A total of 90 specimens obtained in the South and West regions were analyzed in calendar year 2006–2008 and identified five (sub)subtypes (A, A1, D, F2, and G) and nine CRFs (01_AE, 02_AG, 11_cpx, 13_cpx, 14_BG, 18_cpx, 25_cpx, and 36_cpx, and 37_cpx), as either pure strains (80% of all specimens, 72/90) or as URFs (26% of specimens evaluated at more than one locus, 18/70), based on phylogenetic analysis of gag, pol, and env fragments (Figure 1).

It is clear from the analysis of both regions of Cameroon that the genetic character of HIV-1 has continued to shift over the time period studied, and that this evolution is unique for each geographic area. While in the South Region, the proportion of URFs remained relatively constant between 2000–2001 (44%) and 2006-2008 (40%), in the West Region there was a statistically significant decrease of URFs from 60% in time period 2000–2001 to 17% in 2007–2008. Both regions showed increases in the proportion of SGRs over the time period: 25% more SGRs in the South and 7% more SGRs in the West, increases that left both regions with SGRs comprising 100% of the URFs identified. Although most of these changes in URF and SGR proportions were not significant, they may be an indication of growing trends to be closely monitored in future surveys. As only three genomic fragments were studied herein, it should be noted that these are only the minimum proportions of URFs and SGRs possible. Full-length sequencing might have revealed additional recombination in the specimens considered here to be pure (sub)subtypes or CRFs. Therefore, the true frequency of URF’s is likely to be even higher than identified in this study. Samples of interest, such as those with discordant subtype designations in different gene segments, will be further analyzed in order to reveal the true frequency of URF’s.

Recombinant forms of HIV-1 arise as a result of dual infection – the concomitant or sequential infection by two or more strains of HIV-1. The stability of URFs in the South region and the significant decrease of URFs in the West region suggest that the rate of dual infection may have remained constant or decreased over the 7–8 years time period studied. It is also possible that the URFs generated by these infections were poorly transmissible and/or comparably unfit among the co-circulating HIV-1 in these areas. We have found the frequency of dual infection among samples obtained between calendar years 2001–2004 in the urban center of Cameroon to be 16%; studies of dual infection as well as the biologic properties of viruses in these rural villages over the time period studied herein are warranted to better understand the driving factors in the genetic evolution of these viruses.

Although the number of different (sub)subtypes and CRFs identified remained fairly constant in both regions of Cameroon over the time period, it was evident that this did not correspond to static diversity. On the contrary, there were obvious shifts in genetic identity in both regions. In the South, (sub)subtypes A1, F2, H, and K, and CRF09_cpx, identified in 2000–2001, were replaced by CRFs 01_AE, 13_cpx, 14_BG, and 18_cpx in the 2006-2007 specimens. In the West, (sub)subtypes A2, C, G, and H, and CRFs 01_AE and 09_cpx, identified in 2000–2001, were replaced by sub-subtype A1 and CRFs 25_cpx and 37_cpx in 2007-2008. Although most of these shifts in subtype identity and proportion were not significant statistically, they do signify that larger studies of rural villages are necessary to properly characterize these endemic areas. Furthermore, in both regions, the proportion of CRF02_AG had increased at all loci, though only the increase in the West region at gag was significant (48%).

These increases of CRF02_AG suggest that HIV-1 is becoming more genetically homogenous in both the South and West regions. This may appear as particularly encouraging with respect to the env locus, as homogeneity in env would certainly aid in the design and efficacy of novel vaccines. However, considering the abundantly diverse HIV-1 strains still found to be co-circulating in both regions, and the general shift in HIV-1 diversity, especially with the emergence of several SGRs that show high levels of recombination with CRF02_AG variants within the relatively short time period, it is clear that the dominance of CRF02_AG may reduce over time.

The recent study by Brennan et al., in which over 500 specimens obtained from 1996–2004 from urban, HIV-positive blood donors in Yaoundé and Douala were analyzed, found there to be no significant changes in the proportions of all but one subtype (D) over the 8-year period, and that the strain compositions of the URFs identified changed significantly only for the proportion containing subtype G [14]. Despite the small sample size analyzed herein relative to the Brennan et al. study, our survey of these rural areas found significant decreases in subtypes A (at env) and D and CRF01_AE (at gag) in the West region and a significant increase in CRF02_AG (at gag), in addition to the overall significant decrease in URFs, suggesting HIV-1 evolution in these rural areas to be more marked than in the urban centers. These results imply that larger sample sizes in future studies of rural Cameroon may demonstrate an even more dynamic HIV population, and highlights the concern that subtype-specific vaccines may not be relevant in these areas of Cameroon, and that the distribution of viral diversity in these regions must be carefully monitored.

Blood samples from the general population from 82 HIV-positive, asymptomatic, chronically-infected subjects were collected in calendar years 2000 and 2001 in rural villages of the South and West Regions of Cameroon. The 40 samples collected in the South region were from 15 men and 25 women with ages ranging from 21 to 56 years (mean, 37 years). Thirty-four of these specimens were included in a previous study of the South region [2,3,19]. Forty-two specimens (19 men, 22 women, one unavailable) were obtained in the West region, 28 of which were subjects of previous study of the West region [2,3,19]. These study subjects were between 17 and 70 years of age (mean, 36 years). In calendar years 2006–2008, 95 HIV-positive blood samples were collected from additional subjects living in the same regions. The 30 specimens obtained in the South region were from seven men and 21 women (information on two subjects was unavailable) who were between 21 and 62 years of age (mean, 34 years), while the 65 samples from the West region were from 12 men and 24 women (information on 22 subjects was unavailable) who were between 18 and 64 years of age (mean, 35 years). The samples from the South province were obtained from the following villages: Endengue, Minko’o, Yen, Akom, Nkono-Gbas, Bitche, Andoung, Mekas, Olembe, Melombo, Ndtoua, Ebodje, Idondefang, Mabiogo, Bidjouka, Mveng, Djoum and Efoulam. The samples from the West province were collected in the following villages: Biassamba, Makoutam, Makoutmbou, Makpa, Mahoua, Matambou, Nabouobou, Manje Magni, Marapdoum, Tchouadeng, Junitsa, Mapoure and Malantouen. All study subjects were antiretroviral drug naïve. Summarized study subject data is available in Supplementary Table 1 and 2.

Plasma was obtained by Ficoll-Hypaque gradient centrifugation of whole blood. Viral RNA was extracted from plasma using the QIAamp Viral RNA Mini kit (Qiagen Inc, Valencia, CA). 2 μl of RNA was used for one-tube reverse transcriptase polymerase chain reaction (RT-PCR) using the Superscript One-Step RT-PCR for Long Templates kit (Invitrogen, Carlsbad, CA) to amplify a fragment of gag (HXB2 positions 1577–2040, 463 bp), part of the pol gene encoding the protease (PR) enzyme (HXB2 positions 2241-2577, 336 bp), as well as the C2-V5 region of env (HXB2 positions 7001–7647, 646 bp). 2 μl of first-round product was then used in a nested PCR using the Platinum PCR SuperMix High Fidelity system (Invitrogen, Carlsbad, CA). The gag fragment was amplified using the outer primers H1G777 and H1P202 and the inner primers H1gag1584 and G17 [20]. The PR region of pol was amplified using the outer primers NYUPOL6, NYUPOL7 and NYUPOL8 and the inner primers NYUPOL9 and NYUPOL10 [19]. All the primers have previously been described. The amplification conditions for the gag and pol fragments for the one-tube RT-PCR was one cycle of 50 °C for 30 minutes and 94 °C for 2 minutes, followed by 40 cycles of 94 °C for 15 seconds, 50 °C for 30 seconds and 68 °C for 1 minute, ending with a single extension cycle at 72 °C for 7 minutes. For the second-round PCR, the above protocol was used for 35 cycles, beginning with the 94 °C denaturation step. To amplify the C2-V5 region of env the previously described outer primers ED5 and ED12 and the inner primers ES7 and ES8 were used [21]. The amplification conditions for the env fragment for the one-tube RT-PCR was one cycle of 50 °C for 30 minutes, followed by 3 cycles of 94 °C, 55 °C, and 72 °C each for 1 minute. This was followed by 32 cycles of 94 °C for 15 seconds, 55 °C for 45 seconds and 72 °C for 1 minute, ending with a single extension cycle at 72 °C for 7 minutes. For the second-round PCR, the above protocol was repeated without the RT step. Polymerase chain reaction products were directly sequenced at the 5’and 3’ ends using the respective outer primers.

The sequences were automatically aligned with identical regions of reference sequences of all known HIV-1 group M (sub-)subtypes and CRFs from the Los Alamos HIV database using CLUSTAL X^{, and manually cropped using Seaview [13,22,23]. Phylogenetic analyses were conducted using MEGA version 4 software package [24]. Pairwise evolutionary distances were estimated using Kimura’s two-parameter method, and phylogenetic trees were constructed by neighbor-joining [25,26]. Clustering of sequences with a bootstrap value of more than 60% was used for subtyping. All sequences were included in a single phylogenetic tree for each locus in order to identify and omit highly related sequences and avoid study subject resampling. No such sequences were found.}

Differences in the proportions of each (sub)subtype or CRF at each locus, and of URFs and SGRs at each study period for each geographic region were evaluated using a Two-way chi-square test (95% confidence interval).

The sequences generated for this study are available from GenBank with the accession numbers GU047883–GU048094.