Although HIV-1 has spread globally, HIV-2 is confined mainly to West Africa, with declining incidence and prevalence during the past two decades [1,2]. HIV-2-infected individuals progress to AIDS at a lower rate than those infected with HIV-1 . A third of HIV-2-infected individuals from a community cohort in Guinea-Bissau were shown to have undetectable (<100 copies/ml) HIV-2 viral loads, with a mortality rate no higher than that of HIV-uninfected individuals over an 18-year period . Although proviral loads are equivalent in HIV-1 and HIV-2 infection, plasma viral loads are approximately 30-fold lower at similar disease stages in HIV-2 [5–7]. Presumably as a direct consequence of lower viral loads in plasma and genital secretions, both vertical [0–4% without antiretroviral therapy (ART)] [8–10] and horizontal transmission of HIV-2 occurs less frequently [11,12].
Guinea-Bissau is considered to be the centre of the HIV-2 epidemic, with the highest recorded adult prevalence in the 1980s (8%), with up to 20% of those aged over 40 infected [13,14]. How such a poorly transmissible virus reached high levels of prevalence is not fully understood, but unscreened blood transfusions, increased prostitution with displaced soldiers, along with spread via ritual female circumcisions and mass vaccination campaigns during the War of Independence (1963–1974), may have played a role [13,15–17]. The prevalence of HIV-2 has steadily fallen, whereas the HIV-1 epidemic is now an increasing problem in West Africa . Yet HIV-2 transmissions still occur and the host or virus characteristics that determine ongoing HIV-2 spread on a background of reducing community prevalence are unknown.
The widespread availability of HIV-1 sequences and recent advances in molecular phylodynamics have allowed the characterization of HIV-1 transmission networks in Western cohorts [19–21]. The lower numbers of HIV-2-infected individuals and the paucity of sequence data have precluded similar analyses of HIV-2, and no data exist on HIV-2 transmission in rural West Africa. We apply modern phylogenetic and phylodynamic techniques to HIV-2 sequence and socio-demographic data from a well characterized community cohort in rural Guinea-Bissau.
Samples were obtained from Caió, a rural village in Northwestern Guinea-Bissau. Most residents are subsistence farmers, have an animist belief system and frequently travel to neighbouring towns and countries for work, education and family visits. Three serosurveys, each covering approximately 75% of the adult population, were carried out in 1989–1991, 1996–1998 and 2006–2007 to estimate HIV prevalence and incidence [18,22,23]. These age-stratified data from each serosurvey have been published previously . Blood samples were collected and individuals were interviewed using standardized questionnaires addressing risk factors for infection. Incident infections were defined by the presence of an initially HIV-2-seronegative sample and at least one subsequent HIV-2-seropositive sample, although due to the long intervals between serosurveys, a precise date of infection could not be determined. Persons who were diagnosed with HIV-2 infection in the first serosurvey were defined as ‘pre-1989’ cases and likely represented individuals infected during the early growth of the epidemic . Information on sexual risk behaviour came from the most detailed questionnaire used in the 1996–1998 survey . Although 0.7% of the cohort was HIV-1/HIV-2 dually infected in the latest serosurvey , these individuals were excluded in the original studies performed to obtain HIV-2 sequences [24,25]. Therefore none of the participants in the current analysis were HIV-1/HIV-2 dually infected. All participants were ART-naive at the time of sampling. Serological and molecular algorithms used for diagnosis of HIV infection, and CD4 cell count/viral load quantification have been described in detail elsewhere [18,26,27]. Informed consent was obtained from all participants in accordance with the guidelines of the oversight bodies that approved these studies (the Gambia Government/MRC Laboratories Joint Ethics Committee and the Ministry of Health of Guinea-Bissau).
HIV-2 gag (n = 86, 690 bp, p26) and env (n = 70, 1350 bp, C2 to gp41) sequences were available from a subset of participants sampled in 1996–1998, 2003 or 2006–2008 [24,25] (Supplementary methods, http://links.lww.com/QAD/A265). Additional HIV-2 env sequences (n = 68, 360 bp) previously generated from 1991 samples [28,29] were also used in some analyses. In addition, all non-Caió HIV-2 env sequences (covering the same region as the Caió env fragments) from West Africa with location and date of sampling available were used to provide a global background of HIV-2 sequences (n = 41). Due to the paucity of available non-Caió HIV-2 sequences in GenBank, a similar analysis was not possible for HIV-2 gag. All Caió HIV-2 isolates belonged to group A. As only 53 (51%) individuals had both gag and env available, the datasets were analysed independently. HIV-1 CRF02_AG (the most prevalent subtype in Guinea-Bissau ) partial env sequences (830bp) from 56 Caió individuals sampled in 1996–1997, 2003 or 2006–2007  were used to compare HIV-1/HIV-2 population dynamics in Caió. GenBank accession numbers of all sequences used are detailed in Supplementary Table 1 (http://links.lww.com/QAD/A265).
Phylogenetic and phylodynamic analyses
Alignments of gag and env sequences were created using ClustalW2 [31,32] and manually edited using Se-Al (Sequence Alignment Editor, v2.0a11) . Reconstruction of phylogenies and changes in HIV-1 and HIV-2-effective population size (Ne) over time (env) were inferred by Bayesian Markov chain Monte Carlo (MCMC) inference, using BEAST v1.6.1 , under the General Time Reversible model of nucleotide substitution with gamma-distributed rate heterogeneity, a lognormal relaxed molecular clock and the flexible Bayesian Skyline coalescent model. A Maximum Clade Credibility Tree (MCCT) was selected from the sampled posterior distribution for HIV-2 gag and env phylogenies, respectively. For estimation of HIV-2 Ne, the alignment was complemented with 68 env sequences from 1991 and partitioned to allow the region spanned by these additional sequences to evolve under a separate set of model parameters than the rest of the alignment. Further details are specified in Supplementary methods (http://links.lww.com/QAD/A265).
Caió clusters supported by a posterior probability of 1.0 were selected as significant transmission clusters. Branch support alone was relied on for cluster identification, since no consensus exists for defining transmission clusters on the basis of HIV-2 gag and env genetic distance. In addition, we wanted to avoid making restrictive assumptions on the age of transmission events prior to the analysis. Several individuals were sampled for the first time in later serosurveys and found to be HIV-2-infected (27 and 34% in gag and env datasets, respectively). It is therefore impossible to know whether they were infected before or after 1989 and were excluded from analyses comparing incident and pre1989 infections.
Data were analysed using STATA 11 (Stata Corporation, College Station, Texas, USA). Comparisons between normally distributed variables were made by the t-test and non-normally distributed data were compared by the Wilcoxon rank-sum test. Discrete data were compared by the chi-square or Fisher's exact test.
Sequences from 103 HIV-2-infected individuals were used for cluster identification. Study population characteristics are listed in Table 1. Thirty-nine percent of the 103 HIV-2-infected individuals were already infected by 1989, the time of the first serosurvey, and 29% had documented incident HIV-2 infection since this time. The remaining 32% were sampled for the first time in later serosurveys (due to absence from the village or too young age during prior surveys) and found to be HIV-2-infected.
Changes in the HIV-2 and HIV-1-effective population sizes over time
Bayesian Skyline Plots (BSPs) were utilized to reconstruct both HIV-2 and HIV-1 population dynamics in Caió by estimating Ne through time (Fig. 1). Ne reflects the number of infections contributing to onward transmission and is therefore directly related to the rate of transmission (i.e. incidence) (Supplementary methods, http://links.lww.com/QAD/A265). No published data exist comparing the relative dynamics of HIV-1 and HIV-2 in the same community. For HIV-2, the estimated time of the most recent common ancestor (tMRCA) was 1947 [95% confidence interval (CI) 1932; 1962] and the population experienced an exponential growth phase coinciding with the War of Independence (1963–1974) as previously described , but also with the timing of mass smallpox vaccination campaigns and increased access to blood transfusions . The circulating HIV-1 CRF02_AG strains sampled in Caió emerged at a much later date [tMRCA 1973 (95% CI 1956; 1986)], with a rapid growth phase during the same period when HIV-2 growth was stunted. The rate of evolution of HIV-2 env was 2.67 × 10−3 substitutions/site per year (95% CI 1.85 × 10−3, 3.46 × 10−3) compared with 4.1 × 10−3 substitutions/site per year (95% CI 2.39 × 10−3, 5.81 × 10−3) for HIV-1. Interestingly, the growth rates following introduction, as well as the Ne at the origin of the epidemic appear remarkably similar for both viruses. Also, both HIV-1 and HIV-2 have reached a plateau at similar orders of magnitude.
From 86 gag sequences, 50 appeared in 13 transmission clusters – one cluster with seven individuals, four with five individuals, three with four individuals, one with three individuals and four with two individuals. From 111 env sequences, 39/70 appeared in 13 (Caió-only) transmission clusters – one cluster with six individuals, three with four individuals, three with three individuals and six with two individuals (Table 2, Fig. 3). This represents a high proportion of local individuals (58.1% in gag and 55.7% in env) involved in ongoing transmission of HIV-2 in Caió. Because the identified clusters span several decades and our datasets represent only a proportion of the HIV-2-infected population in Caió, it is difficult to infer direct transmission with confidence, with the exception of known sexual partners. Of the 53 individuals included in both env and gag datasets, 50 appeared as concordant clustering (30) and nonclustering (20) infections across both analyses. Two of the nonconcordant events were due to clustering in gag, but not env, due to the addition of non-Caió sequences in the env dataset. Several Caió env sequences clustered with non-Caió sequences (often from Cape Verde) in seven clusters (Supplementary Figure 1, http://links.lww.com/QAD/A265). Frequent HIV-2 migration events between Guinea-Bissau and Cape Verde in this phylogeny were confirmed using the Slatkin-Maddison test  (Supplementary methods, http://links.lww.com/QAD/A265).
Characteristics of clustering and nonclustering HIV-2 infections
No statistically significant differences in risk behaviour characteristics were found between clustering and nonclustering individuals (Table 2). For both phylogenies, clusters contained significantly more known sexual partners (P < 0.001; Table 2). It is not clear to what extent infection with viruses with inherently different replicative capacity accounts for individuals with either undetectable HIV-2 plasma viral load (‘elite controllers’) or progressive HIV-2 infection with detectable viraemia. In both gag and env phylogenies, elite controllers were found to share an MRCA with viraemic individuals within transmission clusters (in some cases these included known sexual partners, Fig. 2).
Incident infections contribute significantly to HIV-2 transmission clusters
Transmission clusters contained significantly more incident infections than pre-1989 cases (Table 2). A high proportion of all incident cases were also found in clusters: 81.5% in gag and 83% in env phylogenies, compared to pre-1989 cases: 36% in gag and 27% in env phylogenies (P = 0.0004 and P = 0.0003, respectively). Incident infections appeared throughout the tree (Supplementary Figure 2, http://links.lww.com/QAD/A265), suggesting there is not a single or few viral lineages that are responsible for ongoing HIV-2 transmissions. Interestingly, only one incident case significantly clustered with a non-Caió env isolate, whereas all others were found within Caió-only clusters.
Whereas these data strongly suggest that after 1989, newly infected individuals were more strongly associated with ongoing HIV-2 transmission than older, prevalent infections in Caió, it is possible that sampling bias (i.e. exclusion of pre-1989 individuals who had died by the time of subsequent sampling dates) may account for this result. A further env phylogeny was reconstructed following inclusion of previously generated sequence data from 68 additional 1991 samples, which were all pre-1989 infections [28,29]. Although the use of these shorter sequences resulted in a phylogeny with poorer resolution, it is clear that the additional pre-1989 cases formed clusters almost exclusively with other pre-1989 cases, which would not be observed if the old infections were contributing to ongoing transmission in the cohort (Supplementary Figure 3, http://links.lww.com/QAD/A265).
We have investigated the phylodynamics of HIV-2 in rural Guinea-Bissau using sequence data from a well characterized community cohort. We used Bayesian inference to analyse population dynamics of HIV-1 and HIV-2. The fact that the two epidemics experienced similar growth rates and patterns following introduction, but at different points in time, may suggest that the population dynamics are determined by high-risk host behaviour at onset rather than ecological or historical factors. Similar conclusions were drawn following comparison of the HIV-1 epidemics amongst men who have sex with men in the US and UK . Alternately, the biological features that make HIV-2 less transmissible than HIV-1 may have been compensated for by a boost in transmission due to iatrogenic spread , allowing HIV-2 to reach epidemic growth rates akin to HIV-1. In the absence of these aids, HIV-2 is declining due to inefficient heterosexual transmission. A third explanation could be that HIV-2 was more virulent and transmissible during the early phases of the epidemic, but due to poorer adaptability to hosts (than HIV-1), a gradual attenuation has occurred over time. We, and others, have previously demonstrated that HIV-2 is under significant evolutionary constraint when compared to HIV-1 [24,37], which may account for HIV-2's disadvantageous competition with HIV-1. Our estimates of the HIV-1 CRF02_AG and HIV-2 group A tMRCA (1973 vs. 1947) are consistent with previous separate analyses using sequences from Guinea-Bissau [13,30]. The similarity between the tMRCA for Caió CRF02_AG isolates and the estimated emergence of CRF02_AG in the Democratic Republic of Congo emphasizes the likelihood that rapid dispersal throughout West Africa occurred at an early stage . HIV-1 and HIV-2 epidemiological data for our cohort are available from 1990 onwards. The similar calculated Ne of the two viruses between 1990 and 1997 is consistent with similar incidence rates during the same period [4.4/1000 person-years of observation (PYO) for HIV-1 and 4.8/1000 PYO for HIV-2] . Although during 1997–2007, HIV-2 incidence fell (1.8/1000 PYO) , the BSP remained at a plateau. This is a common feature of HIV BSPs [36,39–41]. Although this can be attributed to methodological factors (Supplementary methods, http://links.lww.com/QAD/A265), the fact that the HIV-1 and HIV-2 growth curves reached equilibrium at the same level, but at different times, suggest host demographic factors (such as the number of susceptible or infectious hosts) are decisive in these trends.
The rapid rise of the HIV-1 Ne in this region coinciding with plateauing of the HIV-2 Ne lends support to the theory that HIV-1 may have competitively displaced HIV-2, accounting for approximately 30% of the decrease in HIV-2 prevalence [42,43]. Establishing to what extent the near-disappearance of HIV-2 in West Africa is due to low viral load, the presence of HIV-1 or other factors (e.g. risk behaviour, prevention campaigns) may have great relevance to future strategies in curbing the HIV-1 pandemic . As recent interest has emerged in using early ART to halt the spread of HIV-1 [45–47], some have suggested that with inherently low viral loads, HIV-2 could be considered a natural model for what could be achieved in HIV-1 via large-scale early treatment .
We also explored whether viraemic outcome in HIV-2 is associated with particular viral lineages and observed that individuals whose viruses share an MRCA can have undetectable or detectable viral loads. This suggests that host factors are largely responsible for elite control of HIV-2. The observation that known sexual partners within transmission clusters can have discordant viral loads further strengthens this conclusion. A comparative study of host immunity and genetics within such couples could provide valuable insight into what determines the outcome of HIV-2 infection.
Our estimates of individual linkage in clusters (55–58%) are greater than the highest observed in the UK HIV-1 epidemic (36%) . This is not surprising given this is a village cohort and case detection via community serosurveys would identify a greater proportion of all those infected. Viruses from individuals infected since the start of the cohort in 1989 (after the exponential growth of HIV-2) are more frequently (81–83%) found in transmission clusters than those already infected by 1989 (27–36%). Although pre-1989 individuals were involved in transmission events early in the epidemic, they have contributed less to more recent infections. In contrast, newer infections appear to be associated with ongoing HIV-2 transmission in Caió. It is also striking that even in a rural community in Guinea-Bissau, multiple lineages of HIV-2 are evident, with Caió isolates clustering with several external sequences. Notably, many were from Cape Verde, which is another country with Portuguese colonial links. Guinea-Bissau and Cape Verde formed a strategic alliance during the shared war of independence with Portugal and were united by political ties until 1980. A recent phylogeographic analysis has also highlighted the shared ancestry of these two countries during the early dispersal of HIV-2 . The migratory nature of the Caió population in general, but particularly that of commercial sex workers who often seek work in many locations in the sub-region , may also have contributed to this observation. Interestingly, almost all incident cases were involved in Caió-only clusters. This heterogeneity in HIV-2 viruses is likely, therefore, to reflect early events that lead to the exponential increase in HIV-2.
The role of primary infection in transmitting HIV-1 is well documented both in Western and African settings, with up to 50% of primary HIV-1 infections being involved in transmission chains in a Canadian cohort [48,51–53]. The increased risk of transmission during acute/early HIV-1 infection can be explained by the peak in viraemia during this period . Little is known about primary HIV-2 infection. It is conceivable that a similar viraemic peak occurs prior to reaching set points. As these are much lower in HIV-2 than in HIV-1 infection, the main window for HIV-2 transmission may well be during early infection – a hypothesis that is supported by the data above. Individuals in clusters in the gag phylogeny had significantly higher viral loads than nonclustering individuals, although only a nonsignificant trend was observed in the env phylogeny. The significance of this finding is also not clear as the viral loads used are likely to be from samples taken many years after transmission events. Alternately, the higher proportion of new HIV-2 infections found in transmission clusters may reflect both acquisition and forward transmission of HIV-2 during a defined period of increased sexual risk-taking behaviour.
It is estimated that at least 60% of new HIV-1 infections in Africa occur within married or cohabiting couples . In our cohort, 33–42% of viruses in clusters belonged to known heterosexual partners, suggesting that a large proportion of new HIV-2 infections also occur within marriages. This highlights the need for prevention specifically targeting known partners, especially as similar patterns are seen in HIV-1 transmission in Guinea-Bissau (Td.S., unpublished data). Although we examined several other characteristics that may be associated with HIV-2 transmission, we found no association with increased sexual risk behaviour or co-infections. These factors may, therefore, contribute less to the transmission risk than stage of HIV-2 infection or viral load. However, the information available in the analysis was derived from one time point only and under-reporting of sexual risk behaviour cannot be excluded.
The main limitation of our study is the relatively low number of individuals included, compared to country-wide HIV-1 studies such as the ones performed on UK cohorts . Dense sampling of a population with high HIV-2 prevalence would offer an opportunity for a more in-depth HIV-2 transmission analysis, but is unfeasible as most HIV-2 cohorts in West Africa are shrinking [1,18,56]. A similar analysis of HIV-1 transmission in our cohort could help design public health measures to halt an increasing and evolving HIV-1 epidemic in Guinea-Bissau [18,57]. A recent UNAIDS/World Bank collaborative report has highlighted the need to understand HIV-1 transmission dynamics in West Africa, focusing on the likely complexity of transmission within and between risk groups . The long period of time between serosurverys precluded an exact date of infection in our HIV-2-incident cases. However, as our main focus was to compare those who were infected during the early epidemic (i.e. prior to 1989), when HIV-2 spread rapidly, with those who have been infected since (when the HIV-2 epidemic was declining), we are able to draw valid conclusions with the available data. As ART was not available in Caió until 2007, some rapidly progressing HIV-1-infected individuals may also have been missed between serosurveys, leading to underestimates of the HIV-1 Ne. Finally, although our study is unique in including a high proportion of sequences from HIV-2 elite controllers, potential bias could have been introduced via exclusion of individuals in whom PCR was unsuccessful (overall PCR success approximately 75%; 50% in elite controllers ).
In summary, our study in a rural West African community suggests that the population dynamics of HIV-2 and HIV-1 followed similar paths after introduction, although HIV-1 is now displacing HIV-2 as the dominant infection. Despite the poor transmissibility of HIV-2, new heterosexual transmissions still occur. Only time will tell whether these transmission events are sufficient to maintain a low-level epidemic in the region, or whether we will witness a gradual elimination of this sometimes lethal retrovirus from Guinea-Bissau – a country that once had the highest recorded prevalence of HIV-2 infection in the world.
We are grateful to the Caió population for their participation in all the studies. We would like to acknowledge Ramu Sarge Njie for her excellent management of the HIV diagnostics for many of the Caió studies. We would like to thank Nato Gonçalves for all his thorough field work. We would also like to thank Dr J Miguel Azevedo-Pereira and Dr Florence Damond for sharing location and sampling data on their HIV-2 sequences. We are grateful to Professor Peter Aaby for helpful comments and suggestions during preparation of the manuscript.
Justification of author contributions: T.I.dS. carried out HIV sequencing, phylogenetic and phylodynamic analyses and wrote the paper. C.vT. carried out the most recent field study, did the statistical analyses and wrote the paper. C.O. carried out HIV sequencing. A. Jabang helped with sample processing and HIV sequencing. T.V. coordinated the field studies and collection of individual information. M.F.S.vdL. undertook field studies and wrote aspects of the paper. R.A.C. wrote aspects of the paper and provided advice during the design of the study. A. Jaye wrote aspects of the paper and provided advice during the design of the study. S.R.-J. provided supervision of the cohort, provided advice on design of the current study and wrote aspects of the paper. H.W. provided supervision during collection of samples, design of the current study and wrote aspects of the paper. M.C. carried out and provided supervision during viral sequencing, phylogenetic analyses and wrote aspects of the paper. S.H. carried out and provided supervision during phylogenetic and phylodynamic studies, and wrote aspects of the paper.
Funding sources: This work was undertaken via a UK Medical Research Council Clinical Research Training Fellowship (G0701313) awarded to Td.S.
Conflicts of interest
There are no conflicts of interest.
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