Timing of the introduction into Ethiopia of subcluster C' of HIV type 1 subtype C

Viruses circulating in Ethiopia during the 1990s cluster with main subtype C, but a significant subcluster, C', was noted in multiple analyses. This subcluster of subtype C(C') was in a fifty-fifty equilibrium with the main subtype C (Abebe et al., AIDS Res Hum Retroviruses 2000;16:1909-1914). To analyze genetic diversification within the subcluster of HIV-1 subtype C designated C' in the course of the epidemic in Ethiopia, we analyzed 165 env gp120 V3 sequences obtained between 1988 and 1999. We observed a highly significant positive correlation between sampling years of individual sequences and their synonymous distances to the reconstructed common ancestor of the HIV-1 subtype C' subcluster. The extrapolation of the regression line of synonymous distances back to the date when no synonymous heterogeneity was present among the Ethiopian HIV-1 C' population allowed us to estimate 1982 (95% CI, 1980-1983) as the year of the onset of HIV-1 C' genetic diversification and expansion in Ethiopia. These results are in agreement with retrospective epidemiological and serological data, which demonstrated the absence of an HIV-1 epidemic in the Ethiopian population before the 1980s.     

Evidence of the existence of a new circulating recombinant form of HIV type 1 subtype A/J in Cameroon. The European Network on the Study of In Utero Transmission of HIV-1

Several genetic subtypes and circulating recombinant forms (CRFs) of HIV-1 have been identified. The greatest degree of genetic diversity is displayed by variants from Central and West Africa. HIV-1 env C2-V5 and protease sequences were obtained from 15 HIV-1-infected pregnant women, who were selected from a larger cohort study in Yaoundé, Cameroon. Fourteen of 15 virus variants were shown to be recombinant, whereas a single variant appeared to be nonrecombinant subtype A. Five viruses were subtype A/J recombinants, with env genes derived from subtype A and protease genes derived from subtype J. Seven viruses clustered with reference sequences for CRF02 AG(IbNG) in both the env and protease gene fragments, and were thus subtype A/G recombinants. Two variants displayed even more complex recombination patterns. Phylogenetic analyses indicated that the five subtype A/J recombinants might be the first representatives of a previously unrecognized CRF.     

Substitution of hiv type-1 non-B env genes in C2, a subtype B cassette system, results in functional chimeric viruses

Env gene glycoprotein products are essential to viral infectivity and important targets for a host's humoral and cellular immune responses. We have reported the construction of C2, an effective env gene cassetting system for assessing biological properties of HIV-1 subtype B env gene glycoprotein products within a constant genetic background (Zheng NN and Daniels RS: AIDS Res Hum Retroviruses 2001;17:1501-7506). Here we report the ability of C2 to produce chimeric subtype A, C, D, A/E, F, and J HIV-1 and studies of the viruses' biological properties. Virus RNAs were extracted and full-length env genes rescued by RT-PCR. Expression-competent env genes were cloned into the C2 cassette and chimeric recombinant viruses produced by transfecting 293T cells. For each subtype, X4 viruses yielded higher TCID(50) than R5 viruses and the TCID(50) of chimeric viruses were either the same as or lower than their parental viruses. The limited coreceptor usage of R5-tropic parent viruses was retained in the chimeric viruses. Generally, with the exception of the subtype C virus (SE12808), the X4-tropic parental viruses utilized CXCR4 and a wide range of additional coreceptors, while their respective chimeric viruses retained CXCR4 usage but showed a more limited range in respect of other coreceptors. The replication rates of non-B subtype chimeric viruses were generally lower (1.5- to 13.6-fold) than their respective parental viruses with the exception of C2-92UG029, an X4-tropic subtype A chimeric virus. This study demonstrates that C2 is a functional cassette capable of producing infectious chimeric viruses to allow study of the biological phenotypes and functions of HIV-1 subtype B and non-B subtype glycoproteins.     

Genetic diversity of HIV type 1 subtype C env gene sequences from India

Considering the severity of the HIV-1 subtype C epidemic, data on the epidemiology and distribution of HIV subtypes in India are relatively sparse. Keeping this in view, 28 env gene sequences from patients were sequenced and analyzed. The samples were collected over a period of 10 years from 1995 to 2004. Assessment of the interisolate genetic distances of the study isolates, which were all subtype C, showed interisolate distances varying from 2 to 19% (mean: 14%) with the maximum diversity observed in the samples collected in 2003-2004. Analysis of the phylogenetic relationships among subtype C env sequences from six different countries and our study isolates revealed an overall star-like phylogeny with almost all sequences from India forming a monophyletic lineage. A lower diversity within the immunodominant epitopes was found. The data generated from this study should prove valuable for the production of vaccine against subtype C.     

Genetic variability of gag and env regions of HIV type 1 strains circulating in Slovenia

The aim of this study was to investigate the genetic diversity of HIV-1 strains circulating in Slovenia. Proviral DNA isolated from peripheral blood mononuclear cells (PBMCs) of 20 randomly selected HIV-1-infected individuals was classified into subtypes by sequence-based phylogenetic analysis of the env (C2V3) and gag (p24) regions of the viral genome. The phylogenetic tree based on env C2V3 sequences showed that 15 of the 20 samples were subtype B, two A1, one F1, one CRF01_AE, and one CRF02_AG. The phylogenetic analysis of the gag gene yielded identical results expect for one sample that had a discordant subtype; it was identified as subtype A1 in the env and AE in the gag region. Our study confirmed that although subtype B predominates, other subtypes and circulating recombinant forms (CRFs) are also present in Slovenia. The high intrasubtype genetic diversity of subtype B sequences suggests a multiple introduction of subtype B strains into Slovenia.     

Evolutionary history of HIV-1 subtype B and F infections in Brazil

OBJECTIVE: To reconstruct the onset date of the HIV-1 B and F epidemics in Brazil based on virus diversification over time. DESIGN: We studied HIV-1 env V3 sequences (210 nt) with a known sampling year isolated from HIV-1 positive patients from Brazil between 1989 and 1997: 101 subtype B sequences and 41 subtype F sequences. METHODS: HIV-1 V3 env sequences were grouped by year of collection and the relationship between the sampling years of HIV-1 sequences and their genetic distance to the reconstructed common ancestor (intra-population divergence) or to other sequences from the same year (intra-population diversity) was examined by using linear regression analysis. RESULTS: Regression analysis of nucleotide distances, revealed a highly significant positive correlation between sampling years of subtype B and F V3 sequences and their intra-population divergence (P < 0.001) or diversity (P < 0.0001). In both subtype populations, the divergence and diversity increased at a rate of 0.5 and 0.9% per year, respectively. Considering these evolutionary rates, we estimate the onset of the subtype B and F HIV-1 epidemics in Brazil during early 1970s and early 1980s, respectively. CONCLUSIONS: The consistent correlation between divergence and diversity of the V3 sequences with their sampling years indicates that the molecular clock is operational in the evolution of the HIV-1 in Brazil's epidemic, and show that subtypes B and F are evolving at a similar rate over time. The dating results suggest a discontinuous introduction of these subtypes in the Brazilian population.     

HIV type 1 subtypes among bar and hotel workers in Moshi,Tanzania

The HIV-1 prevalence among bar and hotel workers in Tanzania suggests they are a high-risk group for HIV-1 infection. We determined the HIV-1 subtype of 3'-p24/5'-p7 gag and C2-C5 env sequences from 40 individuals representing this population in Moshi. Genetic patterns composed of A(gag)-A(env), C(gag)-C(env), and D(gag)-D(env) were found in 19 (48.0%), 8 (20.0%), and 3 (8.0%) samples, respectively. The remaining 10 samples (25%) had different subtypes in gag and env, indicative of intersubtype recombinants. Among these recombinants, two contained sequences from HIV-1 subsubtype A2, a new genetic variant in Tanzania. Five bar and hotel workers may have been infected with viruses from a common source, based on phylogenetic analysis. The information obtained by surveillance of HIV-1 subtypes in a high-risk population should be useful in the design and evaluation of behavioral, therapeutic, and vaccine trial interventions aimed at reducing HIV-1 transmission.     

Timing of the HIV-1 subtype C epidemic in Ethiopia based on early virus strains and subsequent virus diversification

OBJECTIVE: To trace the introduction of HIV-1 subtype C into Ethiopia based on virus diversification during the epidemic. DESIGN: A set of 474 serum samples obtained in Ethiopia in 1982-1985 was tested for HIV-1. HIV-1 env gp120 V3 and gag or pol regions were sequenced and analysed together with sequences from later stages of the epidemic. RESULTS: None of 98 samples from 1982-1983, one of 193 samples from 1984, and one of 183 samples from 1985 were HIV-1 positive. Phylogenetic analysis of virus sequences from positive samples revealed that they belong to the Ethiopian C, and not the C', cluster. Analysis of 81 Ethiopian C V3 sequences from 1984-1997 revealed that the consensus sequence of the Ethiopian epidemic has been stable over time. Both the 1984 and 1985 V3 sequences, in contrast with three out of 27 (11%) of the 1988 and none out of 51 of the 1992-1997 sequences, had no synonymous substitutions compared to the reconstructed common ancestor of the Ethiopian C viruses. A highly significant correlation between sampling years of the V3 sequences and their synonymous distances to the common ancestor was demonstrated. CONCLUSIONS: The increasing genetic heterogeneity together with stable consensus sequence of the Ethiopian HIV-1 C population demonstrates that evolution of the virus population is characterized by an unbiased expansion around a stationary consensus. Based on the rate of synonymous diversification of HIV-1 strains within the Ethiopian population, we were able to estimate 1983 (95% confidence interval, 1980-1984) as the year of HIV-1 C introduction into Ethiopia.     

Position 22 of the V3 loop is associated with co-receptor usage and disease progression in HIV-1 subtype B isolates

Our goal in this study was to analyze position 22 of the V3 loop associated with co-receptor usage and disease progression in human immunodeficiency virus type 1 (HIV-1) subtype B infection. Bioinformatics approaches were used to compare the amino acid sequence and secondary structure of the V3 loop of the CCR5-tropic virus and CXCR4-tropic virus in HIV-1 subtype B. HIV-1 subtype B V3 amino acid sequence files in the FASTA format were collected from the HIV Sequence Database. The amino acid sequences of different tropism were multiple-aligned with CLUSTAL W program, and the frequencies of the amino acids at each position of the V3 loop sequences of two groups were calculated and sorted in descending order. The secondary structure of the consensus V3 amino acid sequences from CCR5-tropic and CXCR4-tropic viruses were predicted with the APSSP2 method. The amino acids at positions 11, 22, and 25 of V3 were different between the CCR5-tropic virus and CXCR4-tropic virus. The consensus amino acid frequencies were found to be 71.9% S, 66.7% A, and 56.0% D for the CCR5-tropic virus and 50.0% R, 57.1% T, and 26.2% Q for the CXCR4- tropic virus at positions 11, 22, and 25, respectively. There was a strong association between the identity of the residues at position 11, 22, and 25 of the V3 loop amino acid sequence and CD4+ T cell counts of different patients. The change of the residue at position 22 in the R5-tropic or X4-tropic viruses is expected to likely change the secondary structure to be similar to the X4-tropic or R5-tropic viruses. Our study indicates that position 22 of the V3 loop amino acid sequence is significantly associated with viral tropism and disease progression in HIV-1 subtype B.     

An unusual HIV type 1 env sequence embedded in a mosaic virus from Cameroon: identification of a new env clade. European Network on the study of in utero transmission of HIV-1

An atypical HIV-1 strain (CAM001) was identified in a pregnant Cameroonian woman in 1995. HMA subtyping of the env region was unsuccessful, and sequence analyses were performed. Unique sequence motifs were found at the V3 tip (GAGRALHA and GAGRAWIHA), and phylogenetic studies showed that the env C2-V5 sequence branched within group M but remained distinct from all known HIV-1 subtypes, while p17 gag branched with the subtype F sequences. Four other HIV group M viruses, undetermined by HMA, of African origin were found to cluster with CAM001 in the C2-V5 sequences. With the BLAST method, we found in databases three strains whose V3 sequences also clustered with CAM001. These unusual env sequences from eight HIV-1 strains derived from Cameroon formed a separate cluster in HIV-1 group M, which we designated k.     

Human immunodeficiency virus type 1 subtypes among Malaysian intravenous drug users

The HIV-1 genetic variation in 60 infected Malaysian intravenous drug users (IDU) was studied by comparison of the nucleotide sequences and their predicted amino acid sequences in the V3 loop of the external glycoprotein gp120. In this study, HIV-1 B, C and E subtypes were identified among Malaysian IDU, with HIV-1 B being the predominant subtype (91.7%). HIV-1 C and HIV-1 E were minority subtypes among Malaysian IDU. Analysis of the amino acid alignment of the C2-V3 region of the env gene suggests a genetic relationship between Thai and Malaysian B and E subtype strains. This study serves as a baseline for monitoring HIV-1 genetic diversity and spread in Malaysia.     

HIV type 1 subtypes in circulation in northern Kenya

The genetic subtypes of HIV-1 circulating in northern Kenya have not been characterized. Here we report the partial sequencing and analysis of samples collected in the years 2003 and 2004 from 72 HIV-1-positive patients in northern Kenya, which borders Ethiopia, Somalia, and Sudan. From the analysis of partial env sequences, it was determined that 50% were subtype A, 39% subtype C, and 11% subtype D. This shows that in the northern border region of Kenya subtypes A and C are the dominant HIV-1 subtypes in circulation. Ethiopia is dominated mainly by HIV-1 subtype C, which incidentally is the dominant subtype in the town of Moyale, which borders Ethiopia. These results show that cross-border movements play an important role in the circulation of subtypes in Northern Kenya.     

Genetic analysis of human immunodeficiency virus in Abidjan, Ivory Coast reveals predominance of HIV type 1 subtype A and introduction of subtype G

To better understand the molecular epidemiology of HIV genetic diversity in Abidjan, Ivory Coast, we performed a genetic analysis of 170 HIV-1-seropositive specimens representing newly diagnosed tuberculosis patients (n = 143) and women monitored in a mother-to-child transmission cohort study (n = 27). Preliminary screening with RFLP presumptively classified 162 (95.3%) of these as subtype A. The envelope region of 108 specimens was subtyped by sequence analysis: 102 (94.4%) were subtype A, 2 (1.9%) were subtype D, and 4 (3.7%) were subtype G. Subtyping gag and env regions of the genome suggested that five of the six nonsubtype A isolates exhibited a potentially mosaic structure. A comparative phylogenetic analysis of HIV-1 subtype A C2V3 from 27 Ivory Coast and 21 Ugandan sequences revealed a striking clustering among Ivory Coast variants, and an independent segregation from Ugandan subtype A. Despite independent clustering with other subtype A specimens, limited variability of the V3 loop apex was observed; the globally predominant V3 motif, GPGQ, represented 90.1% of the HIV-1 strains. This study demonstrates that clade A is the predominant HIV-1 subtype in HIV-seropositive individuals in Abidjan, Ivory Coast and that these strains are phylogenetically distinct from other subtype A strains observed in East Africa.     

Subtype B and subtype C HIV type 1 recombinants in the northeastern state of Manipur, India

The predominant HIV-1 strain circulating in India is subtype C. However, subtype A and B strains of HIV-1 have also been reported in India. In 1999, the first A/C recombinant strain was reported from Pune in India. Intravenous drug users (IVDUs) from the northeastern region of India have a high HIV-1 seroprevalence. Studies carried out in intravenous drug users in the northeastern region of India have shown that HIV-1 subtype C is the predominant strain infecting IVDUs. Fourteen blood samples were collected from HIV-1-infected individuals from the northeastern region of India and screened by env and gag heteroduplex mobility assays (HMA). Where the env and gag HMA results from a sample yielded different subtypes, sequencing of env and gag PCR products was carried out to confirm the presence of HIV-1 recombinants. Of the 14 samples subtyped, nine samples belonged HIV-1 subtype C (gag C/env C), one to HIV-1 subtype B (gag B/env B), and the remaining were B/C recombinants (gag C/env B). This is the first report of HIV-1 B/C recombinants from India.     

Presence of diverse human immunodeficiency virus type 1 viral variants in Cameroon

Phylogenetic analysis of the gp41 region of 123 HIV-1-seropositive specimens from Cameroon showed that 89 were subtype A (71% of these sequences were IbNg-like), 12 (10%) were subtype D, 11 (9%) were subtype G, 5 (4%; closely related to subtype F2) were subtype F, 1 was subtype H, 2 (1.6%) remained unclassifiable, while 3 were group O. Further analysis of the two unclassifiable specimens in gag(p24), pol(prot), and env (C2V3 or gp41) showed that one (98CM19) was a complex mosaic between subtype A in p24 and subtype J prot, and unclassifiable in env (C2V3 or gp41). The second, 98CM63, clustered distinctly from all known subtypes in p24, prot, C2V3, or gp41. 98CM63 clustered with a specimen from Cyprus and these two geographically and epidemiologically unlinked specimens, with their distinct clustering pattern, may represent a new subcluster of subtype A. In conclusion, these findings confirm the high HIV-1 genetic variability and further suggest the continuous appearance of new viral strains in this population.     

Surveillance of subtype and genetic variation of the circulating strains of HIV-1 in Thailand

Two HIV-1 strains, CRF01_AE and subtype B', were reported in Thailand during the early years of the epidemic. Recently, an intersubtype recombination of HIV-1 strain was found in Thailand. Eight-hundred and twenty-eight samples collected during years 1995-2004 from high-risk groups in Bangkok, northern, northeastern, and southern region of Thailand were studied. HIV-1 env nucleotide sequences were used for phylogenetic analysis of the circulating HIV-1 strain. By single HIV-1 region (env) genotyping, CRFO1_AE was found in 97.3% and HIV-1 subtype B was found in 2.7%. A predominance of CRF01_AE was found in all geographic regions. Parallel analysis of the HIV-1 gag and env genes demonstrated that 2.1% and 4.0% of recombinant HIV-1 strains were found using p17 and p24 region sequences, respectively. The recombinant gag gene was also found in one southern isolate. Phylogenetic analysis of HIV-1 isolated from 20 provinces in 2002 suggested the northern and northeastern isolates were more related than the southern isolates which had the lowest genetic diversity of 0.13. The GPGQ V3 loop tip was also present in isolates from all regions. The molecular epidemiological data from this study may be useful for surveillance design as well as targeting prevention efforts. It also provides information regarding new antigenic regions of circulating strains responsible for the HIV-1 epidemic in Thailand.     

Genetic characterization of incident HIV type 1 subtype E and B strains from a prospective cohort of injecting drug users in Bangkok, Thailand

We obtained specimens from 128 HIV-1 seroconverters identified from 1995 through 1998 in a prospective cohort study of 1,209 HIV-negative injecting drug users (IDUs) in Bangkok, Thailand. Epidemiologic data indicated that parenteral transmission accounted for nearly all infections. HIV-1 DNA from the C2-V4 env region was sequenced, and phylogenetic analyses determined that 102 (79.7%) of the specimens were subtype E and 26 (20.3%) subtype B strains. All subtype B strains clustered with strains often referred to in previous studies as Thai B or B'. The interstrain nucleotide distance (C2-V4) within subtype E strains was low (mean, 6.8%), and pairwise comparisons with a prototype subtype E strain, CM244, showed limited divergence (mean, 5.6%). The subtype B stains showed greater interstrain divergence (mean, 9.2%) and were significantly divergent from the prototype B strain HIV-MN (mean, 13.0%; p < 0.0001). The subtype E strains had significantly lower mean V3 loop charge than did subtype B strains (p = 0.017) and, on the basis of analysis of amino acid sequences, were predicted to be predominantly (91%) non-syncytium-inducing (NSI), chemokine coreceptor CCR5-using (CCR5+) viruses. The subtype B strains had a higher mean V3 loop charge, and a smaller proportion (23%) were predicted to be NSI/CCR5+ viruses. This study demonstrates that most incident HIV1 infections among Bangkok IDUs are due to subtype E viruses, with a narrow spectrum of genetic diversity. The characterization of incident HIV-1 strains from 1995 to 1998 will provide important baseline information for comparison with any breakthrough infections that occur among IDUs in Bangkok who are participating in an HIV-1 vaccine efficacy trial initiated in 1999.     

Apparent founder effect during the early years of the San Francisco HIV type 1 epidemic (1978-1979)

HIV-1 envelope sequence variants were RT-PCR amplified from serum samples cryopreserved in San Francisco in 1978-1979. The HIV-1 subtype B env V3-V5 sequences from four homosexual men clustered phylogenetically, with a median nucleotide distance of 2.8%, reflecting a recent common origin. These early U.S. HIV-1 env variants mapped close to the phylogenetic root of the subtype B tree while env variants collected in the United States throughout the 1980s and 1990s showed, on average, increasing genetic diversity and divergence from the subtype B consensus sequence. These results indicate that the majority of HIV-1 currently circulating in the United States may be descended from an initial introduction and rapid spread during the mid- to late 1970s of subtype B viruses with limited variability (i.e., a founder effect). As expected from the starburst-shaped phylogeny of HIV-1 subtype B, contemporary U.S. strains were, on average, more closely related at the nucleic acid and amino acid levels to the earlier 1978-1979 env variants than to each other. The growing levels of HIV-1 genetic diversity, one of multiple obstacles in designing a protective vaccine, may therefore be mitigated by using epidemic founding variants as antigenic strains for protection against contemporary strains.     

An HIV type 1 subtype A outbreak among injecting drug users in Kazakhstan

Kazakhstan experienced the start of the HIV-1 outbreak among intravenous drug users (IDUs) in 1997. To characterize genetically HIV-1 strains circulating in this country, peripheral blood mononuclear cells (PBMCs) DNA samples (1999-2002) derived from HIV-infected IDUs and their sexual partners in Pavlodar (n = 19), Shymkent (n = 6), and Qaraghandy (n = 18) regions were analyzed by the gag/env heteroduplex mobility assay (HMA). The 366-bp proviral env gene fragments encoding the gp120 C2-V3 region obtained from 16 individuals were sequenced. The results of HMA revealed that all 43 HIV-1 strains studied belonged to gag/env subtype A. The nucleotide sequence analysis showed a marked genetic homogeneity with the mean genetic distance being 3.63 +/- 2.39 (range 0.00-12.13). The mean genetic distance between each sequence within the Kazakhstan set and the East-European IDU subtype A consensus was 2.94 +/- 1.92 (range 0.79-8.48). The data presented thus confirm the spreading of the same IDU subtype A virus in the former Soviet Union.