Mother-to-child transmission of different HIV-1 subtypes among ARV Naïve infected pregnant women in Nigeria.

The rate of mother-to-child transmission (MTCT) of HIV as well as the implications of the circulating multiple subtypes to MTCT in Nigeria are not known. This study was therefore undertaken to determine the differential rates of MTCT of HIV-1 subtypes detected among infected pregnant women before ARV intervention therapy became available in Nigeria. Twenty of the HIV-positive women who signed the informed consent form during pregnancy brought their babies for follow-up testing at age 18-24 months. Plasma samples from both mother and baby were tested for HIV antibody at the Department of Virology, UCH, Ibadan, Nigeria. All positive samples (plasma and peripheral blood mononuclear cells-PBMCs) were shipped to the Institute of Tropical Medicine, Antwerp, Belgium, where the subtype of the infecting virus was determined using the HMA technique. Overall, a mother-to-child HIV transmission rate of 45% was found in this cohort. Specifically, 36.4%, 66.7% and 100% of the women infected with HIV-1 CRF02 (IbNg), G and B, respectively, transmitted the virus to their babies. As far as it can be ascertained, this is the first report on the rate of MTCT of HIV in Nigeria. The findings reported in this paper will form a useful reference for assessment of currently available therapeutic intervention of MTCT in the country.

HIV-1 subtype H near-full length genome reference strains and analysis of subtype-H-containing inter-subtype recombinants.

OBJECTIVE: To characterize near-full-length genomes of two HIV-1 subtype H strains. To extend sequence data to include full env and gag, and analyse and redefine, previously documented subtype H strains. DESIGN: Near-full-length genomes of HIV-1 env subtype H strains VI991 and VI997 were amplified, cloned, sequenced, phylogenetically analysed and compared with a panel of 23 HIV-1 group M reference isolates. The mosaic nature of previously published subtype H strains VI557 and CA13 was reanalysed. MATERIALS AND METHODS: Peripheral blood mononuclear cells (PBMC) from individuals harbouring strains VI991 and VI997 were co-cultivated with PHA stimulated donor PBMC. Near-full-length genomes of VI991 and VI997, and gag and env genes of CA13 and VI557, were amplified by polymerase chain reaction, cloned and sequenced. Intersubtype recombination analyses were performed by similarity plot, bootscanning and phylogenetic analysis. RESULTS: Near-full-length clones of HIV-1 VI991 and VI997 are representative of subtype H. They form a phylogenetic cluster with the only previously described subtype H representative HIV-1 90CF056.1, regardless of the genome region analysed. VI557 is redefined as a gag and env subtype H mosaic virus containing unclassified fragments. CA13 is a complex intersubtype recombinant between subtypes A, H and unclassified strains CONCLUSION: Near-full-length genome analysis identified HIV-1 VI991 and VI997 as two new subtype H representatives. These reagents will allow defining and classifying non-recombinant as well as recombinant HIV-1, eventually helping to solve the puzzle of HIV-1 subtypes.

Genotypic subtypes of HIV-1 in Cameroon.

OBJECTIVE: The only two HIV-1 strains (ANT70 and MVP5180) reported to date from Cameroon are members of the outlier clade (group O). In this study, we assessed the prevalence of group O viruses and other HIV-1 subtypes in Cameroon. DESIGN: A phylogenetic analysis of 18 HIV-1 strains isolated from seropositive individuals from Yaoundé and Douala, Cameroon. METHODS: A 900 base-pair fragment of the env gene coding for V3, V4, V5, and the beginning of gp41 of 17 out of 18 HIV-1 isolates from Cameroon was amplified, cloned and sequenced using polymerase chain reaction. A phylogenetic tree was constructed. RESULTS: The overall env nucleotide sequence divergence among the Cameroon isolates ranged from 6.1 to 27.5%. In a phylogenetic tree, six subtypes were identified when compared with 23 reference strains of different geographic origin. Of these 17 Cameroonian strains, 11 (61%) were of subtype A of which the interpatient distances at the sequence level varied from 6.1% to 18.3% (average, 11.9%). Three (17%) strains were of subtype F, and the other three strains (6% each) belonged to subtypes B, E and H, respectively. The remaining isolate was classified as belonging to group O, on the basis of the sequence of part of the pol gene. A very broad spectrum of different tetrameric amino-acid sequences was observed at the apex of the V3 loop. Eleven strains contained the tetrameric globally predominant GPGQ sequence at the tip of the V3 motif. Two strains had the GPGR sequence typical of the American and European HIV-1 strains. The remaining tetrameric sequences included GPGS, GSGQ, GRGQ, and GLGR. CONCLUSION: These findings on a limited number of viruses suggest extensive env gene diversity of HIV-1 strains from Cameroon, and could have implications for vaccine development in Africa.

Molecular phylogeny of part of the env gene of HIV-1 strains isolated in Côte d'Ivoire.

OBJECTIVES: To examine the genetic variation of HIV-1 isolates in Abidjan, Côte d'Ivoire, and to determine the extent to which phylogenetic trees based on sequence information of part of the env gene containing the principal neutralizing domain are representative for documenting genetic variability. DESIGN: Phylogenetic comparison of 13 HIV-1 strains isolated from patients in Abidjan with previously documented HIV-1 strains of different geographic origin. METHODS: To sequence a 900 base-pair fragment of the env gene containing V3, V4, V5 and the beginning of gp41 of three to four clones per isolate. Phylogenetic tree analysis was performed with the software package TREECON. RESULTS: Eleven HIV-1 isolates of Abidjan were classified as genotype A, while two were classed as genotypes B and D. Intra-genotype A distances at the nucleotide level were a maximum of 14.1%. Inter-genotype distances between genotype A and genotypes B, C, and D varied from 16.0 to 22.6%. Phylogenetic trees, based on sequence data of a 300 base-pair fragment containing the V3 loop, showed significant differences in tree topology and statistical confidence with phylogenetic trees based on sequence data of the 900 base-pair env fragment. CONCLUSIONS: Genotype A Côte d'Ivoire HIV-1 strains, which comprise 11 out of 13 isolates, predominate in Abidjan, which may indicate a local burst of particular variants. Phylogenetic trees should be interpreted with caution when based on a more limited number of nucleotides, such as the V3 region.

Phylogenetic analysis of the env gene of HIV-1 isolates taking into account individual nucleotide substitution rates.

OBJECTIVE: To estimate the relative substitution rate of the individual positions in an alignment of HIV-1 env sequences coding for areas V3, V4, V5, and the beginning of gp41, and to study phylogenetic relationships between HIV-1 strains taking into account these substitution rate estimates. DESIGN: Phylogenetic comparison of 145 HIV-1 strains classified in HIV-1 group M, subtypes A-H and isolated from patients of 24 different geographical origins. METHODS: A new method recently developed for measuring the substitution rates of the individual nucleotides in a sequence alignment was applied to an alignment of env gene sequences. From the resulting substitution rate distribution, an equation was derived that describes the relationship between dissimilarity and evolutionary distance better than equations previously available. Phylogenetic trees were then constructed from matrices of distances computed using this new equation. RESULTS: 'Substitution rate calibration' offers detailed information on the relative substitution rate or variability of the nucleotides in the env gene. A large phylogenetic tree of 145 env gene sequences constructed by neighbour-joining and taking into account the substitution rate spectrum for this gene, clearly shows the existence of the eight subtypes A-H, all supported at a bootstrap level of 90% or higher. Intersubtype distances were between 0.25 and 0.38, which is considerably higher than those found in trees not considering differences in substitution rates among different alignment positions. CONCLUSIONS: Evolutionary distances are seriously underestimated when individual substitution rates are not considered in the estimation evolutionary distances. Furthermore, due to the more accurate estimation of evolutionary distances, naturally occurring HIV-1 intersubtype recombinants could be recognized more easily.

Lack of correlation between V3-loop peptide enzyme immunoassay serologic subtyping and genetic sequencing.

OBJECTIVE: To compare the performance of V3-loop peptide enzyme immunoassay (PEIA) methodologies from four different laboratories for subtyping HIV-1, and to determine the causes for the lack of correlation between V3-loop PEIA serotyping and subtyping by sequencing. MATERIALS AND METHODS: Synthetic peptides derived from the amino-acid consensus sequences of the V3-loop of group M strains representing genetic subtypes A-F as well as reference strains were evaluated in PEIA by four different laboratories for their ability to accurately determine the subtype in a panel of 85 sera obtained from persons infected with known HIV-1 subtypes (28 subtype A, 34 subtype B, four subtype C, 10 subtype D, seven subtype F, one each of subtype H and G). Furthermore, the V3 loop of the corresponding virus was compared with the V3 loop of the peptides used in PEIA. RESULTS: The correlation between HIV-1 subtyping by sequencing and V3-loop PEIA from the different laboratories varied considerably for the different HIV-1 subtypes: subtype A (46-68%), B (38-85%), C (75-100%), D (29-50%), and F (17-57%). A 70% agreement between PEIA and sequencing subtypes was observed for samples with the concordant presence of the same octameric sequences in the V3 loop of the virus and the V3 loop of the peptide used in PEIA; however, only 42% of specimens with different V3-loop octameric viral and peptide sequences yielded concordant results in V3-loop serotyping and genetic subtyping. CONCLUSION: Our results indicate that V3-loop PEIA methodologies used in different laboratories correlate poorly with genetic subtyping, and that their accuracy to predict HIV-1 subtypes in sera of Belgian individuals infected with different HIV-1 subtypes (A, B, C, D, F, G and H) vary considerably. The poor correlation between serotyping and genetic subtyping was partly due to the simultaneous occurrence of subtype-specific octameric sequences at the tip of the V3 loop of viruses belonging to different genetic subtypes.

Interpatient genetic variability of HIV-1 group O.

OBJECTIVE: To analyse the genetic and phylogenetic characteristics of HIV-1 group O viruses. MATERIALS AND METHODS: The env gene, encoding the gp160 glycoprotein, and a partial p24-encoding gag gene fragment of a Cameroonian (CA9) and a Gabonese (VI686) HIV-1 group O virus, isolated from cultured peripheral blood mononuclear cells of symptomatic patients, were sequenced, aligned with other representatives of group O for which the same region has been documented, and genetically and phylogenetically analysed. RESULTS: Phylogenetic analysis of the env gene (gp160) revealed that CA9, VI686, ANT70, and four Ha strains formed a separate cluster, which was supported by 100% of all bootstrap trees. In addition, these seven isolates were part of the same clade in the p24 phylogeny. VAU and MVP5180 may represent two other subtypes. CONCLUSION: We have characterized two group O viruses, originating from Cameroon and Gabon, which show a close evolutionary relationship to ANT70 and four Ha strains based on the entire env gene, suggestive of a first group O subgroup, tentatively named the HIV-1 group O env ANT70 clade or subtype.

HIV-1 subtypes and the HIV epidemics in four cities in sub-Saharan Africa.

OBJECTIVE: To describe the distribution of HIV-1 subtypes in two cities with high HIV prevalence (Kisumu, Kenya and Ndola, Zambia) and two with relatively low prevalence (Cotonou, Benin and Yaoundé, Cameroon), and to examine whether the differences in prevalence of HIV infection could be due to the predominance within the infected populations of subtypes with differing efficiency of heterosexual transmission. METHODS: For around 100 randomly selected HIV-positive sera from the general population and 60 from sex workers in each city, the HIV-1 subtype was determined in the envfragment. For between 19 and 52 of the sera from the general population and 20-32 sera from sex workers, the subtype was also determined in the gag fragment. RESULTS: Over 70% of infections in Cotonou, Yaoundé and Kisumu were with subtype A (by env). However, around one-half of subtype A infections in Cotonou and Yaoundé were found to be the circulating recombinant form CRF02_AG when the gag fragment was also examined. A large number of different HIV strains were found in Yaoundé, including some belonging to group O. Over 20% of infections in Kisumu and around 10% in Yaoundé were with isolated intersubtype recombinant forms. All but a few infections in Ndola were with subtype C and no recombinants were found. CONCLUSIONS: The pattern of distribution of subtypes that we found does not suggest that differences in circulating subtypes play a major role in explaining the differences in prevalence of HIV-1 infection between the four cities. The emergence and spread of recombinants requires close surveillance to adapt testing strategies if needed, to inform vaccine development and to ascertain their role in the future spread of HIV.

Epidemiological and molecular characteristics of HIV infection in Gabon, 1986-1994.

OBJECTIVE: To describe trends in the prevalence of HIV-1 infection in different populations in Gabon, and the molecular characteristics of circulating HIV strains. METHODS: Data were collected on HIV prevalence through sentinel surveillance surveys in different populations in Libreville (the capital) and in Franceville. In Libreville, a total of 7082 individuals (hospitalized patients, tuberculosis patients, pregnant women, asymptomatic adults, prisoners) were recruited between 1986 and 1994. In Franceville, we tested 771 pregnant women and 886 healthy asymptomatic adults (1986-1988). Sera were screened for HIV antibodies by enzyme-linked immunosorbent assay (ELISA) and confirmed by Western blot or line immunoassay (LIA). Reactive samples in ELISA were tested for the presence of antibodies to HIV-1 group O viruses by ELISA using V3 peptides from HIV-1 ANT-70 and HIV-1 MVP-5180 followed by confirmation by LIA and a specific Western blot. Seventeen HIV-1 strains were isolated (1988-1993) and a 900 base-pair fragment encoding the env region containing V3, V4, V5 and beginning of gp41 was sequenced and a phylogenetic tree was constructed. RESULTS: HIV prevalence was relatively low and remained stable (0.7-1.6% in pregnant women, 2.1-2.2% in the general population). The prevalence was also stable among prisoners (2.1-2.6%). Among hospitalized and tuberculosis patients prevalence was higher and increased (1.8-12.7% and 1.5-16.2%, respectively). Only three sera had antibodies to HIV-1 group O. The 17 HIV-1 strains represent six different genetic subtypes including type O. CONCLUSION: Our data from 1986 to 1994 show a stable and low HIV prevalence in Gabon, and a high genetic diversity of HIV-1 strains. This, also observed in Cameroon, is in contrast to that found elsewhere in Africa. Differences in rate of spread of HIV infection are probably explained by interplay between numerous factors. The role of different HIV subtypes in the dynamics of the HIV epidemic should be examined further.

Improved detection of HIV-2 proviral DNA in dually seroreactive individuals by PCR.

OBJECTIVE: To improve the detection rate of HIV-2 proviral DNA in primary uncultured peripheral blood mononuclear cells (PBMC) of HIV-2-seroreactive and HIV-1-HIV-2 dually seroreactive individuals. MATERIALS AND METHODS: Two newly designed HIV-2 PCR primer pairs in the long terminal repeat (LTR) gag and gag-pol regions and a previously described env and LTR HIV-2 PCR primer pairs were tested on samples from 66 confirmed HIV-2-seropositive individuals (The Gambia, 40; Côte d'Ivoire, 17; Guinea-Bissau, nine), 209 dually seroreactive individuals (The Gambia, 82; Côte d'Ivoire, 127), 24 genetically characterized isolated HIV-1 strains (group M subtypes A-H and group O), one simian immunodeficiency virus (SIV) strain cpz, 10 HIV-2 isolates (subtype A, B and unidentified), two SIVsm isolates, and 10 seronegative samples. RESULTS: All HIV-2 primers evaluated showed 100% specificity since there was no amplification observed with 24 HIV-1, one SIVcpz and 10 seronegative samples. One single copy of the HIV-2 genome could be detected with all outer primer pairs as well as all inner primer pairs on one PCR round used. Sensitivity of primers (at least one of the four primer pairs was positive) to HIV-2-seropositive samples was 100% (all nine) in Guinea-Bissau, 71% (12/17) in Côte d'Ivoire, 100% (all 20) in Gambian AIDS patients, and 85% (17/20) in Gambian pregnant women. Doubling the PBMC of dually seroreactive individuals from 7.5 x 10(4) to 1.5 x 10(5) in the PCR revealed the presence of both HIV-1 and 2 proviral DNA in 72% (92/127) in Côte d'Ivoire and 72% (59/82) in The Gambia. By doubling the number of PBMC, HIV-2 detection in dually seroreactive individuals by PCR was increased from 65 to 77% in Côte d'Ivoire and from 67 to 83% in The Gambia. CONCLUSIONS: The use of 1.5 x 10(5) primary uncultured PBMC and the newly designed HIV-2 primer pairs allowed us to document the highest percentage (72%) ever reported of HIV-1-HIV-2 dual infections amongst HIV-1-HIV-2 dually seroreactive individuals in Côte d'Ivoire and The Gambia. Improved detection of HIV-2 proviral DNA, rather than exposure to both viruses, infection with only one virus, or infection with a unique third virus containing epitopes common to both HIV-1 and HIV-2, contributes to a more accurate monitoring of the prevalence of HIV-1-HIV-2 dual infections.

Reanalysis of full-length HIV type 1 group M subtype K and sub-subtype F2 with an MS-DOS bootscanning program.

Five new complete HIV-1 group M genome sequences have been published (Triques et al., AIDS Res Hum Retroviruses 2000;16:139-151). One of these clustered consistently with subtype F sequences, while two others were identified as representatives of a subcluster within the subtype F clade, called F2, and the two remaining sequences were described as a new subtype K. We reanalyzed these sequences by means of bootscanning and phylogeny, using a newly developed MS-DOS bootscanning program. Although our analysis does not contradict the existence of the new subtype K, it also indicates that in some regions the F2 sequences do not cluster with the F1 clade. This suggests that some fragments in the F2 sequences have an uncertain origin, and care should be taken when F2 sequences are used in analyses.

Near full-length genome analysis of HIV type 1 CRF02.AG subtype C and CRF02.AG subtype G recombinants.

HIV-1 CRF02.AG strains are prevalent in west and west-central Africa, suggesting a longstanding presence of these subtype A/G recombinants in the global epidemic. Cocirculation of CRF02.AG strains with other group M subtypes may give rise to HIV-1 recombinants constituting a mosaic genome comprising fragments of three different subtypes. We report on the genetic analysis of the near-full-length genomes of such recombinants (VI1035 and VI1197) as well as CRF02.AG strains in Belgian individuals. VI1035 and VI1197 may be the result of successful "second-generation" recombinations of HIV-1 strains CRF02.AG with, respectively, subtype C (VI1035) and G (VI1197) strains in a dually infected individual.

Study of HIV type 1 gag/env variability in The Gambia, using a multiplex DNA polymerase chain reaction.

A multiplex DNA PCR assay was developed for the simultaneous first-round amplification of HIV-1 gag and env fragments for the heteroduplex mobility assay (HMA). This assay was compared with the conventional amplification assay, using DNA extracted from PBMC samples from 30 HIV-1-seropositive individuals from The Gambia, who were enrolled between 1992 and 1997. From 27 of 30 (90%) samples both gag and env HMA fragments were amplified simultaneously. In one sample only the gag HMA fragment could be amplified by multiplex DNA PCR, and in two samples amplification was negative for both gag and env HMA in multiplex as well as the mono-DNA PCR. Of the 28 Gambian isolates subtyped by gag/env HMA or by sequencing and phylogenetic analysis, the majority (19 of 28; 68%) were intersubtype recombinant. Fifteen of 28 (53%) samples were circulating recombinant form (CRF) CRF02.AG variants. Two isolates clustering with the previously documented Gambian isolate GM4 (previously described as an env GC recombinant) are classified as gag A/env J recombinants.

Genetic and antigenic variability of HIV type 1 in Brazil.

Six Brazilian strains of human immunodeficiency virus type 1 (HIV-1) were isolated from infected individuals residing in different regions of Brazil between 1987 and 1989. Phylogenetic analysis based on an 860-base pair env fragment, including V3, V4, V5, and the beginning of gp41, classified the Brazilian strains significantly in genotype B, with interhost distances between 5.9 and 13.1% (mean value, 10%). Amino acid sequence analysis of the V3 loop revealed that three strains contained the North American/European GPGR motif as the tip of the loop whereas in the other three strains proline (P) was substituted by tryptophan (W), methionine (M), or phenylalanine (F). A consensus peptide, Bra-cons, was designed containing GWGR as the tip of the loop. Serological reactivity to the Bra-cons peptide and other V3 peptides (MN, SF2, HBX2, RF, MAL, ELI, Z6, and a Côte d'Ivoire peptide, CI-cons) was compared for 114 HIV-1-positive sera from Rio de Janeiro. Sixty-nine sera (60.5%) reacted with peptides belonging to genotype B, of which 10 sera also reacted with peptides belonging to genotype A (n = 7) and D (n = 3). Eighteen sera (15.8%) had binding antibodies to the Bra-cons peptide. A high number of sera (n = 43; 37.7%) had no antibodies to any of the V3 peptides tested. This result suggests that HIV-1 variants with aberrant V3 loops may circulate in Rio de Janeiro.

Isolation of simian immunodeficiency viruses from two sooty mangabeys in Côte d'Ivoire: virological and genetic characterization and relationship to other HIV type 2 and SIVsm/mac strains.

OBJECTIVE: To search for the presence of SIV in sooty mangabeys and other monkey species in Côte d'Ivoire, West Africa, and to compare viral isolates with HIV-2 strains from the same region. METHODS: Forty-three captive housed monkeys (28 African green monkeys, 6 sooty mangabeys, 6 baboons, and 6 patas monkeys) were tested for the presence of HIV and SIV antibodies. Virus was isolated from the peripheral blood lymphocytes of seropositive animals and from HIV-2 antibody-positive patients originating from Côte d'Ivoire, Ghana, Senegal, and Belgium. Viruses were characterized by Western blot and radioimmunoprecipitation assay. Proviral DNA was amplified by PCR, cloned, and sequenced to construct a phylogenetic tree. RESULTS: One African green monkey and three sooty mangabeys had antibodies that cross-reacted with HIV-2. From two mangabeys lentiviruses were isolated and designated as SIVsmCI2 and SIVsmCI8. Serological, virological, and sequence data showed that these isolates are members of the HIV-2/SIVsm/SIVmac group of primate lentiviruses. Furthermore, in the phylogenetic tree, these two new viruses form a distinct subgroup that is equidistant to the HIV-2 strains and the previously described SIVsm/SIVmac viruses. CONCLUSION: This study provides additional evidence that sooty mangabey monkeys can be infected with a lentivirus in their natural habitat. Within the SIVsm and SIVmac viruses extensive genetic variation is observed.

Correlation between genetic and biological properties of biologically cloned HIV type 1 viruses representing subtypes A, B, and D.

The relationship between the genetic variability in the V3 loop and the biological characteristics of 38 biological clones from 5 European and 7 African HIV-1 isolates belonging to 3 different subtypes (subtype A, B, and D) was investigated. Seventeen of 19 clones displaying a syncytium-inducing (SI) capacity had a positively charged amino acid located at position 11 and/or 25 in the V3 loop sequence. All 19 non-syncytium-inducing (NSI) virus clones lacked such a positive charge at the same positions (p < 0.001). The mean of net charge in the overall V3 loop sequences of the SI clones was higher than that of the NSI clones (p < 0.001). Within the same strains, the SI clones replicated faster/higher than the NSI clones in peripheral blood mononuclear cells (p < 0.01), but not in CD4+ T cell cultures (p > 0.1). All SI clones but only 5 of 19 NSI clones could replicate in human continuous T cell and monocytic cell lines (p < 0.001). A higher number of positively charged amino acid substitutions was found among the subtype D SI clones. Only one of eight autologous sera tested had the ability to neutralize the contemporaneously isolated NSI clones, but not the SI clones. This study indicates that the V3 loop amino acid sequences of HIV-1 biological clones from different origins belonging to different genetic subtypes are clearly correlated with viral syncytium-inducing capacity, cell tropism, and replication rate.

The neutralization relationship of HIV type 1, HIV type 2, and SIVcpz is reflected in the genetic diversity that distinguishes them.

Neutralizing antibody (NA) patterns in the sera of individuals naturally infected with human immunodeficiency virus (HIV) type 1, HIV-2, and the simian immunodeficiency virus (SIVcpz) to their homologous and heterologous isolates were determined in a peripheral blood mononuclear cell-based neutralization assay. We examined the role of the V3 loop of HIV-1 and SIVcpz in neutralization and the cross-reactivities among them. Cross-neutralization by sera of humans and chimpanzees naturally infected, respectively, with HIV-1 and SIVcpz isolates was more extensive than the infrequent and low-titer cross-neutralizations observed between HIV-1 and HIV-2. Neutralization of 9 of the 16 HIV-1 isolates by 9 of 10 HIV-2 and all 3 SIVcpz antibody-positive sera were weak and sporadic (titer, 1:10-1:160). Twelve of 15 HIV-1 sera neutralized the 2 SIVcpz isolates with titers of 1:10-1:320 but only sporadically neutralized the 6 HIV-2 isolates (titers: 1:10-1:20). The majority of HIV-1 and SIVcpz sera bound to the V3 peptides although their binding capacity did not readily reflect their neutralizing capacity. The HIV-2 sera did not or only weakly bound to the V3 peptides. These results suggest that HIV-1 and SIVcpz share some structural and functional similarities that set them apart from HIV-2.

Development of a one-tube multiplex reverse transcriptase-polymerase chain reaction assay for the simultaneous amplification of HIV type 1 group M gag and env heteroduplex mobility assay fragments.

The emergence of intersubtype recombinant HIV-1 isolates has made it imperative to analyze different regions of HIV-1 genomes. For this purpose a one-tube multiplex RT-PCR, coamplifying first-round amplicons that allow amplification of gag and env heteroduplex mobility assay (HMA) fragments from different HIV-1 group M isolates, was developed, starting with plasma samples. The multiplex RT-PCR assay is sensitive: 115 of 136 (84.5%) samples were positive for both gag and env, positive amplification of the gag fragment was observed in 130 of 136 (95.6%) samples, while for the env fragment 119 of 136 (87.5%) tested positive. The multiplex RT-PCR in combination with gag and env HMA makes large-scale HIV-1 subtyping fast, simple, and more economical.

Genetic variability of HIV type 1 in Kenya.

PIP: The AIDS epidemic is a rapidly growing problem in Nairobi, where the seroprevalence in pregnant women increased from 4% in 1988 to over 10% in 1991. 22 HIV-1-seropositive pregnant women and 1 HIV-1-infected baby (K88) attending the Pumwani Maternity Hospital of Nairobi between 1990 and 1992 were studied as part of a cohort study of maternal risk factors in mother-to-child transmission. A 250-base pair (bp) fragment of the env gene encoding C2V3 was amplified mostly from DNA isolated from primary peripheral blood mononuclear cells and subsequently sequenced. The 23 newly determined HIV env sequences were aligned with 23 previously known sequences of HIV-1 isolates of diverse geographical origin and the sequence of the HIV-1-related chimpanzee isolate SIVcpz-gab, on the basis of primary structure. Distance calculation, tree construction, and bootstrap analysis were realized with the software package TREECON. In the tree, 8 major branches could be observed containing sequences representative of 8 different subtypes A, B, C, D, E, F, G, and H, besides the outlier group O. 19 of 23 Kenyan isolates clustered with D687, Z321, U455, and SF170, which were members of genetic subtype A. Phylogenetic analyses favored positioning of K976 as a divergent A subtype strain. For 4 strains (K29, K88, K98, and K112) the subtype A classification based on the gag gene was also observed on the basis of phylogenetic analysis of the C2V3 coding part of the env gene. The predicted amino acid sequence of the V3 region for these strains was also presented. The finding that among 23 HIV-1 isolates collected in Nairobi, 19 were classified in subtype A versus 3 in subtype D, together with a much larger variation between subtype A strains as compared to subtype D strains, suggests an earlier introduction of a subtype A strain, multiple introductions of subtype A strains, and/or faster diversification of subtype A strains as compared to subtype D strains.^ieng

Genetic variation of HIV type 1: relevance of interclade variation to vaccine development.

Accumulating data in human immunodeficiency virus (HIV)-infected individuals support the hypothesis that in primary human immunodeficiency virus type 1 (HIV-1) isolates of different clades and phenotype (syncytium inducing [SI] and nonsyncytium inducing [NSI]) common antigenic structures must exist that can stimulate the immune response to produce a broad spectrum (cross-clade and cross SI and NSI) neutralization response. Certain vaccination regimens in chimpanzees and human volunteers with clade B SI type HIV-1 derived candidate vaccines induce neutralizing antibodies against intraclade B SI type primary HIV-1 isolates, but not against intraclade B NSI type of viruses. To be effective against the full antigenic spectrum of primary HIV-1 isolates (cross-clade--SI and NSI) candidate vaccines should contain immunogens of primary isolates representative of the whole antigenic spectrum of HIV-1. There is an urgent need to identify these immunogens and to improve their immunogenicity. As long as we have not yet characterized these cross-HIV-1 spectrum conserved immunogens, candidate vaccines against the more prevalent clades C, A, and E should be developed for evaluation in developing countries. In support of the follow-up and evaluation of the hopefully increasing number of phase 1, 2, and 3 HIV-1 vaccine trials in humans, it is considered a high priority to develop a high throughput neutralization assay, to further expand the use of a limited number of key primary HIV-1 isolates as a surrogate for neutralization of the entire HIV-1 antigenic spectrum (cross-clade--SI and NSI), to develop high throughput subtyping as well as a rapid system to monitor the immunogenic relatedness of different HIV-1 clades.