HCV 3b亚型全长序列测定及基线耐药相关置换的分析

目的探讨二代测序法(NGS)在HCV 3b全长序列及基线耐药相关置换(RAS)检测中的应用。方法从1例2016年7月在广州血液中心献血的献血者(HCV RNA阳性)血浆中提取核酸,经序列非依赖性扩增后,构建测序文库并采用illumina Hiseq进行深度测序,然后通过生物信息学方法分析HCV全长序列、病毒基因分型以及针对直接抗病毒药物(DAA)的基线RAS。结果NGS获得8.4 Gb原始测序数据,序列数(reads)超过56 M。经生物信息学分析获得HCV全长序列,平均测序深度为488007,基因分型结果为3b亚型。病毒氨基酸序列里含有12个RAS,分别是NS3区域的Y56H、Q80K、Q80R、A156G,NS5A区域的M28G、Q/A30G、Q/A30K、L31F、L31M、Y93H,以及NS5B区域的S282T和V321A,其中位于NS5A区域的Q/A30K和L31M属于高丰度RAS(99.16%和98.37%),其余10个RAS丰度较低(<0.5%)。结论NGS用于HCV 3b亚型的全长序列检测和基因分型,最终鉴定出基线RAS,对于HCV 3b的流行病学研究和DAA治疗方案的制订有重要意义。

Full-length sequencing and baseline resistance-associated substitution of hepatitis C virus subtype 3b

Objective To investigate the application of next-generation sequencing(NGS)in determining the full-length sequence and baseline resistance-associated substitution(RAS)of hepatitis C virus(HCV)subtype 3 b.Methods Nucleic acid was extracted from plasma of a HCV RNA-positive blood donor,and after sequence-independent amplification,a sequencing library was constructed and NGS was performed using Illumina Hiseq.Bioinformatics methods were used to analyze full-length HCV sequence,viral genotype,and baseline RAS to direct-acting antivirals(DAAs).Results A total of 8.4 Gb data with more than 56 million reads were obtained.The full-length HCV sequence was obtained by bioinformatics analysis,with an average sequencing depth of 488007 and a genotype of 3 b subtype.A total of 12 RASs were identified in HCV amino acid sequence,i.e.,Y56 H,Q80 K,Q80 R,and A156 G located in NS3,M28 G,Q/A30 G,Q/A30 K,L31 F,L31 M,and Y93 H located in NS5 A,and S282 T and V321 A located in NS5 B,among which Q/A30 K and L31 M located in NS5 A had high frequencies of 99.16%and 98.37%,respectively,while the other 10 RASs had low frequencies of<0.5%.Conclusion NGS can be used to determine the full-length sequence and genotype of HCV subtype 3 b and identify baseline RASs,which has great significance in the epidemiological study of HCV subtype 3 b and the development of DAA treatment regimens.