ABSTRACT
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.
Subject(s)
Adult , Humans , Male , Middle Aged , Young Adult , China , Epidemiology , Epidemics , Genetic Variation , HIV Infections , Diagnosis , Epidemiology , Virology , HIV-1 , Classification , Genetics , Homosexuality, Male , Molecular Epidemiology , Molecular Sequence Data , PhylogenyABSTRACT
<p><b>OBJECTIVE</b>To establish a simple and practical method for screening of Env-specific monoclonal antibodies from HIV-1 infected individuals.</p><p><b>METHODS</b>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.</p><p><b>RESULTS</b>Three monocolonal Env-specific antibodies were isolated from one HIV-1 infected individual.</p><p><b>CONCLUSION</b>We can obtain Env-specific antibody by biotin labbled antigen, magnetic beads separating technique coupled with single cell RT-PCR and expression cloning.</p>
Subject(s)
Amino Acid Sequence , Antibodies, Monoclonal , B-Lymphocytes , Allergy and Immunology , HIV Antibodies , HIV Envelope Protein gp120 , Allergy and Immunology , HIV-1 , Allergy and Immunology , Immunomagnetic Separation , Methods , Molecular Sequence Data , Reverse Transcriptase Polymerase Chain Reaction , MethodsABSTRACT
<p><b>OBJECTIVE</b>To analyze the molecular-epidemiological characteristics of HIV-1 strains prevailing among female people living with HIV in Beijing.</p><p><b>METHODS</b>Gag gene fragments from the 100 newly diagnosed female HIV-1 infections during 2006 to 2010 in Beijing were amplified, sequenced, and phylogenetically analyzed.</p><p><b>RESULTS</b>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.</p><p><b>CONCLUSION</b>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.</p>
Subject(s)
Adolescent , Adult , Aged , Child , Child, Preschool , Female , Humans , Middle Aged , Young Adult , Acquired Immunodeficiency Syndrome , Epidemiology , China , Epidemiology , HIV-1 , Genetics , Molecular Epidemiology , PhylogenyABSTRACT
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.
ABSTRACT
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.
Subject(s)
Adolescent , Adult , Humans , Male , Middle Aged , Young Adult , China , Epidemiology , Genotype , HIV Infections , Epidemiology , Virology , HIV-1 , Classification , Genetics , Molecular Epidemiology , Molecular Sequence Data , Phylogeny , Sequence Analysis, DNA , gag Gene Products, Human Immunodeficiency Virus , GeneticsABSTRACT
<p><b>OBJECTIVE</b>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.</p><p><b>METHODS</b>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.</p><p><b>RESULTS</b>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.</p><p><b>CONCLUSION</b>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.</p>
Subject(s)
Humans , AIDS Serodiagnosis , HIV Antibodies , Blood , HIV Infections , Blood , Diagnosis , Viral LoadABSTRACT
<p><b>OBJECTIVE</b>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.</p><p><b>METHODS</b>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.</p><p><b>RESULTS</b>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.</p><p><b>CONCLUSION</b>Five HIV-1 subtypes were identified in Beijing and the surveillance of HIV-1 gene variation should be paid more attention to.</p>
Subject(s)
Humans , China , HIV-1 , Classification , Genetics , Polymerase Chain Reaction , Reverse Transcriptase Polymerase Chain Reaction , env Gene Products, Human Immunodeficiency Virus , Genetics , gag Gene Products, Human Immunodeficiency Virus , GeneticsABSTRACT
<p><b>OBJECTIVE</b>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).</p><p><b>METHODS</b>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.</p><p><b>RESULTS</b>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%.</p><p><b>CONCLUSION</b>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.</p>