The Cross-Sectional Study On Relationship Between Opioid Use And HIV-1 Primary Drug-Resistance And Construction Of A Pseudovirus System For Phenotypic Drug-Resistance Detection

Section one: The cross-sectional study on relationship between opioid use and HIV-1 primary drug-resistanceObjective To investigate primary drug-resistance epidemic situation among untreated HIV-1-infected persons and compare difference of HIV-1 drug-resistance between opioid-abused subjects and non-opioid-abused subjects;to explore the relationship between opioid abuse and HIV-1 primary drug-resistance.Methods All untreated HIV-1-infected subjects met inclusion criteria were collected by convenience sampling during January 2015 to December 2015 from a illicit drug-abuse cohort, which was established at red cross hospital of Nanning city, centers for disease control and prevention(CDCs) of Liuzhou city and Lingshan county. The recruited subjects were divided into two groups,opioid-abuse HIV-infected group(Opioid H1V+) and non-Opioid-abuse HIV-infected group(non-Opioid HIV+), accroding to their drug-abuse status.Demographic characteristics of all subjects were collected. Peripheral blood smaple (5 mL) was collected from each subject. HIV-1 RNA was extracted from blood sample and pol gene was then amplified via RT-PCR. Sequences of pol gene were detected by Sanger sequencing. The drug-resistance information about NNRTIs, NRTIs, PIs was obtained by comparing diversity between HIV database of Stanford University and the sequences we obtained. The comparisons demographic characteristics,drug-resistance mutations, prevalence of primary drug-resistance between two groups were conducted by Chi-square test; the correlation analysis of duration, dose of methadone treatment and HIV-1 drug resistance were conducted by spearman's rank correlation.Results(1)Demographic characteristics of subjectsA total of 243 subjects were enrolled in the investigation, including 134 in Opioid HIV+ group, 109 in non-Opioid HIV+ group. Of all subjects, 146 were male, mainly in Han nationality, the average age was 45.5±11.9. There was no statistically significant difference in composition of gender, nationality, age between two groups(x2=0. 430, P=0.512; x2=1.566, P=0. 211; x2=0.134,P=0.714).(2)The genotypic drug-resistance detection and comparison between two groupsA total of 184 samples were successfully amplified by RT-PCR,containing 101 in Opioid HIV+ group, 83 in non-Opioid HIV+. Of them, 26 were identified as genotypic drug-resistance, primary drug-resistance rate of total subjects is 14.4%(26/184).In Opioid HIV+ group, 19 were identified as genotypic drug-resistance,primary drug-resistance rate is 18.8%(19/101). Of 101 subjects, prevalence of NNRTIs, PIs, NRTIs drug-resistance mutation were 12.9%, 5.9%, 2.0%,respectively, including mutation E138A(5.0%)?K103N(4.0%)?V179D(3.0%)?G190A (1.0%)?V106M (1.0%)?L100I (1.0%) in NNRTIs,V82F (4.0%)?M461 (3.0%) in PIs, T215I/S (2.0%) in NRTIs. Rates of double drug-resistance and double site drug-resistance were 3.0% and 4.0%, respectively.Drug-resistances in 3 antiretroviral drugs were mainly intermediate and low,high drug-resistance only appeared in EFV and NVP of NNRTIs.In non-Opioid HIV+ group, 7 were identified as genotypic drug-resistance,primary drug-resistance rate is 18.8%(19/101). Of 83 subjects, prevalence of NNRTIs, PIs drug-resistance mutation were 4.8%, 3.6%, respectively, including mutation E138A (3.6%). K103N (1.2%) in NNRTIs, V82F (3.6%) in PIs. No double drug-resistance, double site drug-resistance and NRTIs related drug-resistance mutation was found. Drug-resistances in 2 antiretroviral drugs were mainly intermediate and low, high drug-resistance only appeared in EFV and NVP of NNRTIs.Between Opioid HIV+ and Non-Opioid HIV+ groups, primary drug-resistance rate in Opioid HIV+ group was significantly higher than that in non-Opioid HIV+ group(x2=4.044, P=0.044). There was no statistical significant difference in prevalence of PIs and NNRTIs drug-resistance(P>0.05),composition of PIs drug-resistance mutation V82F and NNRTIs drug-resistance mutations E138A, K103N between two groups(P>0.05). There were no correlation between duration, dose of MMT and HIV-1 drug resistance.Conclusion Among untreated HIV-1-infected persons in Guangxi,primary drug-resistance rate in Opioid-abused persons was higher than that in non-Opioid-abused persons.Relative higher rates of drug-resistance in 3 antiretroviral drugs were also observed. The above data suggest that opioid abuse maybe associated with HIV-1 drug-resistance.Section two: Construction of a pseudovirus system for phenotypic drug-resistance detectionObjective To establish a pseudovirus system for phenotypic drug-resistance detection and provide a relatively cheap and easy method for drug-resistance testing.Methods EGFP gene was amplified from plasmid pSV-EGFP and then cloned to backbone plasmid pNL4-3.Luc.E-R- by double enzyme digestion;ENV gene was amplified from HIV-1 RNA isolated from HIV-1-infected persons and cloned to eukaryotic expression plasmid pcDNATM3.1 (+) by double enzyme digestion. The recombinant plasmids were verified by transfection to 293t cells and EGFP or ENV expression. Pseudovirus was produced by co-transfection of two recombinant plasmids to 293t cells.Infection of pseudovirus was determined by co-cultured with TZM-b1 cells and immunofluorescent test.Results(1) Construction of pNL4-3.EGFP.E-R- plasmid. The plasmid was verified by double enzyme digestion and immunofluorescent detection of EGFP expression.Exogenous EGFP gene was cloned to backbone plasmid pNL4-3.Luc.E-R- and the recombinant plasmid expressed immunofluorescent signal in 293T cells, proving that the plasmid was successfully constructed.(2) Construction of pcDNA3.1-env plasmid. ENV gene copies were amplified from 8 HIV-1 positive plasma samples and cloned to pcDNATM3.1(+)respectively. The plasmids were verified by double enzyme digestion and Western blot detection of ENV expression. Ten positive clones were obtained and could be used to construct pseudovirus.(3) Two recombinant plasmids(mass ratio, pcDNA3.1-env:pNL4-3.EGFP.E-R-.=2:1 ) were co-transfected to 293T cells. Cultured supernatants containing pseudovirus were harvested at 48h post-transfection.Fluorescence was observed in TZM-b1 cells after TZM-b1 cells were infected with pseudovirus at 48h post-infection.Conclusion The recombinant pseudovirus carrying EGFP gene was constructed successfully and it could be used for phenotypic drug-resistance detection.