Cloning and characterization of a full-length HIV-1 genome of a prevalent subtype B-Thai strain in Henan Province

Objective To clone, identify and phylogenetically characterize a clade B-Thai HIV isolate representing the most prevalent virus in Henan province. Methods 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. Results 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. Conclusion 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.