Study On The Relationship Between Env Quasispecies And Disease Progression In Primary HIV-1 Infection From MSM Population In Liaoning, China

ObjectiveAIDS (acquired immunodeficiency syndrome, AIDS), is an immune defect disease caused by HIV (human immunodeficiency virus, HIV). The estimated number of people living with HIV was 33.4 million worldwide and 2.7 million new infections in 2009. In china,0.74 million HIV positives cases had been reported,new infections were 48000 in 2009, especially 32.5 percent of which were MSM (men who have sex with men) [1].One of the most important characteristics of HIV is variability. In HIV-1 genome, membrane protein coding genes has the highest various degree, whose env gene difference is 7-20% in the same subtype, the diversity among HIV-1 subtypes may exceed 35% in the viral envelope sequence [2]. When HIV is transmitted from person to person, however, a dramatic evolutionary bottleneck occurs, with 80% of heterosexual infections apparently initiated by a single variant [3]. After transmission, mutational escape and reversion rapidly shape HIV evolution [4].The mutation rate is as high as 1% per year in the env gene region [5]. Within-host HIV-1 evolution is characterized by diversification of the infecting virus population throughout the course of infection. In addition to genetic drift and purifying selection [6-7], HIV-1 can evolve under positive selection from antibodies and cytotoxic T lymphocytes (CTLs) [8]. Study had found that after infection HIV-1 21-28 days, the viral load would reach its peak, and then quickly low to a steady state which was called the viral setpoint [9]. It is now well established that HIV-1 infected individuals with a high viral set point have a more rapid rate of progression to AIDS than those with a low viral setpoint [10-11]. However, little is known about which host and viral factors are responsible for establishing the level of viral setpoint upon HIV-1 infection, and why the same or similar virus infecting different hosts develop different setpoint and prognosis. However, because it is so difficult to establish and maintain a queue of acute HIV infection, at present, the research data in this field is still very limited.In this prospective cohort study, the object was primary HIV-1 infected patients, the method was high-density follow-up to observate the kinetic changes of the levels of viral replication in acute infection, and the research task was gp160 in membrane protein coding region of HIV-1 genome. By way of TA cloning, we could fetch the quasi-species sequence of the earliest time points after infection and setpoint. Then I would obtain virus N-glycosylation sites, coreceptor, genetic diversity, divergence, the variation characteristic of amino acid and analyse the evolution of the virus in different phases. According to the setpoint, I would analysis of these indicators in order to explore the mechanism of setpoint formation in HIV-1 infected patients at the molecular level, to reveal the biological characteristics of HIV-1 in early HIV-1 infection, to find a related factors of protective immunological response, to provide a new way of treatment and vaccine design of HIV infection.Material and Methods1. Study objects12 primary cases that have not received antiviral therapy were studied and diversities of the earliest and set point envelope of each case were analyzed and compared. Viral load set point was defined as the first available measurement 4-24 months after infection.These cases were divided into 2 groups according to the level of set point viral load:high set point group (>4 log10copies/mL)and low set point group(<4 log10copies/mL). 2. CD4+T cells countsCD4+T cell counts were measured using TriTEST CD4FITC/CD8PE/CD3PerCP reagent with 20μl anticoagulated whole blood. After incubation for 15 minutes in the dark at room temperature, FACS lying solution was added and then incubated. CD4+ T cell counts and corresponding ratios were obtained by flow cytometer analysis with FACS MMLTISET software.3. Viral load assayPlasma HIV RNA was quantified with Amplicor HIV-1 Monitor test, version 1.5 (Roche Diagnostics, Rotkreuz Switzerland) by an ultra-sensitive modification.4. RNA extraction, cDNA synthesisHIV-1 RNA from plasma was extracted with RNA Mini Kit (Qiagen, German) according to the manufacture's recommendations. RNA was reverse transcribed into cDNA with a SuperScript(?)ⅢReverse Transcriptase Kit (invitrogen)5. Nested PCR amplificationHIV-1 gp160 gene (3000bp) was amplified with outer primers AEOF (5'-TAGAGCCCTGGAATCATCCGGGAAG-3'HXB25853-5877nt)和AEOR (5'-TTACTACTTGTTACTGCTCCATGT-3'HIV-1HXB2 8936-8913nt), and followed by nested PCR with inner primers UIF (5'-CACCTTAGGCATCTCCTATGGCAGGAA GAAG-3'HIV-1 HXB27850-7879nt)和AEIR (5'-AGCTGGATCCGTCTTGAGATACT GCTCCTAC-3'HIV-1HXB2 8914-8884nt).6. Products PurifiedThe PCR products were confirmed throμgh 1.5% agarose gel electrophoresis in present of 0.5μg/ml ethidium bromide and photographed under ultraviolet illumination. Products were purified with QIAquick Gel Extraction Kit (Qiagen, Germany).7. Construction of gp160 clonesThe amplified gp160 fragments (3000bp) from plasma RNA were cloned into pMDTM18-T Simple Vector (TaKaRa, Japan), and cloning sequences of the quasispecis viruses were confirmed by DNA sequencing by the dideoxy-chain termination method.8. Sequencing analysisSequences were aligned by Contig software. Sequences were edited by Bioedit software. Phylogenetic analysis and Pairwise distance were conducted and compute by MEGA3.1. Cell tropism was predicted with Geno2pheno [coreceptor] software. Analysis of Synonymous substitution and nonsynonymous substitution, Highlighter, N-Glycosite site were analyzed with online tools (http://www.hiv.lanl.gov). Breakpoint was analyzed with Simplot software. HLA-I restricted the CTL epitopes was analyzed with online tools IEDB Analysis Resource.9. Statistical analysisSPSS 15 software was used to make the Statistical analysis. The nonparametric Mann-Whitney U test was used to assess differences number of PNGs and long variation loop. Spearman Rank Correlation was used to correlation analysis.Results一,Characteristic of membrane proteins1,Analysis of the length of env variation loopThe length of V1, V2, V4 of the high viral setpoint group were a little longer than the low viral setpoint group in very early infection,while the length of variation loop of high setpoint group V5 loop were slightly shotter than that of low viral setpoint group. The difference between the two groups was so little that had not reached the significant level.As time goes by, the two groups'(high viral setpoint and low viral setpoint) changes are as follows:the length of V1 was slightly shotter in low setpoint group. The length of V4 and V5 region in both groups had trends of becoming shotter. The difference between the two groups did not reach the significant level. 2,N-glycosylation sites (PNGs)The number of glycosylation sites of V1, V2, V5 of the high viral setpoint group were a little less than the low viral setpoint group in very early infection, while the length of variation loop of high viral setpoint group V3, V4, gp41 extracellular region were slightly higher than that of low viral setpoint group. The difference between the two groups was not statistically significant.As time goes by, the two groups'(high viral setpoint and low viral setpoint) changes are as follows:The number of glycosylation sites of VI and V4 had a decreasing trend in both grops. The number of glycosylation sites of V2 and gp41 extracellular region had a increasing trend in high viral setpoint group, while have a decreasing trend in low viral setpoint group. The number of glycosylation sites of V3 region had a increasing trend in low viral setpoint group. That difference was not statistically significant..3,To predict the result of cell tropismThere were 12 HIV-1 infection cases in primary infection whose virus were mainly CCR5-tropic strains, accounting for 83.3%. All were for the CCR5-tropic in the high viral setpoint group, while 50% in the low viral setpoint, and others were CXCR4. The virus of one patient who was infected by heterosexual transmitted turned from CCR5-tropic in the early stage into CXCR4-tropic at setpoint.二,Evolution characteristics of HIV-1 env region1,Analyse of diversity in the early HIV infected stageThe diversity of individuals FeibigⅡstage in high setpoint group is lower than low setpoint group. While the diversity Feibig IV and VI stage higher in the high viral setpoint group.2,Diverfication rateThe dverfication rates were no significant difference between the high setpoint group and the low (p=0.458) 3,Analysis of nucleotide divergence and amino acid divergenceThe divergence value in high viral setpoint group was higher than the low (P=0.105). It was suggested that log10VL tends to have positively correlated with genetic distance in linear regression model (r=0.162, p=0.157). There were more amino acid divergence in high viral setpoint group than the low (p=0.157), and have the same characteristics compared with genetic divergence.4,dN/dS ratiosdN/dS ratios were relatively high in the high viral setpoint group, and there was significant difference compared with the low (p=0.002). At the same time, we found that dS between the high setpoint group and the low had statistical significance (p <0.001); There was no significant between the two groups with regard to dN (P= 0.308).5,Study on the variations of evolution in homologous virus interpatientAccording to epidemiology and analysis of full-length of env gene, we found that there were two couples who had the homologous transmission. Virus of the one couple was a stringently homologously sigle strain, while the other couple's virus were complicated. One was a single strain, but the other had two strains which were not known when they came into the bodies of patients.Conclusion1. The single strain of the virus, subtype AE of HIV-1, is more common in MSM populations of Liaoning, China.And we also found there was one individual who was infected by two different strains.2. As time goes on, primary HIV infection patients would come to setpoint, whose length of variation loops and number of PNGs had no or little difference. There is no significant difference between the two groups. 3. The variation trends of diversity in primary patients were different according to the different Fiebig stages. But the viral load at setpoint correlated with viral divergent.4. dN/dS and dS correlated with viral load. There is significant difference between the two groups.