Functional complementation of the envelope hypervariable V3 loop of human immunodeficiency virus type 1 subtype B by the subtype E V3 loop.

The hypervariable V3 loop within gp120 of human immunodeficiency virus type 1 (HIV-1) is the major determinant of cell tropism and the entry coreceptor usage of the virus. However, the information obtained thus far has been from only subtype B from North America and Europe, and little is known about other subtypes whose V3 amino acids differ by as much as 50% from subtype B V3. In this study, we examined the functional potential of the V3 element of the HIV-1 subtype E, the most crucial variant causing the AIDS epidemic throughout southeast Asia. A panel of HIV-1LAI recombinants was constructed by the overlap extension method, by which the LAI V3 loop was precisely replaced by that of the subtype E nonsyncytium-inducing (NSI) or syncytium-inducing (SI) variant. All of the recombinant viruses infected peripheral blood mononuclear cells, whereas only those with SI V3 infected MT2 cells, a CD4(+) T cell line. Consistently, the SI V3 recombinants used CXCR4, while the NSI V3 recombinants used CCR5 for infection of HOS-CD4(+) cells. Finally, only the NSI V3 sequence conferred CC-chemokine sensitivity on the parental virus. The data support the notion that the HIV-1 V3 loop consists of a relatively independent domain in gp120 and suggest that the subtype E V3 loop indeed contains the functional element to dictate the cell tropism, coreceptor preference, and chemokine sensitivity of the virus. These findings are of immediate importance in understanding V3 structure-function relationship and for examining phenotypic evolution of HIV-1 subtype E.     

Variants of human immunodeficiency virus type 1, detected in Russia among those infected by the sexual route

We analyzed 52 blood samples obtained independently from among individuals, who had never practiced the intravenous administration of drugs, for the purpose of detecting the subtypes of HIV-1 that circulated during 1999-2002. The study materials were analyzed by the methods of gag/env heteroduplex mobility assay and by env gp120 C2-V3 sequencing. Four viral subtypes (A,B,C and G) and a recombinant gagA/envB were detected in subjects contaminated through heterosexual contacts. Noteworthily, HIV-1 variations of subtype A, which were found in 22 (73.3%) of 30 analyzed samples, were predominant in this risk group. An analysis of nucleotide sequences exposed a high degree of homology between the viruses, detected previously among drug-addicts, and the isolates detected by us in subjects contaminated heterosexually. However, HIV-1, subtype B, detected by us in all 16 studied cases, still continue to circulate among the males infected through homosexual contacts with men.     

Molecular analysis and phylogenetic characterization of HIV in Iran

The rate of human immunodeficiency virus type 1 (HIV-1) infection in Iran has increased dramatically in the last few years. While the earliest cases were found in hemophiliacs, intravenous drug users are now fueling the outbreak. In this study, both the 122 clones of HIV-1 gag p17 and the 131 clones of env V1-V5 region were obtained from 61 HIV-1 seropositives belonging to these two groups in Iran. HIV-1 subtyping and phylogenetic analysis was done by heteroduplex mobility assays (HMA) and multiple clone sequencing. The result indicated all hemophiliacs are infected with HIV-1 subtype B and all intravenous drug users are infected with HIV-1 subtype A. Since intravenous drug abuse is the major transmission route in Iran, HIV-1 subtype A is likely to be the dominant viral subtype circulating in the country. The analysis of genetic distances showed subtype B viruses in Iran to be twice as heterogeneous as the subtype A viruses. In conclusion, this first molecular study of HIV-1 genotypes in Iran suggests two parallel outbreaks in distinct high-risk populations and may offer clues to the origin and spread of infection in Iran.     

Increase of HIV-1 subtype A in Central African Republic.

The concomitant presence of five distinct HIV-1 subtypes and of unclassified HIV-1 was reported in Bangui, Central African Republic (C.A.R.) between 1990 and 1991. This previous study was conducted in individuals belonging to the C.A.R. Armed Forces (FACA) Cohort and in patients from the University Hospital of Baugui. To follow the HIV-1 subtype distribution in Bangui over time, we conducted a cross-sectional surveillance of HIV-1 subtypes between 1987 and 1997 in three groups of individuals in Bangui: 47 men belonging to the FACA Cohort, 38 patients from the CNHUB hospital, and 51 individuals consulting the sexually transmitted diseases (STD) clinic. One hundred and ten HIV-1 were subtyped by heteroduplex mobility assay (HMA) and/or sequencing of env regions encompassing the V3 domain. By comparing the HIV-1 distribution in two time periods (1987-1991 and 1991-1996) in the FACA cohort, we observed a significant increase of subtype A from 43.7% to 83.9%. This subtype distribution does not seem specific to the FACA cohort, in that subtype A accounted for 46.7% of the HIV-1 infections in CNHUB patients in the first time period studied and for 69.6% in the second time period. In STD patients, subtype A infections were predominant in 1995 (72.7%) and 1997 (89.7%). Subtype E viruses could be identified in the second time period, but represented only between 6.5% and 21.8% of the infections in the three groups of individuals studied. Other subtypes (B, C, H) and non-classified HIV-1 in C2-V3 were detected with only a 3.2% to 9.1% frequency for each in the second time period. Phylogenetic analysis excluded infection by a single source for the individuals included in the study. Our data demonstrate an increase in the proportion of HIV-1 subtype A infections in Bangui that raises the question of a preferential transmissibility of specific HIV-1 variants.     

High frequency of non-B subtypes in newly diagnosed HIV-1 infections in Switzerland

HIV-1 subtypes were determined in newly diagnosed residents of Switzerland. Blood was anonymously collected from patients with a first confirmed positive HIV-1 test result. Viral DNA from the env V3-V5 region was amplified by nested polymerase chain reaction (PCR) and screened for subtype B by heteroduplex mobility assay. All amplicons not identified as B were sequenced. From November 1996 to February 1998, 206 samples were analyzed. Main transmission risks were unprotected heterosexual (55.7%) or homosexual (27.1%) sexual contact or intravenous drug use (12.9%). Subtype B dominated in patients of Swiss, other European, American, or Asian citizenship; particularly high frequencies were found in homosexuals (97%) and drug users (94%). Non-B subtypes including A, C, D, E, F, G, H, a possible B/F recombinant, and a sequence related to J were present in 28.2% (95% confidence interval [CI], 22.9%-35.0%). Non-B were frequent in African citizens (95%), heterosexually infected individuals (44%), and women (43%). Heterosexually infected Swiss males harbored non-B strains in 18% and females in 33%. The results document a change in the epidemiology of newly diagnosed HIV-1 infections in Switzerland: predominance of heterosexual transmission and a high frequency of non-B subtypes.     

Biological and genetic characteristics of HIV infections in Cameroon reveals dual group M and O infections and a correlation between SI-inducing phenotype of the predominant CRF02_AG variant and disease stage

In Yaounde, Cameroon, HIV-1 group-specific V3 serology on 1469 HIV-positive samples collected between 1996 and 2001 revealed that group O infections remained constant around 1% for 6 years. Only one group N sample was identified and 4.3% reacted with group M and O peptides. Although the sensitivity of the group-specific polymerase chain reaction (PCR) in two genomic regions was not optimal, we confirmed, in at least 6 of 49 (12.2%) dual O/M seropositive samples and in 1 of 9 group O samples, dual infection with group O and M viruses (n = 4) or with group O or M virus and an intergroup recombinant virus (n = 3). Partial env (V3-V5) sequences on a subset of 295 samples showed that at least eight subtypes and five circulating recombinant forms (CRFs) of HIV-1 group M co-circulate; more than 60% were CRF02_AG and 11% had discordant subtype/CRF designations between env and gag. Similarly as for subtype B, the proportion of syncytium-inducing strains increased when CD4 counts were low in CRF02_AG-infected patients. The V3-loop charge was significantly lower for non-syncytium-inducing strains than for syncytium-inducing strains but cannot be used as an individual marker to predict phenotype. The two predominant HIV-1 variants in Africa, CRF02_AG and subtype C, thus have different biological characteristics.     

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.     

Segregation of human immunodeficiency virus type 1 subtypes by risk factor in Australia

The aim of this study was to determine which human immunodeficiency virus type 1 (HIV-1) subtypes were circulating in Australia and to correlate the subtypes with risk factors associated with the acquisition of HIV-1 infection. DNA was extracted from peripheral blood mononuclear cells, and HIV-1 env genes were amplified and subtyped using heteroduplex mobility analysis, with selected samples sequenced and phylogenetic analysis performed. The HIV-1 env subtypes were determined for 141 samples, of which 40 were from female patients and 101 were from male patients; 13 samples were from children. Forty-seven patients were infected by homosexual or bisexual contact, 46 were infected through heterosexual contact, 21 were infected from injecting drug use (IDU), 13 were infected by vertical transmission, 8 were infected from nosocomial exposure, and 6 were infected by other modes of transmission, including exposure to blood products, ritualistic practices, and two cases of intrafamilial transmission. Five subtypes were detected; B (n = 104), A (n = 5), C (n = 17), E (CRF01_AE; n = 13), and G (n = 2). Subtype B predominated in HIV-1 acquired homosexually (94% of cases) and by IDU (100%), whereas non-subtype B infections were mostly seen in heterosexually (57%) or vertically (22%) acquired HIV-1 infections and were usually imported from Africa and Asia. Subtype B strains of group M viruses predominate in Australia in HIV-1 transmitted by homosexual or bisexual contact and IDU. However, non-B subtypes have been introduced, mostly acquired via heterosexual contact.     

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.     

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.     

山东省某些高危人群HIV-1感染者分子流行病学研究

目的 了解流行于山东境内ＨＩＶ－１毒株的亚型分布及变异情况,分析其来源并推测其流行趋势。方法 采集了２５份ＨＩＶ－１抗体阳性感染者的全血分离单核细胞（ＰＢＭＣ）,提取前病毒ＤＮＡ,经ｎｅｓｔｅｄ－ＰＣＲ扩增ＨＩＶ－１膜蛋白（ｅｎｖ）基因的Ｃ２－Ｖ３区并进行序列测定和亚型分析。结果 ２５例ＨＩＶ－１阳性感染者的ＰＢＭＣ样品中扩增到２４份可用于序列测定的ＨＩＶ－１ｅｎｖ基因片段,经序列测定和基因分析鉴     

Relationship between V3 genotype, biologic phenotype, tropism, and coreceptor use for primary isolates of human immunodeficiency virus type 1.

OBJECTIVES: The predictive value of positively charged amino acids at positions 11 and 25 within the V3 loop region of the human immunodeficiency virus type 1 (HIV-1) envelope gene for the syncytium-inducing (SI) phenotype was assessed. STUDY DESIGN/METHODS: Sequencing was performed on DNA extracted from primary peripheral blood mononuclear cells (PBMCs) and complementary DNA (cDNA) prepared from serial viral isolates from 10 HIV-1-seropositive subjects. Proviral DNA sequencing was also performed on biologic clones from most of these subjects. RESULTS: Positive charge at position 11 and/or 25 in 257 isolate cDNA, PBMC DNA, and biologic clone PBMC DNA sequences was compared with 69 phenotypic determinations, of which 62.3% were SI. V3 genotype was 51.2% sensitive and 85.8% specific for the SI phenotype, with positive and negative predictive values of 62.8% and 79.0%, respectively. Cellular tropism failed to correlate with V3 genotype, coreceptor use, or biologic phenotype. Exclusive use of CCR5 was associated with the nonsyncytium-inducing (NSI) phenotype. Overall, V3 loop charge was higher in SI than in NSI isolates (5.01 and 3.78, respectively; p = 0.0211). CONCLUSIONS: The predictive power of SI phenotype from V3 genotype is relatively weak, especially in a low SI prevalence population. The direct measurement of viral phenotype, cellular tropism, and coreceptor use in HIV-1 isolates is essential for accurate biologic characterization.     

Analysis of gag gene subtypes of HIV-1 variants isolated in Russia by comparative assessment of heteroduplex electrophoretic mobility]

Using heteroduplex mobility assay modified for gag gene analysis (HMA-gag), 37 HIV-1 samples previously genotyped by gag and env nucleotide sequencing were studied. It has been demonstrated that both sensitivity and specificity of HMA-gag were 100%. The gag gene region derived from 20 env subtype A HIV-1 isolates was analyzed by this method. AG recombinant, representing a circulating recombinant form of HIV-1 (AGlbNG) was found among five HIV-1 strains isolated from patients infected through heterosexual contacts in Russia. No novel recombinant forms were found among fifteen HIV-1 variants infected from drug users in 7 cities of Russia. The proposed HMA-gag method extends the potentialities of investigating the genetic variability of HIV-1 and in combination with the previously proposed method for env gene is a convenient approach to search for recombinant forms of this virus.     

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.     

Emerging HIV infections with distinct subtypes of HIV-1 infection among injection drug users from geographically separate locations in Guangxi Province, China

Heroin users from Guangxi province, a southern province of China that borders Vietnam in the south and Yunnan province in China in the west, were studied for prevalence and risk factors for HIV-1 infection. Viral env sequences from HIV-1-positive individuals were also determined for subtypes of HIV-1. The overall HIV prevalence among 227 heroin users was 40%. Most had used drugs for < or = 3 years. Sharing of injection equipment and unprotected sex were significantly associated with HIV-1 infection. Subtypes C and E HIV-1 were detected in infected heroin users and were sharply segregated in two geographic locations: only subtype C was found in a border city with Yunnan province, whereas only subtype E was found in a city bordering northern Vietnam. HIV-1 strains within each subtype were remarkably homogenous, with a mean intersubject DNA distance of 2.32% for subtype E and 1.13% for subtype C, respectively. Phylogenetic analysis of C2-V5 region of Guangxi subtype E env sequences revealed significant clustering with subtype E sequences from southern Vietnam and Cambodia. These results suggest that HIV-1 infection among heroin users in Guangxi represents two emerging epidemics initiated from distinct sources: one from Vietnam and another from Yunnan province. Factors associated with HIV-1 infection were not restricted to injection practices. Unprotected sexual behaviors are likely to increase the probability of HIV transmission beyond this high-risk population. Designing and implementing effective intervention strategies targeted toward both injection drug use and high risk sexual behavior are urgently needed to further reduce HIV-1 spread in China.     

HIV-1 subtyping in Salvador, Bahia, Brazil: a city with African sociodemographic characteristics

To investigate the prevalence of the HIV-1 subtypes in different populations from Salvador, Bahia, Brazil, blood samples from 72 HIV-1-seropositive injecting drug users (IDUs) and 62 individuals infected sexually were analyzed using the heteroduplex mobility assay (HMA). In the IDU group, 89.5% were classified as subtype B, 3% as subtype F, and 7.5% showed a B/F HMA profile. In the sexual transmission (ST) group, 95% were identified as B subtype, 3.4% showed a B/F profile, and 1.6% a B/C/E HMA profile. All Brazilian samples that showed multiple reactivities in the HMA analysis clustered on sequencing with B North American/ European HIV-1 isolates in the phylogenetic analysis, whereas the F subtypes clustered with F Brazilian HIV-I isolates. Serologic reactivities of IDU's sera were examined using a panel of synthetic V3 loop peptides representative of the different HIV-1 subtypes. No difference in serologic reactivity between F and B subtype plasma could be observed. Predominance of HIV-I subtype B was identified in both study groups, whereas subtype F was detected only among IDUs in a frequency lower than described for other Brazilian regions.     

Development of an env gp41-based heteroduplex mobility assay for rapid human immunodeficiency virus type 1 subtyping

The gp120 region of the human immunodeficiency virus type 1 (HIV-1) envelope (env) gene exhibits a high level of genetic heterogeneity across the group M subtypes. The heteroduplex mobility assay (HMA) has successfully been used to assign subtype classifications, but C2V5 primers often fail to amplify African strains. We developed an env gp41-based HMA for which the target sequence is amplified with highly conserved gp41 primers, known to efficiently amplify nucleic acids from HIV-1 group M, N, and O viruses. By using gp41 from a new panel of reference strains, the subtype assignments made by our modified HMA were concordant with those obtained by sequencing and phylogenetic analysis of 34 field strains from 10 countries representing subtypes A to G. Testing of field strains from Nigeria further demonstrated the utility of this modified assay. Of 28 samples, all could be amplified with gp41 primers but only 17 (60.7%) could be amplified with the standard C2V5 primers. Therefore, gp41-based HMA can be a useful tool for the rapid monitoring of prevalent subtypes in countries with divergent strains of circulating HIV-1.     

No association of HIV-1 envelope (C2-V3-C3) sequence pattern with long-term nonprogression

We previously identified a group of 10 long-term nonprogressors (LTNP) with HIV-1 infection. In this study, we have sequenced the envelope gene (C2-V3-C3) from the 10 LTNPs and from a control group of 9 people with rapidly progressing infection (RPI). The 19 individuals' CCR5 genotype and virus phenotype (i.e., syncytium-inducing/non-syncytium-inducing [SI/NSI]) were obtained from a previous study. A phylogenetic tree was constructed containing the 19 envelope sequences together with 42 local control env sequences obtained from other studies. Analysis of the phylogenetic tree did not reveal any relation between the envelope gene (C2-V3-C3) from LTNPs versus RPIs. When data from the CCR5 genotype and the virus phenotype were assembled in the phylogenetic tree, no significant clustering was observed. From alignment of the protein sequences, we found a possible N-glycan in position aa294 in env that was conserved in only 1 of 10 LTNPs; however, it was conserved in 6 of 9 RPIs. Our study could not demonstrate any association between LTNPs and the sequenced envelope gene segment (C2-V3-C3). This lack of association could be due to the relatively small sample size of the data set. Nor did we find any relation between the CCR5 genotype or the SI/NSI phenotype with the sequenced envelope genes from the 19 participants. The possible N-glycan position we have described is an interesting observation that may require further investigation.     

Enhancing of anti-viral activity against HIV-1 by stimulation of CD8+ T cells with thymic peptides.

HIV-1 can be neutralized by soluble factors produced and secreted by activated CD8+ T cells. Production of such anti-viral CD8 factors (including chemokines) can be induced with IL-2 or phytohaemagglutinin (PHA). In addition to PHA or IL-2, we have co-stimulated CD8+ T cells with PHA/IL-2 and a mixture of thymic peptides (TP) of molecular weights below 10 kD. For the activation, CD8+ T cells were purified from peripheral blood mononuclear cells of HIV-1- individuals and any resultant anti-viral activity was monitored using an HIV-1 neutralization assay. Using HIV-1 isolates highly resistant to chemokine inhibition we detected significantly higher levels of HIV-1 neutralizing activity in CD8+ T cell culture supernatants which had been co-activated with TP. When the TP-induced anti-viral activity was monitored, neutralization of both non-syncytia-inducing (NSI) and syncytia-inducing (SI) patient isolates was enhanced by 38% (NSI, PHA +/- TP), 66% (SI, PHA +/- TP), 28% (NSI, IL-2 +/- TP), and 57% (SI, IL-2 +/- TP) compared with the anti-viral activity present in supernatants from CD8+ T cell cultures stimulated only with PHA or IL-2. Peptide sequence analysis of purified TP showed that the TP mixture predominantly contains peptides with homology to human histone and collagen sequences. Our data demonstrate that CD8+ T cells are additionally activated by a mixture of TP. In this way, the production of HIV-1 neutralizing CD8 factors can be enhanced.