Variability of human immunodeficiency virus type 2 (hiv-2) infecting patients living in france

To determine the prevalence of human immunodeficiency virus type 2 (HIV-2) subtypes circulating in France and to identify possible relationships between these subtypes and pathogenesis, we studied 33 HIV-2-infected patients living in France. HIV-2 DNA was directly amplified from peripheral blood mononuclear cells by nested PCR with specific HIV-2 env primers, and the env gene was sequenced. The serological consequences of antigenic variability were studied by using a panel of peptides and by Western blotting. Phylogenetic analysis classified the 33 HIV-2 strains as subtype A (n = 23) or B (n = 10). There were no significant clinical or epidemiological differences between patients infected with either of these two subtypes. There was some evidence for geographical clustering. Subtype A strains from patients originating from the Cape Verde Islands and Guinea Bissau clustered together. The majority of patients infected with subtype B strains originated from the Ivory Coast or Mali. Strains from patients originating in Mali also clustered in subtype A but distinctly from the Cape Verde or Guinea Bissau strains. The subtype B strains showed greater diversity and included some highly divergent strains relative to those previously characterized. The V3 loop of HIV-2 subtypes A and B was found to be quite conserved in comparison with HIV-1. A strong HIV-2 subtype B serological cross-reactivity was found on HIV-1 env antigen by Western blot mostly in the gp41 transmembrane glycoprotein. This could partly explain the double HIV-1 and HIV-2 reactive profiles found in countries where HIV-2 subtype B is prevalent.     

Detection of Phylogenetically Diverse Human Immunodeficiency Virus Type 1 Groups M and O from Plasma by Using Highly Sensitive and Specific Generic Primers

The high degree of genetic diversity within human immunodeficiency virus type 1 (HIV-1), which includes two major groups, M (major) and O (outlier), and various env subtypes within group M (subtypes A to J), has made designing assays that will detect all known HIV-1 strains difficult. We have developed a generic primer set based on the conserved immunodominant region of transmembrane protein gp41 that can reliably amplify as few as 10 copies/PCR of viral DNA from near-full-length clones representing group M subtypes A to H (subtypes I and J were not available). The assay is highly sensitive in detecting plasma viral RNA from HIV-1 strains of diverse geographic origins representing different subtypes of HIV-1 group M as well as HIV-1 group O. Of the 253 group M plasma specimens (subtypes A, 68 specimens; B, 71; C, 19; D, 27; E, 23; F, 33; and G, 12), 250 (98.8%) were amplified by using the gp41 M/O primer set. More importantly, all 32 (100%) group O plasma samples were also amplified with these primers. In vitro spiking experiments further revealed that the assay could reliably detect as few as 25 copies/ml of viral RNA and gave positive signals in HIV-1-seropositive specimens with plasma copy numbers below the limits of detection by all commercially available viral load assays. In addition, analysis of five seroconversion panels indicated that the assay is highly sensitive for early detection of plasma viremia during the "window period." Thus, the highly sensitive assay will be useful for early detection of HIV-1 in clinical specimens from all known HIV-1 infections, regardless of their genotypes and geographic origins.     

A gag gene heteroduplex mobility assay for subtyping HIV-1

A heteroduplex mobility assay (HMA) using 753 and 446 base pair (bp) amplicons of the p17/p24 region of the gag gene of HIV-1 has been developed and validated with reference clones and clinical samples representative of subtypes A, B, C, D, E, G, and H. There was complete concordance between the gag HMA assigned subtype and the subtype known from gag or env sequence data or env HMA. The heteroduplexes from both amplicons can be clearly resolved on either MetaPhor XR agarose or MDE polyacrylamide gels. The MetaPhor XR gel system was the more convenient and is the preferred choice for routine HMA subtyping. This gag HMA provides a rapid, simple and inexpensive method for subtyping HIV-1 based on a genomic region other than the commonly used env gene target. The incorporation of gag HMA into subtype determination algorithms should allow the detection of gag/env recombinant strains of HIV-1.     

克隆中国B,C,E亚型的HIV—1代表株建立适于我国流行株的 …

目的 构建我国人免疫缺陷病毒１型（ＨＩＶ－１）Ｂ、Ｃ和Ｅ各亚型代表株ｅｎｖ基因质粒,用于对中国ＨＩＶ－１进行分型。方法 将与我国ＨＩＶ－１ Ｂ,Ｃ和Ｅ各亚型共享序列最接近的毒株用套式聚合酶链反应（ＰＣＲ）技术扩增包膜（ｅｎｖ）基因,克隆到ｐＧＥＭ－Ｔｅａｓｙ载体中,构建成用于异源双链泳动分析（ＨＭＡ）分型的中国标准亚型质粒,并进行了序列分析和分型的敏感性分析。结果 构建的中国ＨＩＶ－１ Ｂ,Ｃ和Ｅ     

Differentiation of subtypes B and E of human immunodeficiency virus type 1 by polymerase chain reaction using novel env gene primers

Novel sets of env gene PCR primers for distinguishing human immunodeficiency virus type 1 (HIV-1) subtypes B and E were designed. These primers anneal to different regions of the env gene and amplify DNA fragments of distinct sizes in a subtype-specific manner. Blood samples from 11 HIV-1 carriers in Thailand and 46 carriers in Japan were examined by PCR. The new env primers detected HIV-1 proviral DNA in 100% (11/11) and 88% (37/42) of the subtype B and E infection cases, respectively. The env primers also detected proviral DNA in saliva and breast milk samples in seven of 11 cases and two of three cases, respectively. The PCR subtyping results matched completely with those obtained by nucleotide sequencing of the env V3 region. The results suggest that the PCR using the env primers designed in this study may be an accurate and cost-effective method for differentiating subtypes B and E of HIV-1 in a large number of clinical samples. However, subtype E specific primer cross-react with subtype A, C, G, the new primer in this study is useful for regions in South East Asia where subtype E is predominant.     

Genetic characterization of HIV type 1 and type 2 from Bissau, Guinea-Bissau (West Africa)

Previous studies from Guinea-Bissau (West Africa) have demonstrated a unique epidemiology with respect to both HIV-1 and HIV-2 infection. In order to evaluate the prevalence and dynamics of HIV-1 and HIV-2 subtypes in Bissau, the capital city of Guinea-Bissau, a cross-sectional study was set up using serological and molecular techniques. Plasma samples from 103 individuals were screened for HIV-1 and HIV-2 antibodies by ELISA and Western-blot. Seropositive results were confirmed by PCR amplification of proviral sequences in primary peripheral blood mononuclear cells (PBMC) with env and LTR primer sets for HIV-2 and env, LTR and pol primers for HIV-1. A total of 38/103 individuals were HIV-seroreactive (four HIV-1, 15 HIV-2, 19 HIV-1/HIV-2). A total of eight out of 19 dually seropositive specimens showed double PCR amplification of HIV-1 and HIV-2 proviral sequences, accounting for 21% of the infected individuals. In the remaining 11 individuals either HIV-2 or HIV-1 sequences were detected, the majority (n=9) amplifying only HIV-2. These screening data demonstrate a high discrepancy between serology and PCR results for dually seroreactive samples, Western-blot giving an overestimation of double infection. Additionally, HIV-1 strains were subtyped by heteroduplex mobility assay (HMA) on the basis of gp120 sequences. Subtyping of HIV-2 was carried out by DNA sequencing and phylogenetic analysis of env V3 molecular clones. For both HIV-1 and HIV-2 strains circulating in Bissau, our results indicate dominance of subtype A.     

Serotyping and genotyping of HIV-1 infection in residents of Khayelitsha, Cape Town, South Africa

It is estimated that between 5.5 and 6.1 million people are infected with HIV/acquired immunodeficiency syndrome (AIDS) in South Africa, with subtype C responsible for the majority of these infections. The Khayelitsha suburb of Cape Town has one of the highest HIV prevalence rates in South Africa. Overcrowding combined with unemployment and crime in parts of the area perpetuates high-risk sexual behavior, which increases exposure to infection by HIV. Against this background, the objective of this study was to characterize HIV-1 in residents confirmed to be seropositive. Serotyping was performed through a competitive enzyme-linked immunosorbent assay (cPEIA). Genotyping methods included RNA isolation followed by RT-PCR and sequencing of the gag p24, env gp41 immunodominant region (IDR), and env gp120 V3 genome regions of HIV-1. With the exception of a possible C/D recombinant strain, all HIV-1 strains were characterized as HIV-1 group M subtype C. One individual was shown to harbor multiple strains of HIV-1 subtype C. In Southern Africa, the focus has been to develop a subtype C candidate vaccine, as this is the major subtype found in this geographical area. Therefore, the spread of HIV-1 and its recombinant strains needs to be monitored closely.     

HIV-1 diversity in Brazil: genetic, biologic, and immunologic characterization of HIV-1 strains in three potential HIV vaccine evaluation sites. Brazilian Network for HIV Isolation and Characterization

The Brazilian Network for HIV Isolation and Characterization was established for the surveillance of HIV variability in Brazil. Here, we report characterization of HIV strains and virus-specific immune responses from 35 clinical samples collected from three potential HIV vaccine sites. Three genetic subtypes of HIV-1 were identified by heteroduplex mobility assay (HMA) B (in 82.9% of the samples), F (14.3%), and C (2.9%). Phylogenetic analysis based on the C2V3/env DNA sequence from all 25 specimens examined was 100% concordant with HMA results. Four variants of subtype B with different tetrapeptides at the tip of the V3 loop were found: the GPGR motif (North American), GWGR motif (Brazilian B"), and two minor variants, GFGR and GPGS, as previously detected. No significant association was found between HIV-1 subtypes and the mode of transmission or biologic properties of HIV-1 isolates (derived from 88.6% of the specimens). Only 5 of 16 isolates studied were neutralized by the autologous sera. Consistent with previous results, no relation between viral subtype and peptide enzyme-linked immunosorbent assay (ELISA) seroreactivity or neutralization was evident. This study also demonstrated the effectiveness of the collaborative approach followed by Brazilian scientists when addressing a complex subject such as HIV variability.     

Two HIV-1 epidemics in Argentina: different genetic subtypes associated with different risk groups

This study determined the risk behaviors and viral subtypes of HIV-1 found in 134 heterosexual HIV-seroprevalent maternity patients, 41 of their sexual partners (men who have sex with women [MSW]), and 95 homosexual men (men who have sex with men [MSM]) from Buenos Aires, Argentina. Peripheral blood mononuclear cells (PBMCs) were purified from blood and used for DNA extraction, amplification, and genotyping by the envelope heteroduplex mobility assay (env HMA).Most of the women had been infected by having sex with an already infected partner (84%), whereas most of the male partners had been infected via drug use (76%). Both the patients and their sexual partners were poorly educated, only 30% having completed secondary school. The MSM study subjects, however, were significantly better educated and had a lower prevalence of injecting drug use.Env HMA subtype F was found in 77% (103 of 134) of the maternity patients, with similar rates in their partners (73%). Most of the remaining samples were env subtype B. All but one of the couples was concordant in subtype. In the MSM risk group, however, only 10% were env HMA subtype F. Ninety percent of the MSM samples were subtype B.There are at least two independent epidemics of HIV-1 infection in Buenos Aires, Argentina. One, in heterosexual men and women, is dominated by env subtype F whereas the other, in homosexual men, is dominated by env subtype B, as determined by env HMA.     

Screening of HIV-1 isolates by reverse heteroduplex mobility assay and identification of non-B subtypes in Italy

OBJECTIVE: The increasing prevalence of HIV-1 transmission through heterosexual contacts and the growing number of immigrants from non-Western countries, where non-B subtypes and recombinant forms are prevalent, suggest the possible emergence in Italy of a new epidemic wave of HIV-1 non-B subtypes as well as recombinant forms. METHODS: The distribution of HIV-1 subtypes has been evaluated in 63 seropositive individuals residing in Italy, most of whom were infected through a sexual route during the last 5 years. A modified heteroduplex mobility assay (HMA) strategy, reverse HMA (rHMA), has been developed in our laboratory, allowing rapid identification of divergent-from-B-subtype isolates, which have been subsequently characterized by detailed molecular and phylogenetic analyses. RESULTS: Five samples show, on rHMA, an electrophoretic pattern compatible with a non-B subtype classification. Their phylogenetic analysis, performed on both env and gag regions, confirms the rHMA subtyping prediction, given that 3 samples fall into the "A-family" subtype and 2 into the G subtype. The 5 non-B-subtype HIV-1 isolates have been identified among 23 variants (prevalence, 21.74%) isolated during the 2000 to 2001 period in heterosexuals. In parallel, B-subtype isolates show high levels of intrasubtype nucleotide divergence, compatible with a constant HIV-1 molecular diversification. CONCLUSION: The Italian HIV-1 epidemic is still mostly attributable to the B subtype, which shows an increasing nucleotide heterogeneity. Heterosexual transmission and the interracial blending, however, are slowly introducing novel HIV-1 subtypes, and the data indicate that rHMA represents a powerful tool for HIV-1 biomolecular screening in epidemics characterized by a mono-/dual-subtype predominance.     

Study of HIV-1 subtypes in serodiscordant couples attending an integrated counselling and testing centre in Mumbai using heteroduplex mobility analysis and DNA sequencing

Aims: To determine the prevalent subtypes of HIV-1 in serodiscordant couples. Setting: Integrated Counselling and Testing Centre (ICTC), Department of Microbiology. Study Design: Prospective pilot study. Participants: Thirty HIV-1 serodiscordant couples. Inclusion Criteria: a) Documentation of HIV-1 infection in one partner and seronegative status in the other, current history of continued unprotected sexual activity within the partnership, demonstration that they have been in a partnership for at least 1 year and are not currently on highly active antiretroviral therapy HAART; b) willingness of both partners to provide written informed consent including consent to continued couple counselling for 3 months. Materials and Methods: HIV-1 subtyping was carried out by heteroduplex mobility analysis (HMA) by amplifying env region; and DNA sequencing by amplifying gag region. Results: HIV-1 env gene was amplified successfully in 10/30 samples; gag gene, in 25/30 samples; and both env and gag gene were amplified successfully in 5/30 samples. HIV-1 subtype C was detected from 21 samples; subtype B, from 7; and subtype A, from 2. Sample from 1 positive partner was detected as subtype C by env HMA and subtype B by gag sequencing. Conclusion: HIV-1 subtype C was found to be the predominant subtype of HIV-1 in serodiscordant couples attending our ICTC, followed by HIV-1 subtype B and HIV-1 subtype A, respectively. DNA sequencing was found to be the most reliable method for determining the subtypes of HIV-1.     

Discrimination of subtype B and non-subtype B strains of human immunodeficiency virus type 1 by serotyping: correlation with genotyping

The ability of a peptide-based serotyping assay to differentiate human immunodeficiency virus (HIV) type 1 (HIV-1) subtype B infections from non-subtype B infections was investigated with 166 anti-HIV-1- and HIV RNA-positive (by PCR) serum or plasma specimens. The specimens were divided genetically into those infected with subtype B and non-subtype B by application of a screening heteroduplex mobility assay (HMA) that used plasmids for subtypes A and B alone. Specimens that were not clearly infected with HIV-1 subtype B by HMA or for which the two methods had discordant results in distinguishing those infected with subtype B from those infected with non-subtype B were then investigated with a full HMA plasmid panel and, for selected specimens, env sequencing. For the 141 genotyped and serotypically reactive specimens, the correlation between genotyping and serotyping (all subtypes) was 69%. Of the 67 specimens that reacted monotypically as serotype B, 64 were shown to be infected with genotype B (positive predictive value, 96%). Of the 82 specimens that contained genotype B nucleic acid, 64 reacted monotypically as serotype B (sensitivity, 78%), and 4 specimens reacted with a single non-subtype B peptide; the viruses in 14 specimens could not be assigned a serotype. Initial screening results had indicated that 12 samples had results discordant between restricted HMA and serotyping. The V3 loop amino acids of the infecting HIV strains from the seven specimens with discordant serology results were analyzed. For five specimens discordance occurred when the amino acid sequence of the infecting virus closely resembled those of more than one consensus peptide antigen or when the observed V3 crown motif of the strain was atypical for the genetic subtype present. For the other two specimens no explanation for the discordance was identified. Five specimens gave unclear or discordant results in the initial HMA screen, but the results were resolved when the full plasmid panel was used. Serotyping, although of limited sensitivity, distinguishes between subtype B and non-subtype B infections with a high degree of specificity. However, it poorly differentiates the major non-subtype B subtypes, particularly subtypes A and C. When HIV-1 subtype B predominates, serological typing and/or subtype-restricted HMA screening usefully distinguishes between subtype B and non-subtype B infections.     

Role of maternal humoral immunity in vertical transmission of HIV-1 subtype E in Thailand.

The significance of the maternal humoral immune response in relation to vertical transmission of HIV-1 was investigated in 123 mothers infected with subtype E from Thailand. Antibody binding titers to HIV-1 env domains (monomeric gp120, the CD4/gp120 binding site [BS], V3 loop, and gp41) and antibody-mediated neutralization of primary and T-cell line-adapted (TCLA) subtypes B and E HIV-1 isolates were investigated. No correlation between maternal anti HIV-1 antibodies at delivery and vertical transmission of HIV-1 subtype E was found. However, a trend to higher titer antibody-mediated cross-neutralization of a heterologous subtype B TCLA isolate, HIV-1MN, was observed in nontransmitting mothers postpartum. The HIV-1-specific antibody titers in these infected mothers increased significantly from delivery to 6 months postpartum (p < .05), but this was only partially attributable to hemodilution and an additional factor or factors appear to affect humoral immunity to HIV-1 during late pregnancy.     

High genetic diversity of HIV-1 strains in Chad,West Central Africa

The genetic diversity of HIV-1 strains in Chad was documented with a total of 107 samples from patients attending the general hospital in N'Djamena, the capital city of Chad. The genetic subtypes were identified in the V3-V5 env and p24 gag regions by sequence and phylogenetic tree analyses. Of the 107 strains, 78 had the same subtype/CRF designation between env and gag. Four subtypes and three CRFs were found to cocirculate: subtype A, 20.5%; subtype D, 18.7%; CRF02_AG, 13.1%; CRF11_cpx, 13.1%; subtype G, 3.7%; CRF01_AE, 2.8%; and subtype F1, 0.9%. The remaining 29 strains (27%) had discordant subtypes or CRF designations between env and gag; in 15 of these 29 strains, a CRF was involved in the recombination event, and 10 were subtype G in gag and subtype A in env, forming a separate subcluster within subtypes G and A. Subtype D strains represent almost 20% of the HIV-1 strains circulating in Chad and form a separate subcluster in gag and env. Nearly full-length genome sequencing for two such strains (99TCD-MN011 and 99TCD-MN012) revealed that they represent nonrecombinant subtype D variants. Compared with neighboring countries, the genetic subtype distribution of HIV-1 strains in Chad is unique for several reasons: lower prevalence of CRF02, high prevalence of CRF11 and subtype D, and absence of CRF06. These data clearly show that subtype distribution is very heterogeneous in Africa, probably the result of different founder effects.     

Molecular epidemiology of vertical human immunodeficiency virus type 1 transmission in Greece: evidence of non-B subtypes

OBJECTIVES: To investigate the subtype classification of the circulating virus strains among human immunodeficiency virus type 1 (HIV-1)-infected children in Greece. STUDY DESIGN/METHODS: Since the beginning of the acquired immunodeficiency syndrome (AIDS) epidemic in Greece in 1982, 23 children have been reported to be vertically infected with HIV-1. Blood samples were available for 19 of these children, and the C2-C4 env region was successfully amplified by nested polymerase chain reaction (PCR) for 16 subjects. HIV-1 subtype was established by the heteroduplex mobility assay (HMA) in 16 subjects and confirmed by DNA sequencing and phylogenetic analysis in 8 subjects. RESULTS: Most subjects (9; 56%) fell into subtype B. However, a substantial proportion (44%) were classified as subtypes A (3; 19%), C (1; 6%), D (1; 6%), and I (2; 12%). According to epidemiologic information, 5 of 7 children infected with non-B HIV-1 subtypes were born to Greek parents. CONCLUSION: These findings clearly suggest that non-B strains have been introduced into Greece, providing evidence that HIV epidemic in this country will probably change profile over time. In addition, subtype I was identified in 2 HIV-1-infected children, both of whom were born to Greek parents.     

A genotypic resistance assay for the detection of drug resistance in the human immunodeficiency virus type 1 envelope gene

Since it is not clear yet whether enfuvirtide resistance is restricted to gp41, it was decided to develop a genotypic assay for the detection of drug resistance in the entire human immunodeficiency virus type 1 (HIV-1) env gene. Given the increasing prevalence of HIV-1 non-B subtypes in Europe, it is important to evaluate the performance of the assay on a panel of genetically divergent samples. A panel of 1 laboratory and 10 clinical isolates from 10 patients was tested, all enfuvirtide naive and chosen according to the subtype as determined in the pol region (A, B, C, H, CRF01-AE, CRF02-AG, CRF05-DF, CRF11-cpx and U), while their env sequences belonged to subtypes A, B, C, H, A/G recombinant, B/H recombinant, CRF01-AE, CRF02-AG, CRF05-DF and CRF11-cpx. The detection limits of the gp120 and the gp41 PCRs ranged between 500 and 5000 RNA copies/ml plasma. The highest sensitivity was obtained for the laboratory strain, whereas the detection limit for all patient samples, except for the subtype C sample, was 1000 RNA copies/ml. The numerous insertions and deletions in the gp120 gene, that were often present as quasi-species, necessitated the sequencing of cloned PCR products. The gp41 gene displayed less diversity and less insertions/deletions. Especially, the heptad repeat 1 was highly conserved and none of the enfuvirtide naive samples contained any of the already known enfuvirtide resistance mutations at amino acid positions 36-45. This study demonstrates that the assay is able to genotype genetically diverse HIV-1 strains with a good sensitivity.     

Simple, sensitive, and specific detection of human immunodeficiency virus type 1 in clinical specimens by polymerase chain reaction with nested primers.

A simple, sensitive, and specific polymerase chain reaction (PCR) protocol for the detection of human immunodeficiency virus type 1 (HIV-1) is described. We have improved all three PCR steps: sample preparation, DNA amplification, and detection of the amplified product. Some of the improvements have been described previously, but they have never been combined into a complete PCR protocol. Peripheral blood mononuclear cells were lysed directly in a buffer containing sodium dodecyl sulfate, Triton X-100, and proteinase K. This crude cell lysate was amplified in a two-step PCR, first with outer primers and then with inner primers nested within the first primers. The PCR product was visualized by agarose gel electrophoresis and ethidium bromide staining. Thus, we avoided conventional DNA extraction as well as hybridization for the detection of the PCR product. The samples were analyzed with four sets of nested primers (JA4 through JA7, JA9 through JA12, JA13 through JA16, and JA17 through JA20) designed to amplify HIV-1 gag, env gp120, env gp41, and pol sequences, respectively. We were able to amplify HIV-1 sequences in all samples from 90 HIV-1-seropositive individuals with mostly mild symptoms. Of these individuals, 24 were negative in HIV-1 isolation and 9 were selected because they were infected by African and Haitian HIV-1 strains. Eighty-five (94%) individuals were positive with at least three of four primer sets. Samples from 26 healthy blood donors, as well as cells infected in vitro with human immunodeficiency virus type 2 and human T-cell leukemia virus type I, were negative in PCR, thus demonstrating the specificity of the amplification.     

HIV-1 subtyping using gag/env heteroduplex mobility assay and peptide enzyme-linked immunosorbent assay

Two HIV-1 subtypes have accounted for virtually all infections in Thailand: subtype B', found mainly in injection drug users (IDUs), and CRF01_AE (initially subtype E), found in over 90% of sexually infected persons and increasingly in IDUs in recent years. During 1997-1998, 227 blood samples were collected from HIV-1 infected individuals consisting of 92 mothers, 35 children and 100 IDUs. The blood samples were subtyped by heteroduplex mobility assay (HMA) and peptide enzyme-linked immunosorbent assay (PEIA). Using gag and env HMA, CRF01_AE and subtype B' accounted for 96-97% and 3-4% of both the mothers and the children, respectively. In the IDU group, 10% of the plasma samples could only be performed by gag HMA and gave the result as CRF01_AE. CRF01_AE and subtype B' using PEIA accounted for 67% and 33% of the IDUs. There was 100% concordance of the results between gag HMA and env HMA. Ninety-five percentages of concordant results were observed between HMA and PEIA. Of the 6/134 (5%) subjects with discordant results, nucleotide sequencing, used as a gold standard, confirmed the HMA result. In this study, HIV-1 was successfully genotyped by HMA and PEIA. However, a comparison of the subtyping results between HMA and PEIA revealed that HMA was slightly more accurate than PEIA.