单基因组扩增法分析HIV-1 CRF103_01B3′半长基因组准种变异特征  

Quasispecies variation analysis of HIV-1 CRF103_01B 3′half-length genome by single genome amplification

作　　者：代漫 李佳[2] 李茜瑶 刘安 孙丽君 李洁[2] 吕诗韵 黄辉煌 卢红艳[2] 黄春[2] 辛若雷[2] Dai Man;Li Jia;Li Xiyao;Liu An;Sun Lijun;Li Jie;Lyu Shiyun;Huang Huihuang;Lu Hongyan;Huang Chun;Xin Ruolei(Medical Service Division,the Third People′s Hospital of Yibin,Yibin 644000,China;Institute for STD/AIDS Prevention and Treatment,Beijing Center for Disease Prevention and Control,Beijing 100013,China;Clinic Center of Infectious Diseases,Beijing You′An Hospital,Capital Medical University,Beijing 100069,China;Care Clinic for Medicine Department of Infectious Diseases,Fifth Medical Center,PLA General Hospital,Beijing 100039,China)

机构地区：[1]宜宾市第三人民医院医务科,宜宾644000 [2]北京市疾病预防控制中心性病艾滋病防治所,北京100013 [3]首都医科大学附属北京佑安医院感染中心门诊,北京100069 [4]解放军总医院第五医学中心感染病医学部爱心门诊,北京100039

出　　处：《中华微生物学和免疫学杂志》2024年第5期406-413,共8页Chinese Journal of Microbiology and Immunology

基　　金：首都卫生发展科研专项(首发2022-1G-3015);北京市自然科学基金(7202074)。

摘　　要：目的运用单基因组扩增法(single genome amplification,SGA)分析北京报告的HIV-1 CRF103_01B感染者体内3′半长基因组准种变异特征。方法选择2017—2020年北京新诊断或初始抗病毒治疗病例耐药监测中发现的6例CRF103_01B感染者,提取血浆RNA。采用系列倍比稀释法稀释3′端cDNA,巢式PCR扩增CRF103_01B准种病毒3′半长基因组序列。MEGA 11构建Neighbor-Joining(NJ)系统进化树,计算准种病毒基因距离,Highlighter工具可视化准种病毒遗传变异。利用Simplot 3.5软件对准种序列进行BootScan分析,以分析病毒准种间重组。经Geno2pheno在线工具预测病毒嗜性。结果6例CRF103_01B感染者中有5例经男男性行为感染,共获得144条3′半长基因组SGA序列(19~36条/例)。NJ进化树显示6例感染者3′半长基因组准种变异复杂程度不一。急性期HIV-1感染病例BL4748-00准种病毒变异较小,组内基因距离为0.002±0.000,表现出遗传同质性。BL4981-00、BL3150-00和BL3558-00病例准种病毒分别形成至少3个次级进化簇呈现不同的进化方向,其组内基因距离0.031±0.004≥BL3150-00>BL3558-00>BL4981-00≥0.016±0.002。夫妻BL3022-00(女)和BL3023-00准种病毒序列聚集成大单系进化簇(bootstrap值为100%),后者组内基因距离(0.025±0.003)高于前者(0.019±0.002)。BL3558-00病例存在准种病毒间重组现象。6例感染者的准种病毒均为CCR5嗜性。结论CRF103_01B感染者体内准种变异复杂程度与疾病进展有关,急性期病例表现遗传同质性,慢性期感染病例呈现多个进化方向。未发现共感染或超感染现象,但是部分病例存在准种间重组事件。Objective To elucidate the quasispecies variation of 3′half-length genome in HIV-1 CRF103_01B-infected patients in Beijing using single genome amplification(SGA).Methods This study enrolled six CRF103_01B-infected patients who were diagnosed during a drug resistance monitoring for newly diagnosed cases or newly treated cases with antiviral therapy in Beijing from 2017 to 2020.RNA was extracted from their plasma samples,and 3′end of cDNA was diluted by serial dilution method after reverse transcription.Nested PCR was used to amplify the 3′half-length genome sequences of HIV-1 quasispecies.MEGA 11 was used to construct Neighbor-Joining(NJ)tree and calculate the intrahost genetic distance.Genetic variation in HIV-1 quasispecies was visualized by online Highlighter tool.BootScan analysis was performed using Simplot 3.5 software to analyze inter-quasispecies recombination.Virus tropism was predicted by online Geno2pheno tool.Results Among the six CRF103_01B-infected patients,five were men who have sex with men.A total of 1443′half-length genome SGA sequences(19-36 sequences/case)were obtained.The NJ tree based on the 3′half-length genome of HIV-1 quasispecies revealed different degrees of genetic diversity.The HIV-1 quasispecies in BL4748-00 case of acute infection has the least variation with the intrahost distance of 0.002±0.000,showing genetic homogeneity.The quasispecies sequences from BL4981-00,BL3150-00 and BL3558-00 cases formed at least three subclusters,respectively,with different evolutionary directions,and their intrahost distance ranked from 0.031±0.004 to 0.016±0.002(BL3150-00>BL3558-00>BL4981-00).The quasispecies sequences from the couple BL3022-00(female)and BL3023-00 clustered into a large monophyletic cluster(bootstrap value=100%),and the intrahost distance of the latter(0.025±0.003)was higher than that of the former(0.019±0.002).Inter-quasispecies recombination was observed in BL3558-00 case.The quasispecies from the six patients were CCR5-tropic viruses.Conclusions The diversity of qu

关 键 词：HIV CRF103_01B 单基因组扩增 准种变异 男男性行为 

分 类 号：R512.91[医药卫生—内科学]