RESUMO
PIP: The AIDS epidemic is a rapidly growing problem in Nairobi, where the seroprevalence in pregnant women increased from 4% in 1988 to over 10% in 1991. 22 HIV-1-seropositive pregnant women and 1 HIV-1-infected baby (K88) attending the Pumwani Maternity Hospital of Nairobi between 1990 and 1992 were studied as part of a cohort study of maternal risk factors in mother-to-child transmission. A 250-base pair (bp) fragment of the env gene encoding C2V3 was amplified mostly from DNA isolated from primary peripheral blood mononuclear cells and subsequently sequenced. The 23 newly determined HIV env sequences were aligned with 23 previously known sequences of HIV-1 isolates of diverse geographical origin and the sequence of the HIV-1-related chimpanzee isolate SIVcpz-gab, on the basis of primary structure. Distance calculation, tree construction, and bootstrap analysis were realized with the software package TREECON. In the tree, 8 major branches could be observed containing sequences representative of 8 different subtypes A, B, C, D, E, F, G, and H, besides the outlier group O. 19 of 23 Kenyan isolates clustered with D687, Z321, U455, and SF170, which were members of genetic subtype A. Phylogenetic analyses favored positioning of K976 as a divergent A subtype strain. For 4 strains (K29, K88, K98, and K112) the subtype A classification based on the gag gene was also observed on the basis of phylogenetic analysis of the C2V3 coding part of the env gene. The predicted amino acid sequence of the V3 region for these strains was also presented. The finding that among 23 HIV-1 isolates collected in Nairobi, 19 were classified in subtype A versus 3 in subtype D, together with a much larger variation between subtype A strains as compared to subtype D strains, suggests an earlier introduction of a subtype A strain, multiple introductions of subtype A strains, and/or faster diversification of subtype A strains as compared to subtype D strains.^ieng
Assuntos
Variação Genética , Infecções por HIV/virologia , HIV-1/genética , Sequência de Aminoácidos , Sequência Consenso , Feminino , Produtos do Gene env/genética , Genes env , Infecções por HIV/epidemiologia , HIV-1/isolamento & purificação , Humanos , Lactente , Quênia/epidemiologia , Dados de Sequência Molecular , Filogenia , Gravidez , Homologia de Sequência de AminoácidosRESUMO
In view of the genetic diversity of the human immunodeficiency virus Type 1, we assessed the sensitivity and quantification efficiency of the HIV-1 RNA NASBA amplification system with respect to different HIV-1 subtypes and recombinants. Twenty cell culture supernatants representing 17 HIV-1 group M and 3 group O strains were tested, and NASBA RNA loads were compared with results obtained with a RT-PCR based HIV-1 RNA quantitation method, with p24-antigen concentrations and with the infective dose. The current HIV-1 RNA NASBA seemed suitable to quantitate representatives of different HIV-1 M subtypes. Differences between NASBA and RT-PCR loads were observed for certain HIV-1 M strains. Significantly lower RT-PCR loads were measured for most gag A, gag B and gag F strains, whereas NASBA detected lower copy numbers in 1 gag H strain and 1 gag H/env G recombinant. NASBA was not able to quantify 1 HIV-1 group M recombinant. Some of these differences could be explained by the presence and position of mismatches with primers. HIV-1 group O strains were not detectable by both RNA amplification methods. A firm correlation was not observed between the measured RNA loads and either the p24-antigen concentration or the infective dose.
Assuntos
HIV-1/genética , Técnicas de Amplificação de Ácido Nucleico , RNA Viral/análise , Humanos , RNA Viral/genética , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Carga ViralRESUMO
Combination therapy with protease (PR) and reverse transcriptase (RT) inhibitors can efficiently suppress human immunodeficiency virus (HIV) replication, but the emergence of drug-resistant variants correlates strongly with therapeutic failure. Here we describe a new method for high-throughput analysis of clinical samples that permits the simultaneous detection of HIV type 1 (HIV-1) phenotypic resistance to both RT and PR inhibitors by means of recombinant virus assay technology. HIV-1 RNA is extracted from plasma samples, and a 2.2-kb fragment containing the entire HIV-1 PR- and RT-coding sequence is amplified by nested reverse transcription-PCR. The pool of PR-RT-coding sequences is then cotransfected into CD4+ T lymphocytes (MT4) with the pGEMT3deltaPRT plasmid from which most of the PR (codons 10 to 99) and RT (codons 1 to 482) sequences are deleted. Homologous recombination leads to the generation of chimeric viruses containing PR- and RT-coding sequences derived from HIV-1 RNA in plasma. The susceptibilities of the chimeric viruses to all currently available RT and/or PR inhibitors is determined by an MT4 cell-3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide-based cell viability assay in an automated system that allows high sample throughput. The profile of resistance to all RT and PR inhibitors is displayed graphically in a single PR-RT-Antivirogram. This assay system facilitates the rapid large-scale phenotypic resistance determinations for all RT and PR inhibitors in one standardized assay.