H9N2亚型禽流感病毒冷适应株的培育及SPF鸡胚感染后的免疫应答分析  

Development of cold-adapted vaccine of H9N2 subtype avian influenza virus and immune response in infected SPF chicken embryo

作　　者：马鑫鑫 崔宁 历成海 黄庆华[2] 梁立生 杨少华[2] 赵孝民[1] 许传田[2] MA Xin-xin;CUI Ning;LI Cheng-hai;HUANG Qing-hua;LIANG Li-sheng;YANG Shao-hua;ZHAO Xiao-min;XU Chuan-tian(College of Animal Science,Shandong Agricultural University,Taian 271018,China;Institute of Animal Science and Veterinary Medicine Shandong Academy of Agricultural Sciences,Shandong Key Lab of Animal Disease Control and Breeding,Jinan 250100,China;Shandong Rizhao Donggang District South Lake Town,Animal Husbandry and Veterinary Station,Rizhao 276817,China)

机构地区：[1]山东农业大学动物科技学院,山东泰安271018 [2]山东省农业科学院畜牧兽医研究所/山东省畜禽疫病防治与繁育重点实验室,山东济南250100 [3]山东日照东港区南湖镇畜牧兽医站,山东日照276817

出　　处：《中国预防兽医学报》2019年第7期726-733,共8页Chinese Journal of Preventive Veterinary Medicine

基　　金：十三五国家重点研发计划(2016YFD0500201);山东省博士基金(ZR2017BC094);现代农业产业技术体系(CARS-41-Z10);山东省农业科学院农业科技创新工程(CXGC2016A10)

摘　　要：H9N2亚型禽流感病毒(H9N2AIV)冷适应疫苗可以弥补灭活疫苗临床保护效果的不足。为明确宿主对冷适应疫苗的免疫应答效应,本研究将H9N2AIV(A/chicken/Shandong/903/2013,CK/903/2013)接种10日龄SPF鸡胚连续降温传代,获得在25℃能够稳定复制的冷适应株Ca30。实验结果显示:Ca30株具备冷适应性和温度敏感性,在传代过程中病毒HA蛋白出现多个位点的氨基酸突变,在25℃传代20次后已经获得稳定的氨基酸突变。动物感染试验结果显示,以106.0EID50的剂量鼻腔感染1周龄SPF鸡,Ca30株仅在喉气管中低水平复制,病毒接种后第3周SPF鸡血清针对Ca30株、异源病毒株1167的血凝抑制效价均达到最高,分别为8.2log2和5.8log2,接种后第4周针对母本病毒CK/903/2013的血凝抑制效价达到最高值7.6log2。qPCR检测结果显示,Ca30在25℃时能够在鸡胚肺脏、肝脏和心脏中复制,对肺脏的亲和力最强。利用qPCR检测冷适应株Ca30接种鸡胚后各脏器细胞因子的转录水平,结果显示,Ca30株感染鸡胚后,引起鸡胚肺脏中IL-1β、IL-6、IL-12、IL-8、OAS和MDA5转录水平上调,上调4.18~16.47倍;感染后8h引起鸡胚心脏中IL-1β、IL-6、IL-12、IL-8、OAS和MDA5的转录水平上调,之后降低甚至下调;72h时引起鸡胚肝脏中IL-1β、IL-6、IL-8和OAS转录水平上调,上调4.28~6.53倍;在鸡胚脑组织中,各类细胞因子的转录水平变化无明显的规律。本研究培育了一株H9N2AIV冷适应毒株Ca30,为深入研究冷适应弱毒疫苗的免疫保护机制提供了素材。The cold-adapted(Ca)vaccine of H9N2 subtype avian influenza virus(H9N2 AIV)can make up the deficiency of clinical protective effect of the inactivated vaccine.In order to clarify the immune response of the host to the cold-adapted vaccine,H9N2 AIV(A/chicken/Shandong/903/2013,CK/903/2013)was passaged in 10-day-old SPF chicken embryos at gradiently reduced temperatures to develop a cold-adapted Ca strain.The Ca30 strain which could replicate stably at 25℃was obtained in the present study.The results showed that Ca30 strain had cold adaptability and temperature sensitivity.Multiple amino acid mutations in HA were observed during subcultures in SPF chicken embryos and stable amino acid mutations were obtained after 20 passages at 25℃.The Ca30 virus only replicated in the trachea at low level when 106.0 EID50 of Ca30 was inoculated intranasally in one-week-old SPF chickens.Hemagglutination inhibition(HI)titer of chicken serum to strain Ca30 and heterologous strain 1167 reached the highest level of 8.2 log2 and 5.8 log2,respectively,at 3 weeks post-inoculation.The HI titer to parental virus 903 reached the highest level of 7.6 log2 at 4 weeks post-inoculation.Ca30 virus could replicate in lung,liver and heart of chicken embryos at 25℃,and it had the strongest affinity to lung.The expression levels of IL-1β,IL-6,IL-12,IL-8,OAS and MDA5 were up-regulated by 4.18 to 16.47 fold in the lung tissues of chicken embryos after infection of Ca30.The expression levels of IL-1β,IL-6,IL-12,IL-8,OAS and MDA5 were up-regulated at 8h post-infection,and then the expression level was decreased or even down-regulated in the heart tissues.The expression levels of IL-1β,IL-6,IL-8 and OAS were up-regulated by 4.28 to 6.53 fold at 72h post-infection in the liver.The expression of various cytokines in brain were no obvious regularity.In summary,a cold-adapted H9N2 strain was successfully generated,which could provide material for further exploring the immune protection mechanism of cold adapted attenuated vaccines.

关 键 词：H9N2亚型禽流感病毒 冷适应疫苗 细胞因子 荧光定量RT-PCR 

分 类 号：S852.65[农业科学—基础兽医学]