| Objective: (1) To study the genetic variations of V3-V4 and flanking regions of the env gene from the circulating recombinant form (CRF) of human immunodeficiency virus type 1 (HIV-1) in Guangxi. (2) To developed a simple and rapid subtype-screening assay for CRF of HIV-1 in Guangxi. Methods: (1) 50 samples were collected from the prevailing area of HIV-1 in Guangxi. Fragments of the HIV-1 env and gag gene were amplified by nest-PCR from proviral-DNA templates of HIV-1 infected individuals. The PCR products were then directly sequenced by using ABI 310 DNA SEQUENCER. The sequences covering the env V3 region or V3-V4 region of CRF were carried out phylogenetic analysis and amino acid mutations by CLUSTAL, MEGA and dnatools. (2) Proviral DNA from HIV-positive samples were extracted and subjected to first round PCR with universal primers for env and gag region that can detect HIV-1 M group isolates. In the second round PCR, two pairs of subtype-specific primers, respectively detecting subtype C and CRF01-AE, were added into one tube. The PCR products of different subtypes could be distinguished in agarose-gel electrophoresis. Another pair of primers exclusively detecting the recombinant B′/C strains were designed and used. Additionally, all of these samples were subjected to second round PCR with universal primers for two gene regions, then the PCR products were sequenced and analyzed phylogenetically. Results: (1) 46 samples showed that 3 samples (6.52%) were infected with CRF08-BC, and 43 (93.48%) CRF01-AE. Phylogenetic tree analysis showed that 2 CRF08-BC strains were closely related to 98CN006 and 97CNGX6f, and 1 CRF08-BC clustered with C.95IN21068, while 41 CRF01-AE strains clustered with 97CNGX2F and THCM240, and 2 CRF01-AE were closely related to 90CF402. Analyses of nucleic acid and amino acid showed DNA distances in the env V3-V4 regions of 24 samples were closely related to the normal strains in Guangxi, and the env C3 and V4 regions were highly variable compared with V3 region. Analyses of deduced amino acid sequences in the V3 region of 46 samples from the CRF08-BC and CRF01-AE strains showed the CRF08-BC strains had only one type of V3 loop central motif——GPGQ, while the CRF01-AE strains showed seven types——GPGQ(74.4%), GPGR(9.3%), GPGK(4.7%), GPGH(4.7%), GPGA(2.3%), GRGE(2.3%) and RPGE(2.3%). Predictions for the potential use of coreceptors on the basis of the critical amino acids within V3 loop disclosed that 100% of the CRF08-BC strains and 60.47% of the CRF01-AE strains were predicted to be CCR5-using, while 39.53% of the CRF01-AE strains could not be predicted. (2) Phylogenetic analysis 50 samples showed that the env region of 3 samples (6%) were infected with CRF08-BC, 43 (86%) with CRF01-AE, and 4 (8%) remained unclassifiable, while the gag region of 4 samples (8%) were infected with CRF08-BC and 46 (92%) with CRF01-AE. Detection of the subtype-specific primer sets revealed that the env region of 3 were CRF08-BC (100%), 39 were CRF01-AE (90.7%), while the gag region of 4 were CRF08-BC (100%), 45 were CRF01-AE (97.83%), and the primer pairs for CRF07-BC and CRF08-BC amplified 4 samples (100%). Non-specific bands occasionally appeared but did not interfere with interpretation of the results. The average reproducibility of the env region was 100% for CRF08-BC samples, and 93.8% for CRF01-AE samples, while the gag region was 100% for CRF08-BC strains, and 98.3% for CRF01-AE strains. The Comparison between DNA sequencing and the subtype-primers sets showed in the env region the sensitivity of DNA sequencing was 92%, subtype-primers sets was 84%, and the specificities were 100%, while in the gag region the sensitivity of DNA sequencing was 100%, subtype-primers sets was 98%, and the specificities were 100%. The Phylogenetic analysis was consistent with subtype-specific primer sets and the consistent rate was 92% in the env region, while it was 98% in the gag region. Conclusion:(1) Now the predominant strain of the HIV-1 epidemic is subtype CRF01-AE in Guangxi.Based on the analysis of amino acid sequen...
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