Study On Genetic Characteristics Analysis And Minor Drug-Resistant Viral Variants Of HIV-1 In Fujian Province

Human immunodeficiency virus type 1(HIV-1)is characterized by a high genetic diversity due to its extremely high evolution rate,error-prone reverse transcription and presence of recombination during viral replication,while the direction of HIV-1 evolution is determined by host selective pressure,such as immune response and antiretroviral therapy.It has been demonstrated that the high genetic diversity of HIV-1 poses a great impact on the AIDS progression,the efficacy of antiviral agents,and the development of HIV vaccines.Elucidation of the genetic diversity of HIV-1 is therefore critical to AIDS prevention and treatment.In this study,genetic characteristics analysis of HIV-1 isolates was firstly performed in newly infected individuals identified in Fujian Province,so as to understand the genetic diversity of HIV-1 isolates in Fujian Province.Then,a high-throughput sequencing technique for detecting minor drug-resistant HIV-1 viral variants was established to analyze the genetic variation of HIV-1 isolates pre-and post-antiretroviral therapy,and investigate the molecular evolution pattern of HIV-1 CRF01?AE recombinant form under the drug selection pressure.The current study contained two parts.In the first part,a total of 104 plasma specimens were sampled from HIV-1 newly infected individuals in Fujian Province,and the three genes of pol,gag and env were amplified using a nested-PCR assay.Genetic characteristics analysis was performed based on sequencing and epidemiological data.Our findings showed subtype B and four recombinant forms 01?AE,07?BC,08?BC and 55?01B in the HIV-1 newly infected individuals in Fujian Province,in which the recombinant 01?AE(46.91%)and 07?BC forms(35.8%)were predominant.As compared to previous reports,our findings showed a reduction in the proportion of recombinant form 01?AE,and a remarkable increase in the proportion of recombinant form 07?BC.We found sexual contact as the predominant route of HIV transmission in Fujian Province and the proportion of homosexual transmission exhibited a clear-cut rise.There were significant differences seen in the results on subtype distribution of strains between newly infected individuals and newly reported infected individuals.Phylogenetic and homology analysis showed that the HIV-1 isolates in Fujian Province had a close transmission relationship with the isolates from Guangdong,Guangxi,Beijing and Zhejiang provinces.The primary resistance rate of treatment-na(?)ve HIV-1 infected patients was 7.41%,which reaching moderate epidemic level.The results demonstrate gradual changes in the subtype and transmission routes of HIV-1 isolates in Fujian Province with the extension of time.It is therefore considered that dynamic monitoring should be strengthened and the AIDS control strategy should be adjusted timely.In addition,newly HIV-1 infected individuals are recommended as the study subjects.In the second part,we successfully developed a high-throughput sequencing approach for detecting minor drug-resistant HIV-1 variants,which was effective to simultaneously detect 128 specimens.Through the optimization of specimen preparation,sequencing and data analysis,this approach may cover 3671±927 sequences at each base site and achieve 1% threshold for detecting minor drug-resistant HIV-1 variants.By using this approach,a total of 19 treatment-na(?)ve newly HIV-1 infected individuals were selected for detecting primary drug resistance mutations,and 8 major drug resistance mutations were detected in 5 individuals(26.32%,5/19).Our findings demonstrate a high level of HIV-1 drug resistance transmission in Fujian Province,which should be paid much attention.Then,15 patients with virologic failure in first-line antiretroviral treatment were recruited to detect drug resistance mutations.We detected additional 1.7 drug resistance mutations per individual by using high-throughput sequencing relative to conventional Sanger method(P < 0.05),and minor drug resistance mutations consisted of 19.27% of total drug resistance mutations in patients with virologic failure,which can be only detected using high-throughput sequencing.In addition,the additionally detected minor drug resistance mutations remarkably increased the burden of drug resistance in patients developing virologic failure,90.9%(10/11)of the individuals increased the score of resistance to at least two antiretroviral agents,while 81.8%(9/11)increased the grade of resistance to at least one antiretroviral drug.The patients with virologic failure in first-line antiretroviral treatment exhibited comparative low levels of mutations of resistance to protease inhibitors(PIs)relative to newly HIV-1 infected individuals,which is favorable for the transfer to the second-line antiretroviral regimen containing PIs.We screened three suspected,novel drug resistance mutation sites by comparing the high-throughput sequencing results between treatment-na(?)ve and treatment-failure individuals.To demonstrate the emergence and evolution of HIV-1 drug resistance mutation under drug selective pressure during antiretroviral therapy,5 patients harboring HIV-1 recombinant 01?AE form who experienced failure on first-line antiretroviral treatment were enrolled and blood samples were collected during the past 6-to-8-year antiretroviral therapy for detecting the dynamic changes of drug resistance mutations.Our findings demonstrate that the development of HIV-1 drug resistance is a result from the gradual accumulation of multiple drug resistant genes,and the M184V-G190A-TAMs combination,as the basic combination of the drug resistance in the HIV-1 recombinant 01?AE form,may be detected within a short period of time after antiretroviral therapy is initiated.A post-treatment follow-up of HIV-1 drug resistance should be therefore implemented as quickly as possible.Once M184 V mutation emerges,it may appear refractory presence,and is not easy to disappear even if antiretroviral therapy ceases,while G190 A mutation may show adaptive priority relative to other NNRTI mutations.TAMs mutations exhibit competitive development via two pathways,and the HIV-1 recombinant 01?AE form prefers the TAM2 pathway.However,both pathways are not absolutely compatible,and these two pathways may be compatible with the extension of antiretroviral therapy.In addition,the emergence of multi-NRTI resistance mutations that are detected prior to the transfer to second-line regimen,such as Q151M multidrug-resistant complex,T69 insert complex and TAM1 mutations,may make second-line antiretroviral therapy ineffective.