用反向遗传技术致弱基因VIId型鹅源新城疫病毒ZJI株

将新城疫病毒ZJI株基因组cDNA全长分成7个片段,依次连接并克隆至TVT7R转录载体中,构建了含ZJI株全基因组cDNA的转录载体(pNDV/ZJI),pNDV/ZJI与3个辅助表达质粒pCI-NP、pCI-P和pCI-L共转染BSR-T7/5细胞,成功拯救出了具有感染性的新城疫病毒粒子。设计两对引物,经overlapPCR方法将该毒株F蛋白裂解位点的112、115和117位碱性氨基酸突变成弱毒株特征的非碱性氨基酸后,替换pNDV/ZJI上的对应序列,构建了转录载体pNDV/ZJIFM,将pNDV/ZJIFM与3个辅助表达质粒共转染BSR-T7/5细胞,成功拯救出了致弱的基因VIId型鹅源新城疫病毒NDV/ZJIFM,获救病毒的鸡胚最小致死剂量平均死亡时间(MDT)大于120h,同时该病毒的脑内接种致病指数(ICPI)为0.16,上述结果表明,获救病毒的毒力已被致弱,是一个较为理想的疫苗候选株。

Attenuation of a genotype VIId Newcastle disease virus ZJI strain of goose origin by reverse genetics

Based on the complete genome sequence of Newcastle disease virus (NDV) ZJI strain isolated from an outbreak in the goose, seven pairs of primers were designed to amplify cDNA fragment for constructing the plasmid pNDV/ZJI, which contained the full-length cDNA of NDV ZJI strain. The pNDV/ZJI with three helper plasmids, pCI-NP, pCI-P and pCI-L, were then cotransfected into BSR-T7/5 cells expressing T7 RNA polymerase. After inoculation of the transfected cell culture supernatant into embryonated chicken eggs from specific-pathogen-free (SPF) flock, infectious NDV ZJI strain was successfully rescued. The recombinant plasmid pNDV/ZJIFM was generated by converting the multi-basic amino acid sequence of the F0 protein cleavage region in pNDV/ZJI to the non-basic amino acid sequence characteristic of avirulent NDV strain. After cotransfection of the resultant plasmid and the three helper plasmids into BSR-T7/5 cells, the recombinant NDV, NDV/ZJIFM, was generated. The mean death time (MDT) of NDV/ZJIFM was more than 120h and the intrancerebral pathogenicity index (ICPI) was 0.16, indicating that the rescued virus was highly attenuated. This attenuated genotype VIId NDV of goose origin could be a desirable vaccine in controlling the current epidemic of ND.