B型流感病毒NS1蛋白羧基端缺失对病毒致病性的影响分析

Effect of Deletion of the Carboxyl Terminal of the NS1 Protein on Pathogenicity of the Influenza B Virus

本研究利用基因工程技术探索B型流感病毒NS1蛋白羧基端缺失对病毒致病性的影响,并利用反向遗传学技术从体外拯救NS1蛋白羧基端缺失的B型流感病毒,评估去除NS1蛋白羧基端对B型流感病毒致病性的影响,探究B型流感病毒致病性变异的分子基础。通过全基因合成和反向遗传学技术从体外拯救NS1蛋白羧基端发生171个氨基酸缺失的B型流感病毒B/Yamagata/16/88株,命名为B-L5。将实验室之前拯救的母本株B/Yamagata/16/88(以下简称B-S9)和B-L5以3×105 EID5 0的攻毒剂量分别人工感染BALB/c小鼠,通过体重变化、生存率、肺脏病毒滴度等方面进行致病性分析。成功构建了B型流感病毒B/Yamagata/16/88NS1蛋白羧基端缺失株反向遗传平台。母本株B-S9和NS1蛋白羧基端缺失株B-L5均能够人工感染BALB/c小鼠,但不致死,对BALB/c小鼠均呈现低致病性;B-S9感染小鼠后,从攻毒后第3d至第7d,体重持续下降,相反,B-L5感染小鼠后,只在攻毒后第2d出现体重下降,攻毒后第3d体重开始回升;B-S9和B-L5感染小鼠后均能在小鼠肺部进行复制,但攻毒后第3d,B-L5感染小鼠肺脏滴度要比B-S9低7 900倍,攻毒后第6d,B-L5感染小鼠肺脏已检测不到病毒复制。实验结果表明,NS1蛋白羧基端缺失可以明显降低B型流感病毒对小鼠的致病性。B型流感病毒致病性变异的分子基础还有待继续研究,该反向遗传操作系统的建立为B型流感病毒分子机制的研究奠定了基础,同时也为B型流感减毒活疫苗的研究开辟了新途径。

To analyze the molecular basis of the variation of the pathogenicity of the influenza B virus,we rescued a recombinant virus with a deletion in the carboxyl terminal of the NS1 protein using reverse genetics based on the parental virus B-S9 of B/Yamagata/16/88.A mutant strain with a deletion of 171 amino acids in the carboxyl terminal of the NS1 protein was named＂B-L5＂.BALB/c mice were inoculated with 3×105EID50of B-L5 and the parental virus B-S9,respectively.Then,weight changes,survival,and viral titers were documented.During 3days post-inoculation（dpi）to 7dpi,the weight of mice infected with BS9 decreased.However,the weight of mice infected with B-L5 showed weight decreases only at 2dpi,and quickly recovered at 3dpi.B-S9 and B-L5 could replicate in the lungs of BALB/c mice.However,viral titers in the lungs of mice infected with B-L5 were 7900-times lower than those of mice infected with B-S9at3 dpi.Viral titers in the lungs of mice infected with B-L5 were not detected at 6dpi.These results showed that,compared with the parent virus B-S9,the mutant virus B-L5 showed lower pathogenicity in BALB/c mice.Our study suggests that deletion of the carboxyl terminal of the NS1 protein decreases the pathogenicity of the influenza B virus.Establishment of a reverse-genetics system for the B influenza virus will provide a platform for studying its pathogenesis and mechanism of transmission,and for developing live-attenuated influenza B virus vaccines.