High proportion of unrelated HIV-1 intersubtype recombinants in the Mbeya region of southwest Tanzania

BACKGROUND: In Mbeya, a rural region of southwest Tanzania, HIV-1 subtypes A, C and D have been co-circulating since the early 1990s. OBJECTIVE: To define to what extent the co-existence of subtypes has led to recombinant HIV-1 strains and whether there is evidence for epidemic spread of any circulating recombinant form. METHODS: Nine HIV-1-seropositive young adults from Mbeya Town with no evident high-risk behaviour contributed peripheral blood mononuclear cells for this study. Nine virtually full-length-genome-sequences were amplified from this DNA and phylogenetically analysed. RESULTS: Out of the nine samples, two were subtype A (22%), two were subtype C (22%) and five were recombinants (56%): four A/C recombinants and one C/D recombinant. None of the recombinants were related to each other; all of them had different mosaic structures. Most of the genome in the recombinants was subtype C. CONCLUSION: A high proportion of unrelated intersubtype recombinants, none of them apparently spreading in the population, may be present in southwest Tanzania.     

Construction of an infectious HIV type 1 molecular clone from an African patient with a subtype D/C Recombinant Virus

The majority of HIV-1 infections worldwide occur in Africa, where subtype B viruses are rare and intersubtype recombinants are common. Pathogenesis and vaccine studies need to focus on viruses derived from African patients, and infectious HIV-1 molecular clones can be useful tools. To clone non-B subtypes and recombinant viruses from patients, we cultivated HIV-1 from the plasma of a Kenyan long-term survivor. Viral DNA was cloned into a plasmid, which was transfected into COS cells; progeny virus was propagated in PBMCs. Sequence analyses revealed that both the patient's plasma HIV-1 RNA and the cloned DNA genomes were recombinants between subtypes D and C; subtype C sequences comprised the nef and LTR regions. The cloned virus used the CCR5 coreceptor and did not form syncytia in vitro. This infectious HIV-1 subtype D/C recombinant molecular clone obtained from a Kenyan long-term survivor promises to be useful to study pathogenesis and vaccine design.     

Forty-one near full-length HIV-1 sequences from Kenya reveal an epidemic of subtype A and A-containing recombinants

OBJECTIVE: To further define the genetic diversity of HIV-1 in Kenya using approaches that clearly distinguish subtypes from inter-subtype recombinants. DESIGN: Near full genome sequencing and analysis were used, including sensitive new tools for detection and mapping of recombinants. METHODS: Purified peripheral blood mononuclear cell DNA from 41 HIV-1 positive blood donations collected from six hospitals across southern Kenya was used to amplify near full-length genomes by nested PCR. These were sequenced on an ABI 3100 automated sequencer and analyzed phylogenetically. RESULTS: Among 41 near full-length genomes, 25 were non-recombinant (61%) and 16 were recombinant (39%). Of the 25 pure subtypes, 23 were subtype A, one was subtype C and one was subtype D. Most recombinants consisted of subtype A and either subtype C or subtype D; a few contained A2, a recently identified sub-subtype. Two A2/D recombinants had identical breakpoints and may represent a circulating recombinant form. A third A2/D recombinant had the same structure as a previously described Korean isolate, and these may constitute a second A2-containing circulating recombinant form. CONCLUSIONS: In Kenya, 93% of HIV-1 genomes were subtype A or A-containing recombinant strains. Almost 40% of all strains were recombinant. Vaccine candidates tested in Kenya should be based on subtype A strains, but the methods used for evaluation of breakthrough infections during future vaccine trials should be capable of identifying non-A subtypes, the A2 sub-subtype, and recombinants.     

Emerging recombinant human immunodeficiency viruses: uneven representation of the envelope V3 region.

OBJECTIVE: To determine whether the envelope V3 region from HIV-1 subtypes A, C or D had the same probability of being present in intersubtype recombinant genomes. MATERIALS AND METHODS: The envelope C2-C5 and the gag p24-p7 regions from one hundred infants infected perinatally in Tanzania were compared using phylogenetic and recombination analysis. Exact binomial and Fisher's exact tests were used to assess if various genomic regions were more likely to be overrepresented in intersubtype recombinants. RESULTS: Of one hundred HIV-1 positive infants analyzed, twenty-two (22%) showed exclusively subtype A sequence in gag and env. Subtype C accounted for twenty-two infants (22%) whereas nineteen infants (19%) were infected by HIV-1 subtype D. Intersubtype recombinant genomes accounted for thirty-seven infections (37%). The V3 region from subtype A was found in all fifteen A-D recombinants (P = 0.00003) and the V3 region from subtype C was found in all twelve C-D recombinants (P = 0.0002). Conversely, subtype D gag sequences were preferentially represented in the gag of A-D recombinants (P = 0.0003) as well as C-D recombinants (P = 0.002). In A-D recombinants, the V3 region of subtype A was generally surrounded by subtype A C3-C5 sequences. In contrast, the V3 region from subtype C was surrounded by subtype D C3-C5 sequences in C-D recombinants. Significant differences were not found in the number of subtype A or subtype C sequences in A-C recombinants. CONCLUSION: We have shown that several recombinant HIV-1 viruses have been generated and efficiently transmitted to infants in Tanzania. The recombination patterns showed that the V3 region of subtypes A or C was always selected in A-D and C-D recombinants. This selection suggests that the fitness of subtype D-V3 in perinatal transmission may be reduced with respect to V3 from subtype A and/or subtype C. The elevated number of recombinants transmitted perinatally suggests that co-infection or super-infection by two HIV-1 subtypes is not uncommon in this population.     

Among 46 near full length HIV type 1 genome sequences from Rakai District,Uganda, subtype D and AD recombinants predominate

The impact of HIV-1 genetic diversity on candidate vaccines is uncertain. To minimize genetic diversity in the evaluation of HIV-1 vaccines, vaccine products must be matched to the predominant subtype in a vaccine cohort. To that end, full genome sequencing was used to detect and characterize HIV-1 subtypes and recombinant strains from individuals in Rakai District, Uganda. DNA extracted from peripheral blood mononuclear cells (PMBC) was PCR amplified using primers in the long terminal repeats (LTRs) to generate nearly full length genomes. Amplicons were directly sequenced with dye terminators and automated sequencers. Sequences were phylogenetically analyzed and recombinants were detected and mapped with distance scan and bootscan. Among 46 sequences, 54% were subtype D, 15% were subtype A, and 30% were recombinant. All recombinants were individually unique, and most combined subtypes A and D. Subtype D comprised more than 70% of all the HIV-1 genomes in Rakai when both pure subtypes and recombinants were considered. Candidate vaccines based on HIV-1 subtype D would be appropriate for evaluation in Rakai District, Uganda.     

HIV type 1 sequence diversity and dual infections in Kenya

As vertical transmission of HIV-1 is an ongoing problem in East Africa, we analyzed HIV-1 strains of infected mothers, from Kisumu, Kenya. We sequenced the gag and gp120 regions from peripheral blood mononuclear cells (PBMC) of 15 HIV-infected mothers attending an antenatal clinic. PCR, cloning, bootscanning, using the program Simplot, and phylogenetic analyses were conducted to assign subtypes and identify recombinants. Our analyses showed two dual infections from patients who had infections with pure subtypes and recombinants subtype D. In addition, we also noted the presence of subsubtype A1 and A2, as well as unique recombinants in this area. These results imply that the HIV epidemic in western Kenya is a dynamic one and is continually evolving. Therefore, continued monitoring of the epidemic in this region is necessary if a vaccine for the area is to be developed.     

Detection of HIV-1 subtypes,recombinants, and dual infections in east Africa by a multi-region hybridization assay

OBJECTIVE: To enable more rapid and efficient genotyping of HIV-1 in East Africa, where subtypes A, C, and D and their recombinants are co-circulating. DESIGN: Full-genome sequencing of HIV-1 provides complete discrimination of subtypes and recombinant forms but is costly and low-throughput compared to other genotyping approaches. Here we describe the development and evaluation of a Multi-region Hybridization Assay (MHA) for the efficient determination of HIV-1 subtypes A, C, D, recombinants, and dual infections. METHODS: Five genome regions containing clustered mutations distinguishing subtypes A, C, and D were identified and used to design subtype-specific probes. DNA from primary peripheral blood mononuclear cells was used as template for real-time PCR using the fluorescent, subtype-specific probes. RESULTS: A panel of 45 clinical samples from Uganda, Kenya, and Tanzania, previously characterized by full-genome sequencing and including 26 pure subtypes and 19 recombinant strains, was evaluated by MHA. The MHA provided 90% sensitivity and 98% specificity for the three subtypes, efficiently discriminated subtypes from recombinant forms, and detected several dual infections. CONCLUSIONS: Accurate and efficient genotyping of HIV-1 strains in vaccine trial populations in East Africa, ascertainment of dual infections, and elucidation of the genesis of recombinant forms in individuals can be facilitated by the application of MHA.     

Recombination following superinfection by HIV-1

BACKGROUND: There is increasing recognition of recombinant HIV-1 strains globally, but it has been unclear whether recombination results from superinfection during untreated, chronic infection. OBJECTIVE: To search for evidence of recombination and superinfection in Africa, where multiple HIV-1 subtypes facilitate identification of strains. METHODS: Serial blood samples from highly exposed, chronically infected women in Nairobi's Pumwani sex workers cohort were examined. Serial, complete HIV-1 RNA sequence analyses were performed for seven untreated long-term survivors. Sequences were subjected to computational analysis. RESULTS: One woman had evidence of both superinfection and recombination. Complete HIV-1 RNA sequences were first derived from plasma obtained in 1986, when the woman had been HIV seropositive for at least 21 months; this sequence was entirely subtype A. The sequences obtained from plasma in 1995 and 1997, however, were subtype A/C recombinants with a SimPlot demonstrating that the subtype A fragment in 1995 and 1997 was derived from the original 1986 A sequence. Heteroduplex tracking assays demonstrated that the subtype C sequences were not detectable as minor species in 1986. CONCLUSION: Intersubtype recombination took place between the original non-recombinant subtype A strain and the superinfecting subtype C strain in an untreated, chronically infected woman. This finding helps to explain the rising prevalence of recombinant HIV-1 worldwide. Recombination resulting from superinfection with diverse strains may pose problems for eliciting broad immune responses necessary for an effective vaccine.     

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.     

No evidence for recombination between HIV type 1 and HIV type 2 within the envelope region in dually seropositive individuals from Senegal

To investigate the frequency of recombination between HIV-1 and HIV-2 in vivo during dual infection, we performed a retrospective analysis of blood samples from 46 dual HIV-1/HIV-2-seropositive adults for evidence of recombination. HIV viral DNA from peripheral blood mononuclear cells (PBMC) was subjected to two separate nested polymerase chain reaction (PCR) assays using opposing HIV-1 and HIV-2 primer pairs selected to flank a approximately 650-base pair region including the V3 loop of the envelope gene. In the first assay, primers were chosen to amplify recombinants with HIV-1 on the 5' end and HIV-2 on the 3' end, and in the second assay, primers were chosen to amplify recombinants with the opposite orientation. All PCR experiments were run in parallel with positive controls consisting of partial-length env fragments bearing a single central HIV-1/2 recombination site, and appropriate primer-binding sites on each end. The limit of detection for both assays was <10 copies of recombinant product per 150,000 cell equivalents of input PBMC DNA. In all 46 dually seropositive patients in this study, PCR screening of PBMC failed to detect evidence of HIV-1/HIV-2 recombinants in the C2-V5 env region. Although genetic recombination between HIV-1 and HIV-2 may occur, we conclude that any such events within env are exceedingly rare, and do not result in the outgrowth of recombinant strains.     

Accumulation of defective HIV-1 variants in a patient with slow disease progression

Viral population in a long term non progressor carrying CRF02-AG HIV-1 virus variants with a truncated RT gene and attenuated virus replication was analyzed. The proportion of mutant and wild-type RT sequences was determined by clonal analysis of HIV-1 DNA and RNA from blood samples and peripheral blood mononuclear cell (PBMC) culture supernatants. Recombinant HIV-1 strains were generated by reverse genetics to evaluate the replicative capacity of RT variants in PBMC cultures. HIV-1 RNA and DNA sequences in PBMC cultures showed a mixture of stop codons (RT(STOP)), recombinant forms, (RT(RF)), and full length (RT(FL)) strains. In plasma, proportion of HIV-1 RNA sequences with a truncated RT gene fluctuated over time (0% in 2005, 100% in 2007 and 8.3% in 2010), while in proviral DNA was constant (96.5% to 100%). Reconstituted RT(STOP) strains were unable to replicate in PBMC. However, RT(FL) strains could trans-complement the loss of function of RT(STOP) variants. In vivo selection of stop codons in the RT gene resulted in the accumulation of replication-defective virus strains. Nevertheless, the observed release of defective viral particles in plasma was probably the result of viral protein complementation between replication-competent and replication-incompetent HIV-1 variants. The divergence in the proportion of RT(STOP) and RT(FL) variants as well as in the mutation's pattern to antiretroviral drug resistance between HIV-1 plasma RNA and PBMC proviral DNA, suggested that circulating lymphocytes expressing full-length RT might be negatively selected for by a specific T-cell response, possibly contributing to the slow progression to AIDS observed in this patient.     

Circulation of novel HIV type 1 A, B/C, and F subtypes in Argentina

The Argentine HIV-1 epidemic is considered to be represented mainly by subtype B and diverse B/F recombinants, with apparent absence of pure subtype F. In this study we describe three novel HIV-1 variants isolated from four infants born in different and distant provinces of Argentina. Partial analysis of different gene fragments spanning 18.5-40.8% of the HIV-1 complete genome revealed two subtype A HIV-1 strains in siblings, a B/C recombinant with a novel mosaic structure, and a putative subtype F. Characteristic patterns of genomic and amino acid sequences of the newly reported subtype F isolate suggest a closer genetic relationship to Argentine B/F recombinants than any other subtype F strain described so far, while the A and B/C subtypes found correspond to unusual genotypes in Argentina. Understanding the origin, diversity, and spread of HIV-1 strains worldwide will be necessary for the development of an effective vaccine approach.     

Characterization of subtype A HIV-1 from Africa by full genome sequencing.

OBJECTIVE: To improve our understanding of the genetic complexity of HIV-1 subtype A by increasing the number of subtype A isolates that have been sequenced in their entirety. METHODS: Nine HIV-1-seropositive patients from Africa living in Sweden contributed peripheral blood mononuclear cells (PBMC) for this study. Sequencing of the C2-V3 region of env had shown them to be subtype A. DNA from virus cultures was used for the amplification of virtually full-length proviral sequences, and the resulting fragment was sequenced. RESULTS: Six of the nine viral isolates were subtype A throughout the genome, or non-recombinant, and all of these were from east Africa. One virus from the Ivory Coast had the AG(IbNG) genetic form, a recombinant form common in west Africa. Two of the isolates were novel recombinants: one was an A/C recombinant and the other was A/D. Analysis of gag reveals three subclusters within the A subtype: one containing the AG(IbNG) subtype viruses, one containing the AE(CM240) viruses and one containing the non-recombinant A viruses. These genetic clusters have different geographical distributions in Africa. CONCLUSION: The prevailing view of HIV-1 subtype A forming a uniform band across the center of sub-Saharan Africa needs revision. In all probability, the most common subtype in west Africa and west central Africa is the AG recombinant, AG(IbNG), whereas in east central Africa it is the non-recombinant subtype A.     

Characterization of five nearly full-length genomes of early HIV type 1 strains in Ruili city: implications for the genesis of CRF07_BC and CRF08_BC circulating in China

To trace the genesis of HIV-1 CRF07_BC and CRF08_BC, two predominant circulating recombinants among intravenous drug users in China, a retrospective molecular epidemiological investigation (1996-1998) was conducted in Ruili city of Yunnan, where the first AIDS epidemic among IDUs was reported in 1989. Fifty-four HIV-1 env C2V3 sequences were determined and genotyped with 49 subtype B and only 5 subtype C strains. The nearly full-length genome analyses of these five env-based subtype C samples revealed that four of them were actually BC recombinants and only one was pure subtype C. The first identified nonrecombinant HIV-1 subtype C, genetically close to Indian C representatives, provided direct evidence for the hypothesis that subtype C in China was introduced from India. Interestingly, three BC recombinants with subtype B as backbones were identified; one BC recombinant that precisely shared a common subtype B segment (nef region) with CRF07_BC and CRF08_BC was described, which indicated a close evolutionary relationship to these two CRFs. The sequences undoubtedly lead us to a better understanding of the emergence of CRF07_BC and CRF08_BC.     

Preferential in-utero transmission of HIV-1 subtype C as compared to HIV-1 subtype A or D

OBJECTIVE: To determine whether different HIV-1 genotypes present in a single cohort, in Dar es Salaam, Tanzania, showed differences in timing for transmission from mothers to their infants. METHODS: We determined the maternal viral load, transmission time, and the HIV-1 envelope (env) subtype of 253 HIV-1-infected infants enrolled in a randomized double-blind placebo-controlled trial to examine the efficacy of vitamins in decreasing mother-to-child transmission in Tanzania. Classification of HIV-1 positivity in utero was based on PCR results at birth. Infants were classified as intrapartum infected if they scored negative for the sample collected at birth and positive for the sample collected at 6 weeks of age. RESULTS: We found significant differences in the distribution of transmission time according to subtype. A higher proportion of HIV-1 with subtype C env (C-env) was transmitted in utero than HIV-1 with subtype A env (A-env), subtype D env (D-env), or both combined. CONCLUSIONS: The identification of patterns of mother-to-child transmission times among HIV-1 genotypes may be useful in the selection of drug regimens for chemoprophylaxis. Based on our results, the efficacy of regimens administered only at labor may not protect as large a fraction of infants born in geographical regions with subtype C-env epidemics as compared to epidemics in regions where subtypes A-env and D-env predominate in the population.     

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.     

Characterization of B/C recombinants of near full-length HIV type 1 from northeastern India with mosaics identical to ARE195FL but with a different ancestral origin

We have characterized near full-length genomes of three B/C recombinants of HIV-1 from the northeastern state of India. The recombinant viruses showed a backbone of subtype C virus with a single insertion of the subtype B genome in the envelope region. While all of them were distinct from B/C recombinants CRF_07 and CRF_08 circulating in China and CRF_04BR137 circulating in Brazil, two of them presented with break-points identical to the Argentinean B/C recombinant ARE195FL. However, neighbor-joining analysis followed by phylogenetic clustering showed that gp120 belonging to subtype B of all the recombinants clustered with Thai B sequences, while subtype C gag clustered with an Indian subtype C sequence, suggesting a unique ancestral origin of these recombinants.     

Analysis of HIV type 1 diversity in pregnant women from four Latin American and Caribbean countries

Worldwide, the distribution of HIV-1 subtypes and intersubtype recombinants is not homogeneous. In Latin America and the Caribbean, HIV-1 subtype B predominates. However, in the south of Brazil and in countries of the Southern cone (Argentina, Chile, Paraguay, and Uruguay) there is a different distribution of viral subtypes and intersubtype recombinants. The aim of this work was to analyze HIV-1 diversity in a cohort of pregnant women (with primarily heterosexual acquisition of the infection) who were diagnosed with HIV-1 infection during their current pregnancy and who received ARVs during pregnancy for perinatal transmission prophylaxis. Analysis of 121 partial pol sequences from subjects enrolled in Argentina, Brazil, the Bahamas, and Mexico was performed by phylogenetic and recombinant characterization. Different prevalences of subtype B were observed (100% for specimens from Mexico and the Bahamas, 61% for Brazil, and 30% for Argentina). Subtypes C and F were found, along with BC, BF, FC, and CBF recombinants in specimens from Brazilians. A high prevalence of BF recombinants was found (70%) in specimens from Argentina. The different patterns of HIV- 1 subtypes and intersubtype recombinants in South America (Argentina and Brazil) compared to those in Central and North America should be considered in the design of future HIV-1 vaccine trials.