禽流感病毒H9N2与新城疫病毒在鸡成纤维细胞中共感染对病毒复制的影响

doi:10.11843/j.issn.0366-6964.2018.11.025

畜牧兽医学报 ›› 2018, Vol. 49 ›› Issue (11): 2521-2528.doi: 10.11843/j.issn.0366-6964.2018.11.025

禽流感病毒H9N2与新城疫病毒在鸡成纤维细胞中共感染对病毒复制的影响

谢军^1,3,4,5, 孙英杰², 周昌娈², 朱善元⁵, 丁铲^2*, 柏家林^1,3,4*

1. 西北民族大学生物工程与技术国家民委重点实验室, 兰州 730030;
2. 中国农业科学院上海兽医研究所, 上海 200241;
3. 西北民族大学, 甘肃省动物细胞工程技术研究中心, 兰州 730030;
4. 西北民族大学生命科学与工程学院, 兰州 730030;
5. 江苏农牧科技职业学院江苏省兽用生物制药高技术研究重点实验室, 泰州 225300

收稿日期:2018-01-23 出版日期:2018-11-23 发布日期:2019-01-23
通讯作者: 柏家林,E-mail:jlbai@xbmu.edu.cn;丁铲,E-mail:shoveldeen@shvri.ac.cn
作者简介:谢军(1990-),男,江苏泰州人,硕士,主要从事动物分子病毒学研究,E-mail:xjjstz2018@163.com
基金资助:
国家自然科学基金重点项目（31530074）；国家自然科学基金面上项目（31372421）；国家自然科学基金青年基金（31400144）

The Effect of Co-infection of H9N2 Subtype Avian Influenza Virus and Newcastle Disease Virus on Virus Replication in the DF-1 of Chicken Embryo Fibroblasts

XIE Jun^1,3,4,5, SUN Ying-jie², ZHOU Chang-luan², ZHU Shan-yuan⁵, DING Chan^2*, BAI Jia-lin^1,3,4*

1. Key Lab of Bioengineering of the State Ethnic Affairs Commission, Northwest Minzu University, Lanzhou 730030, China;
2. Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Shanghai 200241, China;
3. Gansu Engineering Research Center for Animal Cell, Northwest Minzu University, Lanzhou 730030, China;
4. Faculty of Life Science and Engineering, Northwest Minzu University, Lanzhou 730030, China;
5. Jiangsu Key Laboratory of Veterinary Biotechnologic Drug, Jiangsu Agri-animal Husbandry Vocational College, Taizhou 225300, China

Received:2018-01-23 Online:2018-11-23 Published:2019-01-23

摘要/Abstract

摘要：

临床上禽流感病毒（avian influenza virus，AIV）与新城疫病毒（Newcastle disease virus，NDV）共同感染情况经常发生。为研究AIV与NDV在鸡成纤维细胞DF-1中的共感染对病毒复制的相互影响，首先对NDV和H9N2亚型AIV单独感染DF-1细胞时病毒的复制情况进行了分析，设定以NDV和AIV共感染细胞后12和24 h作为样品采集点，通过Western blot、间接免疫荧光和实时荧光定量PCR检测了两种病毒共感染与单独感染对病毒NP蛋白表达的相互影响。结果显示：Western blot分析表明共感染12和24 h，NDV NP蛋白的表达分别下调了70.6%（P<0.001）和45.7%（P<0.001），AIV NP蛋白的表达分别下调了61.5%（P<0.001）和82.8%（P<0.001）；免疫荧光分析显示共感染组细胞中NDV和AIV的NP蛋白丰度在感染12 h时间点分别下调81.1%（P<0.001）和65.5%（P<0.001），24 h时则分别下调41.2%（P<0.05）和47.4%（P<0.001）；qPCR结果也证实共感染12和24 h组NDV NP的mRNA表达水平分别下调了64.3%（P<0.001）和64.4%（P<0.05），AIV NP的mRNA表达水平分别下调了61.5%（P<0.001）和63.0%（P<0.001）。本研究证明AIV与NDV共感染在细胞水平上可引起病毒复制的相互抑制，为研究NDV和AIV共感染机制在细胞水平上奠定了基础。

关键词: 禽流感病毒, 新城疫病毒, 鸡成纤维细胞DF-1, 共感染, 病毒复制

Abstract:

The co-infection of avian influenza virus (AIV) and Newcastle disease virus (NDV) in Chickens is common clinical incident. To study the effect of co-infection of AIV (H9N2) and NDV (Herts/33) on virus replication in the DF-1 of chicken embryo fibroblasts, the replication of AIV and NDV in the DF-1 was first analyzed respectively and post-infection 12 and 24 h were selected as sample collecting time. The Western blot, immunofluorescence assay and real-time fluorescence quantitative PCR (qPCR) were conducted to compare the replication of AIV and NDV between co-infection and individual infection groups. Compared with the single virus infection group, the intensity band ratio of NDV NP in Western blot analysis in 12 and 24 h post-infection were down-regulated by 70.6% (P<0.001) and 45.7% (P<0.001),while AIV NP was down-regulated by 61.5% (P<0.001) and 82.8% (P<0.001), respectively. The optical intensity band ratio of AIV NP and NDV NP were decreased by 81.1% (P<0.001) and 65.5% (P<0.001) in 12 h post-infection, 41.2% (P<0.05) and 47.4% (P<0.001) in 24 h post-infection. The relative mRNA expression of NDV NP in co-infection in 12 and 24 h post-infection was descended by 64.3% (P<0.001) and 64.4% (P<0.05), while AIV NP was 61.5% (P<0.001) and 63.0% (P<0.001), respectively. These findings indicate that co-infection of AIV and NDV is able to inhibit their replication at cellular level, laying the foundation of further study the mechanism of common infection of AIV and NDV.

Key words: avian influenza virus, Newcastle disease virus, chicken fibroblast DF-1, co-infection, virus replication

中图分类号:

S852.659.5

谢军, 孙英杰, 周昌娈, 朱善元, 丁铲, 柏家林. 禽流感病毒H9N2与新城疫病毒在鸡成纤维细胞中共感染对病毒复制的影响[J]. 畜牧兽医学报, 2018, 49(11): 2521-2528.

XIE Jun, SUN Ying-jie, ZHOU Chang-luan, ZHU Shan-yuan, DING Chan, BAI Jia-lin. The Effect of Co-infection of H9N2 Subtype Avian Influenza Virus and Newcastle Disease Virus on Virus Replication in the DF-1 of Chicken Embryo Fibroblasts[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2018, 49(11): 2521-2528.

参考文献

[1] YOON S W, WEBBY R J, WEBSTER R G. Evolution and ecology of influenza a viruses[M]//COMPANS R W, OLDSTONE M B A. Influenza Pathogenesis and Control-Volume I. Cham:Springer, 2014:359-375.
[2] ENSERINK M, KAISER J. Virology. Avian flu finds new mammal hosts[J]. Science, 2004, 305(5689):1385.
[3] HORIMOTO T, KAWAOKA Y. Strategies for developing vaccines against H5N1 influenza A viruses[J]. Trends Mol Med, 2006, 12(11):506-514.
[4] CLAAS E C J, OSTERHAUS A D M E, VAN BEEK R, et al. Human influenza A H5N1 virus related to a highly pathogenic avian influenza virus[J]. Lancet, 1998, 351(9101):472-477.
[5] SUBBARAO K, KLIMOV A, KATZ J, et al. Characterization of an avian influenza A (H5N1) virus isolated from a child with a fatal respiratory illness[J]. Science, 1998, 279(5349):393-396.
[6] CZEGLÉDI A, UJVÁRI D, SOMOGYI E, et al. Third genome size category of avian paramyxovirus serotype 1(Newcastle disease virus) and evolutionary implications[J]. Virus Res, 2006, 120(1-2):36-48.
[7] MILLER P J, DECANINI E L, AFONSO C L. Newcastle disease:Evolution of genotypes and the related diagnostic challenges[J]. Infect Genet Evol, 2010, 10(1):26-35.
[8] GOUGH R E. Newcastle disease, other avian paramyxoviruses and pneumovirus infections:Avian pneumoviruses[M]//SAIF Y M, BARNES H J, GLISSON J R. Diseases of poultry. 11th ed. Ames:Iowa State University Press, 2003.
[9] LIU X F, WAN H Q, NI X X, et al. Pathotypical and genotypical characterization of strains of Newcastle disease virus isolated from outbreaks in chicken and goose flocks in some regions of China during 1985-2001[J]. Arch Virol, 2003, 148(7):1387-1403.
[10] 韦平,龙进学,阳秀英,等. 传染性法氏囊病病毒快速检测与分型技术的研究[C]//中国畜牧兽医学会家畜传染病分会第十次学术研讨会论文集. 苏州:中国畜牧兽医学会, 2007.
WEI P, LONG J X, YANG X Y, et al. The rapid detection and classification of infectious bursa virus[C]//Chinese Animal Husbandry and Veterinary Society, the 10th Academic Seminar. Suzhou:Chinese Association of Animal Science and Veterinary Medicine, CAAV, 2007. (in Chinese)
[11] NILI H, ASASI K. Avian influenza (H9N2) outbreak in Iran[J]. Avian Dis, 2003, 47(S3):828-831.
[12] GUO Y J, KRAUSS S, SENNE D A, et al. Characterization of the pathogenicity of members of the newly established H9N2 influenza virus lineages in Asia[J]. Virology, 2000, 267(2):279-288.
[13] HAGHIGHAT-JAHROMI M, ASASI K, NILI H, et al. Coinfection of avian influenza virus (H9N2 subtype) with infectious bronchitis live vaccine[J]. Arch Virol, 2008, 153(4):651-655.
[14] ALEXANDER D J. The epidemiology and control of avian influenza and Newcastle disease[J]. J Comp Pathol, 1995, 112(2):105-126.
[15] 中国兽医药品监察所. 兽用生物制品质量标准汇编-2011[M]. 北京:中国农业出版社, 2012.
China Institute of Veterinary Drugs Control. A compilation of quality standards for animal products 2011[M]. Beijing:China Agriculture Press, 2012. (in Chinese)
[16] SCHMITTGEN T D, LIVAK K J. Analyzing real-time PCR data by the comparative C_T method[J]. Nature Protoc, 2008, 3(6):1101-1108.
[17] EL ZOWALATY M E, CHANDER Y, REDIG P T, et al. Selective isolation of Avian influenza virus (AIV) from cloacal samples containing AIV and Newcastle disease virus[J]. J Vet Diagn Invest, 2011, 23(2):330-332.
[18] GE S Q, ZHENG D X, ZHAO Y L, et al. Evaluating viral interference between Influenza virus and Newcastle disease virus using real-time reverse transcription-polymerase chain reaction in chicken eggs[J]. Virol J, 2012, 9(1):128.
[19] COSTA-HURTADO M, AFONSO C L, MILLER P J, et al. Virus interference between H7N2 low pathogenic avian influenza virus and lentogenic Newcastle disease virus in experimental co-infections in chickens and turkeys[J]. Vet Res, 2014, 45(1):1.
[20] PANTIN-JACKWOOD M J, COSTA-HURTADO M, MILLER P J, et al. Experimental co-infections of domestic ducks with a virulent Newcastle disease virus and low or highly pathogenic avian influenza viruses[J]. Vet Microbiol, 2015, 177(1-2):7-17.
[21] 丁德平,韩瑛,陈悦. 干扰素诱生及其抗病毒信号通路研究进展[J]. 中西医结合肝病杂志, 2013, 23(2):121-123.
DING D P, HAN Y, CHEN Y. Research progress of interferon induced and antiviral signaling pathway[J]. Chinese Journal of Integrated Traditional and Western Medicine on Liver Diseases, 2013, 23(2):121-123. (in Chinese)
[22] WULLAERT A, HEYNINCK K, BEYAERT R. Mechanisms of crosstalk between TNF-induced NF-κB and JNK activation in hepatocytes[J]. Biochem Pharmacol, 2006, 72(9):1090-1101.
[23] 孟春春,仇旭升,张向乐,等. 细胞自噬与病毒感染关系的研究进展[J]. 中国预防兽医学报, 2013, 35(11):950-954.
MENG C C, QIU X S, ZHANG X L, et al. The research progress of relationship between cell autophagy and virus infection[J]. Chinese Journal of Preventive Veterinary Medicine, 2013, 35(11):950-954. (in Chinese)

禽流感病毒H9N2与新城疫病毒在鸡成纤维细胞中共感染对病毒复制的影响

The Effect of Co-infection of H9N2 Subtype Avian Influenza Virus and Newcastle Disease Virus on Virus Replication in the DF-1 of Chicken Embryo Fibroblasts

PDF

可视化

摘要/Abstract

引用本文

使用本文

参考文献

相关文章 15

编辑推荐

Metrics

本文评价

[1]	武炜, 王飒, 孟春春, 仇旭升, 廖瑛, 谭磊, 宋翠萍, 刘炜玮, 孙英杰, 丁铲. 应用CRISPR/Cas9敲除HeLa细胞DDX21基因对新城疫病毒复制的影响[J]. 畜牧兽医学报, 2019, 50(7): 1433-1440.
[2]	武炜, 王飒, 孟春春, 仇旭升, 廖瑛, 谭磊, 宋翠萍, 刘炜玮, 孙英杰, 丁铲. 应用CRISPR/Cas9敲除HeLa细胞DDX21基因对新城疫病毒复制的影响[J]. 畜牧兽医学报, 2019, 50(7): 1433-1440.
[3]	申松玮, 王泽源, 范威峰, 祝常椿, 陆游, 石宝兰, 刘雷, 沈海峰, 焦库华, 秦涛, 陈素娟, 彭大新, 刘秀梵. 2017-2018年华东地区H9亚型禽流感病毒分离毒株的抗原差异分析[J]. 畜牧兽医学报, 2019, 50(6): 1230-1238.
[4]	申松玮, 王泽源, 范威峰, 祝常椿, 陆游, 石宝兰, 刘雷, 沈海峰, 焦库华, 秦涛, 陈素娟, 彭大新, 刘秀梵. 2017-2018年华东地区H9亚型禽流感病毒分离毒株的抗原差异分析[J]. 畜牧兽医学报, 2019, 50(6): 1230-1238.
[5]	何世成, 彭志, 王卫国, 唐小明, 鲁杏华, 林源, 邓国华, 王昌建. 2011-2015年环洞庭湖区H3亚型禽流感病毒的分离鉴定与遗传演化[J]. 畜牧兽医学报, 2019, 50(2): 382-389.
[6]	何世成, 彭志, 王卫国, 唐小明, 鲁杏华, 林源, 邓国华, 王昌建. 2011-2015年环洞庭湖区H3亚型禽流感病毒的分离鉴定与遗传演化[J]. 畜牧兽医学报, 2019, 50(2): 382-389.
[7]	胡焱, 段志强, 嵇辛勤, 赵佳福, 邓珊珊, 李世静, 熊建民. GST pull-down验证新城疫病毒基质蛋白与鸡importin β1蛋白的相互作用[J]. 畜牧兽医学报, 2019, 50(1): 126-133.
[8]	胡焱, 段志强, 嵇辛勤, 赵佳福, 邓珊珊, 李世静, 熊建民. GST pull-down验证新城疫病毒基质蛋白与鸡importin β1蛋白的相互作用[J]. 畜牧兽医学报, 2019, 50(1): 126-133.
[9]	高照, 胡娇, 刘秀梵. 两株小鼠致病性差异显著的H5N1禽流感病毒PA蛋白差异互作宿主蛋白研究[J]. 畜牧兽医学报, 2018, 49(9): 1952-1960.
[10]	高照, 胡娇, 刘秀梵. 两株小鼠致病性差异显著的H5N1禽流感病毒PA蛋白差异互作宿主蛋白研究[J]. 畜牧兽医学报, 2018, 49(9): 1952-1960.
[11]	蒋大秀, 马静, 王晓泉, 胡顺林, 刘晓文, 刘秀梵. 2016-2017年华东地区活禽市场7株H9N2禽流感病毒遗传进化分析[J]. 畜牧兽医学报, 2018, 49(8): 1789-1796.
[12]	魏润宇, 罗瑶瑶, 吕艳, 王静静, 郑东霞, 赵云玲, 刘文博, 刘华雷, 王志亮. 4株新出现的Ⅰ类新城疫病毒F基因的变异分析[J]. 畜牧兽医学报, 2018, 49(8): 1781-1788.
[13]	魏润宇, 罗瑶瑶, 吕艳, 王静静, 郑东霞, 赵云玲, 刘文博, 刘华雷, 王志亮. 4株新出现的Ⅰ类新城疫病毒F基因的变异分析[J]. 畜牧兽医学报, 2018, 49(8): 1781-1788.
[14]	蒋大秀, 马静, 王晓泉, 胡顺林, 刘晓文, 刘秀梵. 2016-2017年华东地区活禽市场7株H9N2禽流感病毒遗传进化分析[J]. 畜牧兽医学报, 2018, 49(8): 1789-1796.
[15]	段志强, 邓珊珊, 袁超, 高洪波, 嵇辛勤, 赵佳福, 阮涌. 新城疫病毒M蛋白核定位信号突变降低病毒的致病性[J]. 畜牧兽医学报, 2018, 49(12): 2690-2697.