两株茶尺蠖核型多角体病毒毒株对灰茶尺蠖的毒力及基因组差异

Differences in virulence and genomics of two Ectropis obliqua nucleopolyhedrovirus strains to Ectropis grisescens

为明确茶尺蠖核型多角体病毒(Ectropis obliqua nucleopolyhedrovirus,EcobNPV)毒株对灰茶尺蠖E.grisescens的毒力差异及其机制,以前期筛选获得的高效毒株EcobNPV-QF4和原始毒株EcobNPV-QV为研究对象,采用PCR扩增技术对其进行分子鉴定,通过叶盘法毒力生测试验测定毒力差异,并用定量PCR技术比较病毒拷贝数及基因组测序方法分析基因组差异。结果表明,EcobNPV-QV经分子鉴定存在3个基因型,而EcobNPV-QF4则存在2个基因型,2株毒株的基因型存在差异;EcobNPV-QV和EcobNPV-QF4对灰茶尺蠖的幼虫毒力分别为4.26×10……(6)PIB/头和9.72×10^(7)PIB/头,EcobNPV-QF4对灰茶尺蠖的毒力是EcobNPV-QV的22.8倍;饲毒0~48 h之间EcobNPVQF4在灰茶尺蠖体内的增殖拷贝均显著高于EcobNPV-QV,EcobNPV-QF4比EcobNPV-QV具有更快的繁殖能力;比较2株毒株的基因组,EcobNPV-QF4比EcobNPV-QV长315 bp,与EcobNPV-QV相比EcobNPV-QF4基因组的hr1~hr3区域出现倒位,其中核苷酸还原酶小亚基基因在EcobNPVQF4中重复1次,3个同源重复区的回文序列存在差异但2株毒株仍为α杆状病毒亚家族Ⅱ亲缘种分离株。表明2株毒株的基因组在同源重复区的序列及回文个数间存在差异,推测这可能与导致其对灰茶尺蠖毒力差异有关。

Two strains of Ectropis obliqua nucleopolyhedrovirus(EcobNPV)EcobNPV-QF4 and EcobNPV-QV,which were isolated previously,were compared in this study to verify their difference in virulence to E.grisescens and to find out the reason for the difference.The molecular identification was performed using PCR amplification technique,the virulence difference was determined via virulence bioassay using the leaf disc method,the viral copy numbers were compared by quantitative PCR technique and the genomic differences were compared with genome sequencing.The molecular identification results showed that EcobNPV-QV had three genotypes,while EcobNPV-QF4 had two genotypes,and the genotypes of the two strains were different.Virulence bioassay revealed that the virulence of EcobNPVQF4 to E.grisescens was 9.72×10^(7) PIB/head,22.8 times higher than that of EcobNPV-QV(4.26×10^(6) PIB/head).The proliferative copy number of EcobNPV-QF4 in E.grisescens was significantly higher than that of EcobNPV-QV from 0 to 48 h,which indicated that productivity of EcobNPV-QF4 was stronger than that of EcobNPV-QV.Genomic analysis revealed that the EcobNPV-QF4 genome was 315 bp larger than the EcobNPV-QV genome,and the hr1-hr3 region of EcobNPV-QF4 genome was inverted compared with EcobNPV.The ribonucleotide reductase small subunit duplicated once in EcobNPVQF4,but the two strains were both belong to alphabaculovirus group II.The results showed that there were differences in sequences and palindromes in the homologous repeat region between the two strains,which might have contributed to the difference in virulence of the two strains to E.grisescens.