利用高通量测序技术检测流行性乙型脑炎病毒

Screening for Japanese encephalitis virus in mosquitoes by high-throughput sequencing of viral RNA

目的通过宏转录组和高通量测序方法高效检测和分析蚊虫样品中的流行性乙型脑炎(乙脑)病毒。方法2013年从武汉地区采集蚊虫标本32批(约480只),通过TRIzol加试剂盒的方法对蚊虫进行总RNA提取,对RNA制备RNA文库,应用Trinity、Blast、Seq Man、MAFFT_7、Meg Align、Phy ML等生物学软件对乙脑病毒全基因组核酸序列进行获取及病毒的系统进化分析。结果检测到乙脑病毒1株(WH-JEV株),并分析获得其基因组全长10 947 bp,其中从81位到10 367位为开放阅读框,编码3432个氨基酸。该病毒的测序深度为40X,丰度(TPM)为4.19,对比蚊虫转录组里其他病毒属于低表达量病毒。全基因组序列系统进化分析显示WH-JEV株属于基因Ⅰ型乙脑病毒,而与其最近的LN0716株的核酸序列相似度为99.5%,氨基酸序列相似度为100%。结论利用高通量技术可快速检测出乙脑病毒,并获得相关的全基因组信息。

Objective To use RNA-seq library construction and high-throughput sequencing approach to identify and characterize Japanese encephalitis virus （JEV） within mosquito samples. Methods Thirty-two mosquito pools （around 480 females in total） were collected in Wuhan, Hubei, China. Total RNA was extracted using TRIzol LS reagent and E.Z.N.A. Total RNA Kit and these extractions were further merged into 1 pool for RNA-seq library construction and sequencing. To obtain the full-length genome and reconstruct the phylogenetic tree, we used software including Trinity, Blast, SeqMan, MAFFT_7, MegAlign and PhyML. Results Within the sequencing results, we were able to identify a JEV which we named as WH-JEV strain. The genome of WH-JEV strain was 10 947 nucleotides long, which covers the complete open reading frame from 81 to 10 376 nt of the genome and encodes for a 3432 amino acid polyprotein. The virus has a low abundance （with a coverage of 40X and a TPM of 4.19） comparing to other viruses with the same mosquito RNA library. Subsequent genome analyses suggests that the WH strain belongs to genotype I, and has 99.5% nucleotide identity and 100% amino acid identity with the strain LN0716. Conclusion High- throughput technology could be used to rapidly detect and characterize JEV within mosquitoes.