Search

Objective: To investigate the prevalent status of HIV-1 subtype E epidemic among different populations in Shenzhen, China., Methods: HIV-1 proviral DNA from peripheral blood mononuclear cells (PBMCs) of 3 HIV-infected individuals found in Shenzhen was amplified by nested-PCR. The C2-V3 and its flanking region (350-450 nucleotides) of HIV- 1 env gene were sequenced directly from the PCR products and analysed., Results: These 3 cases were confirmed to be infected with HIV-1 subtype E strain (sz-E). The innergroups distance of sz-E was 2.6%. In comparison with the consensus sequence of international A-E subgroups and some of subtype B epidemic in China, the genetic distances among sz-E and each of A-D subgroups and some of subtype B were more than 24 %. Furthermore, the phylogenetic tree analysis showed that sz-E clustered closely to Econ., Conclusions: These findings suggested that the epidemic time for HIV-1 subtype E, which was mainly prevalent among IDUs population and some professional blood donors, had been about 2-3 years in Shenzhen.

Objective: To clone and express subtype E and subtype B gag gene of the prevalent HIV-1 strain in China., Methods: One HIV-1 positive sample, whose subtype had been determined as sub-type E and one HIV-1 positive sample, whose subtype had been determined as subtype B by sequence analysis of partial env gene were chosen to be amplified by nested PCR. We got two full-length fragments of gag gene and incorporated them first into plin8Pr55 and then into pFastBacl., Results: Sequence analysis showed the cloned gag genes have the complete open-reading frames and have no major deletion and insertion. Using the Bac-toBac system, we obtained the recombinant baculoviruses containing gag genes. Western-blot analysis showed that insect cells infected with gag recombinant baculoviruses expressed HIV-1 gag antigen. Thin section electron microscopy showed that virus-like particles (VLPs) formed in insect cells was infected with gag., Conclusions: The cloning and expression of gag genes of the prevalent HIV-1 stains in China revealed that the cloned genes have intact structure and function and this paves the way for the development of HIV-1 vaccines targeted at the epidemic of HIV-1 in China, also for the study of biological functions of gag gene and development of diagnostic kits of HIV-1.

Objective: To reveal the mechanism of transient infection of T cell by HIV-1 isolates of early stages from Yunnan and Xinjiang, China., Methods: We made these viruses pass on CXCR4 expressing T cell lines and CCR5 expressing U937 cell line. After having observed the biological phenotype, we analyzed sequences of env gene to find genetic mutations of the strains, and used heteroduplex mobility assay (HMA) to show the complexity of the virus groups., Results: Sequence analysis indicated that these viruses are M tropic, NSI strains, correlating with their phenotype; they mutated largely through T cell passage, all showed sequence characteristics deviating from M tropic/NSI to different extents. HMA results indicated the complexity of virus groups was low at the very beginning of the passage and kept high later., Conclusion: We concluded that these viruses tried mutating to different directions to adapt the T cell line but all failed. Although env gene correlates with cell tropism, coreceptor usage and HIV syncytium inducing, it may have relationship with the whole genome, whether these phenotypes are present or not. The results imply that there is no T tropic/SI strain in the M topic/NSI virus pool in early infection, it is generated later following continued infection in vivo.

Objective: To search for possible recombination of HIV-1 strains in China., Methods: The blood samples were collected from the HIV infected individuals in areas where epidemic of more than two subtypes of HIV-1 were found. The HIV env and tat genes were amplified by nested PCR from PBMCs and directly sequenced and analyzed., Results: The sequence analysis were performed on 14 HIV-1 strains collected in the subtype B' and C epidemic areas of China. No evidence of recombination after sequencing of env was found. The recombnant HIV-1 strains of subtype B' and C were found in 10 of the 14 samples tested when the first exon of the tat gene was sequenced. In addition, three subtype B' and one subtype C non-recombinant HIV-1 strains were found among IDUs in Sichuan Province., Conclusions: The recombinant HIV-1 strains of subtype B' and C were firstly identified in Sichuan Province of southwest China and Xinjiang Autonomous Region of western China. Their identical sequences and recombinant patterns indicated the same origin of the recombinant strains, it strongly indicates a close correlation between these two HIV epidemic regions. Since no other subtype of HIV-1 except the recombinant was found in Xinjiang, whereas both subtype B and C as well as B'/C recombinant were found in Sichuan, the recombination was most probably happened in Sichuan Province rather than in Xinjiang.

Objective: To study the prognostic value of virology and immunology markers in Chinese HIV-1 infected people and their correlation., Methods: We studied 190 HIV infected people/AIDS patients' samples from China. Plasma viral load, P24 antigen, virus culture, CD4 positive T lymphocyte (CD4) count and CD8 positive T lymphocyte(CD8) count were tested. Test groups were divided according to clinical staging diagnosis from physicians. The data were analyzed in aspects of their relations with clinical status and correlations among all the above markers., Results: Plasma viral load, CD4 count and CD4/CD8 ratio showed significant difference between asymptomatic stage and symptomatic stages (P < 0.01), also between asymptomatic and AIDS stages(P < 0.001). Virus culture positive rate increased significantly from asymptomatic to symptomatic stage. No significant clinical change was observed associating with P24 antigen positive rate. CD8 count decreased slightly when AIDS occurred (P < 0.001). Close correlation was observed between plasma viral load, CD4 count and CD4/CD8 ratio., Conclusion: Viral load, CD4 count and CD4/CD8 ratio are useful indicators to AIDS prognosis and virus culture can be a supplementary marker. p24 antigen detection seems no diagnostic value to disease progress. The significance of CD8 change will be further studied.

Objective: HIV-1 tat gene is one of its regulatory genes necessary for its replication. This study is to explore whether the variation of tat gene influences the AIDS progress of HIV infected individual., Methods: We carried out investigation on tat gene of HIV among the long term HIV infected non-progressors and the commonly infected individuals. Mononuclear cells of peripheral blood were isolated and cellular DNA was extracted. Nested PCR method was used to amplify tat gene and their sequences were analyzed., Results: Blood were collected from 22 HIV infected individuals, of which 11 cases were infected with B subtype strains based on previous env gene analysis and belonged to long term non-progressors according to clinical features and immunology. The other 11 cases were common HIV infection of which 5 subtype B and 6 subtype C were found. Both long term non-progressor group (subtype B) and the commonly HIV infectedly group (subtype B) had constantly four amino acids different from that of international B subtype consensus sequence The both groups clustered randomly on the phylogenetic tree. In commonly HIV infected subtype C group there were 6 amino acids different from that of international subtype C. The variation inside the tatgene of common subtype C group was small., Conclusions: There was no regular variation and significant difference between long term HIV infected non-progressive group and commonly HIV infected subtype B group. There is no evidence showing that progressive speed of AIDS correlated with the tat sequence profile of HIV-1.

Objective: To clone the HIV-1 env gene and establish the HMA reagents suitable for use with the Chinese HIV strains., Methods: Using Nest-PCR technique, we amplified env gene from representative strains which contain the most proximate consensus of China HIV-1 subtypes B, C and E. The genes were then cloned into pGEM-Teasy vector for HMA subtyping., Result: The env gene of representative strains of China HIV-1 subtypes B, C and E were amplified and cloned into plasmids. The plasmids were used in HMA and compared with those standard env gene of other countries. The heteroduplex bands obtained with ours env genes were more proximate to homoduplex bands. The homogeneity of the corresponding China HIV-1 subtypes with standard TH14(B), IND868(C), MA959 (C) and TH22(E) are 90.2%(B), 88.3%(C), 83.7(C)% and 92.3%(E) respectively., Conclusion: The HMA reagents we established in this study have higher sensitivity in HMA analysis for China HIV-1 strains when compared with the international HMA reagents. The HMA reagents also provide new reference reagents for UNAIDS HIV isolation network.

The recombinant envelope protein of human immunodeficiency virus type 1(HIV-1) of Chinese Yunan epidemic strain was expressed in E. coli for confirming the antigenic domains of the protein. The recombinant prokaryotic expressive plasmid pYNenv was constructed by inserting the env DNA fragment which encode the gp120 of HIV-1 Yunnan strain into pBV220. The env DNA fragment was obtained by nested polymerase chain reaction with the HIV-1 infected patients peripheral blood macrophage and monocyte genomes from Yunan HIV-1 epidemic area as template. The recombinant plasmid pYNenv was confirmed by restriction enzyme analysis. The recombinant protein was expressed in E. coli DH10b by pYNenv after vibration culture at 30 degrees C for 20 hours then at 42 degrees C for 5 hours and confirmed by SDS-PAGE electrophoresis. The protein can specifically react with the serum of HIV-1 infected patient from Yunan epidemic area by Western blot assay. The result showed that the recombinant protein can be used as antigen for detection of HIV-1 membrane glycoprotein antibody as well as for the further study of the role of envelope protein in pathology.

DNA fragments of HIV-1 env gene were amplified by nested-PCR from 17 uncultured peripheral blood mononuclear cells (PBMCs) obtained from HIV-1 seropositive intravenous drug users (IDUs) in Ruili city of Yunnan Province. The C2-V3 region (about 450 bp) of them were sequenced. Sequence analysis showed that there exists two HIV-1 subtypes, B and C, with 5.8% and 2.2% gene divergence inside each subtype. The 12 subtype B strains, were closely related to those found in Thailand, Myanma and Ruili city of Yunnan, and the nucleotide sequence divergence between them ranged from 4.4% to 4.9%; meanwhile, the 5 subtype C strains were most close to those found in India as well as Ruili city, all with a genetic distance of 1.9%. The small divergence among Ruili HIV-1 subtype C strains suggests a recent epidemic. The analysis of V3 loop amino sequence of 12 subtype B HIV-1 reveals that V3-tip motif of 6 samples (50%) is GPGQ and that of 3 samples (25%) is GPGR. In addition, the codon of arginine (R) of all the strains is CGA instead of AGA. This result is in accordance with our previous hypothesis that there is a drift in vivo from GPGR to GPGQ motif on the tipof V3-loop of HIV-1 subtype B strain in this arm with the elapse of time.