| Human Immunodeficiency Virus (HIV) was the pathogen of Acquired Immunodeficiency Syndrome (AIDS). Up to the end 2002, survival HlV-infected individuals have amounted to 42 millions. According to the specialist estimates, the number of HIV-infected individuals and/or AIDS patients is increasing rapidly, it has been one million in China. HIV can be divided into HIV-1 and HIV-2, at least 11 subtypes in HIV-1 main group, of which HIV-1 B' and B/C are prevalent subtypes in China. Systemic phenotype analysis, study on the genome characterization and identify in HIV-1 epitopes may be the basis for the development of candidate vaccines and diagnosis methods, design of anti-HIV agents based on HIV-1 prevalent subtypes in China. Up to now, there have not been a systemic study for HIV-1 B' and B/C strains in China. IN this study, we reported the phenotypic analysis, full-length molecular cloning and major gp41 antigen epitope application of HIV-1 B' and B/C subtype strains in China.Part I. Phenotypic analysis of HIV-1 prevalent Subtypes B' and B/C strains in ChinaAs the single plus stranded RNA virus, HIV is of great variability. The mutation rate is one million times to DNA viruses. It resulted in difference in virus phenotypes. HIV phenotype determines its coreceptor utilization. While HIV entrying into cells, virus isolates are classified into three groups, M-tropic,T-tropic and dualtropic. Early infection with HIV-1 is dominated by M-tropic viruses,the evolution of CXCR4-tropic viruses later in the infection. As to viral factors involved in HIV-1 cell tropism, a specific region of gp!20 protein, the V3 loop, was demonstrated to be a major determinant of coreceptor usage. In this part, one HIV-1 strain was isolated from a HIV-1 infected paid blood donor in Henan province, whose viruses were identified as B' genotype. The cell tropism of the virus was identified. Major results are showed following:A. HIV-1 CNHN24 strain was isolated by PBMC coculturing from a HIV-infected person with phytohemaggluting-stimulated donor PBMC. The phenotype was M-tropic, SI and rapid/high replication. The relationship between cell tropism change and V3 loop mutation was analyzed.B. By coculture of PBMC and MT4 cell, HIV-1 CNHN24 strain turnovered T-tropic, CXCR4-utilization from M-tropic, CCR5-utilization.C. HIV-1 CNHN24 isolate could infect MT4 cell successively, which had been passed 16 generations with high titer of 108 TCID50.D. HIV-1 CNHN24 infected PBMC were cocultured with donor PBMC and/or MT4 cell, HIV-1 RNA and proviral DNA were extracted from the supernatant and PBMC and/or MT4 cell, respectively. V3 loop sequences were absolutely identities for HIV-1 RNA and proviral DNA.E. When HIV-1 CNHN24 turnover its cell tropism, the significant changes have taken place in V3 loop sequence of HIV-1 CNHN24 strain, the mutation rate of nucleic acid sequence was 17%.F. The mutation of V3 loop ammo acid was analyzed on T-tropic turnover from M-tropic, the mutation rate was 9%, showing amino acid mutant at positions 4,11,13,14,24,27,32, of which the changes of amino acid charge were obviously at positions 11,14,27,32.Part II. Full-length genome cloning of subtype HIV-1 B' and B/C in ChinaUp to now, only a few HIV-1 full-length genome sequences of prevalent HIV-1 strains were reported, and no any reports on HIV-1 full-length genome cloning in China. We have developed 3' end and 5' end sequencing of mRMA by RACE technical, designed and synthesized two specific primers according to the sequence for amplify full-length HIV-1 genome. The PCR reaction was performed by using HIV-1 CNHN24 proviral DNA as the template.A. With the optimization of the components of reaction, Long fragment PCR for HIV-1 full-length genome was finally set up.B. The cloning and characterization of a virtually full-length HIV-1 B' subtype was completed first time in Chinese lab.C. The full sequence of the cloned primary isolate CNHN24 has been submitted to GenBank database, search number is AY180905.D. HIV-1 CNHN...
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