The Genetic Variation And Molecular Evolution Study On HIV-1 Strains Circulated In Yunnan Province And Quasispecies During Early HIV-1 Infection

HIV experienced complex origins and spread process in global. Nowadays, HIV - 1 M group has evolved into nine subtypes, 49 CRFs and countless URFs. CRF07-BC,CRF08-BC,CRF01-AE and B′subtype viruses dominate in China and our country is in the stage of rapidly urging epidemic, multiple transmission route and various strains coexist. It is the foundation of HIV prevention and research to understand and grasp HIV evolution characteristics, not only group but also individual. Furthermore, it provides scientific basis for design AIDS vaccine, research and development for drugs and diagnosis reagents.Due to the high degree variation of HIV-1 genome and complex quasispecies, we confront extensively difficult in amplification and sequencing of HIV-1 genome. Previous domestic and international studies mainly adopt the amplification and sequencing of short fragments of HIV-1 genome, it often can't accurately identify the virus genotype and even the recombinant virus.Before the start of our project, GenBank contains a total number of 33 HIV-1 full-length genomes which were obtained from China in more than 20 years. HIV-1 infection during early infection is a special stage, with a unique epidemiology, viral and immunological characteristics. Fast and high-level viral replication, high concentrations of viremia, high risk of transmission during this period result in almost 56-92% of new HIV infections individuals infected by the person during early/primary infection. The evolution of viral quasispecies during this period has an important impact on disease progression and prognosis as well.In this study, we developed two experimental strategies that would allow us to amplify and sequence nearly full-length genome of HIV-1 and complete env gp120 gene of HIV-1 quasispecies from virion RNA in the plasma of infected individuals. First, we use the established method to explore the genetic variation and molecular evolution of HIV-1 on NFLG level in Yunnan Province, where is the epicenter with the most serious epidemic of HIV-1 in China. Second, we research the genetic variation and molecular evolution of HIV-1 gp120 quasispecies of early infectors in MSM (man who have sex with man) populations who present the highest prevalence in new HIV-1 infection.PartⅠThe NFLG level genetic variation and molecular evolution study on HIV-1 strains circulated in Yunnan ProvinceFirst, we developed an experimental strategy that would allow us to amplify and sequence nearly full-length genome of HIV-1 in two halves from virion RNA in the plasma of infected individuals and optimized the conditions for amplification and sequencing. Finally, we successfully obtained 57 HIV-1 NFLG in total of 72 samples which were collected in Yunnan Province in year 2009. Then, we analyzed the NFLG level genetic variation and molecular evolution on the 57 HIV-1 NFLG and part of HIV-1 NFLG selected from HIV DATABASE. MEGA, jpHMM, BEAST v 1.5.4, et al several softwares were used in analysis. The main results are as follows.(1) We identified major circulating subtypes in Yunnan Province. CRF08_BC and CRF01_AE were the main subtypes with the proportion of 45.5% and 40.9%, respectively. However, CRF07_BC and C were in lower prevalence with the proportion of 9.1% and 4.5%, respectively. Then we described the distribution characteristics of the major strains among different populations and locations.(2) Various of unique recombinant forms (URFs) HIV-1strains existed in Yunnan Province. Of the 13 URFs, 9 were B/C URFs, 3 were CRF01_AE/B/C URFs and 1 was CRF01_AE/B URFs.(3) We analyzed the evolutionary relationships of CRF01_AE strains between Yunnan province and its neighbor regions, and then deduced the time and route of CRF01_AE strains transmitted into Yunnan province. First, we confirmed that there no relationships of CRF01_AE strains between Yunnan province and its northeastern neighbor regions, Guangxi province and northern Vietnam. Second, we speculated that the CRF01_AE strains of Yunnan province would be transmitted from its southwestern neighbor regions, Burma and Thailand, through multi-route and multi- point. Genome-specific rates of evolution and divergence times were estimated using a Bayesian Markov chain Monte Carlo framework under various evolutionary models. CRF01_AE strains were introduced to Yunnan province approximately in 1989 -1993 June (early 90s).(4) We deduced the time of origin of HIV-1 C and time of HIV-1 C spread into China on NFLG level. Here we suggest that the most recent common ancestor of subtype C appeared in the middle of the 50s in Africa with the evolutionary rate of 1.86×10-3per site / per year. HIV-1 C strains were introduced into India from Africa around the time the mid-70s (1974). Then, HIV-1 C strains spread to Myanmar and Yunnan in early 80s (1982).(5) We obtained the time of origin of HIV-1 CRF08_BC and CRF07_BC, and their spread route in China. Both CRF08_BC and CRF07_BC originated in Yunnan province approximately in the late 80's with the evolutionary rate of 2.07×10-3per site/per year and 1.83×10-3per site/per year, respectively. Although, CRF08_BC and CRF07_BC originated in almost the same time, they showed distinct spread routes in China.PartⅡThe genetic variation and molecular evolution study on quasispecies of HIV-1 gp120 gene during early HIV-1 infectionFirst, we describe an experimental approach to analyze HIV-1 env gp120 gene as intact genetic units amplified from plasma virion RNA by single-genome amplification (SGA), followed by direct sequencing of uncloned DNA amplicons. Using this method, we obtained multiple single template sequences originated from the same sample. Upon examination, each PCR reaction contains theoretical 0.44 copy number of cDNA template, indicating that amplification was specific and effective. A total of 459 single template sequences were obtained from two time point samples of 12 MSM population subjects in Beijing whose infections were characterized as early infection. In a word, the number of quasispecies sequences obtained from each time point sample ranged from 4 to 35 with an average 19.1±8.6. Then, we analyzed the genetic variation and molecular evolution of gp120 gene based on 459 single template sequences and part of HIV-1 gp120 sequences selected from HIV DATABASE. Hypermut 2.0, MEGA, jpHMM, Highlighter, TreeRate, et al several softwares were used in analysis. The main results are as follows.(1) We identified major circulating subtypes in MSM population subjects in Beijing. CRF01_AE was the main subtype among MSM population subjects in Beijing, with 9 of 12 subjects who were infected by CRF01_AE subtype strains. Apart from CRF01_AE, HIV-1 infection in this population contains the persistent circulation of multiple HIV-1 subtypes and complex new recombinants including two subtype B strains, one CRF07_BC strain and CRF01_AE/C URF strain. Interestingly, we found one subject who was infected by two different subtype strains, subtype B and CRF01_AE/C URF.(2) We identified the transmitted or early"founder"viruses. 9 of 12 subjects were initiated productive clinical infection only by one virus; however, the left 3 subjects were initiated productive clinical infection by two viruses or more than two viruses.(3) We analyzed gp120 gene diversity in early infections. The mean within-patient nucleotide and amino acid diversity of the former time point samples were significantly lower than that of the latter time point samples, suggesting that with the development of the infection process, virus quasispecies diversity in the nucleotide and amino acid levels increased significantly. The mean evolutionary rate was 1.61±0.39E-05 per site/per day, ranging from 1.28 E-05 per site/per day to 2.22 E-05 per site/per day which is very close to previously described. Furthermore, APOBEC-mediated G-to-A substitutions existed in early gp120 sequence. The mean number of APOBEC-mediated G-to-A substitutions of the former time point samples was significantly lower than that of the latter time point samples, suggesting that APOBEC-mediated G-to-A substitutions presented accumulation effect. In a word, APOBEC play a significant role in contributing diversity to HIV-1 and shaping HIV-1 evolution during early infection.(4) The contribution of viral recombination to gp120 gene diversity and virus replication fitness. Comparing the composition of recombinant viruses between the two time-point samples in subject YA– 106 who was infected by two close related CRF01_AE viruses, it indicated that recombination between the same type of virus most likely occurred soon after infection and recombinant strains presented higher replication fitness than non-recombinant strains. However, we did not detect recombinant strain in subject YA-113 who was infected by two distinct subtype viruses, not only the first time point sample but also the latter. Interestingly, two distinct subtype viruses showed distinct replication fitness in subject YA-113 with recombinant subtype virus presenting higher replication fitness than non-recombinant subtype virus in the two time point samples. It illustrated that recombination frequency was higher between same subtype strains than different subtype strains.(5) We studied the characterization of PNGS of CRF01_AE strains during early infection. The mean PNGS number of CRF01_AE strains in the first time point sample was 24.6±1.6, ranging from 22 to 28. There was no significant difference between the first time point sample and the latter time point sample in PNGS number. Moreover, we found no significant difference between early sequences and non-early sequences, not only in PNGS number but also in locations of PNGS.(6) Finally, we analyzed the length of gp120 variable region and the model of selection pressure during early infection. Comparing the length of V1/V2, V4/V5 and V3 between early sequences and non-early sequences, we found no significant difference. Both early sequences and non-early sequences mainly presented GPGQ motif in the V3 top. We found that the coreceptors of all early sequences in our analysis were CCR5 by online V3 loop prediction. 9 subjects presented distinct characteristics of selection pressure, with 4 subjects showing positive selection and 5 subjects showing negative selection. Different strains showed different abundance of positive selection sites and a certain number of distribution areas with the relative concentration of positive selection sites were found in different samples.