禽腺病毒血清4型QHD2021株的分离鉴定和全基因序列分析

Isolation,identification and complete gene sequence analysis of fowl adenovirus serotype 4 QHD2021 strain

为了解河北秦皇岛地区禽腺病毒血清4型(FAdV-4)分离株的遗传变异情况和致病性,本试验对疑似患FAdV-4病死鸡的肝脏、心脏等组织进行PCR检测,将PCR检测为阳性的组织接种鸡肝癌细胞(LMH)进行病毒分离培养,对纯化后的FAdV-4进行全基因测序,对所得的序列进行同源性和遗传进化分析及致病性试验。结果表明:将PCR检测为FAdV-4阳性的肝脏、心脏匀浆于接种LMH细胞后72 h内出现明显细胞病变,初步确定分离毒株为FADV-4毒株,命名为FAdV-4 QHD2021株。全基因测序结果显示,QHD2021株全基因组全长44225 bp,共编码14741个氨基酸。与国外毒株相比短1500 bp左右,但比国内毒株长约500 bp。同源性和遗传进化分析显示,QHD2021株与国内外FAdV-4流行毒株在同一支,与国内SD1601株的同源性最高,为94.3%。序列比对发现QHD2021株与国内流行株、国外经典株相比均出现1段1966 bp的碱基缺失,包括ORF19、ORF27和ORF48的缺失。将经LMH细胞纯化的QHD2021株接种21日龄的SPF雏鸡后,可引起心包积液、肝炎等典型的临床症状,死亡率为100%。病理组织学观察可见QHD2021株感染的SPF鸡肝细胞出现出血坏死、心肌断裂等明显特征性病变,说明FAdV-4 QHD2021株对鸡群的致病性较强。

To understand the genetic variation and pathogenicity of fowl adenovirus serotype 4(FAdV-4)isolated in Qinhuangdao,Hebei Province,PCR detection was performed on liver and heart tissues of dead chickens suspected of FAdV-4 disease.tissues were positive by PCR were inoculated with chicken liver cancer cells(LMH)for virus isolation and culture,the purified FAdV-4 was sequenced for the whole gene,and the sequence obtained was subjected to homology and genetic evolution analysis and pathogenicity tests.The results showed that the FAdV-4 positive liver and heart homogenates detected by PCR showed obvious cytopathic changes within 72 h after inoculation with LMH cells.The isolated strain was identified as fowl adenovirus groupⅠserotype 4 strain and named as FAdV-4 QHD2021 strain.The results of whole gene sequencing showed that the whole genome of QHD2021 strain was 44225 bp,encoding 14741 amino acids.Compared with foreign strains,it was about 1500 bp shorter,but about 500 bp longer than domestic strains.The homology and genetic evolution analysis showed that the QHD2021 strain was in the same branch as the FAdV-4 epidemic strain at home and abroad,and had the highest homology with the domestic SD1601strain,which was 94.3%.Sequence alignment showed that QHD2021strain and domestic epidemic strain had a 1966bp base deletion compared with foreign classic strains,including the deletion of ORF19,ORF27and ORF48.After inoculated 21-day-old SPF chicks with QHD2021strain purified by LMH cells,which caused typical clinical symptoms such as pericardial effusion and hepatitis,and the mortality was 100%.Histopathological observation showed that SPF chicken hepatocytes infected by QHD2021strain showed obvious characteristic lesions such as hemorrhagic necrosis and myocardial rupture.The results showed that FADV-4QHD2021strain was highly pathogenic to chickens.