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

doi:10.11843/j.issn.0366-6964.2023.01.025

畜牧兽医学报 ›› 2023, Vol. 54 ›› Issue (1): 272-280.doi: 10.11843/j.issn.0366-6964.2023.01.025

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

李舜^1,4, 李桂珍², 原耀贤^1,4, 李康健^1,4, 马春全^1,4, 李守军³, 黄良宗^1,4*, 马骏^1,4*

1. 佛山科学技术学院生命科学与工程学院, 佛山 528225;
2. 广东科贸职业学院动物科技学院, 广州 510430;
3. 华南农业大学兽医学院, 广州 510642;
4. 佛科院附属动物医院, 佛山 528225

收稿日期:2022-04-21 出版日期:2023-01-23 发布日期:2023-01-17
通讯作者: 马骏,主要从事虫媒新发病的研究,E-mail:609309908@qq.com;黄良宗,主要从事猪传染病诊断与防控研究,E-mail:13925923069@163.com
作者简介:李舜(1990-),男,河南南阳人,博士,主要从事病原微生物致病性方向研究,E-mail:1030075823@qq.com;李桂珍(1987-),女,广东梅州人,硕士,助教,主要从事动物病理学教学及研究,E-mail:190500657@qq.com;原耀贤(1995-),男,广东佛山人,硕士,主要从事动物免疫病理学方向研究,E-mail:549747922@qq.com
基金资助:
广东省重点领域研发计划项目(2019B020217002)；广东省教育厅高校科研项目(2017KTSCX193)；佛山市科技创新项目(2020001004909)

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

LI Shun^1,4, LI Guizhen², YUAN Yaoxian^1,4, LI Kangjian^1,4, MA Chunquan^1,4, LI Shoujun³, HUANG Liangzong^1,4*, MA Jun^1,4*

1. School of Life Sciences and Engineering, Foshan University, Foshan 528225, China;
2. Guangdong Polytechnic of Science and Trade, College of Animal Science and Technology, Guangzhou 510430, China;
3. College of Veterinary Medicine, South China Agricultural University, Guangzhou 510642, China;
4. Foshan University Veterinary Teaching Hospital, Foshan 528225, China

Received:2022-04-21 Online:2023-01-23 Published:2023-01-17

摘要/Abstract

摘要： 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。

关键词: 犬流感, H3N2, 反向遗传操作系统, PA-X, 致病性

Abstract: 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; TCID₅₀ of 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.

Key words: canine influenza, H3N2, reverse genetic system, PA-X, pathogenicity

中图分类号:

S852.659.5

李舜, 李桂珍, 原耀贤, 李康健, 马春全, 李守军, 黄良宗, 马骏. 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[J]. 畜牧兽医学报, 2023, 54(1): 272-280.

LI Shun, LI Guizhen, YUAN Yaoxian, LI Kangjian, MA Chunquan, LI Shoujun, HUANG Liangzong, MA Jun. Effect of the Length of PA-X Gene on Replication and Pathogenicity of H3N2 Canine Influenza Virus[J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(1): 272-280.

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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

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