Preparation And Evaluation Of HIV-1 Pseudovirus Drug Resistance Quality Control

HIV-1 genotype drug resistance has been widely used in HIV drug resistance monitoring and drug resistance detection after treatment in China.To ensure the accuracy and reliability of the test results,a large number of quality control products are needed for indoor quality control and ability verification.At present,drug-resistant quality control products are mainly prepared directly from patient samples,which has potential biological infection risks.At the same time,with the deepening and standardization of anti-virus treatment of AIDS in China,it is increasingly difficult to obtain patient samples with drug-resistant sites.To solve this problem,this study first tested the samples of HIV-infected pregnant women without anti-virus treatment,screened out the samples with drug-resistant mutations,amplified the full-length target gene fragment of POL region,prepared the quality control product of HIV-1 genotype drug-resistant virus-like particles by cloning,and evaluated its uniformity and stability,providing support for the indoor quality control and ability verification of AIDS drug-resistant testing laboratories.The first part is the analysis of gene subtypes and drug resistance of HIV-infected pregnant women without antiviral treatmentResearch purposes:Through the analysis of gene subtypes and drug-resistant mutations of HIV-infected pregnant women without antiviral treatment in AIDS-prone provinces,the samples with drug-resistant mutations were screened out.Research methods:From June 2016 to June 2019,blood samples of 229 HIV-infected pregnant women who did not receive antiviral treatment were collected,and HIV-1 RNA was extracted.The protease region(PR)and reverse transcriptase region(RT)of HIV were amplified by nested polymerase chain reaction and sequenced.The evolutionary tree was constructed to genotype HIV,and the new recombinant strains explored the recombination mode with simplot software.Use the online tool of Stanford University HIV drug resistance database to analyze drug resistance.Research results:197 of 229 HIV-infected pregnant women were successfully amplified,and the gene subtypes were CRF07_BC 87 cases(44.16%),CRF08_BC 56 cases(28.43%),CRF01_AE 44 cases(22.34%),9 cases(4.57%)and CRF 85.16 cases developed drug resistance,including 13 cases of NNRTIs,3 cases of NRTIs,1 case of PIs,and 1 case of NRTIs and NNRTIs.The second part is the construction of pseudovirus containing HIV-1 genotype drug-resistant virus-like particlesResearch purposes:To construct a pseudovirus containing the full-length drug resistance gene of HIV POL region,and prepare for the subsequent preparation of genotypic drug resistance quality control products.Research methods:A double drug resistance sample(No.CDC3)containing the most drug resistance mutation sites was selected from the first part of drug resistance samples.The gene subtype is CRF07 BC,and the related drug resistance mutation sites are D67DN(NRTIs),K103KNRS,V106VIM(NRTIs),V35R,T39D,S48T,V60I,D121Y,K122E,W153W*,E169D,R211K,V245Q and e248n The resistant drugs were AZT,D4T(NRTIs),RPV,ETR,EFV,NVP,DOR(NNRTIs).RNA was extracted,and the full-length gene of POL region was amplified by nested PCR,followed by sequencing and drug resistance analysis.The recombinant plasmids were packaged by three different cloning techniques,namely recombinant lentivirus plasmid expressing reporter gene,plasmid expressing HIV Gag protein and helper plasmid expressing HIV Rev gene,and then digested and sequenced.293T cells were co-transfected with the above three plasmids,pVSVG envelope plasmid retained in laboratory and POL full-length target gene fragment amplified from drug-resistant samples,and their titers were detected after collecting pseudoviruses.Research results:The sequencing results obtained after PCR amplification of drug-resistant samples,and the drug-resistant results obtained after submitting to Stanford drug-resistant database are consistent with the results of the first part.The results of digestion and sequencing of the three recombinant plasmids and the plasmids expressing envelope protein showed that the plasmid structure was correct.After co-transfection of four plasmids and POL target gene fragment,there were fluorescence signals,which indicated that pseudovirus was successfully constructed,and the titer was 8.38E+8TU/ml.The third part is the evaluation of uniformity and stability of HIV-1 genotype drug resistance quality control productsResearch purposes:Preparation of HIV-1 genotypic drug resistance quality control by pseudo virus and evaluation of its homogeneity and stability.Research methods:The pseudovirus prepared in the second part was added with negative plasma to prepare high,medium and low concentration quality control products.The quality control products were detected by fluorescence quantitative PCR at different storage time,different temperature and after repeated freezing and thawing,and the ct values were statistically analyzed to evaluate the uniformity and stability of the quality control products.Research results:The statistical results of uniformity Ct showed that there was no significant difference(P>0.05),that is,the uniformity was good.Stability results showed that the quality control products could be stored at 37? for 1 day,room temperature for 3 days,4? for 7 days,and-20? and-80? for 14 days.The statistical results of five repeated freeze-thaw experiments showed no significant difference(P>0.05).Research conclusion:Sixteen samples with drug resistance sites and subtype background information were obtained,and one HIV-1 pseudovirus with full-length drug resistance gene fragment was successfully constructed.The quality control product prepared with pseudovirus has good uniformity and stability.