Shimosegawa T . Sequential analysis of amino acid substitutions with hepatitis B virus in association with nucleoside/nucleotide analog treatment detected by deep sequencing
Shimosegawa T . Sequential analysis of amino acid substitutions with hepatitis B virus in association with nucleoside/nucleotide analog treatment detected by deep sequencing
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下濑川 T.
DOI：
10.1111/hepr.12168
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发表时间：
2014
期刊：
影响因子：
4.2
通讯作者：
Matsubara Y
中科院分区：
文献类型：
--
作者：
Ninomiya M;Kondo Y;Niihori T;Nagashima T;Kogure T;Kakazu E;Kimura O;Aoki Y;Matsubara Y
核苷/核苷酸类似物是治疗慢性B型肝炎的主要抗病毒药物。这种治疗的问题是选择耐药突变体。目前，通过桑格法测序的分子克隆进行基因型耐药性鉴定。然而，这种方法既复杂又耗时。这些限制可以通过深度测序技术来克服。因此，我们通过深度测序对一名接受拉米夫定间歇治疗2年的患者的耐药频率进行了序贯分析。在第一例受试者中检测到rtL 180 M和rtM 204 V的拉米夫定耐药突变以及rtT 184 L的恩替卡韦耐药突变。在最后一例受试者中仍检出拉米夫定和恩替卡韦耐药菌株。然而，在深度测序分析中，第一个受试者的rt 180显示76.9%的甲硫氨酸和23.1%的亮氨酸的混合物，rt 204也显示69.0%的缬氨酸和29.8%的异亮氨酸的混合物。在治疗过程中，耐药突变的比例增加。在rt 184时，在58.7%的序列中可检测到耐药变体，其中第一名受试者的亮氨酸被野生型苏氨酸取代。逐渐地，恩替卡韦耐药变体在最后一名受试者中增加了82.3%的亮氨酸。总之，我们证明了一系列核苷/核苷酸类似物耐药的氨基酸取代。我们发现耐药突变体乍一看似乎没有变化，但实际上存在低丰度突变，这些突变可能通过选择显性突变而对核苷/核苷酸类似物产生耐药性。
Taking nucleoside/nucleotide analogs is a major antiviral therapy for chronic hepatitis B infection. The problem with this treatment is the selection for drug‐resistant mutants. Currently, identification of genotypic drug resistance is conducted by molecular cloning sequenced by the Sanger method. However, this methodology is complicated and time‐consuming. These limitations can be overcome by deep sequencing technology. Therefore, we performed sequential analysis of the frequency of drug resistance in one individual, who was treated with lamivudine on‐and‐off therapy for 2 years, by deep sequencing. The lamivudine‐resistant mutations at rtL180M and rtM204V and the entecavir‐resistant mutation at rtT184L were detected in the first subject. The lamivudine‐ and entecavir‐resistant strain was still detected in the last subject. However, in the deep sequencing analysis, rt180 of the first subject showed a mixture in 76.9% of the methionine and in 23.1% of the leucine, and rt204 also showed a mixture in 69.0% of the valine and 29.8% of the isoleucine. During the treatment, the ratio of resistant mutations increased. At rt184, the resistant variants were detectable in 58.7% of the sequence, with the replacement of leucine by the wild‐type threonine in the first subject. Gradually, entecavir‐resistant variants increased in 82.3% of the leucine in the last subject. In conclusion, we demonstrated the amino acid substitutions of the serial nucleoside/nucleotide analog resistants. We revealed that drug‐resistant mutants appear unchanged at first glance, but actually there are low‐abundant mutations that may develop drug resistance against nucleoside/nucleotide analogs through the selection of dominant mutations.