Isolation,identification And Biological Phenotype Of HIV-1 Epidemic Strains In China

Objectives: HIV-1 virus has a high degree of gene polymorphism and significant differences in biological phenotypic characteristics,which directly affects the spread of AIDS and the progression of the disease.At present,the research on HIV-1 in China is mainly focused on viral gene sequence polymorphism,and the study of biological characteristics restricts its research progress to a certain extent because of the need for specific biosafety facilities.Since high gene polymorphism will inevitably lead to changes in the phenotype of HIV,the phenotypic study of different genotypes of HIV strains to determine their pathogenicity and transmission ability is of great significance for elucidating the epidemic characteristics of HIV in China,developing new treatments,and developing broad-spectrum HIV vaccines.The purpose of this study is to isolate and identify the main epidemic HIV-1 strains in China and to comprehensively identify the replication ability and biological phenotypic characteristics of the isolates,to form representative clinical isolates for the main HIV-1 strains in China,and provide technical support for the research and development of AIDS testing reagents,vaccines,and drugs.Methods: In this study,using PBMCs collected from 10 HIV-1 patients from 9 regions of China and 68 clinical isolates of HIV-1 collected in our laboratory from 2005 to 2009,virus isolation and extended culture were carried out by PBMCs co-culture from healthy blood donors,and the clinical isolates of HIV-1 which could be stably subcultured were obtained.The a pproximate full-length genomic sequence of HIV-1 clinical isolates was o btained by overlapping amplification and sequencing of two semi-molecules.The phylogenetic tree was constructed by the Neighbor-joining method in MEGA6.0 software,and the virus genotypes were analyzed.Furthermore,the ML evolutionary tree was constructed by phy ML software,t he phylogenetic relationship of the dominant strains-CRF01＿AE strains was analyzed,and the diversity of CRF01＿AE subtype strains in clinical isolates was dete rmined.The pol and env V3 fragments of clinical isolates were sequenced by the online software ge ne cutter provided in the Los Alamos HIV DATABASES,and the drug resistance mut ation sites and tropism characteristics of clinical isolates were predicted.The tropism of HIV-1 clinical isolates was ver ified by GHOST cells expressing different coreceptors.The HIV replication curve based on antigen detection was used to analyze and compare the replication ability of different PBMCs.Results:(1)A total of 47 virus isolates were obtained,of which 20 strains could replicate with high titer(the content of p24 antigen was higher than that of 10ng/ml).(2)The results of typing showed that the clinical isolates belonged to 26 cases of subtype B(55.3%),18 cases of CRF01＿AE(38.3%),1 case of CRF07＿BC(2.1%),and 2 cases of CRF08＿BC(4.3%).The results of CRF01＿AE further analysis showed that t 16 CRF01＿AE subtype strains from Guangxi belonged to two transmission clusters.(3)Identification of drug resistance mutation sites: 27 of the 47 isolates with full-length pol sequence showed drug-resistant mutations,including 2 strains resistant to protease inhibitors,3 strains resistant to integrase inhi bitors,12 strains resistant to nucleoside reverse transcriptase inhibitors,and 24 strains resistant to non-nucleoside reverse tra nscriptase inhibitors,and 23 of t hem may be moderately and highly resistant.(4)Replication kinetics study: among the 20 strains with p24 antigen content higher than 10ng/ml,11 strains were relatively fast /high type replication strains,and all of them could cause cells to form syncyti al.The replication ability of one subtype B strain was 9.3 times higher than that of reference strain Ⅲ B and 3.4 times that of reference strain p NL4-3.The peak p24 antigen content in the supernatant was usually before or the same number of days before the peak of intracellular DNA copy number.(5)Phenotypic prediction and tropism test: The V3 regions of 47 isolates were predicted by two different online predi ction websites.Among them,31 strains used CXCR4 coreceptor and 1 6 strains used CCR5 coreceptor on Geno2 pheno website,while 27 strains used CXCR4 coreceptor and 20 strains used CCR5 coreceptor on Web PSSM website.The two prediction results of06GXD242,06QSD08 B,06QSD28B,and 07SQD104 were different.The results of the tropism test of 20 high conce ntration strains showed that 14 strains were amphiphilic strains and 6 strains were R5 strains.Co mpared with the predicted results,the CXCR4 strains predicted by the two methods were all CXCR4/CCR5 strains,and the pr edicted CCR5 strains were still CCR5 strains.Conclusion: Forty-seven main epidemic strains of HIV-1 in China with clear epidemiological and biological background were obtained,of which 20 were isolates with high concentration,which had certain biological diversity and representativeness of genotypes.This has laid a certain foundation for the research of pathogenic mechanism and biological co ntrol of HIV-1 in our country.