Molecular Epidemiological Study Of HIV-1 In Yunnan Province Based On Clinical Patients

Yunnan Province,locating in the southwest of China,borders on the Golden Triangle area,which is the famous area for illicit drug production.It is the most important route for drugs to enter the Chinese market and also for HIV-1 to enter China.Most of HIV-1 strains currently circulating in China were firstly introduced or formed in Yunnan,and then spread to Guangxi,Sichuan,Xinjiang,and other regions through drug tranffic or sexually transmitted people,eventually leading to a nation-wide epidemic.Therefore,Yunnan is considered as the HIV-1 epidemic center of China.The prevalence of HIV-1 strains in Yunnan province can affect variation and evolution of HIV-1 strains across the country.A large number of studies have shown that the epidemic of HIV-1 in Yunnan depends on region and population differences,and the epidemic virus strain is constantly changing.There is an urgent need for molecular epidemiological studies on HIV-1 epidemic in Yunnan.In this study,we collected 773 HIV-1 infected people and AIDS patients who were admitted to the Yunnan AIDS Care Center from 2013 to 2015.The demographic information was interviewed with informed consent.HIV-1 major structural gene were amplified and sequenced.Subtypes and phylogenetic relationships were analyzed.Virus resistance information was also illustrated.HCV co-infection status was detected.The nealy full-length genomes of some specific subjects were amplified.The variation and epidemiological characteristics of HIV-1 strains prevalent in Yunnan were demonstrated,the results will to provide basic information for HIV-1 prevention and control in Yunnan and even throughout the country.Objectives:(1)To understand the subtype distribution,phenotypic and variation characteristics of HIV-1 strains prevalent in Yunnan Province;(2)To determine the prevalence and mutation profiles of HIV-1 resistant strains in Yunnan Province,and provide basic information for anti-virus treatment of AIDS in Yunnan Province;(3)To understand the recombination characteristics of HIV-1 strains in Yunnan Province and the evolutionary characteristics of each gene based on the long fragment sequence and the near full-length genome of HIV-1;(4)To demonstrate the status of HCV co-infection and distribution of HCV subtypes among HIV-1 infected idividuals in Yunnan Province.Methods: All HIV-infected individuals(773 cases)admitted to the AIDS care center of Yunnan Province from July 2013 to July 2015 were enrolled into the study.Background information was collected,peripheral blood samples were collected,CD4 cell count was tested.Plasma and Lymphocyte-enriched fluid were separated to fulfill the following studies:(1)Viral RNA was extracted and amplified,gag,pol,env and 3'half genes were amplified by reverse transcriptional nested-PCR.After the sequence was assembled,it was submitted to the online HIV-1 sequence database,the sequence quality was tested through online software.The subtype or recombinant genotype was determined using the NCBI online subtyping software or REGA,jp HMM,and RIP.(2)After aligning the sequence with the HIV-1 subtype reference sequences,phylogenetic tree was constructed using the Kimura 2-parameter model with MEGA6.06 software.The obtained phylogenetic tree was used to confirm the results of subtyping software.The ML-tree was constructed using the GTR+I+G base substitution model with Phy ML3.0 software.The epidemic clusters were identified by using bootstrap value.The phylogenetic relationships of different strains were analyzed.The MCC tree was constructed using the Markov Chains Monte Carlo evolutionary analysis method with BEAST software.The original time of common ancestors of epidemic clusters and the number of effective infections were predicted.(3)Geno2pheno and PSSM softwares were used to analyze the tropism of viruses based on the sequence of the V3 loop in the env gene,the differences in the distribution of different tropism strains were compared.(4)The pol sequence was submitted to Stanford University's drug resistance database for drug resistance prediction.(5)Enzyme-linked immunosorbent assay(ELISA)was used to detect HCV antibodies in plasma specimens of 300 patients who were enrolled before starting antiretroviral treatment.NCR/C,C/E2,and NS5 B gene segments of HCV were amplified and sequenced by reverse transcription nested-PCR from anti-HCV antibody positive speciments.The COMET software and phylogenetic tree analysis were used to determine HCV subtypes and analyze HCV co-infection status among HIV-1 infected individuals.Results:(1)Among the 773 HIV-infected participants,473 had undertaken HAART butfailed to respond to drugs and 300 had not initiated antiretroviral treatment.The patients were from 13 states(cities)in Yunnan Province.Dehong,Lincang,Honghe and Kunming contained the most cases,exceeding 10% of the total separately;HIV-infected people were from multiple ethnic groups,mainly Han(76.1%);heterosexual transmission(73.0%)was the dominant transmission route.(2)596 gag genes(77.1%),586 pol(75.8%),658 env(85.1%),526 gag-pol(68.0%)and 50 near full-length genomic sequences were obtained.(3)HIV-1 subtypes were determined for613 strains.A total of twelve HIV-1 subtypes were identified.The predominant subtypes was CRF08_BC(53.4%),followed by CRF01_AE(19.4%),unique recombinant form URF(12.7%)and CRF07_BC(9.0%).In phylogenetic tree,all of HIV-1 sequences belonging to the same subtype clustered together,while the URF sequences distributed among the subtype reference sequences.Subtypes of the 50HIV-1 strains with near full-length genomic sequences were determined,including 30CRF08_BC,4 CRF07_BC,9 CRF01_AE,and 7 URFs.(4)Phylodynamic analysis of CRF07_BC subtypes was analyzed based on HIV strains prevalent in the whole China.The results showed that CRF07_BC has been introduced into the MSM population,droving its second spreading wave in China(5)The tropism of 658 HIV variants were successfully predicted,including 555 R5 tropic strains,21 R5/X4 tropic strains,and 82X4 tropic strains.For all subtypes,R5 tropic strain was dominant.The distribution of X4 tropic strains in different subtypes was different.The proportion of X4 tropic strains belonging to CRF01_AE strain was significantly higher than other subtypes(P=0.000).(6)1.5% and 38.7% individuals among treatment na?ve individuals and ART-failure individuals,containing drug resistant variants respectively.The dominant drug resistant mutations were M184V/I and 103N/S.(8)Positive rate of HCV antibody in HIV-1 infected patients was 20.67%.54 NCR,53 CE/2 and 53 NS5 B gene sequences were obtained by amplification and sequencing.HCV subtypes were determined successfully in 55 individuals.3 genotypes and 7 gene subtypes were identified,including 3b(45.5%),6n(16.3%),1a(10.9%),3a(10.9%),1b(9.1%),6v(5.5%),6u(1.8%).Conclusions:(1)High diversity and multiple subtypes of HIV strains were identfied in Yunnan Province.Except the dominant CRF08_BC strains,11 virus subtypes were identified.Furthermore,\more than 10% strains belong to URFs.The distribution of HIV-1 subtypes was different in different ethnic groups,transmission routes,and regions,suggesting that Yunnan Province is still the main source of URFs in China.Surveillance is necessary.(2)The genomic schematic of URFs could be divided into B/C or B/C/CRF01_AE depending on the involvement of CRF01_AE strain.B/C URFs mainly occurs in the IDU population,while B/C/CRF01_AE URFs is common in sexually transmitted populations.The proportion of URF strains in Dehong district is as high as 34.7%,supposing the area is the hot spot of HIV recombination and the emergence of new CRFs.(3)The proportion of the CRF07_BC strain in Yunnan is not high,however,it has already spread into the MSM population.The prevalence of CRF07_BC strains in MSM population droves its Second spreading wave in China,therefore,surveillance on CRF07_BC strains in the whole country will be necessary.(4)The prevalence of drug-resistant strains in untreated HIV positive individuals in Yunnan is low,but high level of drug resistance can be identified in patientsexperiencing failed antiviral treatment.Drug resistance should be tested before starting treatment.The dominant drug resistant mutations are mainly M184V/I and103N/S.Efficient second-line drugs should be considered in the subsequent clinical treatment.(5)High ratio of HCV co-infection was identfied among HIV-infected persons.The epidemic subtypes of HCV are complex.The coinfection of HCV might affect the disease progression and treatment effect of HIV-infected persons in the local area and require further monitor.