Of six potential heterosexual transmission pairs (#3/#4, #6/#44, #7/#9, #22/#23, #24/#25, and #81/#82) initially presumed by contact tracing, four pairs (#3/#4, #6/#44, #22/#23, and #81/#82) were strongly supported by phylogenetic reconstruction. One pair (#24/#25) was rejected because the bootstrap value (94.8%) and the genetic distances (0.018) in pol were below the cut-off values, and also the two strains had a separate clustering in env (#25 was located inside the ‘cluster 1’ and #24 was not). One more pair (#7/#9) was rejected because the sequences did not cluster together in pol; however, they clustered together in env despite a low bootstrap value. As one of them (#7) was subtyped as CRF01_AE/B′ in pol, one cannot exclude the possibility that a superinfection with the CRF01_AE variant occurred in this patient, leading to recombination between CRF01_AE and B′ infecting strains and subsequently to the emergence of the recombinant strain as the major infecting virus. As the phylogenetic reconstruction is based on the major sequences analysis over the whole fragment, it rejected this presumed contact tracing relationship at first. To further elucidate the phylogenetic relationships between the two cases #7 and #9, we cut the pol alignment at the breakpoint in which #7 is no more CRF01_AE but B′ and took the alignment segment corresponding to the B′ subtype shared by the two samples over 1252 unambiguously aligned nucleotides. A maximum likelihood analysis under the criteria previously used in this study no more rejected the initially presumed contact tracing relation. The two samples were epidemiologically linked as the genetic distance was 0.005 nucleotides and the bootstrap value 993.
No additional sexual transmission pair was identified by phylogenetic reconstruction. One huge (53 sequences in cluster 1) and one small (three sequences in cluster 2) IDU transmission clusters were identified by phylogenetic reconstruction.
Since the first HIV-1 outbreak in 1989, the mainland China has today a national figure of over 650 thousands of HIV-1-infected people  with diverse subtypes including B, B′, C, CRF07_BC, CRF08_BC, and CRF01_AE [10–14]. Recent studies have reported that CRF01_AE became a dominant subtype in Yunnan province mainly through sexual transmission [6,15]. In present study, we demonstrated a dominant HIV-1 CRF01_AE outbreak (84%) mainly among the IDUs in Hainan, suggesting that this subtype may indeed surpass the previously documented major subtypes (B′, C, CRF07_BC, or CRF08_BC) and lead to a new epidemic in China.
Phylogenetic analysis of viral gene sequences has recently been used to construct direct or indirect epidemiological links in geographically defined populations with acute/primary or chronic HIV-1 infection [16–19]. These recent findings underscore the importance of tracing the molecular linkage of HIV transmission to better understand the population-based epidemic nature/trend and to define an efficient strategy to control the epidemics in the region. Phylogenetic reconstruction of HIV-1 pol gene allows us to identify nearly 80% (66 out of 83) epidemiological links in the representative samples of Hainan's HIV-1-seropositive individuals. In the striking contrast to our previous findings in Xishuangbanna where only small sizes of clusters (2–4) were observed [6,15], a very huge cluster (59) representing 89% of all clustered samples was identified. The finding of significant subclustering of these sequences within CRF01_AE clade both in pol and env raises issues unique to a geographically defined outbreak, such as that occurs on islands. The specific differentiation of this cluster from the other CRF01_AE sequences is most likely due to a founder effect. Founder effects of one particular HIV-1 variant have already been demonstrated in many areas in the world [20–26]. Evidence of a strong founder effect of one particular HIV-1 strain and variant among IDUs was also seen in the Netherlands (subtype B variant), Russia (CRF03_AB and subtype A), China (CRF07_BC and CRF08_BC), northern Vietnam (CRF01_AE variant), and Myanmar (B′ variant). Genetic distances were very low in the cluster 1, indicating a high homogeneity of the strains concomitant with a low evolutionary rate and a fast-spreading epidemic . In our case, of the 59 closely clustered cases, 53 (90%) contracted HIV-1 through IDU transmission as confirmed by contact tracing. Ten percent of the closely related cases were contracted through another mode of transmission thus suggesting that IDUs could potentially serve as a bridge between a high-risk group and a lower-risk population, such as heterosexually active adults that constitute the second HIV-infected group in Hainan. Given that the prevalence of HIV infection is still low (0.1%) in Hainan, an efficient management of local IDUs could be the key strategy for holding back the epidemic in this region.
Phylogenetic means are also widely used to infer or reject transmission links in suspected cases by contact tracing [28–31]. In this regard, our results, in keeping with our previous findings [6,15], strongly support that phylogenetic analysis is a powerful mean for providing molecular evidence to approve or reject close linkage in suspected transmission events, or eventually suggest new linkage of potential transmission. Although the potential sexual transmissions in two couples (#7/#9 and #24/#25) were suggested by contract tracing, phylogenetic analysis rejected such linkages by distinct genotyping and maximum likelihood trees. However, the epidemiological linkage between #7, which was classified as a recombinant, and #9 was subsequently demonstrated in the pol region constituted with the same subtype. It is thus important to carefully check the recombinant nature of the studied strains and to analyze a second genomic region that may bring useful information over the presumed linkage.
Taken together, our findings illustrate that HIV-1 CRF01_AE is becoming a major subtype accounting for current HIV outbreaks in China. Provided that nearly 80% of people with HIV-1 infection are undiagnosed and that there is an increasing mobility of population (vast majority are peasants) in China, a considerable large number of undiagnosed persons with HIV-1 infection may represent potential sources leading to new nationwide HIV outbreaks through sharing syringe needle, unprotected sex, particularly in poorly educated populations. The molecular epidemiological information provided by the present study is critical not only for the design of prevention policies but also for guiding the design of vaccine strategies in the targeting region .
All sequences analyzed in the present study are deposited in EMBL under the accession numbers FM251948-FM252030 and FM865453-FM865531 for the pol and env sequences, respectively.
We thank all participants for their enthusiastic cooperation. We acknowledge Ellène Gozard, Véronique Jagot, Alain Tiafvoon, Ju Yuan, and Liang Yu for technical assistance. This study was supported by Chinese Ministry of Science and Technology (grant 2005AA218020 to W.L.), Institut de Recherche pour le Développement, and Institut de Recherche sur les Vaccins et l'Immunothérapie des Cancers et du SIDA.
W.L. was responsible for the overall study design, organization, data analyses, and writing of the report. W.D., P.F., L.B., N.V., and Q.H. participated in design and implementation of the study. C.Q., M.P., E.D., and J.-M.A. contributed to discussions on study design and interpretation of the findings.
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