Ultra-deep pyrosequencing of hepatitis B virus quasispecies from nucleoside and nucleotide reverse-transcriptase inhibitor (NRTI)-treated patients and NRTI-naive patients.
Ultra-deep pyrosequencing of hepatitis B virus quasispecies from nucleoside and nucleotide reverse-transcriptase inhibitor (NRTI)-treated patients and NRTI-naive patients.
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DOI：
10.1086/597808
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发表时间：
2009-05-01
期刊：
影响因子：
--
通讯作者：
Shafer RW
中科院分区：
文献类型：
--
作者：
Margeridon-Thermet S;Shulman NS;Ahmed A;Shahriar R;Liu T;Wang C;Holmes SP;Babrzadeh F;Gharizadeh B;Hanczaruk B;Simen BB;Egholm M;Shafer RW
B型肝炎病毒（HBV）中出现的核苷和核苷酸逆转录酶抑制剂（NRTI）耐药的动力学尚未得到很好的了解，因为标准的双脱氧核苷酸直接聚合酶链反应（PCR）测序检测仅在耐药突变成为显性后才能检测到耐药突变。为了深入了解NRTI耐药性，我们使用了一种新的测序技术，通过使用标准直接PCR测序和超深度焦磷酸测序（UDPS），对11例NRTI治疗患者的20份血浆样本和17例NRTI初治患者的17份血浆样本中HBV低流行率NRTI耐药突变谱进行了表征。UDPS在5名接受NRTI治疗的患者的10份样本中检测到PCR未检测到的耐药突变，包括拉米夫定耐药突变V173L（在5个样品中），恩替卡韦耐药突变T184S（2个样品）和S202G（在1份样本中），阿德福韦耐药突变N236T（在1个样品中），以及拉米夫定和阿德福韦耐药突变V173L、L180M、A181T和M204V（在1个样品中）。由载脂蛋白B mRNA编辑酶介导的G至A超突变，胞苷脱氨酶的催化多肽样家族估计存在于0.6%的逆转录酶基因中。3例经直接PCR测序检测为G基因型病毒的患者均经UDPS检测为A基因型合并感染。UDPS检测到了低流行率的HBV变异，包括NRTI耐药突变、G → A超突变和低水平的双基因型感染，其灵敏度在以前是不可能的。
The dynamics of emerging nucleoside and nucleotide reverse-transcriptase inhibitor (NRTI) resistance in hepatitis B virus (HBV) are not well understood because standard dideoxynucleotide direct polymerase chain reaction (PCR) sequencing assays detect drug-resistance mutations only after they have become dominant. To obtain insight into NRTI resistance, we used a new sequencing technology to characterize the spectrum of low-prevalence NRTI-resistance mutations in HBV obtained from 20 plasma samples from 11 NRTI-treated patients and 17 plasma samples from 17 NRTI-naive patients, by using standard direct PCR sequencing and ultra-deep pyrosequencing (UDPS). UDPS detected drug-resistance mutations that were not detected by PCR in 10 samples from 5 NRTI-treated patients, including the lamivudine-resistance mutation V173L (in 5 samples), the entecavir-resistance mutations T184S (in 2 samples) and S202G (in 1 sample), the adefovir-resistance mutation N236T (in 1 sample), and the lamivudine and adefovir–resistance mutations V173L, L180M, A181T, and M204V (in 1 sample). G-to-A hypermutation mediated by the apolipoprotein B mRNA editing enzyme, catalytic polypeptide–like family of cytidine deaminases was estimated to be present in 0.6% of reverse-transcriptase genes. Genotype A coinfection was detected by UDPS in each of 3 patients in whom genotype G virus was detected by direct PCR sequencing. UDPS detected low-prevalence HBV variants with NRTI-resistance mutations, G-to-A hypermutation, and low-level dual genotype infection with a sensitivity not previously possible.
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DOI：
10.1073/pnas.93.9.4398
发表时间：
1996-04-30
影响因子：
11.1
作者：
Nowak, MA;Bonhoeffer, S;McDade, H
通讯作者：
McDade, H
影响因子：
11.8
作者：
Lai, CL;Dienstag, J;Condreay, L
通讯作者：
Condreay, L
影响因子：
2.7
作者：
Saldanha, J;Gerlich, W;Heath, A
通讯作者：
Heath, A
影响因子：
3.7
作者：
Gunther, S;Sommer, G;Will, H
通讯作者：
Will, H
影响因子：
4.2
作者：
Shin-I, Tadasu;Tanaka, Yasuhito;Mizokami, Masashi
通讯作者：
Mizokami, Masashi