombinants were identified using the jpHMM tool and phylogenetic analysis. HIV-1 sequences were amplified from 122 of the 130 samples, 118 (90.8%) from the gag region and 78 (60 %) from the env region and 74 samples (56.9%) from both the gag and env regions. Of these sequenced on both regions, 51.4% were subtype A, 9.4% subtype D, 1.4% subtype C, 4.1% subtype G, and 33.7% were discordant and thus possible recombinants, including A1/C, A1/D, A1/A2, and A2/C. The jpHMM tool indicated a further two samples with CD and BD breakpoints within the env gene and one within the gag gene (A1C). An additional sample had an A1D breakpoint in the gag gene, but the envelope was not amplified. HIV-1 subtype diversity in western Kenya should be considered in vaccines designed for clinical trials in this region and this genetic diversity should be continuously monitored.

Phylogenetic diversity and low level antiretroviral resistance mutations in HIV type 1 treatment-naive patients from Cape Town, South Africa.

We analyzed the HIV-1 pol gene from patients in Cape Town to determine the genetic diversity of HIV-1 in the region and to assess the baseline HIV-1 resistance level of treatment-naive patients. Plasma was collected prior to the national antiretroviral therapy (ART) program. RNA was extracted, followed by RT-PCR and automated DNA sequencing of the viral protease (PR) and reverse transcriptase (RT) coding region. Genotyping was done through phylogenetic analysis. The sequences were inspected for resistance-associated mutations against PR and RT inhibitors. A total of 140 pol sequences were analyzed, of which 133 (95%) belong to HIV-1 subtype C, five (3.6%) were subtype B, and one each was subtype G and CRF02_AG. Five sequences (3.6%) had resistance-associated mutations. These include three (2.1%) NNRTI mutations. With the progression of the national ART program, it is important to monitor the resistance profile of naive and treatment-experienced patients.

Molecular analysis of HIV type 1 vif sequences from Cape Town, South Africa.

South Africa has the highest number of HIV-1-infected individuals in the world, with HIV-1 subtype C prevailing. However, HIV-1 subtype C accessory genes are rarely characterized in the country. These genes are important for establishing viral pathogenesis. The Vif protein has been shown to counteract the antiretroviral activity of APOBEC3G/F cytidine deaminases. In this study an additional 50 HIV-1 vif sequences are characterized. These include 48 HIV-1 subtype C and 2 HIV-1 subtype B sequences. Highly conserved HIV-1 subtype C motifs are outlined. The previously identified RLRR (90-93) motif does not seem to be conserved among our newly analyzed sequences. Conserved motifs can be useful for developing new vaccine strategies or antiretroviral drugs.

Complete genome sequencing of a non-syncytium-inducing HIV type 1 subtype D strain from Cape Town, South Africa.

The fast growing HIV-1 epidemic in South Africa is mainly caused by HIV-1 group M subtype C, spreading via heterosexual transmission. In South Africa HIV-1 subtype B and D viruses were responsible for the initial epidemic during the 1980s, primarily in the homosexual population. This study describes the full-length PCR amplification and sequencing of an HIV-1 subtype D strain recovered from plasma from a sample taken during 1990. This is only the second full-length non-syncytium-inducing (NSI) subtype D strain described. Although restricted, the subtype D strain is still being detected in the South African population.

Risk for HIV-1 infection associated with a common CXCL12 (SDF1) polymorphism and CXCR4 variation in an African population.

CXC chemokine ligand 12 (CXCL12), or stromal cell-derived factor 1 (SDF1), is the only known natural ligand for the HIV-1 coreceptor, CXC chemokine receptor 4 (CXCR4). A single nucleotide polymorphism (SNP) in the CXCL12 gene (SDF1-3'A) has been associated with disease progression to AIDS in some studies, but not others. Mutations in the CXCR4 gene are generally rare and have not been implicated in HIV-1/AIDS pathogenesis. This study analyzed the SDF1-3'A SNP and performed mutation screening for polymorphic markers in the CXCR4 gene to determine the presence or absence of significant associations with susceptibility to HIV-1 infection. The study consisted of 257 HIV-1-seropositive patients and 113 HIV-1-seronegative controls representing a sub-Saharan African population belonging to the Xhosa ethnic group of South Africa. The SDF1-3'A SNP was associated with an increased risk for HIV-1 infection (P = 0.0319) whereas no significant association was observed between the occurrence of the SDF1-3'A SNP and increased or decreased plasma levels of CXCL12. Comprehensive mutation analysis of the CXCR4 gene confirmed a high degree of genetic conservation within the coding region of this ancient population.

Sequence analysis of near full-length HIV type 1 subtype D primary strains isolated in Cape Town, South Africa, from 1984 to 1986.

South Africa has one of the fastest growing HIV-1 epidemics worldwide, consisting mostly of subtype C. However, HIV-1 subtype B and subtype D viruses were isolated in the beginning of the epidemic in the early 1980s. This study describes the amplification, cloning, and near full-length genome sequencing of four HIV-1 subtype D primary strains, isolated from 1984 to 1986 in Cape Town, in what seems to have been a small restricted subtype D epidemic in the country.

Novel mutations and SNPs identified in CCR2 using a new comprehensive denaturing gradient gel electrophoresis assay.

A single nucleotide polymorphism (SNP) at codon 64 in the CC chemokine receptor 2 gene (CCR2 V64I) has been associated with a dominant effect of delaying disease progression from human immunodeficiency virus-1 (HIV-1) infection to acquired immunodeficiency syndrome (AIDS). The objective of our study was to design a comprehensive mutation detection assay for the entire coding region of the CCR2A and CCR2B gene transcripts, including all relevant splice site junctions and to identify novel mutations and SNPs within our predominantly African-based population, which could influence an individual's susceptibility to HIV-1 infection and/or progression to AIDS. The mutation detection assay, based on denaturing gradient gel electrophoresis (DGGE), allowed for the complete analysis of five individuals per denaturing gel. Our study cohort consisted of 102 HIV seropositive patients and 144 HIV seronegative controls from the diverse South African population. Application of the CCR2-DGGE assay resulted in the detection of two previously reported CCR2 polymorphisms, namely CCR2 V64I and CCR2 N260N, and 11 novel mutations, including seven SNPs occurring at high allelic frequencies within specific population groups of South Africa. The large number of novel mutations/SNPs identified, using the CCR2-DGGE assay, indicates the importance for comprehensive analysis of all candidate genes in host susceptibility to HIV-1 infection, specifically in the under-studied African-based populations.