新城疫病毒F蛋白碱裂解位点修饰及外源基因的插入对 新城疫LaSota疫苗株致病力的影响

Impact of modification of cleavage site of fusion protein and foreign gene insertion on the virulence of Newcastle Disease Virus LaSota vaccine strain

摘要:

新城疫是危害养禽业发展的重要传染病。新城疫病毒(NDV)具有高度传染性和高致病性, 融合蛋白(F)的F1/F2 裂解位点存在多个碱性氨基酸并由此形成的泛组织嗜性一直以来被认为是NDV致病的主要决定因素。本研究利用已经构建NDV弱毒LaSota疫苗株反向遗传操作平台, 将LaSota病毒F蛋白的碱裂解位点由GGRQGR↓L分别突变为GRRQRR↓F和GRRQRR↓L, 在未加入TPCK胰酶的情况下分别成功拯救出突变修饰LaSota 疫苗病毒株rL-FmF 和rL-FmL, 通过测定鸡胚平均致死时间(MDT)、脑内致病指数(ICPI)和静脉内致病指数(IVPI)等指标对其毒力进行评估, 结果rL-FmF 和rL-FmL的ICPI值由LaSota的0.36分别上升为1.18和1.05, 但MDT均大于90小时, IVPI仍然均为0, 表明碱裂解位点的突变可显著增强致病力。为了检测外源基因插入对病毒致病力的影响, 进一步以rL-FmF为载体, 分别构建并拯救出表达H5亚型禽流感病毒血凝素HA和增强绿色荧光蛋白EGFP基因的重组病毒rL-FmF-HA和rL-FmF-EGFP, 经测定ICPI分别为0.67和1.10, 但MDT均大于90小时, IVPI仍然均为0。结果表明, 对rLaSota病毒F蛋白裂解位点2个非碱性氨基酸突变为碱性氨基酸, 无论F2蛋白氨基端为F或L, 均可显著增强其脑内接种致病力, 接近中发型毒株标准, 但对静脉内接种致病能力均无显著影响, 而对鸡胚致死能力均保持rLaSota病毒缓发型特点(MDT≥90); 外源基因的重组、表达可不同程度致弱病毒, 其致弱程度与外源基因及其表达产物性质有关。结果提示, 影响NDV致病力不仅仅局限于F蛋白裂解位点氨基酸序列; 通过F裂解位点修饰及HA基因插入可以获得致病力较高但基本接近缓发型标准的重组病毒。

Abstract:

Newcastle disease virus (NDV) cause a highly contagious and economically loss in poultry. The amino acid sequence at the protease cleavage site of the fusion (F) protein has been postulated as a major determinant of NDV virulence. In this study, we have examined the role of F protein cleavage site sequence in NDV virulence by use reverse genetics technology. The sequence G-R-Q-G-R-L present at the cleavage site of the F protein of avirulent strain LaSota was mutated to R-R-Q-R-R-F or R-R-Q-R-R-L. The resultant mutated rL-FmF and rL-FmL virus were evaluated by mean death test (MDT) in eggs, intracerebral pathogenicity index (ICPI) and intravenous pathogenicity index (IVPI) tests in chickens. The results showed that the modifica-tion of the F protein cleavage site resulted in a dramatic in crease in virulence from an ICPI value of 0.36 for LaSota to a value of 1.18 for rL-FmF and 1.06 for rL-FmL, respectively. In addition, the mutational viruses showed increase MDT and identical IVPI values to parent virus. the virulence of rescued viruses was greatly enhanced by the amino acid replacements, whether the amino acid on the N terminus of F2 was F or L. On this base, we constructed another two NDVs that expressed a H5 subtype avian influenza virus hemagglutinin (rL-FmF-HA) and green fluorescent protein (rL-FmF-EGFP). the ICPI value of recombinant virus rL-FmF-HA and rL-FmF-EGFP were 0.67 and 1.10, respectively lower than that of rL-FmF. the IVPI of both recombinant vi-ruses still keep 0.00 . The values of MDT for rL-FmF-HA and rL-FmF-EGFP were 117h and 101h, greater than 90h . thus, In-troduction of a foreign gene into NDV genome resulted in growth retardation and attenuation, the decrease degree depended on the nature of foreign protein expressed. These results also indicate that cleavability of the F0 protein is an important determinant for virulence of NDV. NDV can be manipulated in the future for use as a vaccine vector.