The Molecular Epidemiology Of Co-Infections Among HIV And HCV/HPgV-2 In High-Risk Populations Of Honghe State, Yunnan Province

AIDS has been introduced into China since 1985 and in the recent past 10 years its main transmission has changed from by blood transfusion and intravenous drug abuse to sexual behaviors.The percentage of AIDS by sexual transmission had increased from 33.1% in 2006 to 94.6% in 2017 among which 69.2% were transmitted by heterosexuality.It was estimated like that there might be 4 million to 10 million female sex workers(FSWs)in China and 6%-9% of adults aging 18 years to 60 years old might have their experiences as whoremasters.With special vulnerability and extremely high-risk of HIV infection FSWs were usually marginalized and the bridge of transmitting sexually transmitted diseases including HIV from high risk population to normal ones.Thus FSWs and whoremasters should be considered as the key population for the HIV epidemic in China.Hepatitis C virus(HCV)sharing the same transmission media and high risk population with HIV is normally transmitted by blood and skin exposures and common to have co-infection with HIV.HIV infection could increase the risk of infecting HCV,reduce HCV clearance rate in vivo and speed up the Hepatitis C progress.Meanwhile HCV infection could also speed up HIV infection into AIDS and HCV led liver diseases were also the main diseases and causes of death for HIV-infected individuals.As one kind of human virus Human Pegivirus2(HPg V-2),mainly transmitted by blood was firstly reported in 2015 by two separate research teams and it's one of the Pegivirus among Flaviviridae.Up to now only a few researches had verified that it would mainly infect positive HCV carriers,but its pathogenicity and relationship with infections of HIV and HCV were still unclear.Yunnan Province has been the origin source of AIDS epidemic in China while Honghe State,located in southeast of Yunnan Province,is the mostly affected state.In order to clarify the epidemiological status of AIDS in Honghe State,Professor WANG Ning and his research team from Chinese Centers for Disease Control and Prevention had done the epidemiological surveillances of AIDS among underground FSWs and whoremasters into Kaiyuan,Mengzi and Hekou from May 2008 to June 2014.Through series of cross-sectional surveys and prospective cohort studies,many had been found,including emerging infection rates,high-risk factors and their development trends of HIV among FSWs and whoremasters,and vertical transmission risks of HIV positive FSWs.To arrange the molecular epidemiological research of HIV,its co-infections with HCV and HPg V-2 in Honghe State,this graduation thesis was based on Professor WANG Ning's team work and the 771 positive HIV carriers by surveillance there,employing the stored plasmas pecimens and surveillance data,aiming at clarifying the gene mutation characteristics of HIV infection and its co-infections with HCV and HPg V-2 among the local high risk population,the epidemic rules the by molecular level and key chains of transmission to provide evidences for the infection control and prevention of the listed three viruses.Part 1 The Molecular Epidemiology of HIV Infection Among High-Risk Population of Honghe State,Yunnan ProvinceBackground HIV has high variability and Group M of HIV-1 are widely epidemic into 9 subtypes including A-D,F-H,J and K,96(CRF)and numerous(URF).As China's main incoming epidemic region of HIV-1 strains and origin region of recombinant strains,the distribution of epidemic strains in Yunnan Province implied obvious difference from geographical,national and transmission media aspects.There were gaps of lacking the distribution of gene subtypes of HIV-1 and long-term development changes among the high risk population mainly composed of underground FSWs and whoremasters in Honghe State.Methods Selection of HIV antibodies to confirm positive specimens and sort out the background information,extraction and purification of plasma viral RNA,one-step reverse transcriptional nested PCR amplification of the approximately 3100 bp fragments of the HIV-1 pol region and sequencing,below researches had been done:(1)Viral gene subtypes: Employ the method of constructing NJ phylogenetic tree and use online tools COMET and REGA for typing,the determined typing results should be consistent with that of the two tools and their confidence value should be 100%,with epidemiological information to analyze the relationship between gene subtypes and population characteristics and changes over time.(2)Viral gene recombination: Employ jp HMM software for virus gene recombination analysis and construct NJ evolutionary tree with different inserts and standard reference strains,judge the source of recombinant fragments and analyzing the characteristics of recombination patterns in different high-risk groups.(3)Dissemination cluster analysis: Group sequences of different subtypes and cut into the same length,remove known resistance sites,construct ML phylogenetic tree and the criteria for judging transmission clusters is bootstrap>990 and gene distance<0.015.Analyze clustering population characteristics and cluster-related factors.The first specimens of HIV-infected individuals and their viral gene sequences were divided into three groups: 2008-2010,2008-2012,and 2008-2014 to construct ML trees separately and perform clustering analysis,and observe changes in the number and size of transmission clusters over time.(4)Drug resistance analysis: Transmit the splicing edited pol sequence to the HIV Drug Resistance Database in Stanford University(http://hivdb.stanford.edu),and analyze by sequence alignment the location,type,resistance level and sensitivity of drug resistance mutations.(5)The relationship between Honghe strain and Yunnan epidemic strain: The parts of pol gene sequences of 318 HIV-infected individuals sampled in Yunnan Province 2008-2010 were collected,together with the sequences obtained in this study to construct the evolutionary tree,and analyze the relationship between the HIV-1 strains in Honghe State and the epidemic strains in Yunnan Province.Results The HIV-1 pol gene sequences were obtained from 386 specimens,there was no significant difference in the demographic characteristics between successful and failed personnel in the amplification and sequencing,except for the samples sampled in2008-2009 that failed to be amplified and sequenced significantly higher than those sampled at other times.The main acquired results:(1)Gene subtypes of HIV-1 and its population distribution and changes over time: The local epidemic strains of HIV-1 by proportion from high to low were CRF08_BC(62.69%),CRF07_BC(10.88%),new URFs(10.88%),CRF01_AE(8.03%),C(6.99%)and B(0.26%).There was a significant difference in the distribution of HIV-1 subtypes among different populations,and the proportion of CRF08_BC in drug abusers was significantly higher than that of whoremasters and underground FSWs;The CRF01_AE strain appeared almost exclusively in non-drug-abusing whoremasters and underground FSWs;The distribution of HIV-1subtypes had changed significantly over time,and decrease in the proportion of CRF08_BC is most pronounced among sexually transmitted population while the proportions of CRF07_BC and CRF01_AE among underground FSWs were obviously high;The proportion of URF strains among whoremasters were significantly higher than other populations,becoming the main source of new HIV-1 strains.(2)There were significant differences in gene recombination patterns among different new URFs in different populations,B/C were mainly seen among IDU population,the CRF01_AE-involved recombination was mainly seen among non-drug-abusing underground FSWs and whoremasters while the framework of C Subtype with B/C recombination was mainly from CRF08_BC or CRF07_BC.(3)The 386 sequences involved in the analysis formed 32 transmission clusters with their clustering rate20.98% and the sizes of clusters were 2-8,and clusters with 2 sequences dominated(71.88%)while most of the population in the clusters experienced commercial sex.Clustering had obvious correlations with high ages(>50years),single and CRF07_BC infection.Although the number of clusters of each subtype increased with time,no increase was seen in the number of personnel and clustering rate in the cluster and no expansion of transmission clusters was seen.Conclusions (1) CRF08_BC was the prevalent local strain while CRF01_AE was obviously higher among non-drug-abusing underground FSWs and whoremasters than drugabusers.Locally the prevalent strain was transferring from CRF08_BC into CRF07_BC and CRF01_AE,it's consistent with the transferring of main transmission from intravenous drug abuse into sexual behaviors.(2)The main viral recombination form among the IDU population was B/C,and mostly originating from the secondary recombination of CRF08_BC or CRF07_BC.The CRF01_AEinvolved recombination was mainly seen among non-drug-abusing underground FSWs and whoremasters,mostly originating from the secondary recombination of CRF01_AE and CRF08_BC or CRF07_BC.The non-drug-abusing whoremasters were the incubator for new recombinant strains,close monitoring should be considered.(3)The HIV-1 transmission clusters were dominated by small clusters containing 2sequences while most of the population who formed a dissemination cluster had IDU behaviors,speculating that IDU whoremasters and IDU underground FSWs might share the same syringes for drug-abusing while they were engaged in sexual transactions.Expansion of the dissemination cluster over time was not seen,speculating that most of IDU whoremasters and IDU underground FSWs formed relatively long-term stable partners while engaging sexual transactions,few new memberships could join in them and the focus of preventive intervention should be put on reducing the increase of new drug-abusers.Part 2 The Molecular Epidemiology of Co-Infection Between HIV and HCV Among High-Risk Population of Honghe State,Yunnan ProvinceBackground HCV replication and mutation rates are greater than HIV-1,dominating all the RNA virus.Nowadays HCV consists of 7 genotypes and 83 gene subtypes among which Genotypes 1 and 3 are most widely epidemic while Genotypes 4 and 5 are commonly seen in low-income countries.As one heavy-loaded country with HCV infection,China had detected 6 HCV genotypes and 24 gene subtypes.As the high HCV epidemic region,the HCV gene subtypes among drug-abusers in Yunnan Province were complicated with obvious geographical distribution differences.There had been no researches about HCV gene subtypes distribution and long-term changes in Honghe State and among population which were infected with HIV and dominated by underground FSWs and whoremasters.Methods Selection of HIV antibodies to confirm positive specimens and sort out the background information,below researches had been done:(1)Employ ELISA to detect HCV antibody,analyzing the differences of the HCV antibody positive rate among populations.(2)Extract and purify plasma viral RNA.(3)Employ one-step reverse transcription nested PCR to amplify the gene fragments of HCV CE2(1300bp)and NS5B(1000bp)and sequence them.(4)HCV gene subtypes analysis: Quality control of the CE2 and NS5 B sequences and submit them to the COMET HCV alignment website to get the derivation results.Download 53 reference sequences for each gene types and import CE2 and NS5 B fas files separately,construct NJ Tree with MEGA 6,summarize the results of the two methods to determine the genotype and subtype.(5)HCV cluster analysis: Construct ML phylogenetic tree by sequences to determine transmission clusters and the criteria for judging transmission clusters is bootstrap>90% and gene distance<0.05.The first specimens of HIV-infected individuals and their HCV gene sequences were divided into three groups: 2008-2010,2008-2012,and 2008-2014 to perform clustering analysis separately,and observe changes in the number and size of transmission clusters over time.(6)Co-transmission of HIV and HCV: Compare two viral transmission clusters for specimens who obtain both HIV and HCV sequences.Results(1)Five hundred positive specimens of HCV antibody were detected,and that of simple IDU population was 99.25%,showing obviously higher positive rate than non-IDU population(98.12%vs19.47%,P<0.05).The positive rates of HCV antibodies in the simple whoremaster population and the simple underground FSWs were 37.86% and 7.93% respectively.(2)There were 357 HCV RNA amplification sequencing successes among which there were 334 CE2 and 293 NS5 B,and the amplification sequencing success rate was 71.12%.Determinations of HCV genotypes based on CE2 and/or NS5 B regions were listed by proportion from high to low: 3b(37.25%),3a(26.05%),6n(17.37%),1b(10.36%),6a(7.84%)and other subtypes including 6k,6v and 2a.(3)The distributions of HCV gene subtypes showed obvious changes within the 2 periods of 2008-2011 and 2012-2014,and there was the decreasing of 6n among different populations,obvious increasing of 6a among underground FSWs and whoremasters,obvious increasing of 3a among drug-abusing population but decreasing among whoremasters and underground FSWs,obvious increasing of 3b among whoremasters,minimal changes among other populations.(4)Thirty transmission clusters had been verified with the clustering rate of 16.81% and most of them(93.33%)had only 2 sequences.The proportion of all the IDU whoremasters and IDU underground FSWs inside the cluster was 75% while that of simple IDUs was25%,more of the same minorities(85.71%),more of lower education level(66.67%),more of single status(89.34%)and the clustering rates of different HCV gene subtypes showed obvious differences as listed below from high to low: 6a(37.04%),3b(21.37%),3a(17.02%),6n(6.35%)and 1b(5.41%),implying that there were no obvious differences among clustering rates of different high-risk populations.(5)Both of the number of HCV transmission clusters and clustering rates increased over time but the expansion of transmission cluster was not seen,keeping the small dominated clusters with 2 sequences.In specimens among who both HIV and HCV sequences were obtained,6 pairs showed the clustering of both viruses,implying possible co-transmission.Conclusions(1)The highest positive rate of HCV antibody was among the population with IDU behaviors.That the data of positive rate of HCV antibody among sexually high risk populations(underground FSWs and whoremasters)were acquired showed obviously high HCV transmission level as known up to now,considering in-depth monitoring and researches.(2)With complicated gene subtypes the types of HCV strains and their distributions were changing,showing obvious increasing of 3b among whoremasters.(3)Mostly with the transmission pair the clustering rate of HCV was 16.81%,most of the population in the cluster had similar socio-demographic characteristics,and the specimens with unknown basic information showed high clustering rate,implying that those with poor investigating compliance occupied multiple risk factors and they had more infection and transmission risk.The differences of clustering rates among different HCV gene subtypes implied the special epidemic and transmission characteristics among different strains,showing that strains with high clustering rates had more transmission among the drug-abusing population by shared syringes while that with low clustering rates had possible main transmissions by occasional incidents including syringe needle acupunctures.(4)Expansion of the dissemination cluster over time was not seen,speculating that most of local formed relatively long-term stable partners and few new memberships could join in them,and the focus of preventive intervention should be put on reducing the increase of new drug-abusers as the increase of HCV transmission was mainly caused by the increasing syringe-sharing drug-abusing partners.Part 3 The Molecular Epidemiology of Co-Infection Between HIV and HPg V-2Among High-Risk Population of Honghe State,Yunnan ProvinceBackground HPg V-2 is one new type of blood-borne virus through skin,nearly all the confirmed HPg V-2 cases were co-infected with HCV while low epidemic rate also existed among non-HCV infection populations.How is the HPg V-2 epidemic among the special high risk populations in Yunnan Province? Does HPg V-2 affect the infection progresses of HIV and HCV? Both require in-depth researches.Mostly this virus is co-infected with HIV and/or HCV,but its pathogenicity is still inconclusive.Methods Selection of HIV antibodies to confirm positive specimens and sort out the background information,below researches had been done:(1)Detection of HPg V-2 antibody: ELISA was employed to analyze the differences of antibody positive rate among variable populations.(2)Extraction and purification of plasma viral RNA.(3)Obtain HPg V-2 gene fragment and sequence,employ retroviral nested PCR to amplify the 153 bp gene fragment of HPg V-2 NS3 and sequence,compare the positive rates of HPg V-2 RNA among different populations.(4)High-throughput sequencing for the full-length genome of HPg V-2.(5)Sort out the HPg V-2 gene sequences with quality control,determine the kinships of HPg V-2 with other Pegiviruses through phylogenetic analysis,and compare the gene dispersion rate of each gene fragment in the full-length sequence.(6)Detection and comparison of the load capacities of HIV and HCV: All the positive HPg V-2 RNA specimens and doubled negative HPg V-2 RNA specimens were prepared with the same requirements such as positive HIV and HCV RNA,sampling time of the same year and the same anti-virus treatments and transmission routes.Test HIV and HCV loads separately.Results(1)The positive rate of HPg V-2 antibody among HIV-1-infected individuals was 25.29%,and that those with both positive HIV antibody and HCV antibody showed obviously higher positive rate of HPg V-2 antibody than that with positive HIV antibody but negative HCV antibody(27.69%vs20.82%,P<0.05).The co-infection of HPg V-2 and HIV-1 was increasing over time.The positive rate of HPg V-2 nucleic acid among HIV-1-infected specimens was 5.32%,and the positive rate of HPg V-2 nucleic acid among both positive HIV-1 and HCV was higher than that of simply positive HIV-1(6.37%vs3.35%).(2)The positive rates of HPg V-2 antibody and nucleic acid among simple IDU population,simple FSW/whoremaster and PWID/FSW/whoremonger were 38.57% and 6.09%,21.75% and 3.57%,and 19.55% and 6.77% respectively,showing the obviously higher positive rate among simple IDU population than the other groups(both P values <0.05).(3)The average HCV load capacities of positive HPg V-2 nucleic acid group and negative group were 1.11E+06cp/ml and 7.19E+05cp/ml respectively while the average HIV load capacities were 5.36E+04cp/ml and 2.14E+05cp/ml respectively,showing no obvious differences.(4)Seven HPg V-2 full-genome sequences were obtained and the HPg V-2 in Honghe was the sole cluster,the founder effect was remarkable.Three possible transmission clusters were identified among which one might be the co-transmission of HPg V-2 with HIV and HCV.Conclusions(1)That the epidemic rate of HPg V-2 was high in the special high-risk HIV infection population in this research might be correlated with their behavioral exposures to IDU by skin/blood and to frequent sex.(2)HPg V-2 infections were common to see among HCV positive population and usually transmitted by blood and probably by sexual behaviors.(3)The effect of HPg V-2 infection on the duplications of HIV and HCV was not seen.(4)The gene sequence of HPg V-2 was relatively conservative while its strain in Honghe was solely epidemic in regional zones,and its co-infection with HIV and HCV was seldom seen.The high-risk groups where intravenous drugs are intertwined with sexual violence are fertile ground for the production and spread of new viruses,close monitoring and in-depth research should be provided.