2株Ⅰ群禽腺病毒的分离鉴定及致病性分析

Isolation,Identification and Pathogenic Analysis of Two Strains of Fowl AdenovirusⅠ

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500bp的片段,且2株分离株细胞F5代TCID50分别为10-7.75/0.1mL和10-7.60/0.1mL,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。

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 hexongene 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 hexongene showed that the first isolate had the highest homology with FAdV-4(99.57%),and the second isolate had the highest homology with FAdV-8 b(99.18%).Both of these two strains were the group Ⅰ viruses,which were different from the hemorrhagic enteritis of turkeys viruses(in groupⅡ)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 8 bin the serotype genotype,and was named as FAdV-SDE8 b.Our study indicated that both of FAdV-SDC4 and FAdV-SDE8 bstrains 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.