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.     

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.     

A cluster of HIV type 1 subtype C sequences from Ethiopia, observed in full genome analysis, is not sustained in subgenomic regions

The impact of HIV-1 genetic diversity on candidate vaccines is uncertain. One approach to minimize genetic diversity in the evaluation of HIV-1 vaccines is to match the vaccine sequence to the predominant subtype in a vaccine cohort. Over two million Ethiopians are infected with HIV-1, and the predominant subtype is thought to be subtype C. Understanding the phylogenetic relationships between sequences from Ethiopia and within subtype C can help decide what sequence(s) should comprise a candidate vaccine. To that end, nearly full genome sequencing was used to characterize HIV-1 from volunteers who emigrated from Ethiopia. DNA extracted from peripheral blood mononuclear cells (PMBC) was amplified using primers in the long terminal repeats to generate nearly full-length genomes. Amplicons were directly sequenced with dye terminators and automated sequencers. Sequences were phylogenetically analyzed by neighbor joining. The six new Ethiopian sequences were all subtype C, consistent with previous partial and full genome analysis. Together with two other Ethiopian sequences, the new sequences formed a geographic cluster when the complete genome was analyzed. However, subgenomic trees showed only a weak geographic cluster, or none, with respect to Ethiopian strains. Although immunological responses must be considered, from a phylogenetic perspective, there is no compelling support for use of Ethiopian subtype C sequences, compared to other subtype C, as vaccine prototype strains.     

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.     

Genetic evidence of multiple transmissions of HIV type 1 subtype F within Romania from adult blood donors to children

We studied the phylogeny of HIV-1 subtype F viruses from children and adults in Romania in order to (1) clarify whether the Romanian subtype F epidemic was caused by one or several virus introductions and (2) gain insight into the route of spread of the HIV-1 subtype F virus among children and adults in Romania. env (V3), gag (p17/half p24), and pol (prot/half RT) sequences were obtained from three districts in Romania: Tirgu Mures (n = 9, children), Craiova (n = 15, children), and Bucharest (n = 13, adults). Of 37 HIV V3 sequences from Romania, 35 belonged to the genetic subtype F in the neighbor-joining tree, whereas 2 sequences from adults clustered with subtypes A and C. Within the subtype F cluster, no bootstrap-supported subclusters were observed according to geographic area in Romania. Two of the adult V3 sequences that clustered with the children were obtained from individuals who tested HIV seropositive in 1989 and 1990, showing that the subtype F virus was present among adults when the HIV epidemic began among children in Romania. The HIV-1 subtype F viruses obtained from children showed a mean pairwise V3 nucleotide distance of 7.9% and maximum distances of between 18 and 19%; both are higher than previously described. The mean V3 distances (overall, synonymous, and nonsynonymous) were significantly higher for adults than for children. One V3 sequence from the Democratic Republic of Congo clustered within the Romanian sequences, suggesting that the subtype F virus in Romania may originate from this area. Our data also suggest that HIV-1 subtype F was present among Romanian adults before it appeared in 1989 among institutionalized children. The juvenile population was most likely infected with the HIV-1 subtype F virus on more than one occasion, presumably through HIV-contaminated blood (products) obtained from adults.     

Phylodynamic and phylogeographic patterns of the HIV type 1 subtype F1 parenteral epidemic in Romania

In the late 1980s an HIV-1 epidemic emerged in Romania that was dominated by subtype F1. The main route of infection is believed to be parenteral transmission in children. We sequenced partial pol coding regions of 70 subtype F1 samples from children and adolescents from the PENTA-EPPICC network of which 67 were from Romania. Phylogenetic reconstruction using the sequences and other publically available global subtype F sequences showed that 79% of Romanian F1 sequences formed a statistically robust monophyletic cluster. The monophyletic cluster was epidemiologically linked to parenteral transmission in children. Coalescent-based analysis dated the origins of the parenteral epidemic to 1983 [1981-1987; 95% HPD]. The analysis also shows that the epidemic's effective population size has remained fairly constant since the early 1990s suggesting limited onward spread of the virus within the population. Furthermore, phylogeographic analysis suggests that the root location of the parenteral epidemic was Bucharest.     

Highly divergent HIV type 1 group M sequences evident in Karonga District,Malawi in early 1980s

Six divergent HIV-1 partial env and gag genome sequences have been characterized in five subjects in Malawi, from whom blood spot samples were collected between 1982 and 1989, at the time that the AIDS epidemic there was starting. These sequences could not be classified with any of the recognized subtypes or circulating recombinant forms of HIV-1. They showed no consistent and/or supported associations with other subtypes by either env or gag gene phylogenetic analysis. Their genetic distances from defined subtypes suggest that they may be diverse subsubtype C viruses or, alternatively, that they may have mosaic genomes. Bootscanning analyses are consistent with their being mosaic viruses. These sequences highlight early HIV-1 diversity in a population otherwise dominated by subtype C.     

Complete genome sequencing of a non-syncytium-inducing HIV type 1 subtype D strain from Cape Town, South Africa

The fast growing HIV-1 epidemic in South Africa is mainly caused by HIV-1 group M subtype C, spreading via heterosexual transmission. In South Africa HIV-1 subtype B and D viruses were responsible for the initial epidemic during the 1980s, primarily in the homosexual population. This study describes the full-length PCR amplification and sequencing of an HIV-1 subtype D strain recovered from plasma from a sample taken during 1990. This is only the second full-length non-syncytium-inducing (NSI) subtype D strain described. Although restricted, the subtype D strain is still being detected in the South African population.     

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.     

HIV type 1 genetic variability in the northern part of Cameroon.

In 1995, 53 blood samples from Muslim patients with AIDS, or who were thought to have AIDS, were collected in the main hospitals of Adamaoua Province, in the northern part of Cameroon. The variable env C2V3 region of HIV-1 was amplified by nested PCR and phylogenetically analyzed. The results indicated that of 15 amplified samples, 1 belonged to HIV-1 group O, 1 to HIV-1 subtype D, 1 to subtype G, 2 to subtype H, and 10 to subtype A. Furthermore, the northern Cameroonian subtype A could be divided into at least two subclusters as shown by the env tree as well as by two remarkably conserved hexameric amino acid sequences in the apex of V3 (GPGQAF in one subcluster and GPGQTF in the other). This distinction suggests that the HIV-1 subtype A circulating in northern Cameroon evolved from two main sources. More recently, three HIV-1 strains from Nigeria (IBNG) and Djibouti (DJ263 and DJ264), previously reported on the basis of their env C2V3 sequences as subtype A, were found to have a similar A/G mosaic structure alongside their full-length sequence and were tentatively designated as members of a new subtype called "IBNG." Interestingly, within the northern Cameroonian subtype A described, the isolates of the second subcluster clustered distinctly with these A/G mosaic strains, strongly suggesting that they may be members of the IBNG subtype.     

Silent mutation in the V3 region characteristic of HIV type 1 env subtype B strains from injecting drug users in the former Soviet Union

New independent states of the former Soviet Union are facing a rapidly growing epidemic of HIV-1 among injecting drug users (IDUs). This epidemic is caused by three HIV-1 populations, one belonging to HIV-1 subtype A (IDU-A), another to subtype B (IDU-B), and the third being a recombinant of the IDU-A and IDU-B viruses (IDU-A/B, gagA/envB). Each of these populations is characterized by a high level of genetic homogeneity. We identified a unique synonymous nucleotide substitution in the first isoleucine codon at the IHIGPGR motif (ATT), which was observed in the env subtype B V3 sequences derived from IDUs in Russia and the Ukraine. This substitution was observed in none of 179 sequences obtained from IDUs in western Europe, northern America, and Asia. Molecular epidemiological analysis of HIV-1 strains based on this sequence pattern could be useful for tracing the origin and spread of the IDU-B viruses to other countries and risk groups.     

A distinctive clade B HIV type 1 is heterosexually transmitted in Trinidad and Tobago

HIV-1 transmission worldwide is predominantly associated with heterosexual activity, and non-clade B viruses account for the most spread. The HIV-1 epidemic in Trinidad/Tobago and the Caribbean shares many features with such heterosexual epidemics, including a prominent role for coincident sexually transmitted diseases. This study evaluates the molecular epidemiology of HIV-1 in Trinidad/Tobago during a period when abrupt transition from homosexual to heterosexual transmission occurred in the absence of injecting drug use, concomitant with a rapid rise in HIV-1 prevalence in the heterosexual population. Of 31 viral isolates studied during 1987-1995, all cluster with subtype B reference strains. In the analysis of full env genes from 22 early seroconverters, the Trinidad isolates constitute a significant subcluster within the B subtype. The Trinidad V3 consensus sequence differs by a single amino acid from the prototype B V3 consensus and demonstrates stability over the decade of this study. In the majority of isolates, the V3 loop of env contains a signature threonine deletion that marks the lineage of the Trinidad HIV-1 clade B epidemic from pre-1984. No phenotypic features, including syncitium induction, neutralization profiles, and chemokine receptor usage, distinguish this virus population from other subtype B viruses. Thus, although the subtype B HIV-1 viruses being transmitted in Trinidad are genetically distinguishable from other subtype B viruses, this is probably the result of a strong founder effect in a geographically circumscribed population rather than genetic selection for heterosexual transmission. These results demonstrate that canonical clade B HIV-1 can generate a typical heterosexual epidemic.     

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.     

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.     

Rapid spread and genetic diversification of HIV type 1 subtype C in a rural area of southern Mozambique

In this study, we analyzed the human immunodeficiency type 1 (HIV-1) viruses circulating between 1999 and 2004 in antiretroviral-naive women from a rural area of southern Mozambique. Nucleotide sequencing of the HIV-1 long terminal repeat (LTR) U3, envelope (env) C2V3C3, and protease (pr) genomic regions was performed from women sera samples collected in 1999 and 2004. Phylogenetic analysis revealed that all amplified sequences belonged to subtype C. Although env sequences were predominantly CCR5-tropic (R5), CXCR4-tropic (X4) variants were also identified (13%). Both 1999 and 2004 sequences were widely dispersed across multiple clusters and were related to different reference sequences from neighboring countries. Sequences from 2004 showed significantly more nucleotide genetic diversity than sequences from 1999. Importantly, genetic diversification was also observed at the pr and env amino acid level, suggesting that positive selection forces were implicated in the viral diversification. These results indicate the rapid spread and diversification of subtype C virus in Mozambique where HIV-1 prevalence in the Manhi?a antenatal clinic reached 23% in 2004.     

Identification and molecular characterization of subsubtype A4 in central Africa

Phylogenetic analysis on partial env sequences of HIV-1-positive samples obtained from sentinel population groups in HIV serosurveillance studies in 1997 and 2002 in the Democratic Republic of Congo (DRC) revealed a high genetic diversity in the number of cocirculating subtypes/CRFs as well as intrasubtype diversity. A cluster of three strains that formed a distinct and well-separated group within the subtype A radiation was observed. To determine a more precise structure of these viruses, the full-length genomes were sequenced. Phylogenetic tree and bootscan analysis showed that all three newly characterized viruses formed a new nonrecombinant lineage that was more closely related to subtype A. The SUDI (Subtype Distance) program showed that the distances of the newly derived HIV-1 sequences to subtype A references fell in the range of distances previously characterized for subsubtypes. According to current nomenclature rules, we have thus designated this new lineage as subsubtype A4. Analysis of viral sequences from other African countries suggests that the A4 strains seem restricted to DRC where they already circulated at the onset of the HIV/AIDS epidemic.     

The HIV type 1 epidemic in Bulgaria involves multiple subtypes and is sustained by continuous viral inflow from West and East European countries

Little is known about the HIV-1 epidemic in Balkan countries. To fill the gap, we investigated the viral genetic diversity in Bulgaria, by sequencing and phylogenetic characterization of 86 plasma samples collected between 2002 and 2006 from seropositive individuals diagnosed within 1986-2006. Analysis of pol gene sequences assigned 51% of the samples to HIV-1 subtype B and 27% to subtype A1. HIV-1 subtype C, F, G, H, and a few putative recombinant forms were also found. Phylogenetic and molecular clock analysis showed a continuous exchange of subtype A and B between Bulgaria and Western as well as other Eastern European countries. At least three separate introductions of HIV-1 subtype A and four of HIV-1 subtype B have occurred within the past 25 years in Bulgaria. The central geographic location of Bulgaria, the substantial genetic heterogeneity of the epidemic with multiple subtypes, and the significant viral flow observed to and from the Balkan countries have the potential to modify the current HIV-1 epidemiological structure in Europe and highlight the importance of more extensive and continuous monitoring of the epidemic in the Balkans.     

HIV-1 Subtype C Phylodynamics in the Global Epidemic

The diversity of HIV-1 and its propensity to generate escape mutants present fundamental challenges to control efforts, including HIV vaccine design. Intra-host diversification of HIV is determined by immune responses elicited by an HIV-infected individual over the course of the infection. Complex and dynamic patterns of transmission of HIV lead to an even more complex population viral diversity over time, thus presenting enormous challenges to vaccine development. To address inter-patient viral evolution over time, a set of 653 unique HIV-1 subtype C gag sequences were retrieved from the LANL HIV Database, grouped by sampling year as <2000, 2000, 2001-2002, 2003, and 2004-2006, and analyzed for the site-specific frequency of translated amino acid residues. Phylogenetic analysis revealed that a total of 289 out of 653 (44.3%) analyzed sequences were found within 16 clusters defined by aLRT of more than 0.90. Median (IQR) inter-sample diversity of analyzed gag sequences was 8.7% (7.7%; 9.8%). Despite the heterogeneous origins of analyzed sequences, the gamut and frequency of amino acid residues in wild-type Gag were remarkably stable over the last decade of the HIV-1 subtype C epidemic. The vast majority of amino acid residues demonstrated minor frequency fluctuation over time, consistent with the conservative nature of the HIV-1 Gag protein. Only 4.0% (20 out of 500; HXB2 numbering) amino acid residues across Gag displayed both statistically significant (p<0.05 by both a trend test and heterogeneity test) changes in amino acid frequency over time as well as a range of at least 10% in the frequency of the major amino acid. A total of 59.2% of amino acid residues with changing frequency of 10%+ were found within previously identified CTL epitopes. The time of the most recent common ancestor of the HIV-1 subtype C was dated to around 1950 (95% HPD from 1928 to 1962). This study provides evidence for the overall stability of HIV-1 subtype C Gag among viruses circulating in the epidemic over the last decade. However selected sites across HIV-1C Gag with changing amino acid frequency are likely to be under selection pressure at the population level.