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HIV-1 recombinants with multiple parental strains in low-prevalence, remote regions of Cameroon: Evolutionary relics?
© Carr et al; licensee BioMed Central Ltd. 2010
Received: 19 November 2009
Accepted: 28 April 2010
Published: 28 April 2010
The HIV pandemic disseminated globally from Central West Africa, beginning in the second half of the twentieth century. To elucidate the virologic origins of the pandemic, a cross-sectional study was conducted of the genetic diversity of HIV-1 strains in villagers in 14 remote locations in Cameroon and in hospitalized and STI patients. DNA extracted from PBMC was PCR amplified from HIV(+) subjects. Partial pol amplicons (N = 164) and nearly full virus genomes (N = 78) were sequenced. Among the 3956 rural villagers studied, the prevalence of HIV infection was 4.9%; among the hospitalized and clinic patients, it was 8.6%.
Virus genotypes fell into two distinctive groups. A majority of the genotyped strains (109/164) were the circulating recombinant form (CRF) known to be endemic in West Africa and Central West Africa, CRF02_AG. The second most common genetic form (9/164) was the recently described CRF22_01A1, and the rest were a collection of 4 different subtypes (A2, D, F2, G) and 6 different CRFs (-01, -11, -13, -18, -25, -37). Remarkably, 10.4% of HIV-1 genomes detected (17/164) were heretofore undescribed unique recombinant forms (URF) present in only a single person. Nearly full genome sequencing was completed for 78 of the viruses of interest. HIV genetic diversity was commonplace in rural villages: 12 villages each had at least one newly detected URF, and 9 villages had two or more.
These results show that while CRF02_AG dominated the HIV strains in the rural villages, the remainder of the viruses had tremendous genetic diversity. Between the trans-species transmission of SIVcpz and the dispersal of pandemic HIV-1, there was a time when we hypothesize that nascent HIV-1 was spreading, but only to a limited extent, recombining with other local HIV-1, creating a large variety of recombinants. When one of those recombinants began to spread widely (i.e. became epidemic), it was recognized as a subtype. We hypothesize that the viruses in these remote Cameroon villages may represent that pre-epidemic stage of viral evolution.
The geographic location of the origin of the HIV-1 pandemic is Central West Africa, where cross-species transmission of SIVcpz occurred from chimpanzee (Pan troglodytes troglodytes) to human [1–3]. From that transmission event the virus adapted into group M HIV-1 and gradually spread throughout the world. The genetic forms of HIV-1 currently present in Central West Africa, ground zero of the pandemic, may shed light on those early events.
Characterization of HIV-1 genetic diversity in different regions of the world is a challenging, on-going effort. Phylogenetic analyses of viral sequences have revealed distinct monophyletic clusters of strains called subtypes. There are now 9 official subtypes, and over 45 validated circulating recombinant forms (CRF) http://www.hiv.lanl.gov/, and they exist in different patterns in various regions of the world. These patterns have been moderately well described, with strains from most countries now characterized to some degree or another. Cameroon is a location where the genetic diversity has been repeatedly studied; there have been at least 8 scientific reports of the genetic subtypes in Cameroon since 2005 [4–12]. The reason for this intense interest is that the HIV epidemic in Cameroon presents a paradoxical picture: the prevalence of infection is not high by African standards (<10% in rural areas), yet the genetic diversity, including multiple recombinants of complex structure, is extremely high. While all of these studies have reported CRF02_AG as the most prevalent single genetic form in circulation, with estimates ranging from 45% to 61%, the remaining strains have consisted of an array of other genetic forms, both classifiable and not [4–13]. Because of this genetic diversity, partial genome sequencing of relatively small sample sets has hampered the description of the epidemic fully. This report presents the genetic subtypes of more strains from rural Cameroon than have previously been reported, using nearly full genome sequencing to more completely describe the many unique recombinant forms (URF).
Partial Pol Analysis
Nearly Full Genome Analysis
Significant hypermutation was observed in 14.1% of the full genomes, and another 16.7% were otherwise defective. Most of the hypermutated strains were classified using Hypermut http://www.hiv.lanl.gov/, but a few were only partially hypermutated and were discovered by the characteristic G-to-A mutations. From the context of the hypermutations, APOBEC3G is the likely enzyme responsible. In one case, a hypermutated strain also had a 36 aa deletion in the vif gene, suggesting that, for that individual, APOBEC3G was unimpeded by vif. A little over half of the hypermutated strains were URF (6/11, 54.5%), while the rest were both CRF02_AG and the other genetic forms; the URFs, therefore, had a higher rate of hypermutation than the subtypes or CRFs. In addition to 11 hypermutated strains, there were 13 strains with major defects likely to make them functionally dead. Most were frame shift mutations leading to stop codons, but there were 3 strains that had large insertions/duplications in the nef gene. The sizes of the insertions or duplications were 17 aa, 40 aa and 75 aa, respectively. Defective genomes were not more prevalent in URFs than other genetic forms.
While genetic diversity in Cameroon has been described frequently in the literature, this report documents the high degree of genetic diversity using nearly full genome sequencing of samples from very rural sites in Cameroon. The prevalence of infection was relatively low by African standards (4.9%), and CRF02_AG was the predominant strain (66.5%), but about a third of the remaining HIV-1 genotypes detected (57/164) were confined to only one or at most a few persons. As others have shown, Central West Africa is the most likely geographical location for the origin for the HIV-1 pandemic [1–3]. However, counter-intuitively, the prevalence of infection there is lower than the newer epidemics to the east, west and south . We hypothesize that the low prevalence is a reflection of lower transmissibility of HIV in these populations. Even in villages with a large number of unique recombinant forms (URF) such as LE, recombinants not only varied in structure but also in the parental strains involved. Only in one village were there 2 URFs with identical structures, probably sex linked; and the two major parental strains for that recombinant (CRF09_cpx, subtype H) were not even found in the population. Viral loads for these samples revealed no correlation between genetic form (CRF02_AG vs URF, for example) and level of circulating virus (data not shown), but there was a distinct lack of genetically related transmission pairs among the URF from the same village.
A low rate of transmissibility may explain the low prevalence, but it is very difficult to then account for the presence of recombinants with 4 or 5 different subtypes in one strain. Inter-subtype recombinants are generally the result of super-infection with different subtypes and are most commonly seen in populations with heavy exposure to multiple subtypes [16, 17]. There are multiple subtypes in this population, but at a fairly low level. The hospitalized patients and STD clinic attendees would be expected to have the most risk, and the prevalence of infection among them was significantly elevated compared to the rural villages, but the rate of URFs among them was even lower than in subjects from the rural sites. Recent research among subjects in the capital, Yaounde, found that 16% of the subjects were dually infected with either 2 different subtypes or different strains from one subtype . As with the subjects in this study, they were low risk. Further study in these populations is needed in order to discover what the mechanism behind this observation might be.
The genetic complexity of HIV strains from rural Cameroon defies both logic and experience. Multiply recombinant viruses are found in subjects who have a high risk of superinfection with different strains of HIV, such as commercial sex workers or injecting drug users. The village populations in this study, on the contrary, have a low risk of infection as captured by the prevalence, but harbor viruses having 3 or 4 different parental strains. Among the 78 viruses sequenced in full, there were at least 13 different subtypes, sub-subtypes or CRF represented. It is hypothesized that that this diversity may be due to remnants of the viruses predating the epidemic in 1960.
Of the 17 village sites in Cameroon that were selected for this study, 14 were used for the genetic analysis, shown in Figure 1. In a study approved by the IRB of Johns Hopkins University, participants were healthy adults who gave consent to participate, most of them subsistence farmers and hunters . In addition, in-patients at two district hospitals (Ndikinimeki, Lomie) and outpatients at STI clinics in those same locations were enrolled in the study. Finally, HIV-positive blood was collected from the central blood bank in Yaounde, Cameroon, to monitor genotypes among blood donors. Blood was drawn and plasma and peripheral blood mononuclear cells (PBMC) were separated using CPT blood collection tubes (BD, Inc, Franklin City, NJ). The plasma was tested for HIV antibodies by Ortho HIV-1/HIV-2 Antibody capture ELISA (Ortho-Clinical Diagnostics, Rochester, NY) and reactive samples were confirmed by two Western Blots (HIV Blot 2.2, Genelabs Diagnostics, Singapore and Calypte, Cambridge Biotech, Cambridge, MA). Those confirmed positive on both were used for viral load determination and DNA extraction. Viral load was measured using the Roche Amplicor HIV-1 monitor test, v. 1.5 (Roche Molecular Systems, Branchburg, NJ). High molecular weight DNA was extracted from the PBMC using QIAmp DNA extraction kits (Qiagen, Valencia, CA).
The DNA from PBMC was amplified by nested PCR in the pol gene producing a 1.1 kb fragment spanning protease and part of the reverse transcriptase (RT) gene . The first and second round reactions were conducted using 8 ml dNTP (1.25 mM), 5 ml 10× buffer (no MgCl2), 4 ml MgCl2 (25 mM), 0.5 ml of each primer (20 mM), 0.5 ml Ampli Taq gold (Applied Biosystems, Foster City, CA), water and template in 50 ml total reaction volume. First round cycling conditions were: 95°C, 10 min, then 45 cycles of 94°C 30 seconds, 55°C 30 seconds, 72°C 1.5 minutes, then 72°C 7 minutes. The first round primers were: Pro5F (5'-AGAAATTGCAGGGCCCCTAGGAA) and RT3474R (5'-GAATCTCTCTGTTTTCTGCCAG). The second round reaction was with Pro3F (5'-AGANCAGAGCCAACAGCCCCACCA and ProRT (5'-TTTCCCCACTAACTTCTGTATGTCATTGACA). The cycling conditions were the same except that the annealing temperature was 58°C and there were only 30 cycles.
Virtually full-length genomes of HIV-1 were amplified from selected strains based on the results of partial pol sequencing. Limiting template dilution into the first round was performed to decrease the complexity of the sample and allow for direct sequencing of the second round PCR product. The virtually full-length genome was amplified using MSF12b (5'-AAATCTCTAGCAGTGGCGCCCGAACAG) and OFMR1 (5'-TGAGGGATCTCTAGTTACCAGAGTC), followed by F2nst (5'-GCGGAGGCTAGAAGGAGAGAGATGG) and ofm19 (5'-GCACTCAAGGCAAGCTTTATTGAGGCTTA). PCR was performed as described [23, 24], using the Expand Long Template kit (Boehringer-Mannheim) and a hot-start method with a melting wax barrier (Dynawax). Cycling conditions were: 94° for 2 min, then 10 cycles of 94°C for 10 s, 60°C for 30 s and 68°C for 8 min. This was followed by 20 cycles where the annealing temperature was 55°C. The final extension step was 68°C for 10 min. Multiple second round PCR amplifications were combined to provide sufficient template for sequencing.
Template DNA for automated sequencing was prepared as described previously . PCR amplification products of the pol gene and the nearly full-length strains were fully sequenced on both strands by using fluorescent dye terminators and an Applied BioSystems (Applied Biosystems Inc., Foster City, CA) Model 3100 DNA sequencer. DNA sequences were assembled using Sequencher software (Genecodes Inc., Ann Arbor MI) on Macintosh computers. All sequences had at least 2 clear readings in each direction for completion.
A multiple alignment of the Cameroon sequences with selected HIV-1 reference sequences was constructed using MacGDE 1.9.5, software based on Genetic Data Environment (GDE) adapted for Mac OS X [25, 26]. Gaps that were introduced to create the alignment were eliminated in the final analysis. Reference isolates from the different subtypes and circulating recombinant forms from the pandemic, described in the National HIV Database at Los Alamos, NM, were used to classify the Cameroon sequences http://www.hiv.lanl.gov/. Phylogenetic trees were constructed using the neighbor-joining method and the consistency of branching order was evaluated using bootstrap analysis by MEGA3 software . Genetic relationships can be obscured by the presence of recombinant or novel forms in the analysis of HIV-1 strains. To address this, phylogenetic trees were constructed that included only a few aberrant viral sequences at a time. Hypermutated sequences were identified using Hypermut 2.0 software from the National HIV Database http://www.hiv.lanl.gov/ and were deleted from appropriate analyses .
Recombinant analysis was done with bootscanning  and distance scanning  using SimPlot software, version 3.5. The nucleotide positions of recombinant breakpoints were designated relative to HXB-2 (GenBank Accession No: K03455). The significance of the breakpoint assignment was assessed by the bootstrap value of the relevant node in the phylogenetic tree, which was >70% for significance.
Nucleotide sequence accession numbers of the pol gene sequences from Cameroon are available under GenBank Accession No. AY847362-AY847453. The nearly full length genomic sequences are available under GenBank Accession numbers AY371121-AY371170, GQ229529 and GU201494-GU201517.
NDW was supported by awards from the National Institutes of Health Director's Pioneer Award (Grant DP1-OD000370), the WW Smith Charitable Trust, the US Military HIV Research Program, and grants from the NIH Fogarty International Center (International Research Scientist Development Award Grant 5 K01 TW000003-05), AIDS International Training and Research Program (Grant 2 D 43 TW000010-17-AITRP), and the National Geographic Society Committee for Research and Exploration (Grant #7762-04). This research was supported in part by the Global Viral Forecasting Initiative, Google.org, and The Skoll Foundation. Thanks to the entire staff of GVFI-Cameroon for their support and assistance. The Cameroon Ministry of Defense, Ministry of Health, and Ministry of Scientific Research and Innovation provided authorizations and support for this work. The authors express many thanks to the editorial assistance of Este Armstrong.
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