鸭3型甲肝病毒的分离鉴定与VP1基因序列分析  

Identification and VP1 Sequencing of Duck Hepatitis A Virus Type 3

作　　者：焦文龙 傅秋玲[2] 林永强 刘友生 刘荣昌[2] 梁齐章 江南松 万春和[2] 程龙飞[2] 陈红梅[2] 林建生[2] 傅光华[2] 黄瑜[2] JIAO Wenlong;FU Qiuling;LIN Yongqiang;LIU Yousheng;LIU Rongchang;LIANG Qizhang;JIANG Nansong;WAN Chunhe;CHENG Longfei;CHEN Hongmei;LIN Jiansheng;FU Guanghua;HUANG Yu(College of Animal Sciences,Fujian Agriculture and Forestry University,Fuzhou,Fujian 350002,China;Institute of Animal Husbandry and Veterinary Medicine,Fujian Academy of Agricultural sciences/Fujian Provincial Key Laboratory for Avian Diseases Control and Prevention/Fujian Industry Technology Innovation Research Academy of Livestock and Poultry Diseases Prevention&Control,Fuzhou,Fujian 350013,China;Pulike Biological Engineering,Inc.,Luoyang,Henan 471000,China)

机构地区：[1]福建农林大学动物科学学院,福建福州350002 [2]福建省农业科学院畜牧兽医研究所/福建省禽病防治重点实验室/福建省畜禽疫病防控产业技术创新研究院,福建福州350013 [3]普莱柯生物工程股份有限公司,河南洛阳471000

出　　处：《福建农业学报》2024年第8期898-905,共8页Fujian Journal of Agricultural Sciences

基　　金：国家现代农业(水禽)产业技术体系建设专项(CARS-42);福建省农业科学院自由探索科技创新项目(ZYTS2024019);福建省科技计划公益类专项(2022R10260011);福建省农业科学院青年英才项目(YC2019010);福建省农业科学院科技创新团队建设项目(CXTD2021005-1);福建省农业高质量发展超越“5511”协同创新工程项目(XTCXGC2021012)。

摘　　要：【目的】探明引起安徽某鸭场雏鸭肝脏出血和大量死亡的病原及其遗传进化特征。【方法】对安徽省某鸭场的病死雏鸭中采集的出血肝脏开展鸭已知病原核酸检测、病原分离鉴定和动物回归试验,在明确其病原为鸭3型甲肝病毒(Duck hepatitis A virus type 3,DHAV-3)的基础上分析其VP1基因序列分子特征。【结果】细菌分离结果显示,未分离到细菌;经病毒核酸(RT-)PCR检测结果显示,鸭3型甲肝病毒(DHAV-3)核酸阳性,未检测出其他已知引起鸭肝出血的病毒核酸。将该阳性样品经鸭胚进行病毒分离与传代,发现接种后鸭胚发生死亡,胚体全身出血,对第5代尿囊液经RT-PCR检测为DHAV-3,将其命名为AH230225。经测定,该分离株的鸭胚半数致死量(Effective lethal dose 50,ELD_(50))为10^(−4.17)/0.1 mL。动物回归试验表明,该毒株对樱桃谷雏鸭的致死率为80%,且攻毒死亡鸭肝脏和肾脏的剖检病变与临床典型病变相近。对该分离毒的VP1基因核苷酸序列进行同源性分析,显示AH230225株的VP1基因核苷酸序列与AH07株DHAV-3(安徽分离株)的同源性最高,为98.8%,与GenBank登录的10株DHAV-3分离株VP1基因核苷酸序列同源性为90.4%~98.8%,而与DHAV-1和DHAV-2的VP1基因核苷酸序列同源性分别为62.1%~63.0%、64.6%~64.9%;基于VP1蛋白氨基酸序列的遗传进化显示,该分离株与AH07株DHAV-3处于同一小进化分支上,亲缘关系最近;而与SD01株、G株和韩国株(AP-04009、AP-03337)等亲缘关系较远,即远离DHAV-1和DHAV-2进化分支。【结论】引起安徽某鸭场雏鸭肝脏出血和大量死亡的病原为鸭3型甲肝病毒DHAV-3,同时明确了该毒株VP1基因的分子特征及遗传进化规律,为深入研究DHAV-3的致病机制和制定防控措施提供科学依据。【Objective】Pathogen that caused duck liver hemorrhage in Anhui Province was identified and its genetics studied.【Method】A suspected virus strain was isolated from the ducks suffered from liver hemorrhage at a poultry farm in Anhui Province.Pathogenic nucleic acid and an animal regression test were employed to identify the culprit.VP1 of the confirmed duck hepatitis A virus type 3(DHAV-3)were sequenced and analyzed using biological software.【Results】No bacterial pathogens were isolated from the culture of the diseased duck liver tissue.However,the specimens were tested positive for DHAV-3 but free of other viruses commonly known for duck liver hemorrhages by RT-PCR.The duck embryos inoculated with the isolate died with massive hemorrhages,and the 5th generation allantoic fluid detected presence of DHAV-3 by RT-PCR.The isolate was subsequently code-named AH230225 and determined to have the effective lethal dose 50(ELD_(50))of 10^(−4.17)/0.1 mL.In an animal regression test,the Cherry Valley ducklings had a mortality rate of 80%.The dissected lesions in the liver and kidneys of the dead ducks were similar to the typical clinical specimen.The sequenced VP1 nucleotides of AH230225 showed the greatest homology of 98.8%with the DHAV-3 of Anhui isolate,AH07.Its homologies with the 10 strains of DHAV-3 listed in GenBank were 90.4%~98.8%,with DHAV-1,62.1%–63.0%,and with DHAV-2,64.6%–64.9%.It appeared that the VP1 of AH230225 was genetically most closely related to that of AH07 but farther from those of SD01,G,or Korean AP-04009 and AP-03337.In other words,it was distant from DHAV-1 and DHAV-2 on the evolutionary branch.【Conclusion】The virus that caused the liver hemorrhage on ducks at the farm in Anhui was identified to be DHAV-3 with VP1 closely related genetically to that of AH07.

关 键 词：鸭3型甲肝病毒 分离鉴定 VP1基因 

分 类 号：S852.65[农业科学—基础兽医学]