Increased Genetic Diversity and Intersubtype Recombinants of HIV-1 in Blood Donors From Urban Cameroon

Abstract

To the Editor: One of the most significant biologic characteristics of HIV-1 is its broad genetic diversity, which is revealed by the existence of 3 groups (M, N, and O), various subtypes (A-D, F-H, J, and K) and sub-subtypes (A1-A4, F1, and F2), and numerous circulating recombinant forms (CRFs) or unique recombinant forms (URFs) within HIV-1 group M (Los Alamos HIV/AIDS Sequence Database, available at: http://hiv-web.lanl.gov). In areas where CRFs spread widely, second-generation recombinant viruses (SGRs) have been reported to contain the genetic material of at least 1 CRF. The extensive genetic variation, the heterogeneous geographic distribution of HIV-1, and, in particular, the continuous emergence of HIV-1 variants pose potential public health and clinical challenges. It is thus necessary to monitor the evolution of HIV-1 on an ongoing basis. In 2002, we initiated a serologic survey in Douala and Yaoundé, Cameroon, where the pandemic infection of HIV-1 with broad genetic diversity and the natural reservoir of HIV-1 groups M and N have been identified. A total of 240 unlinked plasma samples were collected from 2 blood centers from urban areas in Douala and Yaoundé, Cameroon, and 135 samples were confirmed to be HIV-1-positive by US Food and Drug Administration (FDA)-licensed HIV diagnostic tests (results are to be published elsewhere). Eighty-eight HIV-1-positive samples were successfully amplified by polymerase chain reaction (PCR) and sequenced in gag (p17) and env (gp41) genes. In addition, 3 samples were sequenced in the gag (p17) gene only, and another 36 samples were sequenced in the env (gp41) gene only. The nucleotide sequences have been deposited in the GenBank under the following accession numbers: DQ056863 through DQ056984 (gp41) and DQ056985 through DQ057075 (p17). Phylogenetic analysis based on HIV-1 gag (p17) and env (gp41) sequences indicated that all analyzed sequences were classified as HIV-1 group M. HIV-1 group N or group O viruses were not found in these samples. Table 1 summarizes the genotyping results and indicates that HIV-1 CRF02_AG was dominant (69% in gag, 72% in env, and 60% in gag/env, respectively) in our study, followed by HIV-1 subtypes (15% in gag, 10% in env, and 5.7% in gag/env, respectively) and CRFs or URFs (15% in gag, 18% in env, and 34% in gag/env, respectively). Aghokeng et al1 also reported that 74% of the HIV-1 strains collected from blood donors in 7 localities in Cameroon in 2001 were CRF02_AG based on the sequences of gp41 gene. The prevalence of HIV-1 CRF02_AG is much lower in the general population in Cameroon, however, where HIV-1 CRF02_AG was identified in 20% to 32% of HIV-1-infected persons.2,3 HIV-1 CRF02_AG is also less prevalent in the surrounding countries, such as Congo (2.7%) and Central African Republic (2.6%), although a relatively high prevalence of 26% has been reported in Gabon.4TABLE 1: Genetic Subtypes in the gag and env Regions of HIV-1 Isolates From 2 Cities in CameroonWe also included in our analysis the recently listed reference strains of CRF17 to CRF33 at the Web site of Los Alamos National Laboratory (available at: http://www.hiv.lanl.gov/content/hiv-db/CRFs/CRFs.html). We thus identified CRF19_cpx- and CRF22_01A1-like sequences in 2 and 7 samples in gag (p17) gene, 2 and 4 samples in env (gp41) gene, and 0 and 4 in gag/env genes, respectively. Within the phylogenetic trees, CRF19_cpx-like sequences clustered together to form a separate branch, which is close to the CRF02_AG cluster. The branch of CRF22_01A1-like sequences was closer to the CRF01_AE cluster, but the CRF22_01A1 and CRF01_AE clusters were within the subtype A group of HIV-1. The homology of these CRF22_01A1 sequences was 80% in gag (p17) and 85% in env (gp41), respectively. Until now, CRF22_01A1 has been fully sequenced in 1 isolate (01CM001BBY, accession number AY371159) from Cameroon5 and is reported to contain portions of subtype A1 and to share ancestry with CRF01_AE, but the detailed mosaic structure has not been published. In our study, 4 samples were assigned to CRF22_01A1 in the gag and env genes, suggesting that CRF22_01A1 most likely contains genetic material of subtype A in gag (p17) and the gp41 region and providing further evidence of the existence of the new CRF22_01A1 in Cameroon. Unlike CRF22_01A1, the CRF19_cpx sequence was only identified in gag or env gene in 4 samples analyzed in our study. All 4 of these samples seemed to be infected with HIV-1 recombinant forms of CRF02_AG/CRF19_cpx. It must be noted that the full genomic sequence of CRF19_cpx was first characterized in Cuba but exhibits homology to partial sequences of the viruses from Cameroon, Gabon, Senegal, Chad, Niger, Central African Republic, and Congo.6 These results support the viewpoint that CRF18_cpx and CRF19_cpx may originate in Africa. Parallel phylogenetic analyses in the gag (p17) and env (gp41) genes revealed that 26.1% (23 of 88) of the sequences showed discordant subtypes in the 2 genes analyzed and were considered as URFs. Among them, 22 were identified as containing genetic material of at least 1 CRF in at least 1 gene, and were therefore considered as SGRs. The other URF identified was Agag/Denv. For the 22 SGRs identified in our study, 13 (59%) were the recombinants of CRF02_AG with HIV-1 subtype (A, B, D, and F2) or CRFs (CRF06_cpx, CRF11_cpx, CRF19_cpx, and CRF22_01A1). The remaining were recombinants of CRF06_cpx, CRF11_cpx, CRF13_cpx, and CRF22_01A1 (Table 2). Similar findings have been reported in Burkina Faso and Niger, where CRF02_AG/CRF06_cpx accounts for 6% to 9% of HIV-1 infections.7,8 CRF02_AG/CRF06_cpx and CRF02_AG/CRF09_cpx have been found in Côte d'Ivoire, and CRF07_BC/CRF08_BC has been found in China. Even CRF18_cpx and CRF19_cpx have been found to form new recombinants CRF18/CRF19 in Cuba.6 Our results are in agreement with those of previous studies and indicate that like HIV-1 subtypes in areas in which HIV-1 CRFs are endemic, CRFs are also involved in recombination events. The most striking observation in our study was the extremely high proportion (94%) of HIV-1 recombinants (CRFs or URFs) in our samples. Most of the recombinant forms contain the genetic material of CRF02_AG. The recombinants of CRF02_AG and HIV-1 subtypes A, C, G, H, and J have been reported in rural villages in the center, south, east, and west provinces of Cameroon.2 Consistent with these findings, our study further identified recombinants between CRF02_AG and subtypes B, D, and F2. We could also identify recombinants of CRF02_AG/CRF06_cpx in our study as well as several new URFs containing different CRFs, such as CRF02_AG/CRF11_cpx, CRF02_AG/CRF19_cpx, CRF02_AG/CRF22_01A1, CRF11_cpx/CRF22_01A1, and CRF06_cpx/CRF13_cpx. These results indicate that a second generation of recombinant forms of HIV-1 is emerging as a dominant recombination event in HIV-1 infection and highlight the importance of recombination of HIV-1 subtypes, especially the recombinant CRF02_AG, in blood donors in Cameroon.TABLE 2: Genotype Assignments of Partially Characterized Strains in CameroonIn summary, the results of this study further reveal the complexity of HIV-1 infection in blood donors from urban Cameroon, a region that is characterized by broad genetic diversity, the presence of different HIV-1 subtypes and recombinants, and the continuing emergence of new recombinants of HIV-1 as reflected by the identification of new recombinant strains in our study. The variation of HIV-1 in a population within a limited geographic area suggests the future complexity of the HIV-1 epidemic worldwide and the importance of continually monitoring current HIV-1 assays for sensitivity for HIV variants. Future investigations of HIV-1 variants should include studies on rural populations in Cameroon to evaluate further the extent of viral diversity in blood donors and patient populations. Ana Machuca, PhD* Shixing Tang, MD, PhD* Jinjie Hu, PhD* Sherwin Lee, BS* Owen Wood, PhD* Christopher Vockley, BS* Suresh Gupta Vutukuri, MS† Ranjana Deshmukh, MD, PhD† Bih Awazi, BS‡ Indira Hewlett, PhD* *Laboratory of Molecular Virology Division of Emerging and Transfusion Transmitted Diseases Center for Biologics Evaluation and Research US Food and Drug Administration Bethesda, MD †Haffkine Institute Mumbai, India ‡Mobile Laboratory Sanitation and Hygiene Administration of Health Laboratoire de Santé Hygiène MobileYaounde, Cameroon