首页 >>2014年06期
Miseq高通量测序平台用于一起疑似经性传播HIV的溯源调查
作者:赵琦 时丽丽 蒋岩 温玉洁 潘品良 张桂云 邱茂锋

摘要:

目的  建立分析HIV准种的Miseq高通量测序(简称Miseq测序)技术,并尝试用于一起疑似经性传播HIV的溯源调查。方法  采集疑似有传播关系的2例HIV感染者(编号P1、P2)血液样本,并以同一城市与他们无直接传播关系的2例HIV感染者(编号P3、P4)为对照。提取血浆中RNA,逆转录后进行HIV基因亚型分析和Miseq测序。根据测序结果分析样本中HIV的准种分布,比较使用优势准种数目由少到多(5、20、100、500、全部)时所得的基因离散率及系统进化树。结果  4份样本中HIV的基因亚型相同,均为HIV–1重组亚型(CRF)01_AE。采用Miseq测序,每份样本获得23 788~37 397条有效核酸序列,代表1 229~1 412个独特准种。P2与P1间的平均基因离散率(3.5%~4.5%)远小于与对照样本间(10.3%~19.6%,P<0.01)。系统进化树结果显示,P1和P2的准种聚集在一起,而P3和P4的各自单独聚为一簇;当所用的优势准种数目为20或更多时,P1对P2存在并系关系,提示HIV的传播方向是从P1到P2。结论  Miseq测序在HIV感染溯源调查中可以推断传播关系和传播方向,其操作较简便、检测成本较低,具有较高的实用价值。

关键词:HIV;分子流行病学;疾病传播;新一代测序技术;准种

Abstract:

Objective  An approach for analysis of HIV quasispecies using Miseq high-throughput sequencing platform (hereinafter referred to as Miseq platform) was established and applied to contact tracing for a possible case of HIV sexual transmission.Methods  Four plasma specimens were collected from 2 HIV infections (P1 and P2) suspected to be involved in the sexual transmission and 2 local HIV infections as controls (P3 and P4). The RNAs were extracted from the specimens and then reverse-transcribed into cDNA. After HIV subtyping, Miseq platform was performed to detect and sequence the HIV quasispecies (352 bp) in each specimen. The frequency of quasispecies was counted and ranked. Intrapersonal and interpersonal genetic distance and phylogenetic tree were calculated by using the top 5, 20, 100, 500, and all quasispecies, respectively.Results  The subtypes of HIV from all 4 specimens were CRF01_AE. 23 788 to 37 397 cleaned sequences representing 1 229 to 1 412 unique HIV quasispecies were obtained from these specimens by using Miseq platform. The average genetic distance (3.5%-4.5%) between quasispecies from specimens P2 and P1 was significantly lower than that (10.3%-19.6%) between quasispecies from P2 and the controls (P3 or P4). Phylogenetic tree analysis indicated that sequences from specimens P1 and P2 clustered together while sequences from P3 and P4 exhibited completely independent clusters. When the top 20 or more quasispecies from each specimen were analyzed, sequences from P1 showed a paraphyletic relationship with those from P2, which may indicated that the direction of HIV transmission was from P1 to P2.Conclusion  With the feature of convenient and economic operation, Miseq platform has high practical value in contact tracing of possible HIV transmission.

Key words: HIV;Molecular epidemiology;Disease transmission;New generation sequencing technology;Quasispecies

发表日期:2014/6

引用本文:

图/表:

  • 10.3760/cma.j.issn.0253-9624.2014.06.010.T001:表1 调查对象背景信息及Miseq高通量测序平台获得的核酸序列信息

    10.3760/cma.j.issn.0253-9624.2014.06.010.T001:表1 调查对象背景信息及Miseq高通量测序平台获得的核酸序列信息

  • 10.3760/cma.j.issn.0253-9624.2014.06.010.F001:图1 4份样本中最优势的前20条准种序列占整个HIV准种群总序列数的比例

    10.3760/cma.j.issn.0253-9624.2014.06.010.F001:图1 4份样本中最优势的前20条准种序列占整个HIV准种群总序列数的比例

  • 10.3760/cma.j.issn.0253-9624.2014.06.010.T002:表2 样本间平均基因离散率(%)

    10.3760/cma.j.issn.0253-9624.2014.06.010.T002:表2 样本间平均基因离散率(%)

  • 10.3760/cma.j.issn.0253-9624.2014.06.010.F002:图2 4份样本中HIV准种序列系统进化树

    10.3760/cma.j.issn.0253-9624.2014.06.010.F002:图2 4份样本中HIV准种序列系统进化树

参考文献:

[1]中国疾病预防控制中心. 全国艾滋病检测技术规范(2009年修订版)[S]. 北京:中国疾病预防控制中心, 2009: 45–49.
[2]ChoYK, JungY, LeeJS, et al. Molecular evidence of HIV–1 transmission in 20 Korean individuals with haemophilia: phylogenetic analysis of the vif gene[J]. Haemophilia, 2012, 18(2):291–299.
[3]Salazar–GonzalezJF, BailesE, PhamKT, et al. Deciphering human immunodeficiency virus type 1 transmission and early envelope diversification by single–genome amplification and sequencing[J]. J Virol, 2008, 82(8):3952–3970.
[4]RiederP, JoosB, von WylV, et al. HIV–1 transmission after cessation of early antiretroviral therapy among men having sex with men[J]. AIDS, 2010, 24(8):1177–1183.
[5]OuCY, CiesielskiCA, MyersG, et al. Molecular epidemiology of HIV transmission in a dental practice[J]. Science, 1992, 256(5060):1165–1171.
[6]时丽丽, 赵琦, 蒋岩, 等. 一起疑似输血传播HIV的分子流行病学研究[J]. 中华预防医学杂志, 2013, 47(5):427–430.
[7]RutvisuttinuntW, ChinnawirotpisanP, SimasathienS, et al. Simultaneous and complete genome sequencing of influenza A and B with high coverage by Illumina MiSeq Platform[J]. J Virol Methods, 2013, 193(2):394–404.
[8]DietzJ, SchelhornSE, FittingD, et al. Deep Sequencing Reveals Mutagenic Effects of Ribavirin during Monotherapy of Hepatitis C Virus Genotype 1–Infected Patients[J]. Journal of Virology, 2013, 87(11):6172–6181.
[9]辛若雷, 冯毅, 程春林, 等. 适用于中国HIV–1分型的gag基因引物及其应用[J]. 中华医学杂志, 2009, 89(13):876–880.
[10]ZhouZ, WagarN, DeVosJR, et al. Optimization of a low cost and broadly sensitive genotyping assay for HIV–1 drug resistance surveillance and monitoring in resource–limited settings[J]. PloS One, 2011, 6(11):e28184.
[11]LeitnerT, EscanillaD, FranzénC, et al. Accurate reconstruction of a known HIV–1 transmission history by phylogenetic tree analysis[J]. Proc Natl Acad Sci U S A, 1996, 93(20):10864–10869.
[12]梁浩, 罗皓, 邵一鸣, 等. 广西HIV–1重组毒株env区快速基因分型方法的建立[J]. 中华实验和临床病毒学杂志, 2006, 20(3):282–284.
[13]HedskogC, MildM, JernbergJ, et al. Dynamics of HIV–1 quasispecies during antiviral treatment dissected using ultra–deep pyrosequencing[J]. PLoS One, 2010, 5(7):e11345.
[14]MetzkerML, MindellDP, LiuXM, et al. Molecular evidence of HIV–1 transmission in a criminal case[J]. Proc Nat Acad Sci U S A, 2002, 99(22):14292–14297.
[15]BoutwellCL, RollandMM, HerbeckJT, et al. Viral evolution and escape during acute HIV–1 infection[J]. J Infect Dis, 2010, 202Suppl 2:S309–314.
[16]KeeleBF, GiorgiEE, Salazar–GonzalezJF, et al. Identification and characterization of transmitted and early founder virus envelopes in primary HIV–1 infection[J]. Proc Nat Acad Sci U S A, 2008, 105(21):7552–7557.
[17]HaalandRE, HawkinsPA, Salazar–GonzalezJ, et al. Inflammatory genital infections mitigate a severe genetic bottleneck in heterosexual transmission of subtype A and C HIV–1[J]. PLoS Pathog, 2009, 5(1):e1000274.
[18]AbrahamsMR, AndersonJA, GiorgiEE, et al. Quantitating the multiplicity of infection with human immunodeficiency virus type 1 subtype C reveals a non–poisson distribution of transmitted variants[J]. J Virol, 2009, 83(8):3556–3567.
[19]ScadutoDI, BrownJM, HaalandWC, et al. Source identification in two criminal cases using phylogenetic analysis of HIV–1 DNA sequences[J]. Proc Nat Acad Sci U S A, 2010, 107(50):21242–21247.

用户评论 0条

用户名: 密码: 登陆 注册
 
  • 9
  • 3
  •  4 : 页次:0/0页 共0条记录 5条/每页
    关于我们 | 专家风采 | 会员注册 | 继续教育
    地 址:北京市西城区东河沿街69号正弘大厦511室 邮 编:100052
    电话:010-51322302
    版权所有 中华医学会及中华预防医学杂志编辑部
    京ICP备 07035254 号