| Located in the southwest border of China, Yunnan and Guangxi provinces are areas with the most severly HIV-1epidemic in China. By the end of 2010, the cumulative reported cases infected by HIV-1 in these two provinces are the most in the country. Yunnan is the origin area of HIV-1 epidemic in China, where genetic subtypes are very complicated, and always in the process of dynamic modifications. Subtype B (B') strains were the original strains in Yunnan, then subtype C strains were introduced from India, followed by CRF01AE strains from Thailand. Later on, CRF07BCå'ŒCRF08BC strains emerged and became dominant with subtype B(B') anc C strains vanishing gradually. However, little is known now about the dynamic processes of different HIV-1 subtype origination, transmission, prevalence, and disappearance in Yunnan. Guangxi is region with HIV epidemic growing greatly in China now. Originally , CRF08BC the was main epidemic subtype, which was prevalent in IDUs. However, CRF01AE strains increased quickly in recent years with HIV-1 spreading from IDUs to sexually transmitted population. Nowadays, subtype B (B'),C,CRF08BCå'ŒCRF01AE are simultaneously prevalent in Guangxi. Yunnan and Guangxi are adjacent to each other, both are minority-compact communities and bordering the Southeast Asia countries with severe HIV-1 epidemic. Since Yunnan and Guangxi are similary in location and social demographic situation, it was believed that close linkage should exist between the HIV-1 strains prevalent in them. To anlyze the epidemic situation of HIV-1 in Yunann, Guangxi and even the whole China, it is necessary to characterize the genetic variation and epidemic relationship of HIV-1 strains prevalent in both area comprehensively and systematically. The results will also be valuable for developing the effective vaccines and antiretroviral drugs for limiting HIV-1 spread in China.In our research, 788 and 294 HIV-1 sero-positive blood samples were collected from 15 areas in Yunnan and 13 areas in Guangxi, respectively. full-length gag,pol and partial env (C2V3) genes were amplified by nested-RT-PCR and subsequently sequenced after being purified. In addition, And a few near full-length genomic sequences were also obtained. MEGA 5.03,BEAST v 1.5.4 softwares and some on line sequence analysis software were used to analyse the genetic variation of HIV-1 epidemic strains in Ynnan and Guangxi, as well as the relationship of transmission and evolutionary between these two regions.Partâ… Establishment and use of methods for amplification and sequencing of full-length gag, pol and partial env (C2V3) genes.1. Viral RNA was extracted from the Plasma sampl and subjected to RT-PCR. Primers foramplification and sequencing were designed and optimized. 1584bp full length gag gene, 3147bp full length pol gene and 558bp env (C2V3) fragment were amplified and sequenced. Full length gag and pol genes were connected together as a complete genetic region (location on HXB2:790-5096) for genotype analysis.2. Of 788 specimens obtained from Yunnan, 700 gag , 666 pol and 692 env (C2V3) genes were amplified successfully, while 285 gag, 274 pol and 283 env (C2V3) were amplified successfully from 294 specimens obtained from guangxi. The results showed that viral load of samples affected the success rate of amplification significantly.Partâ…¡Characterization of genetic variation and evolution of HIV-1 strains epidemic in Yunnan province1. Distribution of HIV-1 subtypes epidemic in Yunnan: (1) The subtypes of HIV-1 epidemic in Yunnan were complicated with the order of the constituent ratio as CRF08BC >CRF01AE >CRF07BC >unknown recombinant forms >subtype C > subtype B(B'). (2) The distributions of HIV subtypes in different regions were different, which can be roughly divided into four category: a) CRF08BC dominant area represented by Lincang district and neighbouring Baoshan, Dali districts. In these area, CRF08BC accounted for more than 70% with CRF01AE strains less than one-sixth; b) Area with CRF08BC and CRF01AE coexisting with unknown recombinant forms, represented by Dehong district. In this area, the constituent ratios of CRF08BC and CRF01AE strains were almost the same as about a quarter, with the ratio of unknown recombinant forms as one-third; c) Areas with CRF01AE strains more than CRF08BC strains, represented by Xishuangbanna and Pu'er districts. In these area, the percentage of CRF01AE strains was higher than CRF08BC with sum of them more than 80%; d) Area with CRF08BC, CRF01AE, CRF07BC and unknown recombinant forms coexisting, reprented by Kunming and Honghe districts. In these areas, the constituent ratio of CRF08BC was higher than CRF01AE, and the percentage of CRF07BC in each district were 18.0% and 19.5%, respectively. And there also were some unknown recombinant forms in the two districts. (3) Unknown recombinant forms accounted for more HIV infection in ethnic minorities (17.0%) than in ethnic Han (6.7%)(P<0.01); (4) Subtypes distribution varied significantly in the two primary routes of transmission - heterosexual contact and IDUs. In peoples infected through heterosexual contact, CRF08BC and CRF01AE were the dominant subtypes, accounting for 52.7% and 29.1% infections respectively. However, in IDU peoples, CRF08BC strains accounted for half of infection, while only 4.5% infection were caused by CRF01AE strains, CRF07BC and unique recombinant forms were responsible for 15.5% infections.2. Phylogenetic analysis of CRF08BC strains: (1)CRF08BC strains were not restricted to specific area because no clusters were found among strains in the same regins both in the routes of heterosexual contact and IDUs. (2) CRF08BC strains from the heterosexual contact population of Lincang district distributed among those from other districts. Different to CRF08BC strains from Lincang district, CRF08BC strains from other five districts representing by Xishuangbanna grouped into a relative separate cluster. (3) In the IDUs , six Honghe-IDUs seqences and two Kunming-IDUs seqences clustered with the referenced sequences from Guangxi (97CNGX6F,97CNGX7F,97CNGX9F), which were the earliest CRF08BC strain found in China. The cluster of these strains may predict the close relationship between IDUs from Honghe and Kunming of Yunnan province and those from Guangxi. CRF08BC strains from IDUs in other districts intermixed with those from heterosexual contact population, and no cluster with more than three sequnces in the phyletic tree were identified. This phenomenon suggested that CRF08BC strains in Yunnan and Guangxi were not transmitting in specific district or population.3. Phylogenetic analysis of CRF01AE strains: CRF01AE strains in Yunnan grouped into three obvious clusters in the phylogenetic tree. Cluster 1 included nine strains from Lincang-heterosexual contact population and seven reference sequences from Thailand. Cluster 2 included eleven strains from Xishuangbanna- heterosexual contact population and ten reference sequences from Vietnam and three reference sequences from Guangxi. Cluster 3 included nine strains from Xishuangbanna- heterosexual contact population and three reference sequences from Fujian province. Other seqences distributed in the phylogenetic tree. Based on the reports of epidemic time, it could be predicted that CRF01AE strains in Lincang district might come from Thailand, strains in Xishuangbanna district might come from Vietnam, meanwhile they might have relationship with strains in Gungxi and Fujian.4. Phylogenetic analysis of CRF07BC strains: Forty-two CRF07BC sequences grouped with reference sequences, including 2005 and 1997 Xinjiang reference sequences. Strains from different districts were intermixed, and no clear clusters were found among strains from different district or population.5. Identification and distribution of unique recombinant forms: (1) Sixty-three strains of unique recombinant forms were identified, accounted for 10.2% of total sequences. Three genomic schematic were found: B(B')/C(47 cases), CRF01AE/B(B')/C(12 cases) and CRF01AE/C(4 cases). Three groups of sequences, which demonstrated similar recombinant breakpoints, were identified. The recombinant mosaic of genome belonging to different group were drawn. (2) The distribution of unique recombinant forms in different population: heterosexual contact (30 cases) and IDUs (19 cases) population contained the main part of strains of unique recombinant forms. The percentage of unknown recombinant forms in the IDUs was 15.5%,significantly greater than which in the heterosexual contact population (P<0.01). The genomic schematic of strains in the IDUs were almost B/C recombinants(18/19), while in the heterosexual contact population, CRF01AE/B(B')/C and CRF01AE/C models accounted for 40%. Dehong contained the highest rate of unique recombinant forms, accounting for half of total cases, and most of them were in the IDUs. Most of unique recombinant forms from Kunming and Honghe were in the heterosexual contact population. Although the most specimens were collected in Lincang district, very fewunique recombinant forms were identified. 6. Analysis of genetic distances and selection pressures basing on gag-pol sequences and partial env (C2V3) sequences: (1) The largest genetic distance was observed among subtype B(B') (6.15±2.00)% in gag-pol genes. Relative smaller genetic distances were found among subtype C(3.09±0.94)%, CRF01AE(2.19±0.36)%, CRF07BC(2.77±0.48)% and CRF08BC(1.17±0.31)%. The genetic distances of gag were higher than pol in all subtypes. In the gag coding regions, the genetic distances of P17 and P2P6 were higher than P24 in all subtypes, whereas in the pol coding regions, the orders of the genetic distances in each subtype were not consistent. Comparing to gag and pol genes, env (C2V3) was more variable in all subtypes. (2) Negatie selection pressure was found in gag-pol coding regions because the globleω(dN/dS) was less than 1. Comparation of different segments showed that the globleωs of P17 were highest, follwed by P2P6, and then other regions. The globleωs of env C2V3 were close to 1 in all subtypes. The selection pressures in all epidemic subtypes in Yunnan were similar, no matter gag-pol or env (C2V3) segments.7. Analysis of the V3 loop tip and coreceptor usage: (1) The tip of V3 loop in subtype B(B') was major GPGR, accounted for 94.7% cases, whereas GPGQ accounted for 97.8% in CRF07/08BC and 76.5% in CRF01AE. Six different motifs were defined at the tip of CRF01AE V3 loops, which were more complicated than of other subtypes. (2) Nearly half of subtype B (B') strains might use CCR5 coreceptor, much lower than CRF07/08BC and CRF01AE, in which 98.3% and 88.6% strains might use CCR5 coreceptor, respectively.Partâ…¢Characterization of genetic variation and evolution of HIV-1 strains epidemic in Guangxi1. Distribution of HIV-1 subtypes epidemic in Guangx: (1) The subtypes of HIV-1 epidemic in Guangxi were varied with the order of the constituent ratio as CRF01AE >CRF08BC >CRF07BC >unknown recombinant forms > subtype B(B')> subtype G. (2) CRF01AE strains were dominative in three major transmission routes, which accounted for 79.50% cases in the heterosexual transmission, 58.8% cases in the IDUs, and 71.4% cases in the homosexual transmission. The constituent ratios in this three major routes were significant different. (3) Heterosexual contact was the most important transmission route of HIV-1 in Guangxi. The cases infected thought this route were more than 80% in ten different districts, among which four districts were 100% and three districts were 50-60%. In peoples infected through heterosexual contact, only 6.3% and 10.6% were caused by CRF07BC and CRF08BC strains, respectively.2. Phylogenetic analysis of CRF01AE strains: Among the phylogenetic tree of CRF01AE strains, two major clusters (cluster 1 and cluster 2) and two minor clusters (cluster 3 and cluster 4) were found according to N>3 and Bootstrap >70%. Cluster 1 included some referenced sequences in recent period from Guangxi and Fujian, and no foreign referenced sequences inside. The referenced sequences among cluster 2 were mainly from Vietnam in early period, with three referenced sequences from Guangxi including the earliest strain(97CNGX2F) additionally ,It could infer that strains among cluster 1 were originated from domestic, meanwhile strains among cluster 2 were from Vietnam.3. Phylogenetic analysis of CRF07BC, CRF08BC and B(B') strains: The phylogenetic analysis showed that sequences of CRF08AE strains were mainly derived from Nanning ,which were the clusterd with sequences from other districts. The phylogenetic analysis of CRF07BC was similar to CRF08BC, yet there was a cluster including eight sequences derived from Heterosexual contact population in Nanning, which inferred that local epidemics might exist in Nanning.Two of four subtype B(B') sequences were derived from Hechi, clustered with referenced sequences from Thailand, which indicated that there might be relationship of transmission between two areas.4. Analysis of unknown recombinant forms: Seven strains were identified as unknown recombinant forms, accounted for 2.6% of total cases.Six recombinants get CRF01AE as the parental type, for CRF01AE was the predominant subtype in Guangxi. We found a second-generation recombinant of CRF07BC and CRF08BC. The analysis results revealed that the majority of the gag-pol was CRF07BC with two 794bp and 782bp CRF08BC fragments inserted in pol.5. Analysis of genetic distances and selection pressures of gag-pol sequences and partial env (C2V3) sequences: (1) The genetic distance of gag-pol sequences in subtype B(B') was the highest(4.41±2.18)%, then the order of it from high to low was CRF08BC(2.72±0.52)%, CRF07BC(2.37±1.04)%, and CRF01AE(2.09+1.16)%. The genetic distances of gag were higher than pol in all subtypes except for subtype B(B'). In the gag coding regions, the genetic distances of P17 and P2P6 were higher than P24 in all subtypes, whereas in the pol coding regions, the orders of the genetic distances in each subtype were not identical. Env C2V3 was the most variable region in all subtypes. (2)All of gag-pol coding regions were suffered the negative selection pressure. The selection pressures in all epidemic subtypes in Guangxi were similar .6. Analysis of the V3 loop tip and coreceptor usage: (1)The tip of V3 loop in subtype B(B') was major GPGR, accounted for 83.3% cases, whereas at the tip of CRF07/08BC and CRF01AE V3 loops, 100.0% and 71.2% cases contain GPGQ, respectively. Seven different motifs were defined at the tip of CRF01AE V3 loops, which were more complex than of other subtypes.(2) Coreceptor usage in CRF07/08BC and CRF01AE strains both were CCR5, accounted for 100.0% and 91.5%, respectively.Partâ…£Identification of the original time of HIV epidemic in Guangxi and analysis of phylogentic relationships of them with those epidemic in Guangxi The main subtypes of HIV-1 strains epdimic in Guangxi and Yunan were CRF01AE,CRF07BC and CRF08BC. However, the distributions of subtypes in these two areas were widely divergent. The major subtype in Yunnan was CRF08BC, followed by CRF01AE and CRF07BC, meanwhile, CRF01AE was perdominant in Guangxi, much more than CRF07BC and CRF08BC. The results of our studies were inconsistent with previous reports, indicated that the epidemic subtypes in Guangxi might be changing. The transmission relationships between Guangxi and Yunnan were further revealed by using phylogenetic analysis, and the origin times of different HIV subtypes in Guangxi were deduced by using the coalescent theory of Bayesian.1.The phylogenetic relationships of CRF01AE epidemic in Guangxi and Yunnan,and the origin time of CRF01AE in Guangxi: two major clusters (Cluster A and Cluster B) in the gag-pol phylogenetics tree of CRF01AE were identified in Guangxi. Phylogenetic analysis showed that Cluster B might originate from Vietnam, which have some certain transmission relationships with Yunnan epidemic strains, meanwhile the origin of Cluster A was unknown, which have little relationships with Yunnan epidemic strains. Evolutionary rate of CRF01AE in gag-pol gene was 2.68×10-3(95%HPD 2.15-3.12×10-3) nucleotide substitutions/site/year.The date of origin for CRF01AE in Thailand was deduced to 1982.4(95%HPD 1980.1-1986.3), and then CRF01AE was transmitted to northern Vietnam and Guangxi in 1996.3(95%HPD 1994.7-1997.0).Cluster A of Guangxi CRF01AE might be introduced from Fujian province with multiple transmitted time-points, which might emerge in China earlier than strains originated from northern Vietnam to Guangxi. Evolutionary rate of Cluster B was 2.56×10-3(95%HPD 2.26-2.88×10-3) nucleotide substitutions/site/year, and it might spread from northern Vietnam to Guangxi in 1993.1(95%HPD 1991.4-1995.1). The founder strain might be introduced into Congzuo which bordered with northern Vietnam in about 1993, and then transmitted into neighbouring districts, and then midland such as Nanning. It was introduced into Hechi in about 1996.2. The transmission relationships of CRF07BC between Guangxi and Yunnan: three dependable clusters were found among the phylogenetic tree. But only one cluster was consist of both Guangxi and Yunnan sequences, however, the strain derived from Yunnan was infected through occupational exposure. So very far phylogenetic relationship was found among CRF07BC strains from Yunnan and Guangxi, suggesting less possibility of directly transmission of CRF07BC strains between the two provinces.Evolutionary rate of CRF07BC gag-pol was 1.84×10-3 ( 95%HPD 1.49-2.19×10-3 ) nucleotide substitutions/site/year. We deduced that CRF07BC was introduced into Guangxi in 1991.4 (95%HPD 1990.0-1994.6). Parts of epidemic strains in Nanning might be introduced in about 2004, with uncertain sources.3. The transmission relationships of CRF08BC between Guangxi and Yunnan: Most of Gunagxi CRF08BC sequences were clustered with referenced sequences of Guangxi in 1997, only few were clustered with sequences derived from Yunnan. As a result of founder effect, Gunagxi CRF08BC strains were relatively independent of Yunnan.Evolutionary rate of CRF08BC gag-pol was 2.44×10-3(95%HPD 2.10-2.78×10-3) nucleotide substitutions/site/year. We deduced that the origin date of CRF08BC in China was 1993.0( 95%HPD 1990.3-1994.6) , while the date of introduced into Guangxi was 1997.3(95%HPD 1996.1-1997.4). As Guangxi CRF08BC sequences were clustered with sequences derived from Wenshan and Honghe in Yunnan,it could infer that CRF08BC strains in Guangxi were originated from Wenshan and Honghe. |
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