Objective To study the prevalence of HIV-1 drug-resistant strains in antiretroviral therapy-naive HIV-1 infectors,and provide background information for HIV-1 drug resistance survey and clin-ical antiretroviral therapy in Beijing in 2008. Methods Referring to the guidelines for HIV drug resistance threshold survey(HIVDR-TS) of WHO, collecting 60-70 plasma samples of HIV-1 infectors who were detec-ted in 6 months and not more than 25 years,we detected HIV-1 pol genotype and genetic mutations associated with drug resistance,counted the prevalence of drug-resistant strains, and evaluated the prevalent level. Re-Sults Of 61 plasma samples answering for the standards, 50 were successfully sequenced and genotyped pol sequence. The major infection route was homosex, which accounted for 62%. B, CRF01_AE, and CRF07_ BC were major genetic subtype, which accounted for 42%, 28% and 26%, respectively. One Pl-resistant strain was found, the incidence of which was 2% (1/50). One NRTI-resistant strain was found, the inci-dence of which was 2% (1/50). No NRTI-resistant strain was found, the incidence of which was 0. The in-cidence of drug-resistant strains in the protease (PR) region was 2%, and the incidence of reverse tran-scriptase (RT) region was also 2%. Both of the prevalence were classified as low level ( <5% ). Conclu-sion PR, RT-resistant HIV-1 strains were found in drug-naive infectors, and the prevalence was low in Beijing. Current antiretrovirai therapy regiments were still feasible. Most of the AIDS patients did not need to test drug resistance before antiretroviral therapy.

Objective To predict the CTL epitopes of Tat exon 1 region in HIV-1 CRF07_BC strains, which were prevailing in China. Methods Total of 236 plasma samples were from the 3rd National HIV Molecular Epidemic Survey (NMES3). All the subjects were infected with HIV-1 CRF07_BC viruses. The tat exon 1 region was amplified by reverse transcription reaction and nested polymerase chain reaction (nested-PCR), then the PCR products were sequenced. The distribution of CTL epitopes of this region were predicted by on-line software BIMAS HLA Peptide Binding Predictions and statistics software. Results To-tal of 236 CRF07_BC strains were from 16 provinces, mainly in intravenous drug asers(58.9%)and then sex(25.0%). It was showed that there were 12 CTL epitopes of 236 Tat exon 1 region of CRF07_BC strains mainly located in proline-rich region, cysteine-rich region and core-region. Those epitopes were banded by 5 HLA presenting molecules in genotype(A * 2501 ,A * 2902, B * 15,B * 5301 and Cw * 1203) and 6 HLA presenting molecules in serotype (B53, B58 ,B57 ,A3 ,A68 and Cw12). The frequency of single amino acid substitution was more than 50% in 7 CTL epitopes. Conclusion The CTL epitopes in Tat exon 1 of CRF07 _BC strains were located in different functional regions, and there were some amino acid variations in them.

Objective: To explore drug resistance of different viral loads, and investigate the relationship between drug resistance and CD4⁺T cell counts in patients with HIV antiretroviral therapy (ART) in China from 2003 to 2015. Methods: Data were extracted from the Chinese National HIVDR Surveillance database from 2003 to 2015. For this study, the data collected were as follows: having received ART for ≥12 months; 18 years or older; demographic characteristics, information of ART, CD4⁺T cell counts, viral load (VL) and HIV drug resistance of a total of 8 362 patients were collected. Multi-variables non-conditional logistic regression model was used to study the relationship between viral load, HIV drug resistance and CD4⁺T cell counts. Results: Participants with age of (41.8±10.5) years were enrolled in this study. Among them, 59.9% (5 009 cases) were men. The percentage of CD4⁺T cell counts <200 cells/μl in the total population was 17.9% (1 496 cases), the highest was in VL ≥1 000 copies/ml with drug resistance, which was 43.0% (397/923) , followed by VL 50-999 copies/ml with drug resistance, which was 31.1% (69/222), and the lowest was in VL 50-999 copies/ml without drug resistance 13.2% (273/2 068). Compared to VL 50-999 copies/ml without drug resistance, VL<50 copies/ml, VL 50-999 with drug resistance, VL≥1 000 copies/ml without drug resistance, and VL ≥1 000 copies/ml with drug resistance, the OR (95%CI) of CD4 <200 cells/μl were 0.9 (0.7-1.0), 3.2 (2.3-4.4), 2.6 (2.1-3.2), and 4.9 (4.0-5.9), respectively. Among 222 patients with VL 50-999 and HIVDR, the most frequent antiretroviral drugs were EFV and NVP, both of which were NNRTI, and whose percentage both were 94.1% (209 cases). The most frequent mutations were M184V/I (NNRTI), and the percentage was 26.1% (58 cases). The second one was K103N (NNRTI), and the percentage was 22.5% (50 cases). The percentage of V32L/E (PI) and V82A (PI) were lower, they were 0.9% (2 cases) and 0.5% (1 case) respectively. Conclusion Decreased CD4⁺T cell counts were associated with HIV drug resistance at low viraemia. In the case of low viral load, the most vulnerable were the NNRTI antiviral drugs such as EFV and NVP.

Objective:To understand influencing factors on the deaths of HIV/AIDS patients receiving antireviral treatment in Butuo county of Liangshan Yi Autonomous Prefecture (Liangshan) from 2010 to 2019, to provide data for drug replacement and sustainable antiviral treatment strategy.Methods:A matched case-control study was used to collect basic and follow-up information on AIDS death patients receiving antiviral treatment in Butuo county of Liangshan from 2010 to 2019. The control group was formed by sampling twice the number of cases. The logistic regression model was used to analyze the risk factors affecting mortality.Results:In 3 355 patients of HIV/AIDS treated with antiviral therapy, 1 179 cases in the death group and 2 176 cases in the control group. Including 81.34% were 30-49 years old, 69.09％males, 99.55% Yi nationality, 91.12% were married or cohabitated, 95.77% had junior high school education or below, and 88.41% peasants. Amultivariate logistic stepwise regression model showed that among the death risk factors, age ≥50 years old was 5.08 times (95% CI:3.05-8.48) that of the 18-29, female was 0.70 times (95% CI: 0.52-0.94) than male, the transmission rate of intravenous drug use was 1.43 times (95% CI: 1.06-1.91) that of heterosexual transmission, CD4 +T lymphocyte (CD4) count ≥350 cells/μl before treatment was 0.38 times (95% CI: 0.30-0.48) that of CD4 <200 cells/μl before treatment, the most recent antiviral treatment regimen containing LPV/r was 0.04 times (95% CI: 0.01-0.18) than that of stavudine (d4T) + lamivudine (3TC) + nevirapine (NVP)/efavirenz (EFV) regimen, drug resistance was 3.40 times (95% CI: 2.13-5.42) of non-drug resistance, non-viral load and non-drug resistance test results were 12.98 times (95% CI: 10.28-16.40) of non-drug resistance. Conclusions:Age, gender, transmission route, CD4 before treatment, the latest antiviral treatment program, and drug resistance test after antiviral therapy were the influencing factors of HIV/AIDS death in Butuo county. It is necessary to expand the coverage of viral load and drug resistance test to change the antiviral therapeutic schedule scientifically and carry out publicity and education on the compliance of patients with antiviral treatment and medical staff training in order to reduce the mortality of patients with antiviral treatment.

Objective:To investigate the prevalence of pretreatment drug resistance and the genetic polymorphism of CRF08_BC strains among HIV-1 patients in China.Methods:This cross-sectional survey involved the plasma samples of HIV patients in a national pretreatment HIV drug resistance survey conducted in 2018. RNA was extracted from the samples. The fragments containing protease and partial reverse transcriptase (PR/RT) regions were obtained and sequenced. Drug resistance was analyzed using Stanford HIVdb Program. Differences in polymorphic mutations between drug-resistant and non-drug-resistant HIV-1 strains were analyzed by Chi-square test or Fisher′s exact test. The association between drug-resistant and polymorphic mutations was evaluated using CorMut R package. Molecular transmission networks were constructed using HIV-TRACE software. Results:Totally 465 partial pol sequences were obtained from individuals with CRF08_BC infection in 25 provinces and cities. The total pretreatment drug resistance rate was 17.8% (83/465). The pretreatment drug resistance rates to non-nucleoside reverse transcriptase inhibitors (NNRTIs), nucleoside reverse transcriptase inhibitors (NRTIs) and protease inhibitors (PIs) were 16.6% (77/465), 1.1% (5/465) and 0.9% (4/465), respectively. The resistance rate to rilpivirine (RPV) was the highest (15.7%, 73/465). The most common mutation was E138A (11.6%, 54/465). There were six polymorphic mutations (S162C, K102Q, T200A, V179E, I202V, T200M) that co-variated with E138A. The molecular transmission network showed that patients infected with CRF08_BC strains carrying the resistant mutations at position E138 mainly gathered in clusters in Yunnan and Sichuan, and the highest degree of connection was in Lincang, Yunnan. Conclusions:In China, HIV-1 CRF08_BC-infected patients showed a high rate of pretreatment resistance to one of the second-generation NNRTIs, namely RPV. Further researches were warranted to evaluate the impacts of co-mutations of the E138A mutation and polymorphic sites on HIV resistance and replicative capacity.

Objective:To explore the resistance to protease inhibitors (PI), nucleoside reverse transcriptase inhibitors (NRTIs), non-nucleoside reverse transcriptase inhibitors (NNRTIs) and integrase inhibitors (INSTIs) of HIV-1 CRF55_01B and the transmission of drug-resistant strains among HIV-1 CRF55_01B infected patients before antiviral treatment in China.Methods:HIV-1 RNA was extracted from plasma samples of the patients infected with CRF55_01B in the national surveillance of HIV drug resistance before antiviral treatment in 2018. A 1 056 bp gene fragment of protease/reverse transcriptase (PR/RT) region and an 846 bp gene fragment of integrase (IN) region were obtained and sequenced. Drug resistance was analyzed by using all drugs included in the Stanford University HIV db Program, HIV-1 molecular network analysis was performed with software HIV-TRACE and polymorphism mutations of CRF55_01B integrase gene region were analyzed.Results:A total of 178 samples from 26 provinces, municipalities and autonomous regions in China were analyzed, and 170 sequences of CRF55_01B PR/RT region and 170 sequences of IN region of corresponding samples were obtained. The drug resistance rate was 15.3% (26/170). The drug resistance rates of PIs, NRTIs, NNRTIs and INSTIs were 1.2% (2/170), 1.2% (2/170), 15.3% (26/170), 0.6% (1/170), respectively. The level of drug resistance was mostly low. NNRTIs drug resistance mutations were mainly V179D/E co-appeared with other mutations, and 84.1% (143/170) of the infected patients carrying V179D/E alone showed potential drug resistance. INSTIs drug resistance mutation was G163R, and showed low resistance to EVG and RAL. The molecular network access rate was 30.0%(51/170)according to the 0.9% gene distance threshold. The resistant strains were transmitted between men with homosexual transmission and heterosexually transmitted people, and both carried resistant mutations E138G and V179E. In the integrase region, CRF55_01B and CRF01_AE and B subtypes showed high mutation frequency difference in 5 sites (T215A、G134N、I135V, K136R and L101I/V).Conclusions:Before antiviral treatment, CRF55_01B infected patients in China had a high resistance to NNRTIs. Strains carrying both E138G and V179E resistance mutations were transmitting in clusters. The prevalence of CRF55_01B integrase inhibitor resistant strains is low, but some genetic polymorphisms with high mutation rate in the integrase gene region have potential influence on drug sensitivity. The influence of drug resistance of new recombinant strains on antiviral therapy in China needs to be further monitored and analyzed.

Objective:To establishment an assay for HIV-1 intact proviral DNA assay through droplet digital PCR (ddPCR).Methods:DNA was extracted by culturing 8E5 cells, a Tlymphocyte cell line containing a single copy of integrated HIV-1 provirus. Serial diluting DNA were prepared by amplified 1-fold, 5-fold, 25-fold, 625-fold, 3 125-fold, and 15 625-fold across the HIV-1 Ψ region, env region, and eukaryotic chromosome 10 RPP30 regions, and the linear relationship was calculated and the minimum detection concentration. DNA solution of 5 μl, 3.1 μl, 2.5 μl was added to the ddPCR mixture respectively, with each dilution undergoing two batches of detection, and each was repeated four times. The intra-batch variation coefficient was detected, while the inter-batch variation coefficient was detected by the same DNA amount and different DNA amounts to determine the stability; 8E5 cell was used to detect the intact proviral content in cells.Results:The linear fitting goodness of Ψ region, env region and RPP30 region are R2≥0.999, R2≥0.993, R2≥0.996 in 6 dilutions of DNA, respectively. At a 3 125-fold dilution, the lowest positive droplets were detected in the Ψ region, env region and RPP30 region were 3, 2 and 2, respectively, the detected concentrations were 2.37 copies/μl, 1.21 copies/μl and 1.58 copies/μl. The ddPCR repeatability experimental detecting DNA showed that the Ψ region of the intra-batch variation coefficients ranged from 0.66% to 3.43%, with the inter-batch variation coefficients of the same DNA at 3.19%, 4.3% and 3.45% respectively, and the inter-batch variation coefficients of the different DNA at only 4.35%. The env region of the intra-batch variation coefficients ranged from 0.7% to 3.20%, with the inter-batch variation coefficients of the same DNA at 3.18%, 4.52% and 3.4% respectively, and the inter-batch variation coefficients of the different DNA at only 4.02%. The RPP30 region of the intra-batch variation coefficients ranged from 0.91% to 2.91%, with the inter-batch variation coefficients of the same DNA at 3%, 4.55% and 3.37% respectively, and the inter-batch variation coefficients of the different DNA at only 3.98%. The proportion of 8E5 cells containing defective provirus and the proportion of intact provirus were calculated to be approximately 90% and 45%, respectively. Conclusions:Droplet digital PCR used to detect HIV-1 intact proviral DNA, showed strong stability and provided a technical means for HIV-1 infection reservoir detection.

OBJECTIVETo study resistance evolution pathway of HIV-1 CRF_BC under drug selection pressure, and compare with B subtype.

METHODSBased on the reverse transcriptase region of CRF_ 97BC HIV-1 from 588 treatment-naive and 274 treatment patients, selection pressure based method was used to select resistance-associated mutations, and Bayesian network was used to construct the resistance evolutionary pathway under antiretroviral therapy. Meanwhile, it was constructed that the resistance evolutionary pathway for B subtype with the same regimens using the data from HIV resistance database, and made a comparison with CRF_07BC.

RESULTSThe major resistance mutations for CRF_07BC were identified including K103N, Q197K, V179D and Y188L. While for B subtype, the major resistance mutations include M184V, K103N,Y181C, T69N,G190A, K238T,Y188H and P225H. Much difference was observed between these two classes. However, the classical TMA1 (41L, 210W and 215Y) and TMA2 (67N, 70R and 219E/Q) pathways exist in both pathways. As different from B subtype, the predicted major drug resistance mutations for CRF_07BC did not contain TAM-related mutations, and nucleoside reverse transcriptase inhibitor-related mutations and non-nucleoside reverse transcriptase inhibitor-related mutations were mutually depending on each other.

CONCLUSIONHIV-1 CRF_07BC showed distinctive resistance evolutionary pathway, the mutations K103N,Q197K,V179D and Y188L were the major resistance mutations, and different resistance evolutionary pathways were observed between HIV-1 CRF_07BC and B subtype.

Anti-HIV Agents ; pharmacology ; Bayes Theorem ; Drug Resistance, Viral ; genetics ; Evolution, Molecular ; HIV-1 ; drug effects ; enzymology ; genetics ; Humans ; Mutation ; RNA-Directed DNA Polymerase ; genetics

Objective: To analyze the change trend of HIV genetic subtypes and compare the first CD₄⁺T cell counts of newly diagnosed HIV infected patients in Liuzhou from 1998 to 2012, and provide a reference for AIDS prevention and control. Methods: Newly diagnosed HIV-infected patients from 1998 to 2012 in Liuzhou were selected through national HIV/ADIS comprehensive response information management system. Their plasma samples were used for RNA gene extraction, amplification, sequencing and genotyping. Coharan-Armitage trend test was used to analyze the ratio trend of genetic subtypes and phylogenetic clusters of HIV and Wilcoxon Rank Sum Test was used to compare the first CD₄⁺T cell counts (CD₄) of the different subtype HIV infected patients. Results: A total of 1 877 newly diagnosed HIV infected patients were included in the study. From 1998 to 2012, the proportions of CRF01_AE and CRF01_AE (Cluster 1) increased from 78.4% (76/97) to 91.5% (1 441/1 574), from 63.9% (62/97) to 74.0% (1 164/1 574), and the proportion of CRF07_BC decreased from 17.5% (17/97) to 4.6% (72/1 574), respectively (Z=4.632, P<0.001; Z=2.455, P=0.014; Z=-5.943, P<0.001). The median and interquartile range of the first CD₄ of the patients infected with subtype CRF01_AE (Cluster 1), CRF01_AE (Cluster 2), CRF07_BC and CRF08_BC were 230 (83-375), 215 (48-351), 365 (254-503) and 334 (206-479) cell/μl, respectively. The first CD₄ levels of the patients infected with subtype CRF01_AE (Cluster 1) or CRF01_AE (Cluster 2) were significantly lower than those of CRF07_BC (Z=-4.795, P<0.001; Z=-4.238, P<0.001). Conclusion The genetic subtypes of HIV were mainly CRF01_AE in newly diagnosed HIV-infected patients and this subtype proportion was in increase and the first CD₄ levels of the patients were low in Liuzhou during 1998 to 2012.