PA-X基因的长度变化对H3N2犬流感病毒复制能力和致病性的影响

Effect of the Length of PA-X Gene on Replication and Pathogenicity of H3N2 Canine Influenza Virus

H3N2犬流感病毒(canine influenza virus,CIV)已在中国多地的犬群中流行,是禽流感跨宿主感染并形成新分支的近期案例。研究表明,PA-X基因与甲型流感病毒适应新宿主的能力相关,且其长度能够影响甲型流感病毒的复制及致病能力。为了解PA-X基因的长度变化对H3N2 CIV复制能力及致病力的影响,本研究利用H3N2 CIV的8质粒操作系统,拯救了三株重组H3N2 CIV毒株:PA-X基因表达大小为232个氨基酸多肽的亲本病毒CIV_PA-X_232;对PA编码区第191、192位氨基酸的密码子进行改造,PA-X基因不表达蛋白的重组病毒CIV_PA-X_Knock;对PA+1编码区第232位氨基酸进行突变,PA-X基因表达大小为252个氨基酸多肽的重组病毒CIV_PA-X_252。通过比较3株重组病毒的聚合酶活性,在MDCK细胞中的复制效率及对小鼠致病性的差异,来评价表达不同长度的PA-X基因对H3N2 CIV的影响。结果显示,CIV_PA-X_252和CIV_PA-X_Knock的聚合酶活性显著(P<0.05)高于CIV_PA-X_232,且CIV_PA-X_252的聚合酶活性显著(P<0.05)高于CIV_PA-X_Knock;CIV_PA-X_252和CIV_PA-X_Knock在MDCK细胞中的复制能力显著(P<0.05)强于CIV_PA-X_232;小鼠攻毒试验结果显示,3株重组病毒对小鼠的体重变化无明显影响,且对小鼠无致死性;仅能在感染后第1天的小鼠肺中检测出3株重组病毒的TCID50,且病毒滴度无显著差异;3株重组病毒均能对小鼠肺造成病理损伤,其中,CIV_PA-X_252对小鼠的致病性最强,CIV_PA-X_232次之,CIV_PA-X_Knock对小鼠的致病性最弱。上述结果表明,PA-X基因的延长能够提高H3N2 CIV在MDCK细胞中复制能力和对小鼠肺造成病理损伤的能力,但该变化可能对H3N2 CIV在小鼠肺内的复制能力没有影响。本试验初步探究了PA-X基因的长度变化对H3N2 CIV的影响,为后续H3N2 CIV致病机制的研究提供了初步的参考。

H3N2 canine influenza virus(H3N2 CIV)circulate in dogs from multiple area in China,which is a novel branch formed by trans-host infection of avian influenza.Many research showed that the PA-X gene may play roles in the adaptation of influenza A viruses to new hosts,and can impact the replication efficient and virulence of influenza A virus by its length changing.To better understand the function of PA-X gene in H3N2 CIV,eight-plasmid operating systems were used to rescue three recombinant viruses,the parental virus CIV_PA-X_232 strain(232 aa),the CIV_PA-X_Knock strain,and the CIV_PA-X_252 strain(252 aa).To explore the effect of the length change of PA-X gene on H3N2 CIV,we compared the viral polymerase activity,replication efficiency in MDCK cells,and pathogenicity in mice of the three recombinant viruses.In vitro,the polymerase activity of CIV_PA-X_252 and CIV_PA-X_Knock were significantly(P<0.05)higher than that of CIV_PA-X_232,and that of CIV_PA-X_252 was higher than that of CIV_PA-X_Knock;the replication ability of CIV_PA-X_252 and CIV_PA-X_Knock were significant(P<0.05)higher than that of CIV_PA-X_232 in MDCK cells.In vivo,three recombinant viruses had no significant difference in the body weight of mice and were not lethal;TCID50of three recombinant viruses could be only detected in the lung of infected mice on 1 dpi,and the titer of CIV_PA-X_252 and CIV_PA-X_Knock was slightly higher than CIV_PA-X_232 with no significant difference;three recombinant viruses all caused lung pathological damage in mice,in terms of pathogenicity,CIV_PA-X_252>CIV_PA-X_232>CIV_PA-X_Knock.These results showed that the expression of PA-X protein with different size can affected the H3N2 CIV in transcription,replication,and the pathogenicity of mice.This study preliminarily explored the effects of PA-X gene within different length by H3N2 CIV,providing a reference for subsequent studies on the pathogenesis of H3N2 CIV.