O型口蹄疫云南太保减毒(YNTBa)株3A蛋白突变对病毒毒力的影响

Foot-and-mouth disease virus (FMDV) is a highly contagious viral disease agent. Review of existing research indicates the non-structural protein 3A of FMDV is a virulence factor in cattle and host range determinant of FMDV. In our previous study, we found that three amino acid sites in 3A protein played an important role during the attenuation process of the FMD attenuated ZB vaccine strain. But another FMDV serotype O Yunnan Taibao attenuated strain (YNTBa), a rabbit-passaged attenuated strain, had been not successfully used as a live FMD vaccine strain owing to its attenuation not completely. The three key amino acid sites in the YNTBa via suckling rabbit-passaged attenuation didn`t mutation in the appropriated positions of 3A protein of ZB strain though contrasted 3A protein sequences of ZB and YNTBa. We suspect that these mutations are likely to be the major determinants of attenuation, and the host factors of specific binding partner for 3A protein is critical role for mediating the interaction that may contribute to virulence attenuation in cattle. In this project, a chimerical virus in which the 3A protein coding region of the full-length cDNA clone of YNTBa strain will replaced by that of 3A gene of ZBatt vaccine virus, and some mutants bearing above mutations in 3A protein will derived by site-directed mutagenesis. The viral biological characters of the chimerical virus and those mutants will assay to explore the effects on virulence of mutations in 3A protein. Furthermore, the host factor which proved that 3A protein of FMDV interacted with DCTN3、KTN1、ZG16B、UBQLN1、WHAMM and BAG6, will also explore how they interact with those mutates virus. The project can help understand the role of 3A protein on the virulence of YNTBa and the relationship of 3A protein mutation and virulent attenuation. The project will highlight the important of 3A-host interaction in FMDV virulence and reveal the pathogenic mechanism of FMDV.

口蹄疫病毒（FMDV）3A蛋白是病毒毒力相关因子,我们前期研究发现FMD弱毒疫苗ZB株减毒过程中，3A蛋白内3个氨基酸位点突变发挥了重要作用。O型FMD云南太保减毒(YNTBa)株减毒不完全，而该毒株在3A蛋白内相关位点没有发生突变。本项目以现有YNTBa株感染性克隆分子为骨架，根据FMD弱毒疫苗ZB株研究结果，对YNTBa株3A基因进行替换和突变多个位点，构建嵌合病毒和一系列特定变异位点的突变病毒，测定这些病毒的生物学特性，验证3A蛋白突变对病毒毒力的影响；进一步，利用突变病毒，研究FMDV和3A蛋白互作宿主因子（DCTN3、KTN1、ZG16B、UBQLN1、WHAMM和BAG6蛋白因子）之间的相互作用，阐明3A蛋白突变对YNTBa减毒的影响，探索3A-宿主因子相互作用对病毒毒力变异可能的作用机制，为揭示FMDV致病机理提供科学依据。

FMDV基因变异所引起的致病性、抗原性及对宿主嗜性等的改变是病毒毒力变异的分子基础。为了研究FMDV毒力变异机制，本研究首先利用FMD兔化弱毒疫苗ZB株感染性克隆分子为骨架，构建L蛋白重组病毒rZBatt-L、3A蛋白重组病毒rZBatt-3A和3A蛋白中一系列点突变病毒(A51G、E78G、H80C、K84N、A97P、D107N、R127I)，比较其与亲本病毒rZBatt生物学特性。结果表明，FMD弱毒疫苗ZB株L蛋白中的N2D、M143L和E147G突变不改变病毒的生物学特性，对病毒毒力不产生明显的影响；3A蛋白置换改变ZB病毒对牛原代肾细胞（BK）的复制及感染性，导致宿主细胞因子KTN1、UBQLN1和BAG6表达与亲本病毒rZBatt差异显著（P<0.05）；3A蛋白H80C的突变导致了病毒在BK细胞产生CPE，H80C突变是毒力变异的关键氨基酸位点。其次，利用FMD兔化减毒YNTBa株感染性克隆为骨架，用来源于弱毒疫苗ZB株的3A蛋白片段置换YNTBa株中的相应片段，构建重组病毒rTB-ZB-3A，比较其与亲本病毒rYNTBa生物学特性。结果表明，病毒感染BK细胞后，rTB-ZB-3A不能产生CPE，而亲本病毒rYNTBa感染则能产生CPE；10TCID50剂量接种乳鼠二者毒力差异显著（P<0.05）；与rYNTBa比较，rTB-ZB-3A感染BK细胞导致KTN1、UBQLN1、WHAMM和BAG6 4个蛋白mRNA表达下调，差异显著（P<0.05）。结果提示，YNTBa株3A蛋白置换导致了病毒毒力减弱，3A蛋白改变FMDV对天然宿主细胞的复制及感染性。再次，分析YNTBa感染BK细胞后转录组表达差异，结果显示，病毒感染8 h和24 h的实验组与对照组分别筛选出436和113个表达变化≥2倍的差异基因，差异基因主要对应于细胞分化、DNA转录、细胞因子产生和调亡等基因，与细胞代谢、核糖体、吞噬体、蛋白消化吸收、细胞分化、细胞转录调控等相关，涉及AGE-RAGE、TNF、IL-17、TGF-beta、PI3K-Akt、MAPK、GnRH、Estrogen和Oxytocin等多条信号通路。研究对了解FMDV 3A-宿主因子相互作用、病毒宿主嗜性改变，解析FMDV的毒力变异机制提供理论依据，促进FMD疫苗研究。