The Evolution of HIV-1 Diversity in Rural Cameroon and its Implications in Vaccine Design and Trials

West-Central Africa is an epicenter of the HIV pandemic; endemic to Cameroon are HIV-1 viruses belonging to all (sub)subtypes and numerous Circulating Recombinant Forms (CRFs). The rural villages of Cameroon harbor many strains of HIV-1, though these areas are not as well monitored as the urban centers. In the present study, 82 specimens obtained in 2000 and 2001 from subjects living in the rural villages of the South and West Regions of Cameroon were subtyped in gag, pol, and env and compared to 90 specimens obtained in 2006–2008 in the same regions, in order to analyze HIV-1 evolution in these rural areas. It was found that in the South Region, the proportion of unique recombinant forms (URFs) remained constant (∼40%), while the amount of URFs containing fragments of a CRF increased by 25%. (Sub)subtypes A1, F2, H, and K, and CRF09_cpx, identified in 2000 and 2001, were replaced by CRFs 01_AE, 13_cpx, 14_BG, and 18_cpx in 2006–2008. In the West Region, (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 2006–2008. The proportion of URFs in the West Region dropped significantly over the time period by 43%. In both Regions, the proportion of CRF02_AG increased at all loci. These findings demonstrate that the evolution of HIV-1 is distinct for each endemic region, and suggests that the proportion of URFs containing CRF fragments is increasing as the genetic identity of the virus continues to shift dramatically. This highlights the concern that subtype-specific vaccines may not be relevant in Cameroon, and that the distribution of viral diversity in these regions of Cameroon must be carefully monitored.


Introduction
As an epicenter of the HIV pandemic, the West-Central African country of Cameroon is host to one of the broadest genetic arrays of HIV viruses [1][2][3][4][5][6][7][8].In addition to HIV-2 and HIV-1 group N and O viruses, all the group M subtypes have been found in Cameroon, and many Circulating Recombinant Forms (CRFs) of HIV-1 were first identified there [9][10][11][12].CRFs arise from the transmission of Unique Recombinant Forms (URFs), which are the result of recombination brought about by dual HIV-1 infection -the concomitant or sequential infection by at least two distinct HIV-1 strains.Complicating the genetic landscape in Cameroon and elsewhere is the phenomenon of Second-Generation Recombinants (SGRs): URFs that contain fragments of one or more CRFs (such as CRF11_cpx, CRF22_01A1, and CRF36_cpx), as opposed to the initially-identified CRFs, which are generally mosaics of pure subtypes (such as CRFs 01_AE through 10_CD) [13].Recently, we have found the frequency of dual infection in the urban center of Yaoundé to be 16% (~11% incidence per year) in samples obtained between 2001 and 2004.This finding confirmed earlier data that had commonly identified URFs as the HIV-1 strains circulating among the HIV-positive population in the urban cores as well as certain rural areas [3,6,14,15].This rate of dual infection coupled with the extremely diverse nature of the pandemic in Cameroon is cause for concern, as the design of relevant, effective vaccines may be severely compromised by such a genetic milieu.
A substantial body of data on HIV diversity in Cameroon has come from studies of the urban centers [6,14,16,17].Phylogenetic study of the gag and env loci amplified from specimens obtained from subjects living in the cities of Yaoundé and Douala in 2002 by Machuca et al., found 60% of samples to be CRF02_AG, and 26% to be URFs; as well, 12 subtypes and CRFs were identified [6].Recently, a comparative study of over 500 specimens obtained from calendar year 1996 to 2004 from urban, HIV-positive blood donors in Yaoundé and Douala found there to be no significant changes in the proportions of all but one subtype (increase in subtype D) over this time period, and that the strain compositions of the URFs did not shift significantly except in the case of a decrease in subtype G fragments [14].
Though rural settings comprise the majority of the country's population, and in certain rural areas HIV prevalence has been found to be more than double the national rate, little data on HIV diversity in these regions exists, particularly data obtained in the last decade [1,18].Most recently, a survey of rural villages of the East Region was conducted on samples obtained in 2000, which found CRF02_AG to be predominant among the gag, pol, and env gene fragments studied, and half of the specimens studied to be URFs, nearly all of which were SGRs containing CRF02_AG fragments [15].Intriguingly, this survey led to the discovery of two novel CRFs in Cameroon, CRFs 36_cpx and 37_cpx, demonstrating the unique importance of proper monitoring of HIV diversity in these rural areas [10,11].Similarly, another study conducted using a limited number of samples from villages in the East, Center, Southwest, and South Regions obtained in 2000 found CRF02_AG and subtype A viruses to be dominant, with numerous other subtypes co-circulating in these rural areas [2].Further study of samples obtained in 2000-2001 from rural areas of the East, Center, South, Southwest, and West Regions also identified extremely broad subtype diversity, and in particular, revealed that URFs were exceedingly common (63% of samples) in villages of the West Region [3].
To date, no study of the evolution of HIV subtype diversity in the rural countryside has been reported.As such there is little information on the genetic landscape of current HIV infections in such regions with record high HIV-1 viral diversity -a concerning fact with regards to appropriate vaccine development.In the present study, the HIV-1 genetic subtypes infecting individuals in calendar year 2000-2001 in the rural villages of the South and West Regions of Cameroon were compared to those in calendar year 2006-2008 in the same geographic areas, in order to analyze HIV-1 evolution over this time period.A total of 82 specimens obtained in the South and West regions were analyzed in calendar year 2000-2001 and identified nine (sub)subtypes (A, A1, A2, C, D, F2, G, H, and K), and 6 CRFs (01_AE, 02_AG, 09_cpx, 11_cpx, 18_cpx, and 36_cpx) as either pure strains (67% of all specimens, 55/82) or as URFs (52% of specimens evaluated at more than one locus, 27/52), based on phylogenetic analysis of gag, pol, and env fragments (Figure 1).
In the West Region over the 7-8 year period, (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 2006-2008.The proportion of URFs in the West Region dropped significantly over the time period by 43%.Significant decreases were found in subtypes A (at env) and D and CRF01_AE (at gag), and a significant increase in CRF02_AG (at gag) was identified.

Discussion
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 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.

Phylogenetic Analysis
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 neighborjoining [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).

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

Conclusion
In this report, we observed that CRF02_AG in the rural South and West regions of Cameroon remained dominant over a 7-8 period, that various co-circulating HIV-1 subtypes and CRFs identified in 2000-2001 were replaced by others by 2006-2008, and that the proportions of SGRs in both regions increased.Importantly, these SGRs were mainly variants of CRF02_AG that had recombined with other HIV-1 subtypes.These findings demonstrate that even in this short time period, the genetic identity of the virus continues to shift dramatically, suggesting that the viral diversity in these rural regions may become more complex, that any subtype specific vaccine may not be relevant in such regions of Cameroon, and that the distribution and evolution of viral diversity in these regions of Cameroon must be monitored.Therefore, a vaccine that would be effective in these regions of the world must target conserved antigenic regions shared by diverse HIV-1 strains.

Figure 3 .
Figure 3. Proportion of CRF02_AG at each locus, South and West regions.Statistically significant increase (p < 0.05) is denoted by an asterisk.

Figure 4 .
Figure 4. Proportion of Unique Recombinant Forms (URFs) and Second-Generation Recombinants (SGRs), South and West regions.(a) Percentages of URFs among total specimens.(b) Percentages of SGRs among total URFs.Statistically significant decrease (p < 0.05) is denoted by an asterisk.