白纹伊蚊感染登革病毒后病毒特异性piRNAs分析

摘要

Objective To screen and analyze Dengue virus-derived vsRNAs and piRNAs in Aedes albopictus.Methods Female adults Aedes albopictus were collected 2-4 days post-emergence and were injected with Dengue type 2 virus NGC strain,while control group were injected with equal volume of physiological saline.Total RNA was isolated,and small RNAs ranging up to 30 nt in length were excised and analyzed using Illumina HiSeq 2000.After removing adaptor sequences and contaminated reads,Clean reads were aligned to Dengue virus genome and its complementary sequence by SOAP2.The length,strand ratio nucleotide bias and genome distribution of Dengue virus-derived vsRNAs and vpiRNAs were further analyzed.Results Total,3835 vsRNAs and 395 vpiRNAs unique tags were obtained,among them 94.99％ vpiRNAs were derived from virus genome strand,and distributed across the viral genome,but with an enrichment at several "hot-spots".Except weak bias for adenine at position 10 (10A)in the sense molecules as the feature of secondary piRNA,other signature piRNA characteristics were not observed,including a preference for a uridine at their 5'-end,which were main characteristics of primary piRNAs,and a significant 10 nt overlap (Ping-Pong)between sense and antisense piRNA.Conclusion Dengue virusderived piRNAs were indentified in Dengue virus infected Ae.albopictus.%目的 筛选并分析白纹伊蚊感染登革病毒后病毒特异性vsRNAs与vpiRNAs.方法 白纹伊蚊羽化后2～4d雌性成蚊显微注射DENV-2病毒NGC株,对照组注射生理盐水.8d后提取样品总RNA,分离小RNA,通过illumina Hiseq 2000测序仪进行测序分析.以SOAP2软件与DENV-2基因组和互补序列进行比对.对病毒特异性的vsRNAs与vpiRNAs的长度、碱基与链偏好性、基因组分布进行分析.结果 对于感染蚊虫,我们共计获得3835个特异的DENV-2来源的vsRNAs,其中395个特异性的vpiRNAs.94.99％ vpiRNAs主要来源于病毒的正链基因组.vpiRNAs在DENV-2基因组上的分布也具有偏向性,最高峰位于病毒非结构蛋白NS5编码区域的3'末端.第10位碱基具有较强的腺嘌呤偏好性,但第1位碱基不具有尿嘧啶偏好性;也无典型“乒乓”扩增循环的特征.结论 白纹伊蚊感染登革病毒后,体内出现病毒特异性vsRNAs与vpiRNAs,对其鉴定与分析将为在白纹伊蚊抗病毒免疫研究提供理论基础.