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Sequential analysis of amino acid substitutions with hepatitis B virus in association with nucleoside/nucleotide analog treatment detected by deep sequencing
Author(s) -
Ninomiya Masashi,
Kondo Yasuteru,
Niihori Tetsuya,
Nagashima Takeshi,
Kogure Takayuki,
Kakazu Eiji,
Kimura Osamu,
Aoki Yoko,
Matsubara Yoichi,
Shimosegawa Tooru
Publication year - 2014
Publication title -
hepatology research
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.123
H-Index - 75
eISSN - 1872-034X
pISSN - 1386-6346
DOI - 10.1111/hepr.12168
Subject(s) - nucleotide , nucleoside , virology , nucleoside analogue , hepatitis b virus , amino acid substitution , biology , virus , genetics , gene , mutation
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 S anger 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 rtL 180 M and rtM 204 V and the entecavir‐resistant mutation at rtT 184 L 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.