Abstract
We identified a novel HIV-1 circulating recombinant form (designated CRF57_BC) from a total of four patients with no obvious epidemiologic linkage in western Yunnan (Dehong prefecture) in China. Two strains (09CN.YNFL37 and 10CN.DHFL17) were identified in this study. An additional two strains (341 and 1439) were found among strains reported in a previous study. CRF57_BC was composed of subtype B and subtype C, with one subtype B segment inserted into the gag region of the subtype C backbone. Subregion tree analysis showed that the B regions originated from a Thai B lineage and the C regions were from an India C lineage. The emergence of CRF57_BC may reflect the continual generation of various forms of intersubtype recombinants in western Yunnan.
HIV-1 can be divided into four major phylogenetic groups: the M group, N group, O group, and the newly defined P group, of which the M group is the most widely epidemic in the world. In recent years, many cases of viral recombination between M group subtypes have been reported, and the circulation of recombinant strains is salient for HIV-1 prevention and control. In China, the main circulating strains are CRF01_AE, CRF07_BC CRF08_BC, and subtype B, according to the third national HIV-1 molecular epidemiology survey.1 As the putative place of origin and introduction for these strains within China, the government of Yunnan province has devoted much effort to HIV prevention and control. However, the ongoing generation of multiple forms of HIV-1 intersubtype recombinants is still occurring; new recombinant form strains have continued to be reported in this area.2,3 Here we identify another circulating recombinant form (CRF) strain (CRF57_BC) found in high-risk populations in western Yunnan and characterized its genomic features.
Blood plasma from three HIV-positive patients (YNFL37, DHFL17) in Dehong prefecture, Yunnan was collected (Table 1). The patients were diagnosed as HIV-1 positive in 2009 and recruited as part of a national HIV-1 drug resistance survey in Yunnan. The study was approved by the institutional review boards of the National Center for AIDS/STD Control and Prevention of China. Near full-length genome (NFLG) amplification and sequencing were performed as previously reported.4 Recombination breakpoints were determined using RIP and jpHMM (www.hiv.lanl.gov) and SimPlot.4 Breakpoint confirmation and the origin of each segment were then analyzed by phylogenetic trees. The standard subtype reference alignment file including all HIV-1 group M, CRF01_AE, CRF07_BC, CRF08_BC, and CRF31_BC was downloaded from the Los Alamos National Laboratory HIV Database (www.hiv.lanl.gov). Nucleotide sequences were first aligned by using Clustal W, and then adjusted manually using BioEdit.5 Phylogenetic trees were constructed with MEGA 5.0 by using the neighbor-joining method with 1,000 bootstrap replications to confirm the subtype of mosaic fragments.6
Table 1.
Demographic and Epidemiologic Characteristics of Study Subjects Harboring CRF57_BC
| Strain name | Year of sampling | Geographic origin (prefecture, province) | Sex | Age | Risk factor | Accession number | Reference/remark |
|---|---|---|---|---|---|---|---|
| 341 | 2007 | Dehong, Yunnan | Male | 35 | Injecting drug user | HM776939 | Liu et al.7 |
| 1439 | 2009 | Dehong, Yunnan | Male | 30 | IDU | JX679207 | Li et al.3 |
| 09CN.YNFL37 | 2009 | Dehong, Yunnan | Female | 18 | Heterosexual | KC870044 | This study |
| 10CN.DHFL17 | 2010 | Dehong, Yunnan | Male | 27 | Heterosexual | KF250400 | This study |
NA, not available.
An extensive database search revealed that two recombinant strains, 341 and 1439, show high-level of homology with the two strains in this study3,7 (Table 1). The four strains formed a distinct monophyletic cluster, distantly related to all known HIV-1 subtypes/CRFs (Fig. 1). Recombination analysis shows that the sequences were composed of subtypes B and C, with one region (II) of subtype B inserted into the subtype C backbone (I, III) (Fig. 2). The breakpoint positions refer to HXB2 coordinates, and were located by HIV Sequence Locator (www.hiv.lanl.gov/content/sequence/LOCATE/locate.html). Therefore, the map of the recombinant genome is as follows: C (790–1,200 nt), B (1,225–1,808 nt), and C (1,863–9,417 nt). Subregion segments were verified by phylogenetic analysis, which was well confirmed by bootstrap values above 0.7. The analysis also indicated that the parental origin of subtype B regions is probably from a Thai B lineage and the subtype C regions originate from an India C lineage (Fig. 3). The recombinant structure is distinct from any known CRFs to date. Therefore, these new recombinants are now designated CRF57_BC.
FIG. 1.
Phylogenetic tree analysis of the near full-length genome (NFLG) sequences of the four isolates. All HIV-1 group M reference sequences were used to construct the neighbor-joining phylogenetic tree. Node stability was assessed by bootstrap analysis with 1,000 replications, and bootstrap values above 0.7 are shown at the corresponding nodes. All CRF57_BC isolates in this study are marked gray. The scale bar represents 2% genetic distance.
FIG. 2.
Recombination breakpoint analyses of CRF57_BC. (A) Bootscan plots of CRF57_BC with subtype B (RL42), subtype C (95IN21068), and subtype J (04CMU11421) as reference genotypes. The parameters of SimPlot bootscan analysis were set as the default except for a window size of 300 and step size of 30. (B) Genomic map of CRF57_BC. The mosaic map was generated using the Recombinant HIV-1 Drawing Tool (www.hiv.lanl.gov/content/sequence/DRAW_CRF/ recom_ mapper.html).
FIG. 3.
Phylogenetic analyses of three mosaic segments defined by bootscanning. The phylogenetic trees of the three mosaic segments were constructed with MEGA 5.0 using the neighbor-joining method. The subtype references of the M group are derived from the Los Alamos HIV Sequence Database. Node stability was assessed by bootstrap analysis with 1,000 replications, and only bootstrap values above 0.7 are shown at the corresponding nodes. The sequences of CRF57_BC are marked in bold black.
All of China's major prevalent HIV-1 strains are found in Dehong, Yunnan province, commonly considered the epicenter for HIV/AIDS in China. Previous studies have demonstrated that both CRF07_BC and CRF08_BC originated from Dehong prefecture2,8 and were spread widely to other parts of the nation through drug trafficking routes. The coexistence of multiple strains along with a sustained active epidemic increases the probability of recombination. However, in contrast to the nationwide transmission of CRF07_BC and CRF08_BC,1 the newly formed URFs and CRFs were found circulating on only a small scale in Dehong. This may be due to the relatively limited mobility of the local population, compounded by currently strict controls on drug trafficking that further impede high-risk contact and transmission through other means (e.g., heterosexual transmission).
In this study, we characterized a novel HIV-1 circulating recombinant form CRF57_BC, consisting of different recombinant breakpoints between the subtype B and subtype C strains, from three donors without obvious epidemiologic linkage in Dehong prefecture, Yunnan province, China. The emergence of the new CRF may further complicate the development of effective vaccines to limit the HIV-1 epidemic in China. In addition, whether recombination may confer selective advantages over parental viruses remains to be investigated.
Sequence Data
The nucleotide sequences of the two isolates 09CN.YNFL37 and 10CN.DHFL17 have been submitted to GenBank with accession numbers KC870044 and KF250400, respectively.
Acknowledgments
This work was supported by the National Major Projects for Infectious Diseases Control and Prevention Grants (2012ZX10001-008 and 2012ZX10001-002), the National Natural Science Foundation of China Grants (81020108030 and 81261120393), the State Key Laboratory for Infectious Disease Development Grant (2012SKLID103), and the International Development Research Center of Canada Grant (104519-010). We would like to thank Brian Foley from the Los Alamos National Laboratory for advice on CRF nomenclature.
Author Disclosure Statement
No competing financial interests exist.
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