| Objective:Acquired Immune Deficiency Syndrome（AIDS）,referred to as AIDS,is a serious infectious disease caused by the Human Immunodeficiency Virus（HIV）.It mainly invades human CD4+T lymphocytes and makes the human body gradually loss of immune function,and eventually death due to various opportunistic infections and malignancies,and there is no cure at present.HIV has a high degree of variability,according to serological reaction and gene sequence differences can be divided into two types:HIV-1 and HIV-2,in which HIV-l is divided into four groups:M,O,N and P.In addition,there are nearly 100 kinds of circulating recombinant forms（CRFs）and unique recombinant forms（URFs）.Highly active antiretroviral therapy（HAART）is one of the most important breakthroughs in the field of AIDS.Although most patients can achieve completely virological suppression through HAART,some patients still have virological failure.Drug resistance is one of the main causes of virological failure.In recent years,with the continuous promotion and updating of global antiviral drug use and the popularization of drug resistance monitoring,the use of antiviral drugs has been limited by the discovery of drug resistance and the increasing trend of drug resistance in some areas,especially in low-and middle-income countries（LMICs）.At the same time,there is a long way to go to achieve the goal of 90-90-90 set by the Joint United Nations Programme on HIV/AIDS and the World Health Organization by 2020.Therefore,a better understanding of the mechanism of HIV drug resistance is of great significance to improve the efficacy of antiviral therapy and the development of better antiviral drugs.The most common method of HIV drug resistance testing is genotypic drug resistance testing.At present,the interpretation standard for the genotypic drug resistance of different subtypes and recombinant strains of HIV is mainly based on the research data of subtype B strains of HIV-1.However,many studies have shown that the difference of drug target genes among subtypes may affect the pathway of drug resistance mutation（DRM）.At the same time,the difference of gene polymorphisms among subtypes can also affect the occurrence and development of drug resistance in the host,and finally make different subtypes of strains show specific drug resistance characteristics.These studies suggest that the drug resistance criteria summarized according to subtype B strains may not be completely suitable for HIV-1 non-B strains.CRF01＿AE is one of the earliest identified and most widely affected recombinant HIV-1 strains in the world.CRF01＿AE is also one of the main epidemic strains of HIV-1 in China.However,at present,there is a lack of systematic research on the specific drug resistance-related mutations,the effects of polymorphism on treatment outcomes and the association between mutations in CRF01＿AE strains.In this study,we analyzed the natural polymorphisms of the pol gene of CRF01＿AE HIV-1 strains in a cohort of patients with a large antiretroviral therapy in Liaoning Province,and compared the natural polymorphisms between patients with treatment failure（TF）and treatment success（TS）.Through the comparison of the baseline of treatment and the mutations of pol gene at the TF time point of patients with CRF01＿AE infection who failed first-line treatment,the characteristics of acquired drug resistance mutations of CRF01＿AE strains were confirmed.In addition,we further explored the role of potential new DRMs by co-mutation analysis and next generation sequencing（NGS）.The purpose of this study is to further improve the interpretation of drug resistance genotypes of HIV-1 CRF01＿AE strains,and to provide a scientific basis for improving the level of drug resistance monitoring and treatment management of patients with antiviral therapy in China.Methods:1.Study subjectsFrom January 2002 to December 2017,2034 HIV-1 infection patients with baseline drug resistance genotypes were selected from the cohort of CRF01＿AE long-term antiretroviral therapy in Liaoning Province for pol-RT gene polymorphism analysis,of which 1330 patients were treated with first-line HAART,that is,two Nucleoside Analogue Reverse Transcriptase Inhibitors（NRTIs）plus one Non-Nucleoside Analogue Reverse Transcriptase Inhibitors（NNRTIs）.The effect of natural polymorphisms on treatment outcome was analyzed by comparing the pol-RT sequences of 105 TF patients and the pol-RT sequences of 1148 TS patients.42 TF patients who received first-line antiviral therapy of Tenofovir（TDF）/Lamivudine（3TC）/Efavirenz（EFV）were selected to carry out comparative analysis and co-mutation analysis of pol-RT mutations before and after self-sequences.Finally,4 patients with both Y181C and L228R at TF time point were selected for temporal analysis.2.Sequence quality assessment,phylogenetic and genotypic resistance analysesHIV-1 pol sequences（covering protease region 1-99 amino acids and reverse transcriptase region 1-240）were obtained by monitoring genotypic resistance in 2034patients with CRF01＿AE infection.Amino acids are automatically compared with the reference sequences downloaded from the Los Alamos HIV database（https://www.hiv.lanl.gov/）by ClustalW of Mega v7.0 and then manually corrected.The Fast Tree v2.1.9 software is used to construct the phylogenetic tree by the maximum likelihood method.The maximum likelihood tree was constructed from the pol sequences of 42 TF patients at baseline and TF time point by Mega v7.0.The US Stanford University HIV Drug Resistance Database（https://hivdb.stanford.edu/）was used to identify DRMs,and use the HIVdb algorithm（version 8.8）（https://hivdb.stanford.edu/hivdb/by-mutations/）to explain DRMs.3.Polymorphism analysisIf the amino acid of a certain point in a subtype is different from that of the standard strain HXB2 of subtype B,and the proportion is more than 1%,then this site is defined as the natural polymorphic site of this subtype.The mutation frequencies of the 240amino acid sites in the coding region of reverse transcriptase（RT）gene in the baseline HIV-1 pol sequence of CRF01＿AE patients were compared with the corresponding amino acid sites of subtype B strains without antiviral treatment in the drug resistance database of Stanford University.At the same time,the mutation frequencies of 240amino acid sites in the RT region of the baseline HIV-1 pol sequence in 105 TF patients and 1148 TS patients were compared.4.Co-mutation analysisThe baseline and TF point sequences of 42 patients with first-line treatment failure were analyzed by using CorMut software package（Version 1.25.0）based on the R language（Version 3.5.2）.The standard strain HXB2 of HIV-1 subtype B was used as the reference sequence.The amino acid sites at position 1-240 in the coding region of HIV-1 pol-RT gene were selected as the analytical range.The positive selection mutation was determined based on the selection pressure（Ka/Ks）,in which the Ka/Ks value greater than 1 was the positive selection,and the logarithmic ratio（LOD）was higher than 2,which was considered the positive selection was significant.Conditional selection pressure（cKa/Ks）was used to measure the correlation between positive selective mutations,where cKa/Ks>1 and LOD>2 indicated that there was a co-variation relationship.5.Temporal analysis of Y181C/L228R mutations by deep sequencingViral RNA was extracted from plasma according to the operation instructions of QIAamp Viral RNA Mini Kit.In order to observe the relationship between the occurrence and development of amino acid mutations at 181 and 228 sites in HIV-1pol-RT gene,the coding region of amino acid 105-256 of RT gene was amplified.After the PCR products were purified and quantified,the deep sequencing library was constructed,and the library was standardized and detected by computer.On the Windows operating system,using Oracle VM Virtual Box-5.2.22 software package to build a virtual environment,running QIIME2 Core-2018.4（1525276946）to analyze the data package generated by deep sequencing,reporting the sequences and number of HIV-1 quasispecies in each sample,and counting the mutation rates of amino acids at position 181 and 228.6.Statistical analysisThe mutation rates of HIV-1 pol-RT gene involved in this study were statistically analyzed by binomial distribution.Wilcoxon and McNemar tests were used to compare the mutations in the sequences of 42 first-line TF patients before and after autologous treatment.Other statistical calculations were carried out by using SPSS 20.0 software.P<0.05 indicates that the difference is statistically significant.Results:1.Characteristics of transmitted drug resistance and gene natural polymorphism of CRF01＿AE strains1.1 Transmitted drug resistance rate of CRF01＿AE strains Of the 2034 patients with CRF01＿AE infection,40 cases had transmitted drug resistance mutations at the baseline of treatment,with a transmitted resistance rate of2.0%.Common transmitted DRMs included K103N,G190S,K101E,T215S,K65R and K219Q.1.2 Natural polymorphisms of amino acids encoded by pol-RT gene of CRF01＿AE strainsFifty-three sites（53/240,22.08%）found in the RT region（1-240 amino acids）were polymorphisms,of which 9 sites（40,68,69,98,103,118,179,210 and 238）were known drug resistance-related sites.Then,these sequences were compared with the natural polymorphism in the RT region of the untreated subtype B strains in Stanford HIV drug resistance database（https://hivdb.stanford.edu/cgi-bin/RTMutSummary.cgi,accessed in 04/08/2019）by the binomial distribution,and found that the mutation rate of thirty-one sites（4,5,6,8,11,28,32,35,36,39,40,43,88,103,104,105,111,118,123,135,172,173,174,177,179,200,203,207,211,214,238）of CRF01＿AE strains was significantly higher than that of subtype B strains（|Z value|≥3）,and were the natural polymorphic sites of the subtype-specific amino acid of CRF01＿AE strains.Five polymorphic sites（40,103,118,179,238）were drug resistance-related mutation sites listed in HIV genotype drug resistance detection and evaluation system of the Stanford University in the United States,and were the subtype-specific drug resistance-related polymorphism sites of CRF01＿AE strains.1.3 Characteristics of natural polymorphisms of two CRF01＿AE lineagesThrough the comparison and evolutionary analysis of 2034 pol-RT gene sequences of CRF01＿AE,it was found that these sequences mainly belonged to two lineages of CRF01＿AE strains.Through binomial distribution analysis of natural polymorphism differences between lineages,it was found that there were differences in natural polymorphism sites and drug resistance-related mutation sites between them.2.Effect of natural polymorphism of CRF01＿AE strain on treatment outcomeAmong the 2034 CRF01＿AE infected patients,a total of 1330 patients received first-line ART,including TF in 105 cases（7.9%）and TS in 1148 cases（86.3%）.Through binomial distribution comparison,it was found that there were significant differences in a total of 13 sites between TF and TS patients（|Z value|≥3）,including polymorphic site 75 and 189 found only in TF patients,and 4 polymorphic site 4,5,8,21,32,49,105,165,169,171 and 204 found only in TS patients.The mutation rate of site 75 in TF patients was significantly higher than that in TS patients（|Z value|≥3）.3.Characteristics of common acquired DRMs and potential new DRMs in CRF01＿AE infected patients failed TDF/3TC/EFV first-line treatmentPhylogenetic analysis showed that in 42 CRF01＿AE patients who failed the first-line TDF/3TC/EFV treatment,the baseline and TF time points of each patient were clustered in the phylogenetic tree.Wilcoxon test was used to compare the DRMs of RT sequence at baseline and TF time point in 42 patients.It was found that the number of DRMs at TF was significantly higher than that at baseline（Z=-5.604,p<0.001）.By binomial distribution and McNemar test,the mutation frequencies of amino acid sites in RT sequence at baseline and TF time points were compared,and found that the mutation rates of 14 sites increased significantly in TF of which 13 sites were known drug resistance-related sites,including 7 NRTI-related sites and 6 NNRTI-related sites.The NRTI-related DRMs detected in TF were K65R（57.1%）,M184V/I（47.6%）,S68G（26.2%）,A62V（14.3%）,K70/E/R（9.5%）and Y115F（9.5%）.NNRTI-related DRMs detected in TF included G190S/C（66.7%）,K101E/N/Q（52.4%）,V179D/I/A/T/E（45.2%）,Y181C（42.9%）,K103R/N/S（42.9%）and V106M（23.8%）.Among them,unknown mutation V75L was detected at site 75 of drug resistance-related site,and its mutation rate was significantly increased when treatment failed(Z value=2.494,p<0.05;p McNemar=0.008).In addition,a new mutation,L228R,was detected at site 228 of the non-DRM site in Stanford University HIVdb algorithm,and its mutation rate was significantly increased when treatment failed(Z value=2.306,p<0.05;p McNemar=0.063).It is speculated that V75L and L228R may be potential new DRMs of CRF01＿AE strains.4.Analysis of the relationship between new drug resistance related sites and known drug resistance sitesIn order to explore whether there was a correlation between potential new DRMs and known DRMs,the co-mutation analysis software package was used to analyze the association between mutations at 14 sites with a significant increase in mutation rates when first-line treatment failed.Among them,9 known DRMs（K65R,V106M,Y115F,V179T,Y181C,M184V and G190S）and 2 potential new DRMs（V75L and L228R）were positive selective mutations（Ka/Ks>1,LOD>2）.Through conditional selective pressure analysis,it was found that there were 28 unequal correlations between these mutations（cKa/Ks>1,LOD>2）.Among them,the known DRM Y181C and G190S had the strongest association(cKa/KsY181C-G190S=22.86,LOD=inf).The correlation between L228R and known DRM Y181C was stronger than that of other mutations(cKa/KsY181C-L228R=6.00,LOD=4.09).5.L228R appears at the same time or accompanied by Y181CThe longitudinal plasma samples of four patients with CRF01＿AE infection with both Y181C and L228R mutations were analyzed by deep sequencing in order to observe the timing changes and the relationship between Y181C and L228R.The results showed that Y181C and L228R were detected only at TF under drug selection pressure.Under the pressure of drug selection,Y181C was first detected in the other patient at 1 month after treatment,and some strains carrying L228R were detected at 6 months after treatment.The fourth case could not be analyzed because of the failure of sequencing.Conclusions:The transmitted resistance rate of CRF01＿AE HIV-1 strains in Liaoning area was low,and the high-level natural polymorphism of CRF01＿AE strains had little effect on the treatment outcome.V75L and L228R may be new mutations related to drug resistance of CRF01＿AE,and L228R may be the compensatory mutation of Y181C. |
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