Berry, Neil John; (1996) Detection, quantification and genetic analyses of human immunodeficiency virus type 2 (HIV-2) infections in the Gambia, West Africa. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Infection with Human Immunodeficiency Virus type 2 (HIV-2) has been investigated in collaborative studies with the MRC unit, Fajara, in The Gambia, West Africa using virological, serological and molecular techniques. Serum antibody levels of anti-HIV-2 were measured with competitive serological assays using culture-derived viral antigens which were applied in field studies in West Africa. These were subsequently replaced with a recombinant β-galactosidase fusion protein of an immunodominant region of the transmembrane glycoprotein (gp36) of HIV-2. Competitive EIAs were demonstrated to be a specific and sensitive means of detecting anti-HIV-2 and, in most cases, for speciation of HIV-1 and HIV-2 infections and for the detection of related non-human primate lentiviruses. A recombinant HIV-2 p26 antigen was produced as a glutathione-S-transferase fusion protein by PCR amplification and cloning into the pGEX-3X expression vector. These studies yielded a soluble recombinant antigen which was used as an immunogen to raise a polyclonal anti-p26- specific reagent. An assay for the detection of p26 antigens was also developed. Partial nucleotide sequence of the p26 gene of four Gambian HIV-2 strains (CBL.20-23) indicated these to be subtype A strains. Nested PCR assays were applied to HIV-2 pro viral DNA detection using oligonucleotide sequences in the Long Terminal Repeat (LTR), pol, and vpx regions of the HIV-2 genome. In a cohort of Gambian seropositive individuals, HIV-2 provirus was detected in DNA extracted from PBMCs in 84/86 (97%) of individuals with LTR primers and 39/41 (95%) in a similarly conserved region of pol (integrase). LTR sequences and recombinant Pfu DNA polymerase, rather than Taq polymerase, were applied in a radiometric incorporation assay for quantification of HIV-2 proviral DNA and evaluated by an end-point limiting dilution method. A wide range of proviral DNA levels were found in HIV-2-infected individuals where increased proviral loads, expressed as copies per 105 CD4-positive lymphocytes, were strongly associated with CD4 depletion. Nested HIV-1 and HIV-2 PCR primers were also used to identify dual infections. Amplification and direct sequence analysis of the U3 region of the HIV-2 LTR, from either proviral DNA or viral RNA, indicated a high level of sequence conservation of four cis-acting elements in vivo. A PCR-based approach to genotyping HIV-2 strains was also developed, capable of differentiating HIV-2 subtype A strains from subtype B or SIV-like viruses based on U3 sequence-specificity. Subtype A viruses were found to predominate in the Gambian samples analysed.

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