[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.


CONCLUSIONS
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.
Feng
Fu-min
FM
Institute of Microbiology and Epidemiology, Academy of Military Medical Science, Beijing 100071, China.
Bao
Zuo-yi
ZY
Zhuang
Dao-min
DM
Liu
Si-yang
SY
Li
Lin
L
Li
Jing-yun
JY
D000595:Amino Acid Sequence; D001483:Base Sequence; D001782:Blood Donors; D002681:China; D003001:Cloning, Molecular; D004279:DNA, Viral / Q000235:genetics; D005260:Female; D016679:Genome, Viral; D015658:HIV Infections / Q000821:virology*; D015497:HIV-1 / Q000145:classification / Q000235:genetics*; D006801:Humans; D007963:Leukocytes, Mononuclear / Q000821:virology*; D010802:Phylogeny; D016364:Reading Frames; D017422:Sequence Analysis, DNA; D017385:Sequence Homology
D004279:DNA, Viral