人源H7N9流感病毒促进Ⅰ型干扰素产生机制的研究

Study on the mechanism of human H7N9 influenza virus promoting typeⅠinterferon production

为阐明不同来源H7N9流感病毒诱导Ⅰ型干扰素(IFN-Ⅰ)产生水平差异及其机制,本研究选取人源流感病毒A/Anhui/1/2013(AH/1)株和禽源流感病毒A/Pigeon/Shanghai/S1421/2013(PG/S1421)株作为模式病毒株,利用C57BL/6小鼠进行致病性试验。结果显示,AH/1株感染可以致小鼠发病死亡,而PG/S1421株则不会致小鼠发病。采用间接免疫荧光试验和流式细胞试验分别对两株病毒在A549细胞和小鼠腹腔巨噬细胞中的感染效率进行检测,结果显示两株病毒在两种细胞中的感染效率无显著差异。将该两株病毒分别感染小鼠腹腔巨噬细胞,在感染后不同时间点收集培养上清,并裂解细胞收取细胞总蛋白。采用ELISA方法对细胞培养上清IFN-Ⅰ含量进行测定。结果显示,感染后不同时间,AH/1株和PG/S1421株均可以诱导IFN-Ⅰ产生,但相较于PG/S1421株,AH/1株感染可以诱导细胞产生更多的IFN-Ⅰ;利用western blot对细胞总蛋白中IFN-Ⅰ产生信号通路的上游感受器分子视黄酸诱导基因Ⅰ(RIG-Ⅰ)的表达水平进行检测。结果显示,在PG/S1421株感染细胞的12 h内,胞内RIG-Ⅰ分子的表达量无显著变化,而AH/1株感染后,胞内RIG-Ⅰ分子的表达量增加,并且表达量随时间延长而上升。以上试验结果表明,AH/1株感染可以通过上调RIG-Ⅰ表达进而诱导宿主细胞产生更多IFN-Ⅰ。本研究阐明了人源H7N9流感病毒促进IFN-Ⅰ产生的初步机制,为进一步解析人源H7N9流感病毒促进IFN-Ⅰ产生的精细分子机制以及免疫逃逸机制奠定了基础,为H7N9流感防控提供参考依据。

In order to elucidate the difference in the production of interferonⅠ(IFN-Ⅰ)induced by different isolates of H7N9 influenza viruses and its mechanism,human influenza virus A/Anhui/1/2013(AH/1)strain and avian influenza virus A/Pigeon/Shanghai/S1421/2013(PG/S1421)strain were used as model virus strainsto evaluate the their pathogenicity in C57 BL/6.The results showed that micemorbidity were oberved in AH/1 infection group and occurred in PG/S1421 infection group.There was no significant difference in infection rate in the detection of the infection efficiency of the two strains in the cells by indirect immunofluorescence assay.Mouse peritoneal macrophages were infected with the two viruses,and the culture supernatant was collected at different time points after infection,and the cells were lysed to collect the total cell protein.Cell culture supernatants were assayed for IFN-Ⅰcontent by ELISA.The results showed that AH/1 and PG/S1421 could induce IFN-Ⅰproduction at different time points after infection,however,compared to PG/S1421,AH/1 infection could induce more IFN-Ⅰin cell.Western blot was used to detect the expression of retinoic acid-inducible gene I(RIG-Ⅰ),an upstream sensor of the IFN-Ⅰproduction signaling pathway.The results showed that the intracellular RIG-Ⅰexpression did not change significantly when infected by PG/S1421 within 12 hours,but increased when infected by AH/1 and was getting higher after infection with times.The above results showed that AH/1 infection could induce more IFN-Ⅰby up-regulating the expression of RIG-Ⅰ.This study clarified the preliminary mechanism of human H7N9 influenza virus on promoting IFN-Ⅰproduction and laid the foundation for further analysis of the precise molecular mechanism of human H7N9 influenza virus promoting production of IFN-Ⅰ,immune escape mechanism,and provided reference data for the control of H7N9 virus.