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.     

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.     

Diversity in HIV-1 envelope V1-V3 sequences early in infection reflects sequence diversity throughout the HIV-1 genome but does not predict the extent of sequence diversity during chronic infection

Differences in the extent of genetic diversity have been observed in human immunodeficiency virus type-1 (HIV-1) envelope sequences early in infection, and this has been linked to gender and to modifiable exogenous factors such as hormonal contraceptive use and genital tract infections. But it is unclear whether envelope diversity is indicative of diversity in other regions of the viral genome, and thus whether it adequately reflects whether multiple or a single virus initiated the infection. Here we show that six women with homogeneous envelope V1-V3 sequences during primary infection also had homogeneous gag and polymerase (pol) sequences at the same time. On the other hand, six women with multiple envelope sequences had diverse gag and pol genotypes during a similar interval after infection. This suggests that envelope sequences reflect sequence diversity throughout the viral genomes present early in infection and thus provide an indication of whether a single virus or multiple viruses initiated the infection. Analysis of HIV-1 sequences from about 3 years after infection revealed that the level of diversity and diversification was similar between the women in the two groups.     

Characterization and selection of HIV-1 subtype C isolates for use in vaccine development

HIV-1 genetic diversity among circulating strains presents a major challenge for HIV-1 vaccine development, particularly for developing countries where less sequence information is available. To identify representative viruses for inclusion in candidate vaccines targeted for South Africa, we applied an efficient sequence survey strategy to samples from recently and chronically infected persons residing in potential vaccine trial sites. All 111 sequences were subtype C, including 30 partial gag, 26 partial pol, 27 V2-V3 env, and 28 V5-partial gp41 sequences. Of the 10 viruses cultured from recently infected individuals, 9 were R5 and 1 was R5X4. Two isolates, Du151 and Du422, collected within 2 months of infection, were selected as vaccine strains on the basis of their amino acid similarity to a derived South African consensus sequence The selection of recently transmitted R5 isolates for vaccine design may provide an advantage in a subtype C R5-dominant epidemic. The full-length Du422 gag and Du151 pol and env genes were cloned into the Venezuelan equine encephalitis (VEE) replicon particle (VRP) expression system. Du422 Gag protein expressed from the VRP accumulated to a high level and was immunogenic as demonstrated by cytotoxic T lymphocyte responses in mice vaccinated with gag-VRPs. Optimization of codon use for VRP expression in human cells did not enhance expression of the gag gene. The cloned Du151 env gene encoded a functional protein as demonstrated by fusion of VRP-infected cells with cells expressing CD4 and CCR5. Genes identified in this study have been incorporated into the VEE VRP candidate vaccines targeted for clinical trial in South Africa.     

Genotypic and phenotypic analysis of HIV type 1 primary isolates from western Cameroon.

In this study we report the molecular and biological characteristics of 19 HIV-1 primary isolates obtained in April 1999 from 47 HIV-1-infected individuals living mainly in western Cameroon. Discontinuous portions of gag, pol, and env were amplified by polymerase chain reaction and directly sequenced. Phylogenetic analysis of these sequences showed that all were of HIV-1 group M with the following genotypes: A(gag)/A(pol)/A(env) (n = 4), A(gag)/AG(pol)/AG(env) (n = 2), AG(gag)/A(pol)/AG(env) (n = 1), AG(gag)/U(pol)/AG(env) (n = 1), AG(gag)/AG(pol)/AG(env) (n = 6), G(gag)/G(pol)/G(env) (n = 3), F2(gag)/F2(pol)/F2(env) (n = 1), and a novel A(gag)/J(pro/rt)/A(int)/U(env) complex recombinant (n = 1). This A/J/U recombinant shared the same gag-pol cross-over point with known CRF02.AG viruses and 99CMBD6, an AG recombinant from our panel of isolates. The biological phenotype of most of the isolates correlated with the clinical status of the patient. Six isolates were syncytium inducing (SI) on MT-2 cells whereas 13 isolates were of the non-syncytium-inducing phenotype (NSI). Coreceptor usage by these isolates determined on GHOST cells correlated with their biological phenotype, as all SI isolates used CXCR4 and all NSI isolates used CCR5. Our results show a high predominance of subtype A (mainly CRF02.AG-like viruses) in western Cameroon and fewer HIV-1 subtypes compared with other parts of Cameroon. Genetic variability was, however, not reflected in the biological characteristics of the isolates. The presence of a novel A/J/U complex recombinant from this region further emphasizes the role of recombination in the global evolution of HIV.     

Identification of a genetic subcluster of HIV type 1 subtype C (C') widespread in Ethiopia

Others and we have previously shown that subtype C is the predominant HIV-1 subtype and the major cause of AIDS in Ethiopia. The present study shows that subtype C in Ethiopia has a genetic subcluster, designated C', has not increased in frequency, or spread geographically, over the period 1988 (%C' = 23/53) to 1996-1997 (%C' = 26/50). There is no association of the HIV-1 subtype C or subcluster C' with geographic location, time of sample collection, or risk group in Ethiopia. Of 105 randomly collected samples representing 7 different towns in Ethiopia, all but 2 (1 subtype A from Addis Ababa, 1997 and 1 subtype D from Dessie, 1996) belong to subtype C.     

Near full-length clones and reference sequences for subtype C isolates of HIV type 1 from three different continents

Among the major circulating HIV-1 subtypes, subtype C is the most prevalent. To generate full-length subtype C clones and sequences, we selected 13 primary (PBMC-derived) isolates from Zambia, India, Tanzania, South Africa, Brazil, and China, which were identified as subtype C by partial sequence analysis. Near full-length viral genomes were amplified by using a long PCR technique, sequenced in their entirety, and phylogenetically analyzed. Amino acid sequence analysis revealed 10.2, 6.3, and 17.3% diversity in predicted Gag, Pol, and Env protein sequences. Ten of 13 viruses were nonmosaic subtype C genomes, while all three isolates from China represented B/C recombinants. One of them was composed primarily of subtype C sequences with three small subtype B portions in gag, pol, and nef genes. Two others exhibited these same mosaic regions, but contained two additional subtype B portions at the gag/pol overlap and in the accessory gene region, suggesting ongoing B/C recombination in China. All subtype C genomes contained a prematurely truncated second exon of rev, but other previously proposed subtype C signatures, including three potential NF-kappa B-binding sites in the viral promoter-enhancer regions, were found in only a subset of these genomes.     

A new human immunodeficiency virus type 1 circulating recombinant form from Tanzania

It is becoming increasingly important to identify and to study human immunodeficiency virus type 1 (HIV-1) circulating recombinant forms (CRFs) with evidence of epidemic spread, since mosaic strains arise frequently, especially in populations where multiple subtypes cocirculate. We describe the almost complete nucleotide sequence of 3 subtype C and D recombinant viruses, selected from a pool of 13 D(gag)-D/C/D(env) perinatally infected infants from Dar es Salaam, Tanzania. All three genomes had cross-over points with approximately the same genomic localization. The subtype C-like sequences were located within pol, vif, vpr, vpu, the first exons of rev and tat, V3, and the U3-R regions of the LTR. Phylogenetic analyses of the full-length genomic sequences from these viruses showed the formation of a distinct subcluster on the HIV-1 subtype D branch. The pattern of recombination of genomes belonging to this new CRF, named CRF10_CD, might have resulted from independent recombination events occurring at high frequency or from a single source that originated earlier in this population. Future surveys will be needed to determine the potential of this CRF for epidemic spread.     

Genetic and biological properties of HIV type 1 isolates prevalent in villagers of the Cameroon equatorial rain forests and grass fields: further evidence of broad HIV type 1 genetic diversity

To understand the evolution of HIV-1, the genetic and biological characteristics of viruses that infect persons living in regions in which the virus has been evolving for several decades must be studied. Thus, we investigated teh genetic subtypes, coreceptor usage, and syncytium-inducing ability of viruses in 47 HIV-1-infected blood samples from individuals living in rural villages in the equatorial rain forest and grass field regions in Cameroon. Heteroduplex mobility analysis (HMA) of gag (part of p24 and p7) and env (C2V5) or sequence and phylogenetic analysis of gag (part of p24 and p7), pol (protease), and env (C2V5), revealed a broad HIV-1 group M genetic diversity. Subtype analysis revealed genetic evidence of seven subtypes (A, C, D, F, G, H, and J) and three circulating recombinant froms (CRFs) (CRF01_AE, CRF02_AG, and CRF11_cpx). Only 15 (32%) of the 47 samples analyzed revealed a concordant subtype in all three genes (gag, pol, and env), while discordant subtypes and CRFs were identified for the remaining 32 (68%) samples. Two patterns of HIV-1 diversity could be discerned in two provinces. While more concordant subtypes in gag, pol, and env genes were identified in villages of South province (10 of 13, 77%), the HIV-1 diversity in the West province was characterized by intersubtype recombinants (63%). Five new intersubtype recombinants were identified including Agag Jpol Genv, Ggag Upol Aenv, AGgag Jpol Aenv, Agag AGpol Henv, and Cgag AGpol AGenv. All of the 40 viruses tested used the R5 coreceptor, of which four also used the X4 coreceptor. Four viruses were able to induce syncytia in MT-2 cells, however, syncytium induction did not correlate with coreceptor usage. This study further reveals the complexity of HIV-1 infection in rural Cameron and points to the future of the global epidemic, which may be characterized by more genetically diverse viruses.     

Identification of human immunodeficiency virus type 1 subtypes B and F B/F recombinant and dual infection with these subtypes in Argentina.

DNA sequences encoding the third variable region (V3) of human immunodeficiency virus type-1 (HIV-1) envelope glycoprotein gp120 were obtained from 18 infected individuals residing in different regions of Argentina. Proviral DNA representing the env V3 region was obtained by PCR from uncultured peripheral blood mononuclear cells (PBMC) and genetic heterogeneity was examined by phylogenetic analysis. Sequences representing the gag p17 region were also obtained for a subset of these samples. Moreover, 1 sample that it was not possible to classify according to initial phylogenetic analysis was further analysed by molecular cloning of both V3 and p17 regions. Phylogenetic analysis according to different methodologies were performed comparing obtained sequences with a set of reference sequences representing previously characterized HIV-1 subtypes. The recombinant identification program (RIP) was used to study the presence of possible recombinant sequences. Phylogenetic analysis demonstrated that viruses representing both subtypes B and F are circulating among HIV-1 infected individuals in Argentina. In addition, RIP analysis showed that an initially unclassified sequence exhibited similarities to subtypes B and F in different fragments of the V3 region. Separate phylogenetic analysis of each of these fragments revealed divergent clustering, suggesting that this sequence harbours a point of recombination within the V3 loop. Interestingly, we also identified a dually infected individual with viruses belonging to subtypes B and F, as demonstrated by molecular cloning analysis of the env V3 and the gag p17 regions. Taken together, our study shows that both subtypes B and F are circulating in different regions of Argentina. Moreover, the data presented here show that dual infections with subtypes B and F can occur, and consequently B/F recombinant sequences are arising in the region.     

High prevalence of diverse forms of HIV-1 intersubtype recombinants in Central Myanmar: geographical hot spot of extensive recombination

OBJECTIVES: To investigate the molecular epidemiology and genetic structure of HIV-1s causing the epidemic in Central Myanmar and to explore the genesis of HIV epidemic in this area. DESIGN: A molecular epidemiological investigation was conducted in 1999-2000 in the city of Mandalay among high-risk populations and the structural features of circulating HIV-1s were analyzed. METHODS: HIV-1 genotypes of 59 specimens were screened based on gag (p17) and env (C2/V3) regions. Near full-length nucleotide sequences of HIV-1 isolates with subtype discordance were determined and their recombinant structures were characterized. RESULTS: Three lineages of HIV-1 strains, including CRF01_AE (27, 45.8%), subtype B' (Thailand variant of subtype B) (15, 25.4%) and subtype C (8, 13.6%), were distributed in Mandalay, while substantial portions (9, 15.3%) of specimens showed various patterns of subtype discordance in different regions of HIV-1 genomes. The study on six HIV-1 isolates with subtype discordance revealed that they were highly diverse types of unique recombinant forms (URFs) comprised of various combinations of three circulating subtypes. One URF was a particularly complex mosaic that contained 13 recombination breakpoints between three HIV-1 subtypes. Approximately half of recombinants showed 'pseudotype' virion structures, in which the external portions of envelope glycoproteins were exchanged with different lineages of HIV-1 strains, suggesting the potential selective advantage of 'pseudotype' viruses over parental strains. CONCLUSION: The study revealed the unique geographical hot spot in Central Myanmar where extensive recombination events appeared to be taking place continually. This reflects the presence of highly exposed individuals and social networks of HIV-1 transmission.     

Subtypes A,C, G,and recombinant HIV type 1 are circulating in Bangladesh

We analyzed the genetic diversity of HIV-1 circulating in Bangladesh by direct sequencing and subsequent phylogenetic analysis of the V3 region of the env gene and p17 fragment of the gag gene from nine unrelated patients. The sequences from one sample grouped into subtype A, five samples grouped into subtype C, and one grouped into subtype G. In addition, two patients appeared to be infected with different recombinant viruses consisting of subtype A and unclassifiable viral sequences. Epidemiological analysis revealed heterosexual transmission in the majority of cases. Furthermore, most subjects had a history of traveling, either to India or to the Arabian Peninsula. This study shows that several HIV-1 subtypes are circulating in Bangladesh, and we conclude that there must have been several introductions of HIV-1 into the Bangladeshi population.     

Full-length gag sequences of HIV type 1 subtype C recent seroconverters from Pune, India

Although HIV-1 subtype C is the most prevalent subtype worldwide, data on subtype C viruses are rather limited. Very little information is available on the complete HIV-1 subtype C gag sequences from India. We report full-length gag (p55) sequences from six Indian early seroconverters. The samples were collected within few weeks of seroconversion and may represent immunologically naive viruses. The comparison of p55 sequences with other Indian and non-Indian subtype C sequences as well as with nonsubtype C sequences obtained from the Los Alamos database revealed gag as a well-conserved region of the HIV genome (range: 84-95%). The phylogenetic tree indicated that the sequences compared here cluster together within clade C. Two epitopes in the p24 region of the gag gene were subtype C specific while many epitopes in the same region were also present in other clades. The data on HIV-1 subtype C full-length gag sequences would be useful in the design and evaluation of effective subtype C-based HIV vaccines.     

Spreading of HIV-1 subtype G and envB/gagG recombinant strains among injecting drug users in Lisbon,Portugal

We have evaluated the genetic diversity of HIV-1 strains infecting injecting drug users (IDUs) in Lisbon, Portugal. Heteroduplex mobility assay and/or phylogenetic analysis revealed that env (C2V3C3 or gp41) subtype B is present in 63.7% of the 135 viral samples studied, followed by subtypes G (23.7%), A (6.7%), F (5.2%), and D (0.7%). Similar analysis of gag (p24/p7) performed on 91 of the specimens demonstrated that 49.5% of the infections were caused by subtype G viruses; other gag subtypes identified were B (39.5%), F (3.3%), A and D (1.1.% each), and the recombinant circulating form CRF02_AG (5.5%). Discordant env/gag sub-types were detected in 34.1% of the strains and may reflect the presence of dual infections and/or recombinant viruses. The presumptive B/G recombinant form was highly predominant (21 of 31). The genetic pattern of HIV-1 subtype B and G strains is suggestive of multiple introductions and recombination episodes and of a longstanding presence of both subtypes in the country. C2V3C3 amino acid sequences from IDU-derived subtype G viruses presented highly significant signatures, which distinguish the variants from this transmission group. The unusually high prevalence of subtype G sequences (34.1%), independent of the geographic origin of the infected individuals, makes this IDU HIV-1 epidemic unique.     

Sequence analysis of env C2/V3, gag p17/p24, and pol protease regions of 25 HIV type 1 isolates from Ho Chi Minh City, Vietnam

Env C2/V3, gag p17/p24, pol protease, and RT regions of HIV-1 isolates recently obtained from 25 HIV-1 seropositive individuals from Ho Chi Minh City (Vietnam) were studied, and genes subtypes were determined by DNA sequence analyses. Twenty-three isolates out of 25 were identified as belonging to subtype E, now recognized as circulating recombinant form 1 (CRF01_AE). The motif at the top of the V3 loop (generally GPGQ) was then preceded by an isoleucine or a methionine (M) residue; the M residue might be a local signature of Vietnamese E isolates compared to Thai E viruses. Two isolates (8%) were shown to be intersubtype recombinants: one E/B and one CRF02_AG(IBNG)/D. The polymorphism of pol protease was considered only for CRF01_AE isolates and is clearly different from that recorded for B viruses with substitutions at positions 13, 35, 36, 41, 69, and 89.     

Evidence of a novel B/C recombinant exhibiting unique breakpoints of near full-length HIV type 1 genome from Northeastern India

Despite the predominance of the HIV-1 clade C in India, the presence of other subtypes and recombinants has been reported. Here we report the identification of a novel HIV-1 B/C recombinant isolated from Northeast India and characterized near full length genome of the recombinant virus. Bootscan analysis of the nearly full-length genome showed a unique mosaic structure consisting of a subtype B backbone with three subtype C genome insertions. Breakpoint analyses revealed insertion of fragments belonging to subtype C at positions 1853-2223 in gag and 3025-3759 and 3998-5073 in pol. Phylogenetic analysis revealed that the segments of subtype B clustered with sequences of subtype B viruses reported from Thailand whereas segments of subtype C clustered with sequences of subtype C viruses reported from India. We report the mosaic structure that is distinct to HIV-1 B/C recombinant viruses reported to date.