Sub-subtype A1 Research Articles

The Evolution of HIV-1 Diversity in Rural Cameroon and its Implications in Vaccine Design and Trials

West-Central Africa is an epicenter of the HIV pandemic; endemic to Cameroon are HIV-1 viruses belonging to all (sub)subtypes and numerous Circulating Recombinant Forms (CRFs). The rural villages of Cameroon harbor many strains of HIV-1, though these areas are not as well monitored as the urban centers. In the present study, 82 specimens obtained in 2000 and 2001 from subjects living in the rural villages of the South and West Regions of Cameroon were subtyped in gag, pol, and env and compared to 90 specimens obtained in 2006–2008 in the same regions, in order to analyze HIV-1 evolution in these rural areas. It was found that in the South Region, the proportion of unique recombinant forms (URFs) remained constant (∼40%), while the amount of URFs containing fragments of a CRF increased by 25%. (Sub)subtypes A1, F2, H, and K, and CRF09_cpx, identified in 2000 and 2001, were replaced by CRFs 01_AE, 13_cpx, 14_BG, and 18_cpx in 2006–2008. In the West Region, (sub)subtypes A2, C, G, and H, and CRFs 01_AE and 09_cpx, identified in 2000–2001, were replaced by sub-subtype A1 and CRFs 25_cpx and 37_cpx in 2006–2008. The proportion of URFs in the West Region dropped significantly over the time period by 43%. In both Regions, the proportion of CRF02_AG increased at all loci. These findings demonstrate that the evolution of HIV-1 is distinct for each endemic region, and suggests that the proportion of URFs containing CRF fragments is increasing as the genetic identity of the virus continues to shift dramatically. This highlights the concern that subtype-specific vaccines may not be relevant in Cameroon, and that the distribution of viral diversity in these regions of Cameroon must be carefully monitored.

Comparison of the Generic HIV Viral Load ® assay with the Amplicor™ HIV-1 Monitor v1.5 and Nuclisens HIV-1 EasyQ ® v1.2 techniques for plasma HIV-1 RNA quantitation of non-B subtypes: The Kesho Bora preparatory study

The implementation of cost effective HIV-1 RNA quantitation assays in resource-poor settings is of paramount importance for monitoring HV-1 infection. A study comparing the analytical performance of three HIV-1 RNA assays (Generic HIV Viral Load ®, Amplicor™ v1.5 and Nuclisens EasyQ ® v1.2) was performed on 160 plasma samples from 160 consecutive antiretroviral treatment naive HIV-1-infected pregnant women assessed for eligibility in the Kesho Bora trial aimed at prevention of mother-to-child transmission of HIV-1 in three African countries (Burkina Faso, Kenya and South Africa). Correlation and agreement of results of the three assays were assessed for plasma HIV-1 RNA quantitation in specimens harbouring mainly sub-subtype A1, subtype C, and circulating recombinant form (CRF) 02_AG and CRF06_cpx. Good degrees of correlation and agreement were observed between these HIV-1 RNA assays. However, nine (9/160, 5.6%) strains detectable with the Generic HIV Viral Load ® assay were not detected by either the Amplicor™ ( n = 7) or EasyQ ® ( n = 2) test. One strain (0.6%) was missed with the Generic HIV Viral Load ® assay. Further, concordantly positive plasma samples harbouring CRF02_AG and CRF06_cpx yielded significantly higher HIV-1 RNA concentrations when tested by Generic HIV Viral Load ®, as compared to Amplicor™ v1.5 (mean differences, +0.33 and +0.67 log 10 copies/ml; P = 0.0004 and P = 0.002, respectively). The Generic HIV Viral Load ® assay accurately quantified the majority of the non-B HIV-1 subtypes assessed in this study. Due to its low cost (∼10 US $/test), this assay performed with open real-time PCR instruments is now used routinely in the Kesho Bora trial and may be recommended in other African settings.

Quantifying Differences in the Tempo of Human Immunodeficiency Virus Type 1 Subtype Evolution

Human immunodeficiency virus type 1 (HIV-1) genetic diversity, due to its high evolutionary rate, has long been identified as a main cause of problems in the development of an efficient HIV-1 vaccine. However, little is known about differences in evolutionary rate between different subtypes. In this study, we collected representative samples of the main epidemic subtypes and circulating recombinant forms (CRFs), namely, sub-subtype A1, subtypes B, C, D, and G, and CRFs 01_AE and 02_AG. We analyzed separate data sets for pol and env. We performed a Bayesian Markov chain Monte Carlo relaxed-clock phylogenetic analysis and applied a codon model to the resulting phylogenetic trees to estimate nonsynonymous (dN) and synonymous (dS) rates along each and every branch. We found important differences in the evolutionary rates of the different subtypes. These are due to differences not only in the dN rate but also in the dS rate, varying in roughly similar ways, indicating that these differences are caused by both different selective pressures (for dN rate) and the replication dynamics (for dS rate) (i.e., mutation rate or generation time) of the strains. CRF02_AG and subtype G had higher rates, while subtype D had lower dN and dS rates than the other subtypes. The dN/dS ratio estimates were also different, especially for the env gene, with subtype G showing the lowest dN/dS ratio of all subtypes.

Identification and Characterization of CRF02_AG, CRF06_cpx, and CRF09_cpx Recombinant Subtypes in Mali, West Africa

Abstract Multiple HIV-1 subtypes and circulating recombinant forms (CRFs) are known to cocirculate in Africa. In West Africa, the high prevalence of CRF02_AG, and cocirculation of subtype A, CRF01_AE, CRF06_cpx, and other complex intersubtype recombinants has been well documented. Mali, situated in the heart of West Africa, is likely to be affected by the spread of recombinant subtypes. However, the dynamics of the spread of HIV-1 recombinant subtypes as well as nonrecombinant HIV-1 group M subtypes in this area have not been systematically assessed. Herein, we undertook genetic analyses on full-length env sequences derived from HIV-1-infected individuals living in the capital city of Mali, Bamako. Of 23 samples we examined, 16 were classified as CRF02_AG and three had a subsubtype A3. Among the remaining HIV-1 strains, CRF06_cpx and CRF09_cpx were each found in two patients. Comparison of phylogenies for six matched pol and full-length env sequences revealed that two strains had discordant subtype/CRF designations between the pol and env regions: one had A3(pol)CRF02_AG(env) and the other had CRF02_AG(pol)A3(env). Taken together, our study demonstrated the high prevalence of CRF02_AG and complexity of circulating HIV-1 strains in Mali. It also provided evidence of ongoing virus evolution of CRF02_AG, as illustrated by the emergence of more complex CRF02_AG/A3 intersubtype recombinants in this area.

Identification of a Possible Ancestor of the Subtype A1 HIV Type 1 Variant Circulating in the Former Soviet Union

The HIV-1 subsubtype A1 epidemic of the former Soviet Union (FSU) was caused by an A1 variant apparently derived from a single introduction event. We identified an A1 variant highly related to the A1 lineage circulating in the FSU in a patient from Conakry, Republic of Guinea, who was diagnosed with HIV-1 when he moved to Italy in 2002. The most probable route of infection was two blood transfusions received in his country of origin in 1998. Full-length (9781 bp) molecular characterization revealed that this strain was evolutionarily parental to, but distinct from, the A1 lineage circulating in the FSU. Similar results were obtained analyzing partial genome sequences. A full-length sequence similarity plot and bootscanning analysis supported this evidence and excluded any potential recombination events with other HIV-1 variants. This viral strain represents the first evidence of an African patient infected by an A1 subtype related to the A1 variants spreading in FSU countries. The identification of this distinct A1 variant may support the origin of the Russian A epidemic from West Africa.

High Prevalence of Unique Recombinant Forms of HIV-1 in Ghana: Molecular Epidemiology From an Antiretroviral Resistance Study

In Ghana, programs to expand antiretroviral access are being implemented. In this context, the dynamic genetic evolution of HIV-1 requires continuous surveillance, particularly when diverse genetic forms co-circulate. Phylogenetic and antiretroviral resistance analyses of HIV-1 partial pol sequences from plasma RNA samples from 207 Ghanaian individuals were performed. 66% of infections were CRF02_AG, whereas 25% were unique recombinant forms (URFs). All 52 URFs were characterized by bootscanning. CRF02_AG was parental strain in 87% of URFs, forming recombinants with genetic forms circulating in minor proportions: CRF06_cpx, sub-subtype A3, CRF09_cpx and subtypes G and D. Two triple recombinants (CRF02_AG/A3/CRF06_cpx and CRF02_AG/A3/CRF09_cpx) were identified. Antiretroviral resistance analyses revealed that six individuals, five of which were antiretroviral drug-experienced, harbored mutations conferring high level of resistance to reverse transcriptase inhibitors. No major resistance mutations were identified in the protease, although insertions of one and three amino acids were detected. The high frequency of URFs detected probably reflects a significant incidence of coinfections or superinfections with diverse viral strains, which increases the genetic complexity of the HIV-1 epidemic in West Africa. Monitoring of HIV-1 drug resistance might provide data on the implications of intersubtype recombination in response to antiretrovirals.

Diversity of Human Immunodeficiency Virus Type 1 Subtypes in Kagera and Kilimanjaro Regions, Tanzania

A strategy to prevent the spread of HIV-1 worldwide is complicated by the high genetic diversity of the virus. To gain a better understanding of the HIV-1 genetic diversity in Tanzania, a molecular epidemiological investigation was conducted in Kagera and Kilimanjaro regions. While several studies have addressed HIV-1 subtypes in Tanzania, this is the first study to describe the virus subtypes circulating in Kagera. The Kagera region is the epicenter of the HIV-1 epidemic in Africa, and it was therefore of interest to compare the prevalence of HIV subtypes in this region and Kilimanjaro. Blood samples were obtained from 246 HIV-1-infected pregnant women attending antenatal clinics. Plasma HIV-1 RNA was extracted, amplified, and sequenced in the env C2V3 and/or pol regions from 209 samples. Based on the analysis of env C2V3 and pol sequences, 47.4% had concordant subtypes, 19.1% were discordant indicating recombination, and for 33.5% sequences were obtained for only one region. The distribution HIV-1 subtypes based on the phylogenetic analysis of paired env C2V3/ pol sequences in Kagera region was A/A (27.8%), C/C (29.6%), D/D (16.7%), and unique recombinant forms (25.9%), and in Kilimanjaro region was A/A (32.9%), C/C (25.9%), D/D (10.6%), CRF10_CD (1.2%), and unique recombinant forms (29.4%). The env C2V3 subsubtype A2 and env C2V3/pol CRF10_CD were also observed indicating that these recombinants are circulating in Tanzania. The high diversity of HIV-1 subtypes and the high prevalence of recombinants demonstrated in this study necessitate expanded and continuous monitoring of the epidemic in Tanzania. The trend may have implications for current national control strategies against the HIV-1 epidemic.

Characterization of HIV Type 1 Reverse Transcriptase Mutations in Infants Infected by Mothers Who Received Peripartum Nevirapine Prophylaxis in Jos, Nigeria

This study was carried out to characterize HIV-1 reverse transcriptase (RT) mutations in vertically infected infants in Jos, Nigeria. DNA was extracted from peripheral blood mononuclear cells of 102 infants, aged 0 to 6 months, born to HIV-1-infected mothers who had received peripartum single-dose nevirapine prophylaxis. PCR-based diagnosis revealed that 14 infants (13.7%) were infected with HIV-1. Phylogenetic analyses of RT revealed wide viral diversity, with CRF02_AG, subtype G, subsubtype A3, CRF06_cpx, and a subtype D recombinant present in the population. Four of 13 (31%) infants had NNRTI resistance mutations--V179I (2 infants), Y181C, and V179E. Intriguingly, subtype G sequences did not have NNRTI mutations but rather carried a Q207N mutation, which may undergo negative selection under drug pressure. Our data suggest wide diversity for vertically transmitted HIV-1 viruses in Nigeria and highlight the potential significance of transmitting rare mutations in subtype G.

Genetic Diversity of HIV-1 Non-B Strains in Sicily: Evidence of Intersubtype Recombinants by Sequence Analysis ofgag,pol, andenvGenes

The molecular epidemiology of HIV-1 strains in Sicily (Italy) was phylogenetically investigated by the analysis of HIV-1 gag, pol, and env gene sequences from 11 HIV-1 non-B strains from 408 HIV-1-seropositive patients observed from September 2001 to August 2006. Sequences suggestive of recombination were further investigated by bootscanning analysis of various fragments. Overall, we identified several second-generation recombinant (SGRs) strains, which contained genetic material of CRF02_AG in at least one gene. Notably, three individuals were found to be infected with subsubtype A3, and one of them showed genetic recombination with subsubtype A4. The current study emphasizes the genetic analysis of gag, pol, and env genes as a powerful tool to trace the spread of complex HIV-1 recombinant forms, and highlight the genetic diversity of HIV-1 non-B strains in Italy.

Diversity of HIV in Rural Burkina Faso

: On introduction of a program for prevention of mother-to-child transmission (PMTCT) of HIV in Nouna, rural Burkina Faso, we determined HIV prevalence in this region to be 3.6%, which is significantly lower than the 7% reported for 2 major cities of Burkina Faso. Forty-three samples from drug-naive pregnant women and patients before introduction of antiretroviral therapy (ART) were genotypically characterized in gag, pol, and env regions. One individual each was infected with HIV-2 or dually infected with HIV-1 and HIV-2. The most dominant HIV-1 subtypes were CRF02_AG and CRF06_cpx, similar to what has been observed in other West African countries. A discordant genotype was observed in almost half of the analyzed samples, with most putative recombinants deriving from CRF02_AG and CRF06_cpx. Recently reported strains like the CRF09_cpx and the sub-subtype A3 as well as some unique recombinant forms of HIV like D/D/CRF02_AG and CRF02_AG/CRF02.AG/CRF_09cpx were also detected. Analysis of drug resistance-associated polymorphisms detected the nonnucleoside reverse transcriptase inhibitor (NNRTI) resistance mutations K103N/E and V118I in 1 individual each, suggesting transmission of drug-resistant viruses or prior use of antiretroviral drugs. Resistance-associated polymorphisms (K20I and M36I) were prevalent in the complete protease (PR) region, but no primary drug resistance mutations were detected. Analysis of the HR1 and HR2 regions of gp41, important for T-20 sensitivity, revealed no known resistance mutations but several polymorphisms of unknown importance. Monitoring for drug resistance mutations among naive subjects is important in this area on introduction of antiretroviral drugs.

Circulating and Unique Recombinant Forms of HIV Type 1 Containing Subsubtype A2

HIV-1 strains containing subsubtype A2 are relatively rare in the pandemic but have been repeatedly identified in Kenya, where candidate vaccines based in part on subtype A, but not A2 strains, may be evaluated. Among the most recent is CRF16_A2D, a circulating recombinant form (CRF) whose prototypes are complete or partial HIV-1 sequences from Kenya, Korea, and Argentina. Using samples from blood bank discards in Kenya and complete genome sequencing, this report further documents CRF16_A2D and related recombinants and identifies a second CRF, CRF21_A2D. The two A2-containing CRFs, and two recombinants related to CRF16_A2D, share common structural elements but appear to have been independently derived. Concerted selection may have influenced the emergence and spread of certain A2-containing strains in Kenya. The second complete subtype C sequence from Kenya is also reported here. Monitoring of A2-containing recombinants and subtype C strains, both relatively rare in Kenya, may be informative in the course of cohort development and evaluation of candidate vaccines.

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.

Genetic analysis of human immunodeficiency virus type 1 nef in portugal: Subtyping, identification of mosaic genes, and amino acid sequence variability

Extending our previous genetic characterization of human immunodeficiency virus type 1 (HIV-1) strains circulating in Portugal, we here report the first phylogenetic and putative amino acid sequence variability analyses of nef accessory gene. Viral sequences (n = 53) were amplified by nested PCR from proviral DNA purified from peripheral blood mononuclear cells of HIV-1 infected individuals (n = 49). Phylogenetic inference analysis demonstrated a distribution of the viral sequences between subtypes A (sub-subtype A1), B, D, F (sub-subtype F1), G, H, and J, with subtypes G and B accounting altogether for more than half of the genotypes found. A significant number of the proviral DNA sequences analyzed (18.4%) were shown to correspond to intragenic nef recombinants, with the majority having the typical CRF02_AG nef structure. In addition, three novel intragenic recombinant structures were found (B/G/B, CRF02_AG/H, and D/G). From phylogenetic analysis, it was concluded that part of the non-recombinant nef genes might have actually been amplified from mosaic viruses: CRF06_cpx, CRF14_BG, and a new envA/nefJ recombinant. While comparing all the putative Nef sequences, significant amino acid sequence variability was observed. However, most of the described nef functional motifs were relatively well conserved in the majority of the sequences analyzed and numerous amino acid changes fell outside these regions. The results presented unambiguously endorse the high level of complexity of HIV-1 epidemics in Portugal.

Genetic Diversification and Recombination of HIV Type 1 Group M in Kinshasa, Democratic Republic of Congo

As the HIV-1 pandemic becomes increasingly complex, the genetic characterization of HIV strains bears important implications for vaccine research. To better understand the molecular evolution of HIV-1 viral diversity, we performed a comparative molecular analysis of HIV strains collected from high-risk persons in Kinshasa, Democratic Republic of Congo (DRC). Analysis of the gag-p24, env-C2V3 and -gp41 regions from 83 specimens collected in 1999-2000 revealed that 44 (53%) had concordant subtypes in the three regions (14 subsubtype A1, 10 subtype G, 8 subtype D, 5 subtype C, 2 each subsubtype F1 and CRF01_AE, and one each of subtypes H and J, and subsubtype A2, while the remaining 39 (47%) had mosaic genomes comprising multiple subtype combinations. Similar multisubtype patterns were also observed in 24 specimens collected in 1985. Sequence analysis of the gag-pol region (2.1 kb) from 21 discordant specimens in the gag-p24, env-C2V3 and -gp41 regions in 1985 and 1999-2000 further confirmed the complex recombinant patterns. Despite the remarkable similarity in overall subtype distribution, the intra- and intersubtype distances of major subtypes A1 and G increased significantly from 1985 to 1999-2000 (p=0.018 and p=0.0016, respectively). Given the complexity of HIV-1 viruses circulating in DRC, efforts should focus on the development of vaccines that result in cross-clade immunity.

Molecular Epidemiology of Human Immunodeficiency Virus Type 1 Sub-Subtype A3 in Senegal from 1988 to 2001

The global human immunodeficiency virus (HIV)epidemic is characterized by significant genetic diversity in circulating viruses. We have recently characterized a group of viruses that form a distinct sub-subtype within the subtype A radiation, which we have designated HIV type 1 (HIV-1) sub-subtype A, circulating in West Africa. A prospective study of a cohort of female sex workers (FSW) in Dakar, Senegal over an 18-year period indicated that an A3-specific sequence in the C2-V3 region of the env gene was found in 46 HIV-1-infected women. HIV-1 sub-subtype A3 appeared in the FSW population as early as 1988 and continued to be transmitted as of 2001. We also found that HIV-1 A3 is not confined to the FSW cohort in Senegal but is also circulating in the general population in Dakar. Furthermore, analyses of viral sequences from a few other West and Central African countries also demonstrated evidence of HIV-1 A3 sequence in isolates from HIV-1-infected people in Ivory Coast, Nigeria, Niger, Guinea Bissau, Benin, and Equatorial Guinea. Overall, because of the evidence of sub-subtype A3 in the general population in Senegal, as well as in a few neighboring West and Central African countries, along with the increasing incidence of infection with A3-containing viruses in the Dakar high-risk FSW population, we feel that HIV-1 sub-subtype A3 viruses are important to distinguish and monitor.

Distinct Human Immunodeficiency Virus Type 1 Subtype A Virus Circulating in West Africa: Sub-Subtype A3

Phylogenetic analyses demonstrate significant diversity in worldwide circulating strains of human immunodeficiency virus type 1 (HIV-1). Detailed studies have revealed a complex pattern of intersubtype recombinations, as well as evidence of sub-subtypes circulating in various populations. In this study, we characterized an HIV-1 strain that had previously been identified as a distinct subcluster within the subtype A radiation based on partial sequence data. These viruses were of particular interest given that we recently found that their prevalence was significantly higher in dually infected individuals compared to women who were singly infected with HIV-1. Five viruses isolated from commercial sex workers in Dakar, Senegal, were full-length PCR amplified and sequenced. Phylogenetic analyses indicated that, whereas three of these viruses were closely related and clustered overall within the HIV-1 subtype A radiation, they were distinct from previously characterized sub-subtype A1 and A2 viruses. The clustering pattern was maintained in the individual gag, pol, and env regions of the genome. Distance calculations between these viruses, which we termed A3, and other reference sub-subtype A1 and A2 viruses fell in the range of distances between previously characterized sub-subtype groups. In addition, we found evidence of two A3-containing recombinants in our cohort. These recombinants are mosaics composed of sequence from both sub-subtype A3 and CRF02_AG, the major circulating recombinant form in this West African population. Based on phylogenetic analyses, we propose that the group of viruses found in the Dakar sex worker cohort, previously referred to as HIV-1 A subcluster 2, be referred to as HIV-1 sub-subtype A3.

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.

Full-length genome analysis of HIV-1 subtype C utilizing CXCR4 and intersubtype recombinants isolated in South Africa.

The HIV-1 epidemic in South Africa is largely due to subtype C viruses, which preferentially use CCR5 as a coreceptor for infection. We describe full-length genome sequences of two CXCR4-utilizing HIV-1 subtype C viruses and two intersubtype recombinants from South Africa. Three of the viruses (99ZACM4, 99ZACM9, and 99ZASW7) were isolated in 1999 from AIDS patients in Johannesburg, and a fourth virus (98ZADu178) was isolated in Durban in 1998 from an asymptomatic female sex worker. Isolates 99ZASW7 and 99ZACM9 from Johannesburg were subtype C throughout the genome, 99ZASW7 used the CXCR4 coreceptor, and 99ZACM9 used both CCR5 and CXCR4. Isolate 98ZADu178 from Durban was a novel recombinant between subsubtype A2 and subtype C. The third isolate from Johannesburg, 99ZACM4, was a complex, novel recombinant with multiple breakpoints and contained segments of subtypes A, C, D, G, and K. These results establish the presence of intersubtype recombinants in South Africa, indicating that ongoing surveillance for other subtypes and recombinants is necessary.