2株Ⅰ群禽腺病毒的分离鉴定及致病性分析

doi:10.16431/j.cnki.1671-7236.2018.12.030

摘要/Abstract

摘要：

为了解山东省禽腺病毒（fowl adenovirus，FAdV）毒株的基因遗传演化情况及致病性，本试验对山东省两家疑似暴发鸡包涵体肝炎和心包积液综合征鸡场采集的病料（肝脏、脾脏）进行PCR鉴定，并将鉴定为FAdV的2份阳性病料提取病毒液，接种鸡肝癌细胞（LMH）进行毒株传代培养和细胞病变（CPE）观察、PCR检测、TCID₅₀测定、鸡胚致病性试验、SPF鸡回归试验、病毒hexon部分基因扩增及序列分析。结果显示，试验成功分离到2株FAdV，2株分离株细胞传第1代即可观察到CPE，PCR均可扩增出大小为500 bp的片段，且2株分离株细胞F5代TCID₅₀分别为10^-7.75/0.1 mL和10^-7.60/0.1 mL，均对7日龄SPF鸡胚有强致病性；第1分离株和第2分离株对21日龄SPF鸡攻毒死亡率分别为80%和15%。hexon基因核苷酸同源性分析结果显示，第1分离株与FAdV-4株同源性最高（99.57%），第2分离株与FAdV-8b株同源性最高（99.18%）。这2株FAdV分离株均与Ⅰ群禽腺病毒同源，与火鸡出血性肠炎病毒（HEV）所在的血清Ⅱ群及减蛋综合征病毒（EDSV）所在的血清Ⅲ群禽腺病毒位于不同分支。第1分离株基因型为C型，血清型为4型，命名为FAdV-SDC4株；第2分离株基因型为E型，血清型为8b型，命名为FAdV-SDE8b株。综上所述，FAdV-SDC4和FAdV-SDE8b株属于近几年国内流行毒株。本研究结果可为鸡包涵体肝炎、心包积液综合征的防控工作和疫苗株的选择提供科学依据。

关键词: Ⅰ群禽腺病毒; 分离鉴定; hexon基因; 致病性

Abstract:

In order to understand the genetic evolution and pathogenicity of fowl adenovirus (FAdV) in Shandong province,we conducted two henneries suspected outbroke inclusion body hepatitis and pericardial effusion syndrome of pathogenetic materials (liver and spleen) by PCR identification,preliminary considered they were two FAdV virus strains.The positive pathogenetic materials were grinded by the extract of inoculate LMH cells,then we subculture the strain viruses.The CPE observation,PCR analysis,TCID₅₀ calculation,chicken embryos pathogenicity test,SPF chicken regression analysis,and the phylogenetic analysis of hexon gene were performed in this study.We successfully isolated two FAdV virus strains,and found that the CPE could be found in the first generation,and the PCR analysis showed the bands (500 bp) were in the target locations.The F5 TCID₅₀ was 10^-7.75/0.1 mL for the first strain and 10^-7.60/0.1 mL for the second strain,and they both had strong pathogenicity to 7 days old SPF chicken embryo.When the 21 days old SPF chickens were infected by these two strains,the death rates were 80% and 15%,respectively.The nucleotide homology analysis of hexon gene showed that the first isolate had the highest homology with FAdV-4 (99.57%),and the second isolate had the highest homology with FAdV-8b (99.18%).Both of these two strains were the group Ⅰ viruses,which were different from the hemorrhagic enteritis of turkeys viruses (in group Ⅱ) and the egg drop syndrome viruses (in group Ⅲ).The first isolate was type C in the genotype,type 4 in the serotype,and was named as FAdV-SDC4;While the second isolate was type E in the genotype,type 8b in the serotype genotype,and was named as FAdV-SDE8b.Our study indicated that both of FAdV-SDC4 and FAdV-SDE8b strains were the epidemic strains in China in recent years,and our analysis laid the basis for the management to inclusion body hepatitis and pericardial effusion syndrome,and could contribute to the vaccine research to these diseases.

Key words: fowl adenovirus Ⅰ; isolation and identification; hexon gene; pathogenicity

中图分类号:

S852.65

王颖, 任国鑫, 金鑫, 张万林, 张庆霞, 张瑜, 袁娟, 杨文廷, 蒋伟, 周振成. 2株Ⅰ群禽腺病毒的分离鉴定及致病性分析[J]. 《中国畜牧兽医》, 2018, 45(12): 3563-3571.

WANG Ying, REN Guoxin, JIN Xin, ZHANG Wanlin, ZHANG Qingxia, ZHANG Yu, YUAN Juan, YANG Wenting, JIANG Wei, ZHOU Zhencheng. Isolation,Identification and Pathogenic Analysis of Two Strains of Fowl Adenovirus Ⅰ[J]. , 2018, 45(12): 3563-3571.

参考文献

[1] SAIF Y M主编.苏敬良,高福,索勋译.禽病学[M].第11版.北京:中国农业出版社,2005. SAIF Y M,wirter.SU J L,GAO F,SUO X,translator.Poultry Diseases[M].11th Edition.Beijing:China Agriculture Press,2005.(in Chinese)
[2] 尹燕博.我国Ⅰ群禽腺病毒主要流行血清型及其防控[J].中国家禽,2018,40(3):1-5. YIN Y B.Investigation on dominant circulating serotypes of groupⅠfowl adenovirus in China and the control[J].China Poultry,2018,40(3):1-5.(in Chinese)
[3] 李海英,尹燕博,徐守振,等.Ⅰ群禽腺病毒12个血清型毒株Hexon蛋白全基因序列测定和酶切位点分析[J].中国兽医学报,2012,32(1):33-43. LI H Y,YIN Y B,XU S Z,et al.Analysis of avian adenovirus (FAV) hexon gene group Ⅰsequence and restriction enzyme cutting site from 12 serotype strains[J].Chinese Journal of Veterinary Science,2012,32(1):33-43.(in Chinese)
[4] PICHLA S L,DRINKER M,ZHOU X,et al.Structure-based identification of a major neutralizing site in an adenovirus hexon[J].Journal of Virology,2007,81(4):1680-1689.
[5] GANESH K,SURYANARAYANS V,Raghavan R,et al.Nucleotide sequence of L1 and part of P1 of hexon gene of fowl adenovirus associated with hydropericardium hepatitis syndrome differs with the corresponding region of other fowl adenoviruses[J].Veterinary Microbiology,2001,78(1):1-11.
[6] 万春和,刘荣昌,陈翠腾,等.鸭腺病毒A型Hexon基因克隆与序列分析[J].中国畜牧兽医,2017,44(10):3042-3048. WAN C H,LIU R C,CHEN C T,et al.Cloning and sequence analysis of hexon gene of duck adenovirus A[J].China Animal Husbandry & Veterinary Medicine,2017,44(10):3042-3048.(in Chinese)
[7] NICZYPORUK J S.Phylogenetic and geographic analysis of fowl adenovirus field strains isolated from poultry in Poland[J].Archives of Virology,2016,161(1):33-42.
[8] GRGIC H,KRELL P J,NAGY E.Comparison of fiber gene sequences of inclusion body hepatitis (IBH) and non-IBH strains of serotype 8 and 11 fowl adenoviruses[J].Virus Genes,2014,48(1):74-80.
[9] VERA P F,MORALES A,CORTES D V,et al.Clinicopathological characterization and genomic sequence differences observed in a highly virulent fowl aviadenovirus serotype 4[J].Avian Pathology,2015,45(1):73-81.
[10] YE J,LIANG G,ZHANG J,et al.Outbreaks of serotype4 fowl adenovirus with novel genotype,China[J].Emerging Microbes & Infections,2016,25(5):e50.
[11] 刘金华,甘孟侯.中国禽病学[M].第2版.北京:中国农业出版社,2016. LIU J H,GAN M H.China Poultry Diseases[M].2nd Edition.Beijing:China Agriculture Press,2016.(in Chinese)
[12] 王婉,王彦红,刘伊,等.鸡包涵体肝炎病原检测及病理学观察[J].畜牧兽医学报,2015,46(2):273-278. WANG W,WANG Y H,LIU Y,et al.Pathogenic detection and pathlogical observation of inclusion body hepatitis in chicken[J].Acta Veterinaria et Zootechnica Sinica,2015,46(2):273-278.(in Chinese)
[13] MENG F,DONG G,ZHANG Y,et al.Co-infection of fowl adenovirus with different viruses in a chicken flock[J].Poultry Science,2018,97(5):1699-1705.
[14] 张誉文,焦鹏涛,陈果,等.心包积水综合征流行原因分析[J].中国家禽,2017,39(1):63-66. ZHANG Y W,JIAO P T,CHEN G,et al.The analysis of epidemic causes of hydropericardium syn-drome[J].China Poultry,2017,39(1):63-66.(in Chinese)
[15] 张坦,郑杰,张发明,等.鸡包涵体肝炎病毒山西株的分离与鉴定[J].中国畜牧兽医,2013,40(4):194-197. ZHANG T,ZHEN J,ZHANG F M,et al.Isolation and identification of inclusion body hepatitis virus in Shanxi[J].China Animal Husbandry & Veterinary Medicine,2013,40(4):194-197.(in Chinese)
[16] 牛登云,沈元,王蕊,等.2015年我国Ⅰ群禽腺病毒分子流行病学调查[J].中国家禽,2016,38(9):65-68. NIU D Y,SHEN Y,WANG R,et al.Molecular epidemiological survey of group Ⅰ fowl adenoviruses in China in 2015[J].China Poultry,2016,38(9):65-68.(in Chinese)
[17] 国纪垒,刁有祥,程彦丽,等.Ⅰ群禽腺病毒血清10型的致病性[J].中国兽医学报,2013,33(8):1179-1183. GUO J L,DIAO Y X,CHEN Y L,et al.Pathogenicity of fowl adenoviruses Ⅰ serotype Ⅹ[J].Chinese Journal of Veterinary Science,2013,33(8):1179-1183.(in Chinese)
[18] ZHAO J,ZHONG Q,ZHAO Y,et al.Pathogenicity and complete genome characterization of fowl adenoviruses isolated from chickens associated with inclusion body hepatitis and hydropericardium syndrome in China[J].PLoS One,2015,10(7):e0133073.
[19] PANIGRAHI S,SWAIN K,ROUTRAY A,et al.Inclusion body hepatitis and hydropericardium syndrome in poultry:A brief review[J].ARC Journal of Animal and Veterinary Sciences,2016,2(4):12-16.
[20] SCHACHNER A,MATOS M,GRAFL B,et al.Fowl adenovirus-induced diseases and strategies for their control——A review on current global situation[J].Avian Pathology,2018,47(2):111-126.
[21] VERA H A,MORALES G A,CORTES E D,et al.Clinicopathological characterization and genomic sequence differences observed in a highly virulent fowl aviadenovirus serotype 4[J].Avian Pathology,2016,45(1):73-81.
[22] 国纪垒.Ⅰ群禽腺病毒山东株的分离鉴定及其致病性研究[D].泰安:山东农业大学,2012. GUO J L.Pathogenicity of fowl adenovirusesⅠShandong strains[D].Tai'an:Shandong Agricultural University,2012.(in Chinese)
[23] RUAN S,ZAHO J,YIN X,et al.A subunit vaccine based on fiber-2 protein provides full protection against fowl adenovirus serotype 4 and induces quicker and stronger immune responses than an inactivated oil-emulsion vaccine[J].Infection Genetics and Evolution,2018,61(6):145-150.