一株鹅源禽腺病毒4型的分离及致病性

Isolation and Pathogenicity of a Goose Derived Fowl Adenovirus Type 4

2023年,昆明一朗德鹅场部分雏鹅出现软脖子症状,伴有零星死亡,病变疑似禽心包积液综合征(hydropericardium syndrome,HPS)。本研究旨在对本次雏鹅病因进行分子病毒学诊断和病原的致病性研究。采集肝病料接种鸡肝癌细胞(leghorn male hepatocellular cells,LMH):1)观察细胞病变效应(cytopathic effect,CPE),取培养液负染、电镜观察病毒粒子形态;2)靶标4型禽腺病毒(serotype 4 fowl adenovirus,FAdV-4)的衣壳蛋白编码基因hexon基因进行PCR诊断,对扩增基因序列进行遗传演化分析;3)以5×10^(5.5)TCID_(50)/羽的剂量皮下注射感染10日龄健康泰州鹅,进行动物回归感染以确证其致病性。结果显示,将病料上清接种LMH细胞48~72 h后CPE明显,细胞圆缩、间隙变大、随后碎裂、逐渐脱落,电镜观察见二十面体无囊膜病毒粒子;PCR检测FAdV-4阳性,分离纯化病毒,命名为YNKM2023株;hexon基因的遗传演化分析显示,分离株与国内已有的鸡、鸭FAdV-4流行毒株相似性高达99.8%~99.9%,与印度分离株相似性为99.5%~99.6%。接种感染后,泰州鹅未出现典型的临床症状,但感染后3 d开始出现HPS的特征性病变,感染后6 d检测到中和抗体。综上,本研究成功分离到鹅源FAdV-4/YNKM2023株,该毒株与国内已有的鸡鸭流行毒株高度同源,尚未发生明显的基因漂移。分离株可以感染鹅,病毒可以在雏鹅体内复制引起组织器官病变并产生免疫应答,对鹅表现出一定的致病性,这有助于了解FAdV-4在鹅群的流行情况及其对雏鹅的致病性。

In 2023,some goslings at a Longde Goose Farm in Kunming showed symptoms of soft neck accompanied by sporadic deaths,with the pathology suspected to be hydropericardium syndrome(HPS).The purpose of this study was to carry out molecular virological diagnosis and pathogenicity research on the etiology of gosling.Liver samples from diseased goslings were collected and inoculated with leghorn male hepatocellular cells(LMH).1)The cytopathic effect(CPE)was observed.The culture medium was negative stained and the morphology of virus particles was observed by electron microscope.2)The hexon gene encoding the capsid protein of the target serotype 4 fowl adenovirus(FAdV-4)was subjected to PCR diagnosis,followed by genetic evolutionary analysis of the amplified gene sequence.3)At a dose of 5×10^(5.5)TCID_(50)per bird,subcutaneous injection was administered to healthy 10-day-old Taizhou geese for animal retroinfection to confirm its pathogenicity.The results showed that,after the inoculation of LMH cells with the supernant of disease material for 48 to 72 hours,clear cytopathic effects are observed,characterized by cell rounding,increased intercellular spaces,subsequent fragmentation,gradual detachment.Under electron microscopy,non-enveloped icosahedral viral particles were observed.The PCR test result showed positivity for FAdV-4,the virus was isolated and purified,and named strain YNKM2023;Genetic evolution analysis of the hexon gene revealed a high similarity(99.8%~99.9%)with domestically prevalent chicken and duck FAdV-4 strains,and a similarity of 99.5%~99.6%with strains isolated from India.After being infected,Taizhou geese did not exhibit severe characteristic clinical symptoms,but began to show characteristic lesions of HPS three days post-infection,with neutralizing antibodies detected six days post-infection.In conclusion,this study successfully isolated the goose-origin FAdV-4/YNKM2023 strain,which is highly homologous to the existing chicken and duck epidemic strains in the country,with no apparent genetic drift occurring.The isolated strain can infect geese,replicate in goslings causing tissue organ lesions,and trigger an immune response,showing a certain pathogenicity to geese.This contributes to understanding the prevalence of FAdV-4 in goose populations and its pathogenicity to goslings.