To the Editors:
The molecular epidemiology of HIV-1 is characterized by a complex and evolving genetic diversity. This results from a high rate of mutations and recombination and a high turnover of virions in HIV-1-infected individuals. Based on phylogenetic analysis, HIV-1 is classified into 3 known groups of HIV-1 (M, N, and O); a new group named as P has recently been identified in a Cameroonian patient.1 The great majority of HIV-1 sequences belong to the M group, composed of 9 genetic subtypes (A-D, F-H, J-K), 6 subsubtypes (A1, A2, A3, A4, F1, and F2), and at least 48 circulating recombinant forms (CRFs). A myriad of unique recombinant forms have also been identified (http://www.hiv.lanl.gov/content/sequence/HIV/CRFs/CRFs.html). Most genomes of CRFs are highly complex intersubtype recombinants.2 In sub-Saharan Africa, most HIV-1 types and subtypes cocirculate, although their geographical distribution is not uniform.3 In west Africa, CRF02_AG accounts for the majority of HIV-1 infections. In Cameroon, the majority (>80%) of HIV-1 infections among blood donors are caused by CRFs and unique recombinant forms with 80% of the recombinant strains containing segments of subtype G.4
To date, a number of studies have been performed to determine the prevalence of dual infection in Cameroonian populations.5-7 We set out to characterize HIV-1 subtypes and to determine the frequency of HIV-1 intersubtype and intrasubtype dual infections in antenatal attendees and blood donors from northwestern Cameroon. The study protocol was approved by the Ethical Review Board of the Ministry of Public Health of Cameroon and the National Science and Ethics Committee.
This is a cross-sectional clinical cohort study in a population with low-risk sexual behavior. A total of 120 participants in the northwestern region of Cameroon were enrolled in this study following their informed written consent. Sera were screened by a particle agglutination test (Serodia-HIV; Fujirebio, Tokyo, Japan). Proviral DNA extraction, amplification, and sequence analysis were performed using a modification of previously reported methods.7 Of the 51 seropositive specimens subjected to nested polymerase chain reaction (PCR) using universal primers (Unipol-5 and Unipol-6 for first round, and Unipol-1 and Unipol-2 for second round) and a Super Mix premixed reagent (Invitrogen, Carlsbad, CA). The partial pol was amplified using first round PCR primers Unipol-5 (5′-TGGGTACCAGCACACAAAGGA ATAGGAGGAAA-3′, at position 3434-3765 of HIV-1LAI) and Unipol-6 (5′-CCACAGCTGATCTCTGGCCT TCTCTGTAATAGACC-3′, at position 4483-4516 of HIV-1LAI) and second round primers Unipol-1 (5′-AGTGGATTCATAGAA AGCAGAAGT-3′, at position 4052-4074 of HIV-1LAI) and Unipol-2 (5′-CCCCTATTCCTTCCCCTTCTTTTAAAA-3′, at position 4363-4388 of HIV-1LAI) were used to generate sequence fragment.7 The PCR products were purified using the QIAquick PCR Purification Kit (Qiagen, Hilden, Germany) and inserted into Invitrogen TA-cloning vector (Invitrogen, Carlsbad, CA) for cloning and sequencing. Between 10 and 20 colonies per sample were analyzed for evidence of intrasubtype and intersubtype dual infections. Only proviral DNA amplification led to the identification of dual infections in most patient samples. Attempts to identify dual infections from reverse-transcribed and amplified HIV RNA were unsuccessful in most of our specimens. It is therefore possible that some of the sequences amplified are archived viruses that do not significantly contribute to a productive infection.
The nucleotide sequences were manually refined in Se-Al (http://tree.bio.ed.ac.uk/software/), and electropherogram sequence assemblages were obtained with Genetyx Mac 10.0 and Genetyx MacATSQ v3.0 (Software Development, Tokyo, Japan).8 Subtype reference sequences of HIV-1 group M available from the Los Alamos Sequence database were aligned to the generated sequences using ClustalX. The MEGA version 4.0 software package was used to perform phylogenetic analysis, and the pairwise evolutionary distances were estimated using the HKY85 model.9,10 The phylogenetic trees were constructed using neighbor joining method, and the reliability of tree topologies was estimated by bootstrap analysis (1000 replicates).11,12 Epidemiologically linked transmissions were defined as those with bootstrap values >80%. For clarity of the figure, only 2 sequences were included in the tree for this letter, but an analysis was also performed including all clones per patient. An analysis including a good sampling of subtypes and including all the clones per patient may also reveal intrasubtype recombination, and only consensus sequences derived from intersubtype dual infections were included and deposited to GenBank with the accession numbers HQ714903 to HQ714956 (Fig. 1).
The results show that 11 HIV-1 strains are cocirculating in the northwestern region of Cameroon. HIV-1 group M CRF02_AG infections (35.0%), subtype A (4.0%), C (6.0%), D (4.0%), G (27.0%), H (4.0%), U (2.0%), CRF11.cpx (4.0%), CRF22.cpx (2.0%), CRF25.cpx (2.0%), and CRF37.cpx (10.0%). Four intersubtype (8.0%) dual infections were identified among the 51 samples analyzed (1 H/G, 1 C/CRF25.cpx, 1 H/CRF37.cpx, and 1 C/CRF37.cpx). We identified 3 potential transmission pairs (Fig. 1, unfilled circles), which were supported by epidemiological data. Moreover, there are 2 subclusters of 10 new Cameroonian and 1 outlier sequence.
In a study of HIV-infected blood donors in several regions of Cameroon from 1996 to 2004, subtype G accounted for 4.5% of HIV-1 infections.13 Our findings confirm an earlier report that showed a relatively high proportion of subtype G and CRF02_AG in western Cameroon.14 Identification of HIV-1 subtype H strains in our cohort is of substantial interest. Although subtype H has been formally recognized for many years, only 4 near full-length genomes and 2 recombinants with subtype H segment have been reported to date.15 The most recent data available on the distribution and prevalence of subtype H suggest that it represents <0.2% of global HIV infections and is most prevalent in central Africa (3.14%).4 This could indicate that dual infections observed are a major event among this heterosexual population.The relatively high frequency of intersubtype dual infections with subtype G seems to correlate with the high prevalence of subtype G (CRF02_AG) viruses in northwestern Cameroon.16
In conclusion, dual infections with divergent HIV-1 strains are more common than previously thought and are important because this information provides a more rational basis for developing AIDS vaccines and for predicting the global evolution of HIV in the future.
Nicaise Ndembi, MPhil, PhD*†
Shizuka Iwamoto, BSc‡
Charlotte Ngansop, BSc*
Philippe Lemey, PhD§
Alashle Abimiku, PhD†
Dora Mbanya, MD*
Lazare Noche Kaptue, MD*‖
Eiji Ido, PhD‡¶
*Université de Yaoundé-I, Yaoundé, Cameroon; †Institute of Human Virology, University of Maryland School of Medicine, Blatimore; Institute of Human Virology-Nigeria, Abuja, Nigeria; ‡Center for Emerging Virus Research, Institute for Virus Research, Kyoto University, Japan; §Rega Institute, Katholieke Universiteit Leuven, Leuven, Belgium; ‖Université des Montagnes, Bangangté, Cameroon; ¶Department of Molecular Virology, Tokyo Medical and Dental University, Yushima, Bunkyo-ku, Tokyo, Japan
The authors are grateful to the participants and Laboratory staff at St Elizabeth General Hospital. They also thank Prof Frances Gotch, Dr Jean Carr, and Dr Catherine Brennan for reviewing the manuscript. This work was partly supported by Grant-in-Aid from the Ministry of Education, Culture, Sports, Science and Technology of Japan.
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