Charlotte Ngansop

The prevalence of diverse HIV-1 strains was stable in Cameroonian blood donors from 1996 to 2004.

Objective:The HIV epidemic in Cameroon is characterized by a high level of strain diversity despite a relatively low prevalence of infection. In this study, HIV strains infecting blood donors in Cameroon were characterized to determine the prevalence of subtypes and intersubtype recombinants and if strain prevalence was changing over time. Methods:From 1996 through 2004, 676 HIV-infected blood donations were collected at blood banks in Douala and Yaoundé, Cameroon. A subset of the HIV-1 group M strains (n = 574) were classified based on phylogenetic analysis of viral sequences from the gag p24, pol integrase, and env gp41 regions. Results:HIV-1 group M accounted for 97.3% (n = 658) of infections, whereas group O was present in 2.2% (n = 15) and HIV-2 in 0.4% (n = 3). Within the group M infections, 14 subtypes and circulating recombinant forms (CRFs) and unique recombinant forms (URFs) were identified. Overall, CRF02_AG accounted for 58.2% of infections, URFs 14.8%, and levels of subtypes, A, B, C, D, F2, and G, and CRFs, 01, 06, 09, 11, 13, 22, and 37, varied from 0.2% to 6.1%. Evaluation of HIV strains present in the donor population over this 9-year period showed no substantial changes in the proportion of infections caused by each subtype and CRF, the percentage of intersubtype recombinants, or the strain composition of the URFs. Conclusions:HIV-1 strain diversity in Cameroon did not significantly change, suggesting a mature and relatively stable epidemic.

Four new HIV-1 group N isolates from Cameroon: prevalence continues to be low.

Analysis of 3555 HIV-seropositive specimens, collected in Cameroon from 2002 to 2006, led to the identification of four HIV-1 group N infections based on differential seroreactivity to HIV env-derived peptides and proteins and confirmation by nucleic acid amplification. Group N prevalence continues to be low accounting for only 0.1% of HIV infections in Cameroon. Near full-length genomic sequences were obtained from viral RNA or proviral DNA by PCR amplification of overlapping fragments for three isolates, 06CM-U14296, 06CM-U14842, and 02CM-SJGddd. Two genome segments, partial pol and env-nef, were obtained from viral RNA for the fourth isolate, 02CM-TIM0217. With the four group N isolates identified in this study and group N sequences previously reported, eight near full-length and five partial genome sequences are now available. Despite genetic divergence from HIV-1 group M and O, all of the group N infections evaluated by five commercial HIV immunoassays were detected.

Genetic diversity of HIV type 1 in rural eastern Cameroon.

To monitor the presence of genotypic HIV-1 variants circulating in eastern Cameroon, blood samples from 57 HIV-1-infected individuals attending 3 local health centers in the bordering rural villages with Central African Republic (CAR) were collected and analyzed phylogenetically. Out of the 40 HIV-1 strains with positive polymerase chain reaction (PCR) profile for both gag and env-C2V3,12 (30.0%) had discordant subtype or CRF designation: 2 subtype B/A (gag/env), 1 B/CRF01, 2 B/CRF02, 1 CRF01/CRF01.A, 2 CRF11/CRF01, 1 CRF13/A, 1 CRF13/CRF01, 1 CRF13/CRF11, and 1 G/U (unclassified). Twenty-eight strains (70.0%) had concordant subtypes or CRF designation between gag and env: 27 subtype A and 1 F2. Out of the remaining 17HIV-1 strains negative for PCR with the env-C2V3 primers used, 10 (58.8%) had discordant subtype or CRF, and 7 (41.2%) had concordant one based on gag/pol/env-gp41 analysis. Altogether, a high proportion (22/57, 38.6%) of the isolates were found to be recombinant strains. In addition, an emergence of new forms of HIV-1 strains, such as subtype B/A (gag/env), B/CRF01 and B/CRF02, was identified. The epidemiologic pattern of HIV-1 in eastern Cameroon, relatively low and high prevalence of CRF02 and CRF11, respectively, was more closely related to those of CAR and Chad than that of other regions of Cameroon, where CRF02 is the most predominant HIV-1 strain. These findings strongly suggest that this part of Cameroon is a potential hotspot of HIV-1 recombination, with a likelihood of an active generation of new forms of HIV-1 variants, though epidemiologic significance of new HIV-1 forms is unknown.

Evaluation of HIV type 1 group O isolates: identification of five phylogenetic clusters.

HIV-1 group O strains have a level of genetic diversity similar to that of strains in group M; however, group O has not been readily classified into genetic subtypes. Phylogenetic classification of group O has been hindered by the limited sequence information available. To facilitate phylogenetic analysis, we sequenced the gag p24 (693 nt), pol p32 (864 nt), and env gp160 (approximately 2700 nt) genes from 39 group O-infected specimens. These specimens include 32 plasma samples collected in Cameroon between 1996 and 1999, 2 specimens collected in the United States, and 5 infections previously isolated in Equatorial Guinea. Phylogenetic analysis of HIV-1 group O sequences resulted in the identification of five clusters that are maintained across gag, pol, and env, generally supported by high bootstrap values, and approximately equidistant from each other. In addition to the group O clusters, several isolates branch independently and are equidistant from the other group O isolates. Cluster I comprises greater than 50% of the group O isolates and is a diverse set of isolates that is subdivided into subclusters. The average intra-, sub-, and intercluster distances for group O are similar to the corresponding distances for group M subtypes. The five group O clusters have characteristics similar to those of group M subtypes. Thus the data presented may form the basis for classification of group O into subtypes. However, full-length genomes representing each group O cluster will be required to formalize a group O subtype classification.

Natural Infection of Wild-Born Mandrills (Mandrillus sphinx) with Two Different Types of Simian Immunodeficiency Virus

We found a novel primate lentivirus in mandrill (Mandrillus sphinx). To clarify the evolutionary relationships and transmission patterns of human/simian immunodeficiency virus (HIV/SIV), we screened blood samples from 30 wild-born healthy Cameroonian mandrills. Five (16.7%) of them were seropositive for SIV. Three SIV strains were isolated from the five seropositive mandrills by cocultivation of their peripheral blood mononuclear cells (PBMCs) with PBMCs of rhesus macaques, a human T cell line (M8166), and/or a cynomolgus macaque T cell line (HSC-F). One of the newly isolated SIV strains was intravenously inoculated into two rhesus macaques and resulted in chronic infection. In the SIV-infected macaques at 45 weeks after inoculation, we observed a mild decline in the number of peripheral CD4(+) lymphocytes, lymphadenopathy, and blastic follicular dendritic cells with mild follicular hyperplasia in the peripheral lymph nodes. A phylogenetic analysis based on the pol sequence showed that the newly found SIVs from Cameroonian mandrills did not cluster with SIVmndGB1, which is the former representative strain of SIVmnd. The SIVmnds from Cameroon formed a new, independent lineage that branched before the root of the HIV-1/SIVcpz lineage with 996 of 1000 bootstrap replications. They clustered host specifically, and exhibited about 16.9% diversity at the level of nucleotide sequence among Cameroonian SIVmnd strains. These results indicate that the SIVmnds isolated in Cameroon are a novel type of SIVmnd and have infected Cameroonian mandrills for a long time. We therefore designated the Cameroonian SIVmnd as SIVmnd type 2 and redesignated SIVmndGB1 as SIVmnd type 1. To date, M. sphinx is the only primate species other than humans that is naturally infected with two different types of SIV.

HIV type 2 intergroup recombinant identified in Cameroon.

A unique HIV-2 intergroup recombinant strain was identified in Cameroon. The virus, CM-03-510-03, was amplified from blood collected from a 47-year-old female patient in Douala, Cameroon in 2003 who was seroreactive for HIV-2. A near full-length genome 9089 nucleotides in length was amplified from proviral DNA. The genome for CM-03-510-03 is composed of segments of HIV-2 groups A and B with four recombination break-points and has open reading frames for all the structural and regulatory genes. A comparison of CM-03-510-03 to the only previously reported HIV-2 intergroup recombinant shows that the two strains share one recombination breakpoint but are otherwise distinct from each other. Similar to HIV-1, HIV-2 intergroup recombination is an indication that coinfection with more than one strain has occurred in individuals and is a mechanism that increases strain genetic diversity.

HIV Type 1 Infection in Pygmy Hunter Gatherers Is from Contact with Bantu Rather Than from NonHuman Primates

To investigate the route of zoonotic transmission of HIV-1, we isolated three and seven HIV-1 strains from 449 Pygmy hunter gatherers and 169 neighboring Bantu, respectively, in southern Cameroon. Phylogenetic analysis based on pol-integrase and env-C2V3 sequences revealed that strains from Pygmies were 1CRF02_AG/CRF02_AG, 1 subtype G/CRF02 AG (pol/env), and 1 CRFll_cpx/CRF11_cpx, and that those from Bantu were 2 CRF02_AG/CRF02_AG, 1 CRF02_AG/CRF01_AE/A, 1 CRF02_AG/subtype A, 1 G/A, 1G/CRF02_AG, and 1 unclassified fH. CRF02_AG and CRF11_cpx have been identified in Cameroon. The results suggest that HIV-1 has been introduced into Pygmies through their neighboring Bantu rather than directly from nonhuman primates.

Near Full-Length Genome Characterization of an HIV Type 1 CRF25_cpx Strain from Cameroon

Recombinant forms of HIV-1 contribute significantly to the ongoing epidemic. In the present study, we characterized the near full-length genome of one candidate HIV-1 CRF25_cpx strain originating in Cameroon, 06CM-BA-040. Viral RNA was extracted from plasma, and the genome was obtained using RT-PCR amplification to generate 10 overlapping fragments. Bootscanning, recombination breakpoint analysis, and phylogenetic trees confirmed that 06CM-BA-040 had a genomic structure consistent with two available CRF25_cpx reference sequences. The CRF25_cpx mosaic composition consisted of nine segments derived from subtypes A and G as well as unclassified (U) regions. Subtype G and CRF25_cpx clusters diverged from each other with long branch lengths but were distinct from other known subtypes with high bootstrap support (94%). The epidemiological significance of CRF25_cpx strains is unknown; however, the availability of additional genomic sequences will improve our understanding of the overall genetic diversity within this recombinant form of HIV-1.

High Frequency of HIV-1 Dual Infections in Cameroon, West Central Africa

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. 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. In sub-Saharan Africa, most HIV-1 types and subtypes cocirculate, although their geographical distribution is not uniform. 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. To date, a number of studies have been performed to determine the prevalence of dual infection in Cameroonian populations. 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 lowrisk 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. 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’-TGGGTACCAG CACACAAAGGA ATAGGAGGAAA-3’, at position 3434–3765 of HIV-1LAI) and Unipol-6 (5’-CCACAGCTGATCTC TGGCCT 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’-CCCCTAT TCCTTCCCCTTCTTTTAAAA-3’, at position 4363–4388 of HIV-1LAI) were used to generate sequence fragment. 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). 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. The phylogenetic trees were constructed using neighbor joining method, and the reliability of tree topologies was estimated by bootstrap analysis (1000 replicates). 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. Our findings confirm an earlier report that showed a relatively high proportion of subtype G and CRF02_AG in western Cameroon. Identification of HIV-1 subtype H strains in our cohort is of substantial interest. Although subtype H has been formally recognized for many The authors have no conflicts of interest and competing financial interest.

HIV type 1 group M subtype G in Cameroon: five genome sequences.

Near full-length viral genome sequences were obtained for five putative subtype G candidates identified in HIV-infected Cameroonian blood donors, based on partial genome sequences for the gag, pol, and env regions. Phylogenetic analysis of the genome sequences shows that all five strains are pure subtype G with no indication of intersubtype recombination. The Cameroon subtype G sequences did not form a geographically based subcluster and were intermixed within the subtype G branch with isolates from several different countries. HIV-1 group M subtype G accounts for only 4.5% of HIV infections in Cameroon. However, genome segments of subtype G are present in 67% of all infections and 80% of infections due to intersubtype recombinant strains in Cameroon. The addition of five subtype G genome sequences to the HIV database may contribute to a better understanding of the origins and classification of HIV-1 subtypes and CRFs.