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Cameroon is a West African country where high genetic diversity of HIV-1 has been reported. The predominant CRF02_AG is involved in the emergence of more complex intersubtype recombinants. In this study, we sequenced the full-length genome of a novel unique recombinant form (URF) of HIV-1, 02CAMLT04 isolated in blood donors in urban Cameroon. Phylogenetic tree and bootscan analysis showed that 02CAMLT04 was complex and appeared to be a secondary recombinant derived from CRF02_AG and CRF22_01A1. The genomic composition of 02CAMLT04 strain showed that it is composed of three segments; twenty four percent of the genome is classified as CRF02_AG, spanning most of the envelope gene. The remaining seventy six percent of the genome is classified as CRF22_01A1. In addition, the sequence analysis of 13 full-length sequences from HIV-1 positive specimens received from Cameroon between 2002 and 2010 indicated that five specimens are pure CRF22_01A1 viruses, and six others have homology with CRF22_01A1 sequences in either gag, pol or env region where as 6% of strains contain portions of CRF22_01A1. Further study demonstrated that CRF22_01A1 is a primary prevalence strain co-circulating in Cameroon and is involved in complex intersubtype recombination events with subtypes (D or F), subsubtypes (A1 or F2) and CRFs (CRF01_AE or CRF02_AG). Our studies show that novel recombinants between CRF22_01A1 and other clades and recombinant forms may be emerging in Cameroon that could contribute to the future global diversity of HIV-1 in this region and world wide.
The genetic diversity of HIV-1 is very broad in Cameroon where all group M clades and several circulating recombinant forms CRFs (in particular CRF01_AE, CRF02_AG, CRF06_cpx, CRF09_cpx, CRF11_cpx, CRF13_cpx, CRF22_01A1, CRF36_cpx and CRF37_cpx), in addition to groups O, N and P viruses, have been identified1–10. Among the broad genetic diversity of HIV-1 strains, CRF02_AG represents over 65% of HIV infections with an additional 26% classified as unique recombinant forms (URFs)8, 10. Co-circulation of different subtypes and CRFs in Cameroon results in the continuing emergence of new intersubtype recombinants or even M/O recombinants11. Therefore, Cameroon is an ideal area to investigate the genetic diversity of HIV-1 and its impact on the global pandemic. In 2002, we initiated a molecular and serologic epidemiology survey in blood donors in Douala and Yaoundé, in Cameroon to investigate HIV-1 genetic diversity, study virologic and immunologic characteristics of the viruses, evaluate the performance of US FDA licensed HIV-1 assays in the presence of numerous HIV-1 variants, and identify samples to serve as candidate reference reagents for diagnostics. We found that several group M subtypes (A, B, C, D, F2 and G) and CRFs (CRF02_AG, CRF06_cpx, CRF11_cpx, CRF13_cpx, CRF19_cpx and CRF22_01A1) were circulating in this population. A new URF strain, 02CAMLT04, had CRF22_01A1gag-CRF02_AGenv genotype12, and a novel strain, 02CAMLT72 isolated in Douala, was designated as pure CRF22_01A113.
CRF22_01A1 strain was initially identified in Cameroon in 20014 and circulating in this country for many years. Brennan’s studies reported that the percentage of CRF22_01A1 in concordant specimens was 6.6% (1996–1999), 4.7% (2000–2002), and 6.6% (2003–2004) over the 9-year period14, however, molecular, epidemiological and evolutionary study of CRF22_01A1 phylogenetic association with different HIV-1 subtypes was not described. Here, we report 14 full-length genomic sequences of HIV-1 specimens isolated in Cameroon from 2002–2003 and 2006–2010. Of these 14 specimens, 5 were pure CRF22_01A1, 6 were determined to be recombinants of CRF22_01A1 with CRF02_AG. These new data support involvement of CRF22_01A1 in the emerging diversity of HIV-1 in Cameroon.
For HIV characterization studies, 441 blood samples from HIV-1-seropositive individuals were collected at Douala (LT), Bamenda (LPH), Buea (BDHS), Limbe (LB), Yaounde (ARC) and a few villages in Cameroon (NYU) in 2002–2010. A total of 208 viruses were isolated and genotyped based on partial sequence analysis, 63% of them were CRF02_AG, 26% were URFs, and 6% were CRF22_01A1 containing recombinants12,15. In this study, 13 viruses containing CRF22_01A1 genome and 3 URF strains were selected for full-length sequence analysis, however nucleic acid was available for only 14 of them which were completely sequenced and characterized.
Viral RNA was extracted from the plasma samples using QIAamp Viral RNA Mini kit (Qiagen, Valencia, CA). Then, reverse transcription, polymerase chain reaction (PCR) and sequencing were performed as previously described6, 7. The nearly full-length genomic sequences reported in this study have been deposited in the GenBank database (accession numbers: EU743964, JN864047-JN864059).
Nucleotide sequences were aligned with HIV-1 reference strains of different subtypes, subsubtypes and CRFs from Los Alamos HIV database (A1_SE8538, A1_PS1044, A1_92UG037, A1_Q23 17, A1SE7535, A1_92RW008; A2_97CDKTB48, A2_94CY017; B_HXB2 LAI IIIB, B_671_00T36; C_BR025, C_ETH2220; D_ELI, D_94UG114; F1_VI850, F1_93BR020_1; F2_MP255, F2_02CM.0016BBY; G_DRCBL, G_92NG083; H_VI997, H_VI991; J_SE7887, J_SE7022; K_EQTB11C, K_MP535; 01_90CF4071, 01_90CF11697, 01_93TH051, 01_93TH253, 01_CM240; 02_pBD6_15, 02_IBNG)16 using Clustal W method17 as implemented in Vector NTI. The phylogenetic reconstructions were also performed with MEGA 4 software package using the neighbor-joining method18. To analyze the recombinant structure of the new viruses, the SimPlot 3.5.1 software was used to determine the percentage of similarity between selected pairs of sequences and to calculate bootscan plots19. The regions that did not cluster with any of the known subtypes were submitted to BLAST analysis (http://hiv-web.lanl.gov/BASIC-BLAST) to find the closely related sequences of other HIV-1 strains. The viral nucleotide positions correspond to the reference strain HXB2 (GenBank Accession Number K03455) used throughout this manuscript.
Near full-length genome sequencing was completed for 14 viruses of interest. One virus, NYU488, was collected from Bapile located in the East Province of Cameroon, and thirteen viruses were collected from the Southwest Province of Cameroon (Figure 1A). Phylogenetic analysis of these sequences confirmed their relatedness, as they all clustered together in the CRF22_01A1, CRF02_AG or subtype G radiation with a high bootstrap value (BV). As shown in Figure 2, six strains (02CAMLT04, LPH27MF, LB005, LB011, LB013 and LB054) were deeply clustered in the CRF22_01A1 radiation (BV, 100%) revealing a close relationship, 3 strains (LB045, NYU488 and ARC087) clustered between CRF22_01A1 and CRF01_AE suggesting they are CRF22_01A1 recombinants. 4 strains (ARC007, LB052, BDSH129 and LT66) clustered with CRF02_AG (BV, 93%) and LT31 clustered with subtype G (BV, 100%). These sequences were further analyzed by bootscanning to determine the phylogenetic mosaic structure. The 02CAMLT04 sequence was plotted against references restricted to the subsubtype A1, F2, subtype B, C, H, K, CRF22_01A1 and CRF02_AG. The genome of 02CAMLT04 was divided into three segments at the two breakpoints of nt 6951 and nt 9002, and the neighbor-joining trees were further built for each segment (Figure 3). The first segment extended from the gag to the beginning of the env gene (nt 789–6951), and clustered with CRF22_01A1 (BV, 100%). The second segment representing most of the env gene (nt 6952–9002) clustered with CRF02_AG radiation (BV, 100%) and the rest of the segments covering the nef gene (nt 9003–9408) clustered with CRF22_01A1. A significant portion (76%) of the 02CAMLT04 genome was in perfect alignment with CRF22_01A1 reference sequences except that most of the env gene clustered with CRF02_AG. These results demonstrated that the 02CAMLT04 virus was generated by the recombination of CRF22_01A1 and CRF02_AG. To our knowledge, this is the first report of a novel URF of CRF22_01A1 recombinant with CRF02_AG in full-length sequence.
Bootscan analysis of these viruses revealed the presence of CRF22_01A1 homology, the overall structures of these CRF22_01A1 containing viruses are diagrammed in Figure 4. Five sequences (LPH27MF, LB005, LB011, LB013 and LB054) clustered phylogenetically with CRF22_01A1 references throughout the entire genome demonstrating that these viruses share the same genomic mosaic and can be designated as pure CRF22_01A1. Two viruses (ARC087 and NYU488) shared identical recombinant breakpoints and were largely CRF22_01A1 (80% of whole genome spanning mostly pol to nef genes) and 20% for CRF02_AG. LB045 virus was divided into 4 segments with a complex CRF02/CRF22/CRF02/CRF22 recombinant, about 65% of the LB045 genome was CRF22_01A1. BDSH129 virus was almost entirely CRF02_AG (84%) with a small piece of CRF22_01A1 (16%) spanning the gag gene and the beginning of the pol gene. LB052, a triple CRF02/CRF22/CRF02 recombinant, was also predominantly CRF02_AG (76%) with a small portion of CRF22_01A1 fragment spanning the most of env gene from the beginning. ARC007 virus was a subtype B/CRF02 recombinant strain. LT66 and LT31 viruses were a complex F2/CRF02/F2/CRF02/F2 and CRF01/G/CRF02/G recombinant, respectively (data not shown).
To further understand the involvement of CRF22_01A1 in genetic recombination, the blast search of HIV sequence database against 6162 nt of 02CAMLT04 segment (nt 789–6951) yielded four high search score HIV-1 strains 01CM.0130NY, 01CM.1152NG, 02CM.3163MN and 01CM.4008HAN which were originally identified in Cameroon and classified as 01AF2U, A1U, AF2 and 01DU, respectively20. Phylogenetic analysis showed that 01CM.0130NY virus clustered with high confidence to CRF22_01A1 radiation (BV, 100%, Figure 2), 02CM.3163MN and 01CN.1152NG clustered with other CRF22_01A1 containing recombinant(BV, 85%). 01CM.4008HAN was a unique strain clustered between CRF01_AE and subsubtype A1. Further bootscanning analysis showed that they were recombinants of CRF22_01A1 with HIV-1 subtypes F, D, subsubtype A1, F2 or CRFs, all of these strains had a unique mosaic pattern combining CRF22_01A1 (Figure 4). The detailed phylogenetic composition of CRF22_01A1 containing recombinants described above is listed in table (Supplemental Content).
We identified five pure CRF22_01A1 and six CRF22_01A1 containing URFs strains in Cameroon. We also reclassified four CRF22_01A1 containing recombinants from the HIV Sequence GenBank (Figure 4). Nine unique recombination patterns were identified; breakpoints were found throughout the genome, the most definitive breakpoints were at the beginning of the pol, accessory or beginning of the env region. CRF22_01A1 fragments may be identified in any region across the HIV whole genome. Table 1 summarizes the subtype/CRF assignments of recombinants containing CRF22_01A1 fragments evaluated in samples collected from 2000–2010. Since the identification of CRF22_01A1 in Cameroon, it may be emerging as a dominant HIV-1 variant in this population that could act as a parental subtype for recombination events. The emergence of more complex intersubtype recombinants containing fragments of CRF22_01A1, such as CRF36_cpx, have been reported in Cameroon7. Reanalysis of the CRF36 genome including CRF22_01A1 reference sequences showed that the CRF36_cpx contains the fragment of CRF22_01A1, CRF01_AE and CRF02_AG. Brennan et al found that 22.4% of HIV-1 URFs were CRF22_01A1 in the blood donor samples collected in 1996–2004 in Cameroon14. Our recent study of samples collected from 2006 to 2008 in Cameroon also identified three CRF02gag/CRF22pol/CRF22env strains12,13. These studies highlight the complexity of recombinants between CRF22_01A1 and CRF02_AG in Cameroon (Table 1). CRF02_AG appeared to be the dominant virus in the population and accounted for 60–68% of HIV-1 infections with an additional 26% classified as URFs2, 21–23. This high degree of recombination could be due to preferential founder effects, and the subsequent introduction of the other genetic form, such as CRF22_01A1 or CRF02_AG, frequently resulting in recombinants containing fragments of CRF22_01A1 or CRF02_AG.
One interesting finding in this study is that all of the recombinants of HIV-1 characterized displayed a fairly similar breakpoint at the accessory gene and env region which spans from vif to gp120 gene. For example, a breakpoint of 02CAMLT04 genome in nucleotide position nt 6911 in gp120 was also observed around similar position in 02CAMLT72 (nt 7015), 01CM.1152NG (nt 6963), LB052 (nt 6099), LB045 (nt 6261), 01CM.4008HAN (nt 6088) and 01CM.0130NY (nt 6668). The segment from one third of the env to the 5’ end of the nef region (nt 7016–8895) in 02CAMLT72, showed a CRF22_01A1 virus derived from subsubtype A1, but was replaced by CRF02_AG in a similar region of 02CAMLT04 virus, suggesting that this region may be a hotspot for recombination between CRF22_01A1 and other subtypes/CRFs. The exact prevalence of the CRF22_01A1 intersubtype recombinants is still unclear. However, according to the current study, given that the CRF22_01A1-containing recombinants were possibly introduced to the population as early as 2000, CRF22_01A1 could be more broadly distributed in Cameroon even outside this country (Figure 1B). Although the CRF22_01A1 strain was prevalent over the 10-year period, the numbers of reported CRF22_01A1 cases were limited since reference strains of CRF22_01A1 were not well classified until now. The complexity of CRF22_01A1 strains in Cameroon seemed to increase during the 2002–2010 period. The CRF22_01A1 strain has also been identified in Saudi Arabia24, 25, Equatorial Guinea26, 27, Central African patients28 and USA29 indicating that the CRF22 strain was spreading to different geographic regions.
It has been well known that HIV-1 CRFs behave like a pure subtype of HIV-1 and are able to recombine with other subtypes or CRFs during HIV-1 viral reverse transcription when co-infection or super-infection occurs. We document here that CRF22_01A1 is circulating in Cameroon and is involved in complex intersubtype recombination events with subtypes (D or F), subsubtypes (A1 or F2) and CRFs (CRF01_AE or CRF02_AG). CRF22_01A1 recombinants seem to co-circulate at higher proportions than its pure prototype. Our findings further demonstrate the dynamic evolution of emerging variants in Cameroon which could potentially impact the phlyogenetic nature of the epidemic in this region in the future.
The authors wish to acknowledge Drs. Mingjie Zhang, Krishnakumar Devadas and Robin Biswas for review of the manuscript. The findings and conclusions in this article have not been formally disseminated by the Food and Drug Administration and should not be construed to represent any Agency determination or policy.
Source of Funding; Supported by The National Heart, Lung, and Blood Institute for funding part of this work through an IAA – National Heart, Lung and Blood InterAgency Agreement BY1-HB-5026-01.
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Table. Phylogenetic analysis of CRF22_01A1 containing recombinant segments