[Molecular epidemiological characteristics of HIV-1 strains isolated from newly diagnosed MSM subjects (2006-2010) in Beijing, China].

This study aims to analyze the molecular epidemiological characteristics of HIV-1 strains prevailing among men who have sex with men (MSM) in Beijing, China. The pol gene fragments from 250 newly diagnosed HIV-1-infected MSM individuals during 2006-2010 in Beijing were amplified by RT-nested PCR, sequenced, and phylogenetically analyzed. HIV-1 pol gene from 189 individuals were amplified and analyzed; 81 (42. 9%), 3 (1. 6%), 2 (1.0%), 88 (46. 6%), and 15 (7.9%) individuals were infected with HIV-1 subtypes B, B', C, CRF01_AE, and CRF07_BC, respectively. The subtypes B and CRF01_AE could both be grouped into two clusters, and CRFO7_BC strains shared high homology and were presumed to originate from a common ancestor. The HIV-1 circulating in MSM in Beijing had a lower genetic diversity than in heterosexuals. The HIV-1 epidemic (2006-2010) in MSM in Beijing was actually a rapid spread of HIV-1 CRF01 AE and B, or rather native strains of the two viruses.

[Molecular epidemiological characteristics of HIV-1 B'/C strains in Beijing].

OBJECTIVE: To analyze the molecular epidemiological characteristic of HIV-1 B'/C strains prevalent in Beijing. METHODS: Plasma samples were collected from 200 newly diagnosed HIV-1 B'/C individuals reported during 2006 to 2010 in Beijing. The gag gene fragments were amplified from RNA template extracted from plasma with reverse transcription (RT) and nested polymerase chain reaction (PCR). And the sequences were analyzed by phylogenetic methods and Entropy analysis. RESULTS: A total of 159 sequences were successfully amplified from the gag genes of which 147 was CRF07_BC and 12 CRF08_BC. There were 3 main sub-clusters in CRF07_BC phylogenetic tree and they were named as sub-cluster IDU-Max (89 sequences), sub-cluster IDU-Min (22 sequences) and sub-cluster MSM (34 sequences) based on transmission.No international reference strain was closely related with these three sub-clusters except for one strain identified in Taiwan. All CRF07_BC recombinant strains were remarkable for their low interpatient diversity in gag genes (3.7%, 3.3% and 2.0% for isolates from IDU-Max, IDU-Min and MSM respectively).When compared with sub-cluster IDU-Max, there were 32 and 41 significantly different sites of nucleotide polymorphism compositions in sub-clusters IDU-Min and MSM. CONCLUSION: This is the first report of describing the existence of three main epidemic sub-clusters in CRF07_BC strains prevalent in Beijing. And IDU-Max sub-cluster is the dominant strain. The CRF07_BC in Beijing are less diverse than other strains and may be derived from a common ancestor.

[Identification of Env-specific monoclonal antibodies from Chinese HIV-1 infected person by magnetic beads separating B cells and single cell RT-PCR cloning].

OBJECTIVE: To establish a simple and practical method for screening of Env-specific monoclonal antibodies from HIV-1 infected individuals. METHODS: Human B cells were purified by negative sorting from PBMCs and memory B cells were further enriched using anti-CD27 microbeads. Gp120 antigen labbled with biotin was incubated with memory B cells to specifically bind IgG on cells membrane. The memory B cells expressing the Env-specific antibody were harvested by magnetic beads separating, counted and diluted to the level of single cell in each PCR well that loading with catch buffer containing RNase inhibitor to get RNAs. The antibody genes were amplified by single cell RT-PCR and nested PCR, cloned into eukaryotic expression vectors and transfected into 293T cells. The binding activity of recombinant antibodies to Env were tested by ELISA. RESULTS: Three monocolonal Env-specific antibodies were isolated from one HIV-1 infected individual. CONCLUSION: We can obtain Env-specific antibody by biotin labbled antigen, magnetic beads separating technique coupled with single cell RT-PCR and expression cloning.

[Molecular-epidemiological characteristics of HIV-1 isolated from newly diagnosed female subjects in Beijing, 2006 - 2010].

OBJECTIVE: To analyze the molecular-epidemiological characteristics of HIV-1 strains prevailing among female people living with HIV in Beijing. METHODS: Gag gene fragments from the 100 newly diagnosed female HIV-1 infections during 2006 to 2010 in Beijing were amplified, sequenced, and phylogenetically analyzed. RESULTS: Eighty-two HIV-1 gag gene fragments were amplified and analyzed. 1 (1.22%), 1 (1.22%), 3 (3.66%), 23 (28.05%), 8 (9.76%), 2 (2.44%), 1 (1.22%), 18 (21.95%), 3 (3.66%), 1 (1.22%), 14 (17.07%), 4 (4.88%) and 3 (3.66%) individuals were infected with HIV-1 subtypes A1, A2, B, B', C, D, G, H, CRF01_AE, CRF02_AG, CRF07_BC, CRF08_BC and B'/C recombinants respectively. CONCLUSION: The subtypes circulating in female HIV infections in Beijing were more diverse than in male and the proportions of B' and rare subtypes were relatively high. Surveillance programs on HIV-1 genetic diversity should be strengthened.

Genetic diversity of HIV type 1 isolated from newly diagnosed subjects (2006-2007) in Beijing, China.

The purpose of this study was to investigate the genetic diversity of HIV-1 circulating in Beijing and its molecular epidemiological linkages with regard to risk factors of viral transmission. HIV-1 from plasma samples of 280 diagnosed individuals (2006-2007) was characterized. The gene fragments of gag, pol, and env from the infected plasma samples were amplified by reverse transcriptase polymerase chain reaction (RT-PCR), sequenced, and phylogenetically analyzed. From the 280 plasma samples analyzed, a total of 496 sequences were successfully amplified from the gag, pol, and env genes. Nine HIV-1 group M subtypes or CRF including A1, B, B', C, CRF01_AE, CRF02_AG, CRF06_cpx, CRF07_BC, and CRF08_BC, and six new B'/C recombinants were identified. CRF07_BC was found to be the most dominant subtype (32.5%) followed by CRF01_AE (25.0%), B (20.0%), and B' (15.7%). The data from this study indicate the existence of multiple HIV-1 subtypes or CRFs in Beijing and may be proven useful in the development of vaccine candidates in the future.

The prevalence of drug resistance mutations among treatment-naive HIV-infected individuals in Beijing, China.

To investigate the prevalence of HIV-1 genotypic mutations for drug resistance among patients in Beijing, blood samples from 145 newly confirmed (2006-2007), treatment-naive HIV-1-infected individuals were analyzed. Seven subtypes or CRF were subsequently determined and scored by the Stanford HIV Drug Resistance algorithm: CRF01_AE HIV-1 (27.6%), subtype B' (24.1%), CRF07_BC (21.4%), subtype B (20.7%), CRF08_BC (3.4%), subtype C (2.1%), and CRF06_cpx (0.7%). Eleven of the 145 subjects studied were found to harbor the strains resistant to either protease inhibitors (PIs) (3.4%), or nucleoside reverse transcriptase inhibitors (NRTIs) (2.1%), or nonnucleoside reverse transcriptase inhibitors (NNRTIs) (3.4%). Although the prevalence of drug resistance was relatively low among the treatment-naive HIV-1 patients in Beijing in comparison to those in industrialized countries, we will continue monitoring newly infected subjects for any potential alteration of the prevalence pattern to ensure the success of the ongoing scale-up of antiretroviral treatment.

[Genotypic resistance mutations to antiretroviral drugs in newly confirmed human immunodeficiency virus infectors in Beijing].

OBJECTIVE: To examine the prevalence of drug resistance mutations among the treatment-naive HIV (human immunodeficiency virus) infectors living in Beijing so as to provide the basal information for clinical antiviral treatment. METHODS: HIV pol genes from plasma samples of 150 treatment-naive HIV-infected patients were amplified, sequenced and phylogenetically analyzed. And the drug-resistance associated mutations in protease and reverse transcriptase regions were analyzed with Stanford University HIV Drug Resistance Database. RESULTS: A total of 111 pol gene sequences were obtained. The overall prevalence of drug resistance was 8.1% (9/111), corresponding to 3.6% (4/111) for protease inhibitors, 1.8% (2/111) for nucleoside reverse transcriptase inhibitors and 3.6% (4/111) for non-nucleoside reverse transcriptase inhibitors. No drug resistance mutation was identified in 17 intravenous drug users. CONCLUSION: The prevalence of drug resistance is relatively high in the newly confirmed HIV infectors in Beijing. Regular surveillance and monitoring of drug-resistant HIV should be implemented.

Sequence analysis of the gag-pol gene of human immunodeficiency virus type 1 of intersubtype (B'/C) recombinant strain in Beijing, China.

Little is known about the molecular and biological properties of HIV-1 intersubtype B'/C in Beijing. To fill the gap, we sequenced and analyzed the gag-pol genes from 39 HIV-1 B'/C recombinant infectors in Beijing, China during 2007. The results show that 36 CRF07_BC and 2 CRF08_BC isolates have a structural profile identical or nearly identical to CRF07_BC or CRF08_BC according to sequences in the gag-pol regions. The CRF07_BC circulating in injecting drug users (IDUs) and heterosexuals forms a diverse phylogenetic tree and most isolates from homosexuals cluster together. However, all the B'/C recombinant strains were remarkable for their low interpatient diversity in gag-pol genes (3.1, 3.0, and 2.2% for isolates from IDUs, heterosexuals, and homosexuals, respectively). We identified I7V, E91G, N242T, and K361R in the gag gene and R290I (HXB2 positions) in the pol gene as signature amino acid substitutions characteristic of HIV-1 CRF07_BC from the Beijing lineage. In addition, one new B'/C recombinant was detected. These results may contribute to an understanding of HIV-1 in Beijing.

[Genetic characteristics of HIV-1 CRF01_AE strains circulating in Beijing].

OBJECTIVE: To analyze the genetic characteristics of HIV-1 CRF01_AE strains prevailing in Beijing. METHODS: Plasma samples were collected from the newly diagnosed HIV-1 individuals being reported during 2006 to 2008 in Beijing. Gag gene fragments were amplified from RNA template which were extracted from plasma by RT and nested PCR methods. 105 CRF01_AE sequences were analyzed by phylogcnetic methods and characterized through calculating the genetic distance and Entropy analysis. RESULTS: There were four main sub-clusters in the phylogenetic tree. We named them as sub-clusters Homo-Max (67 sequences), Hetero (6 sequences), Mix (8 sequences) and Homo-Min (18 sequences) respectively, based on the mode of transmission. It was found that no international reference strain was closely related to the sub-cluster Homo-Max, Hetero or Homo-Min, including 91 samples. The strains in sub-cluster Mix consisting 8 cases that were closely related to the strains identified in Thailand and Vietnam. Genetic distance analysis on gag genes showed that the diversity of sub-clusters Homo-Max and Homo-Min was obviously less than that of the sub-cluster Hetero or Mix. When compared with sub-cluster Mix, there were 37, 29 and 11 significantly different nucleotides polymorphism compositions sites in sub-clusers Homo-Max Homo-Min and Hetero. CONCLUSION: This was the first report describing that four main epidemic sub-clusters were existed in CRF01_AE strains prevailing in Beijing. The virus with sub-cluster Homo-Max was the dominant strain in this region with shorter period of circulation and higher proportion seen in the HIV-infected persons. The virus in sub-cluster Mix was highly homologic with the CRF01_AE strains from Thailand and Vietnam.

[Subtype and sequence analysis of gag genes among HIV-1 strains circulating in Beijing during 2007].

To investigate the subtype distribution and sequence characteristics of HIV-1 strains prevalent in Beijing during 2007 and to analyze the relationship between distribution of HIV-1 subtypes and transmission routes, we collected the anti-conglutinated whole blood samples from HIV-1 newly infected individuals in Beijing during 2007 and separated plasma specimens from the aamples. RNAs were extracted and the gag genes from the various samples were amplified by RT-PCR and nest-PCR. The PCR products were sequenced directly and phylogenetic analyses of gag genes were performed using the MEGA2 software. Among 200 HIV-1 plasma samples,161 gag HIV-1 gene fragments were amplified and analyzed. Seven HIV-1 subtypes or circulating recombinant forms (CRFs) of HIV-1 including A1 (1 strains), B (35 strains), Thai B (19 strains), C (3 strains), CRF01_AE (49 strains), CRF07_BC (51 strains), CRF08_BC (3 strain) were identified circulating in Beijing. The gene divergences inside the subtypes were different, with 7.7%, 6.5%, 6.7%, 6.7%, 5.5%, 4.3%, 5.8%, in subtype A1, B, Thai B, C, CRF01_AE, CRF07_BC, CRF08_BC, respectively. Subtypes CRF07_BC and CRF01_AE were predominant in Beijing account for 31.7% and 30.4% among samples. Seven HIV-1 subtypes exist in Beijing and the surveillance of HIV-1 gene variation should be paid more attention.

[Viral load test conducive to excluding negative subjects from suspects in HIV antibody detections].

OBJECTIVE: To study whether plasma viral load testing is helpful to exclude ones free from Human immunodeficiency virus (HIV) infections from suspects in HIV antibody detections. METHODS: 19 Specimens, which showed disconcordant results of the two HIV EIA testing (S/CO < 6) and indeterminated results of Western blot (WB) test, were selected. Viral load of the specimens were detected. A six-month follow up survey in detecting HIV antibody was conducted in these subjects. RESULTS: None of these 19 cases was observed to be positive HIV viral loads and there was no any progress in WB bands development during the follow-up period. The possibility of HIV infection could be excluded. CONCLUSION: When the specimens react with very low intensity in both EIA and WB, negative viral load result is conducive to exclude negative subjects from suspects in HIV antibody detections.

[Subtype and sequence analysis of gag and env genes among HIV-1 strains circulating in Beijing residents during 2006].

OBJECTIVE: To investigate the subtype distribution and the prevalence of sequence characteristics of HIV-1 strains in Beijing residents during 2006 and to analyze the relationship between distribution of HIV-1 subtypes and transmission routines. METHODS: Blood samples from 32 new confirmed HIV-1 infected individuals from Beijing residents in 2006 and separated plasma specimens were collected. RNAs were extracted and the gag and env gene were amplified by RT-PCR and nest-PCR. PCR products were sequenced directly and phylogenetic analyses of gag and env gene were performed using the MEGA2 software. RESULTS: Among 32 HIV-1 plasma samples, 22 gag and 4 env gene fragments were amplified and analyzed. Five HIV-1 subtypes or circulating recombinant forms(CRFs) of HIV-1 including Thai B (2 strains), B (9 strains), C (2 strains), CRF07_BC (5 strains), CRF01 AE (4 strains) were identified being circulated in Beijing. The gene divergences of gag gene inside the subtypes were 6.6%, 4.3%, 6.8%, 4.9% and 3.0% in subtype B, Thai B, C, CRF01_AE and CRF07_BC respectively. Subtypes B were predominant in Beijing, accounted for 40.9% among 22 samples. CONCLUSION: Five HIV-1 subtypes were identified in Beijing and the surveillance of HIV-1 gene variation should be paid more attention to.

Field evaluation of alternative testing strategies for the detection of HIV infection in Beijing.

OBJECTIVE: To identify a cost-efficient alternative antibody testing strategy for screening and confirmation of HIV infection by rapid simple tests (RSTs) and enzyme-linked immunosorbent assays (ELISAs). METHODS: Four RSTs (RST1, RST2, RST3, and RST4) and five ELISAs (ELISA1, ELISA2, ELISA3, ELISA4, and ELISA5) were evaluated in two phases by using banked and serum specimens prospectively collected at regional hospitals and voluntary counseling and testing (VCT) centers in Beijing. A total of 200 banked serum specimens were included in the first phase, including 62 HIV-positive, 127 HIV-negative and 11 indeterminate specimens. All specimens were tested by four RSTs and five ELISAs respectively. The second phase involved prospective testing of 389 routine specimens, including 92 HIV-positive, 287 HIV-negative, and 10 indeterminate specimens. All the specimens were tested by two RSTs (RST2 and RST4) and three ELISAs (ELISA1, ELISA3, and ELISA4), which were selected for their respective excellent sensitivity and/or specificity. Western blot (WB) was used as a gold standard for confirming the reactivity of all the specimens. RESULTS: Sensitivity, specificity, and efficacy were calculated for each assay in two phases. In the first phase, four assays (ELISA4, RST2, RST3, and RST4) had a specificity of 100%. For the determination of efficacy, ELISA4, RST2, and RST4 were selected in the second phase. ELISA1 and ELISA3 which have a sensitivity of 95.9% and 93.2% respectively also entered this phase. In the second phase, all the five assays (ELISA1, ELISA3, ELISA4, RST2, and RST4) had a sensitivity and specifity of over 90%. ELISA1 had a sensitivity of 99% and ELISA4 a specificity of 99%. CONCLUSION: The sensitivity ELISA1 and the specificit of ELISA4 are comparable to ELISA/WB standard strategy. Application of this alternative testing strategy provides a cost-effective method for determining HIV prevalence in Beijing.