Study On Genetic Characterization Of HIV-1 Full-length Sequences In Primary Infection From And MSM Cohort In Liaoning Province, China

ObjectionAIDS is the greatest threat to human society as one of infectious diseases. Because there is a high level of genetic variability in HIV-1,although antiviral drugs have been widely used in AIDS treatment, but for years AIDS prevention and vaccine study did not make any breakthroughs. The latest assessment report showed that MSM population had become an important transmission mode of HIV-1 infection, which we should focus our attention and control.The genetic background of China's population in Europe and America and Africa race significantly different,and their immune response most likely to have new features.But HIV-1 molecular epidemiology study is still very limited, targeted MSM in China infected with HIV-1.For vaccine and treatment of HIV, there is the lack of pathogenic immunological support study such as these basic information.understanding biological and genomic characteristics and the relationship between viral evolution and dissemination of the founder virus in HIV-1 primary infection in vivo, is a necessary prerequisite for HIV-1 prevention work in this future high-risk groups.This study selected 21 primary HIV-1 infection cases of MSM populations in Liaoning province, China._Using SGA/S method to get the virus complete genome sequence,we analyzed primary HIV-1 infection prevalence trends and the level of genetic variation of early virus infection from the level of the virus genome-wide,identified early virologic characteristics of primary infection,predicted earlyI HLA-I restricted CTL epitopes and analyzed its amino acid polymorphism.We analyzed immune response characteristics related to different progression of HIV-1 infection.This study would support important data to the prevention, diagnosis, monitoring and treatment of HIV-1 infection,in China.Materials and methods 1.Study populationLiaoning MSM high-risk cohort of 800 cases began to receive voluntary counseling and testing by 1.5 months of follow-up from October 2008.From this cohort,21 primary infections were enrolled. The enrollment standard:serological ELISA test,WB test,and pooling PCR for the serological negative detection,one or more of the following conditions are prmary infections,①HIV antibody change to be positive in 6 months②HIV antibodies negative but HIV RNA positive③HIV antibody weakly positive, but can be repeated within two weeks, and OD values were higher than the previous④clinical symptoms of early HIV infection, HIV antigen positive and WB less than four protein bands. Whole blood samples were EDTA-Collected,plasma samples were aliquot within 4 hours of collection and stored at-80℃.2.RNA extractionFor plasma specimens containing>10,000 RNA copies/ml, plasma RNA was extracted by using a QIAamp(?)Viral RNA Mini Kit(Qiagen).Samples with vRNA loads <10,000 copies/ml were concentrated by centrifugation at 23,600g for 1h at 4℃prior to the same extraction. RNA was recovered in a final elution volume of 60ul.3.cDNA synthesisReverse transcription of RNA to single-stranded cDNA was performed by using SuperScript(?)ⅢReverse Transcriptase Kit (invitrogen).The resulting cDNA was used immediately for PCR or kept frozen at-80℃until further analysis.4.Two-halves Nested-PCR using SGA/SAccording to a Poisson distribution, the cDNA dilution that yields PCR products in no more than 30% of wells contains one amplifiable cDNA template per positive PCR more than 80% of the time. cDNA was endpoint diluted in 8 well such that fewer than 3 PCRs yielded an amplification product. Near full-length 9.8kb were amplified with two fragments using Platinum(?)Taq DNA Polymerase High Fidelity (Invitrogen)5. SequencingViral genes were directly sequenced by using dideoxy Terminator chemistry.The sequences were determined by using an ABI 3730x1 genetic analyzer and edited by using the Contig program, version 9.0. All chromatograms were carefully inspected for sites of ambiguous sequence (double peaks).Sequences were aligned by using Bioedit program,version 3.3. All trees were constructed by using the neighbor joining method using Kimura's correction.6.Cell tropismBy using PSSM web tool, cell tropism of infected strains was predicted.The virus was judged as CCR5 tropism while in the V3 loop the first 11-25 amino acids was the S /GXXXGPGXXXXXXXE/D. If the 11 and 25 amino acids were R or Q,the virus was judged as CRCX4 tropism.7.The number of amino acid and PNGSs in EnvAnalyzing the amine acid length polymorphism in Env partial regions and the number of N-GlycoSite was performed by using Bioedit program and N-GlycoSite web tool in HIV DataBase.Gotting three-dimensional HIV-1 protein structure model in the RCSB Protein DataBank,the three-dimensional HIV-1 protein structures of our samples were constructed.8.Prediction of HLA-I-restricted CTL epitopeBy using web tool IEDB Analysis Resource,according to HLA-I information, HLA-I-restricted CTL epitopes were predicted and analyzed while IC50<50.9.Statistic analysisThe data of viral load were enrolled after logarithm transforming. Statistical correlation was determined by Spearman test and statistical significance was determined by Wilcoxon test using SPSS(version 16.0).P<0.05 was considered statistically significant.Result1.Demographic information and clinical featuresThe experiment included 21 cases of Liaoning MSM population of primary HIV-1 infection, of which 19 cases were CRF01-AE subtype, and 1 CRF07-BC subtype, and 1 B subtype. Except for 1 case had history of heterosexual contact, the rest are MSM contact. The four cases which had the transmission relationship have been observed longitudinally.2.SGA/S 22 nearly full-length sequences and 5 3'terminal half-molecule sequences were obtained from 21 primary infection cases by this method. A total of three defective sequences in all were amplified, two missing near the 500bp bases,one missing the V3 motif, accounting for 6% of the total number of the remaining sequences.In addition, in all only 2 sequences contained mixed bases, accounting for 4.3%.3.Viral evolution and transmissionThe phylogenetic analysis based on 3'terminal half-molecule sequences showed that 19 CRF01AE subtype sequences clearly divided into two gene clusters, namely with Liaoning early separated sexually transmitted HIV-1 strains and China southeast coast endemic strain.Comparing gag, env, and full-length gene sequences between the two pairs CRFOIAE subtype cases owing transmission relationship and other CRF01AE cases in this experiment, phylogenetic trees showed in a pair of these cases the sample point sequences focused on a gene cluster;but sequences in another pair were located in different gene cluster. Combined with the laboratory of clinical epidemiological data, the preliminary view that there is one cases may be infected with another CRFOIAE strain during the window period was suggested.4.Env protein polymorphism and the number of glycosylation siteThe number of PNGSs in V1V2 and V1 to V4 regions were negatively correlated with set point viral load in 19 CRFOIAE subtype primary infections.And compared with 25 HIV-1 CRFOIAE sexual chronic infections in Asia selected from HIV Database,the number of amino acids from 15 CRFOIAE cases who were on the same cluster increased in the trend in V1V2 region and V1 to V4 region, the number of PNGSs in V1 to V4 region increased in the trend,and the differences were up to the level of significance.To understand the mutations of amino acid have any effect on viral protein conformation, a sample of 21 cases of C4 domain protein three-dimensional structure diagram were constructed by HIV-1 gp160 protein three-dimensional model 3jwdA as a template. gp120 C4 glycosylation sites N448 appears aspartic acid substitution in one case, resulting in gp120 C4 domain protein structural changes; and compared to cases with wel N488 glycosylation sites, viral load fell rapidly, set point virus level is very low and CD4+T cells remained at a higher level. Also a pair of cases had been identified with the spread of the sample length polymorphism exists in the gp120 V4 region, had led to differences in protein structure, and the viral load level between had significant difference.5.HLA-I-restricted CTL epitope and polymorphism prediction in Gag, Pol, and Nef proteinAccording to set point viral load, there were not significant differences in the number of epitopes in Gag,Pol and Nef; and the number of epitopes in Pol were correlated to the load level.The number of HLA-I restricted CTL epitopes in a pair of early samples infected by the same strain had significant differences with the level of viral load-related. In 78 amino acid polymorphism sites of 21 samples predicted CTL epitopes, amino acid mutation rate of 2,5 and 9 sites were high above 25%.Conclusion1. SGA/S method could access single strain of primary HIV-1 infection in vitro.2. In Liaoning Province MSM population, HIV-1 primary infection with subtype CRF01AE is main, in addition to CRF07BC and B subtype.3. In HIV-1 primary infection of Liaoning Province MSM population, certain amino acids length polymorphism,and special sites of variation would change the protein structure, and affect the immune recognition.4. MSM population in the Liaoning Province were infected mainly by HIV-1 with CCR5 tropic; although there was also the spread of CXCR4-tropic strains, but the viral load levels are relatively low5. The predicted HLA-I restricted CTL epitope diversity was evident in primary HIV-1 infection of Liaoning Province MSM,and in individual cases changes of the number in CTL epitopes were associated with disease progression.