Cloning and characterization of a full-length HIV-1 genome of a prevalent subtype B-Thai strain in Henan Province / 中华实验和临床病毒学杂志

ABSTRACT

<p><b>OBJECTIVE</b>To clone, identify and phylogenetically characterize a clade B-Thai HIV isolate representing the most prevalent virus in Henan province.</p><p><b>METHODS</b>Peripheral blood mononuclear cells (PBMCs) from an HIV-1 infected patient in Henan Province were separated, and co-cultivated with phytohemagglutinin-stimulated healthy donor PBMCs. Proviral DNA was extracted from productively infected PBMCs. The full-length HIV-1 genome was amplified by using the LA Tag long template PCR system. Primers were positioned in conserved regions within the HIV-1 long terminal repeats. Purified PCR products were T-A ligated into a pWSK29-T vector(CNHN 24 clone). Three recombinant clones containing virtually full-length HIV-1 genome were identified by PCR. The full-length genome was sequenced by using the primer-walking approach. Nucleotide sequence similarities were calculated by the local-homology algorithm. Phylogenetic trees of gag, pol and env reading frames were constructed using the Phylip software.</p><p><b>RESULTS</b>HIV-1 C3V4 sequences indicate that the epidemic in this area was B-Thai subtype. V3 loop multiple amino acid sequence alignments showed amino acid alterations at nine positions. The 9,010 bp genomic sequence derived from isolate CNHN 24 contained all known structural and regulatory genes of an HIV-1 genome. No major deletions, insertions, or rearrangements were found. The highest homologies of the gag, pol, vpr, and vif reading frames to the corresponding clade B-Thai RL 42 sequences were 95.42%-97.08%. Phylogenetic trees showed the closest relationship of CNHN 24 and RL 42.</p><p><b>CONCLUSION</b>The cloning and characterization of a virtually full-length HIV-1 B-Thai subtype in central China was completed in our laboratory. The data should be helpful to future studies on the genetic diversity of HIV-1.</p>