中国农业科学

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最新录用鸡腺病毒血清8a型毒株的分离鉴定及致病特点

李慧昕,宋文平,韩宗玺,刘胜旺*
  

  1. 中国农业科学院哈尔滨兽医研究所 / 兽医生物技术国家重点实验室,哈尔滨 150069
  • 发布日期:2022-10-09

Isolation and Pathogenicity of Fowl Adenovirus Serotype 8a Strain

LI HuiXin, SONG WenPing, HAN ZongXi, LIU ShengWang*   

  1. Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences/State Key Laboratory of Veterinary Biotechnology, Harbin 150069
  • Online:2022-10-09

摘要: 【目的】鸡腺病毒(fowl adenovirus, FAdV)在鸡群中流行广泛且血清型众多,不同血清型毒株对鸡的致病能力尚不完全清楚。本研究对FAdV-8a型分离株的致病特点进行研究,为了解血清8a型毒株的致病能力、进而对鸡群中不同血清型FAdV的防控提供科学依据。【方法】2017从发病鸡群采集肝脏组织,组织匀浆后无菌处理,接种鸡胚分离病毒PCR鉴定,初步确定分离毒株为鸡腺病毒为确定分离株分类情况对病毒的全基因组进行测序及序列分析,经基因组序列比对hexon基因遗传演化分析,从分子水平确定病毒的种/血清型。为了解分离株的致病特点和致病能力3010日龄SPF鸡随机分成2组,经滴鼻和点眼途径人工感染从临床发病(发病率和死亡率)、病毒血症、排毒、循环抗体消长规律及其与中和抗体相关性感染后5d鸡剖检症状及病理组织学检测、病毒的组织分布及组织嗜性等方面评价分离株JL/17040810日龄SPF鸡的致病。【结果】对分离株的全基因组序列进行测定和分析表明基因组序列与FAdV-8a型参考毒株TR59株同源性最高基因组结构和编码基因特点与该毒株高度一致。基于全基因组序列进行遗传演化分析,分离株处于FAdV-E种的进化分支TR59株亲缘关系较近同时对分型基因hexon基因进行遗传演化分析,分离株划分为8a血清型。综合分离株基因组特征及遗传演化分析,确定分离株JL/170408FAdV-E种、血清8a型毒株。分离株感染10日龄SPF鸡,感染后3~13d是临床发病高峰期,但不引起鸡只死亡。感染后3d开始出现病毒血症经呼吸道和消化道排毒,病毒血症持续时间长达51d之久,此时感染鸡的抗体未全部转为阳性且循环抗体滴度较低(平均S/P<1),低水平的循环抗体不能有效清除病毒。感染后54d,感染鸡抗体滴度出现高峰(平均S/P>2),有效清除感染鸡体内病毒,表现为病毒血症消失和排毒明显下降。对感染后5d的鸡剖检和病理组织学检测,未见明显病理组织学变化,但是荧光定量PCR可检测到15个组织中有不同程度的病毒载量,病毒的组织嗜性广泛且对肌胃有一定的偏嗜性。对循环抗体监测发现感染鸡抗体阳转出现的较晚感染后15d部分鸡只抗体阳性,感染后24—51d,循环抗体处于相对平稳状态,但此时感染鸡抗体未全部阳性。感染后54d出现了循环抗体滴度的高峰提示鸡只可能发生再次感染,到感染后63d,循环抗体滴度开始下降2只鸡的抗体转为阴性。对感染后63d的血清进行中和抗体测定,并与循环抗体相比较,两者之间无明显相关性。【结论】FAdV-8a型分离株JL/170408可单独致10日龄SPF鸡发病,但不引起死亡,为低致病力毒株。JL/170408的组织嗜性广泛,对肌胃具有偏嗜性,感染后鸡只排毒时间长,排毒具有反复性。


关键词: 鸡腺病毒, 血清8a型, 基因组特征, 致病性

Abstract: 【ObjectiveFowl adenovirus (FAdV) circulates in chicken flocks with multiple serotypes, while there is less information about the pathogenicity of all serotype strains. To understand the capability of causing disease as a primary pathogen to chicken of the FAdV-8a strain, we carry out the experiment for evaluating the pathogenic characteristics of this serotype strain, which will help making the control strategy for breeding. MethodIn 2017, liver tissue was collected form the diseased flocks. The liver tissue homogenates were inoculated into the embryo egg for isolating the virus. The isolate was determined as a FAdV strain after PCR detection. To classify the isolate, genomic sequencing and the genetic evolution based on the complete genome sequence and the hexon gene sequence were performed. To clarify the pathogenicity, thirty 10-day-old SPF chicks were randomly divided into 2 groups and exposed to the isolate JL/170408 via nasal inhalation and eye droppings. The clinical syndrome (including morbidity and mortality), viremia, virus shedding, circulating antibody, postmortem examination and histopathological detection at 5 days post infection (d.p.i.), viral distribution and the tropism on tissues were performed to evaluate the pathogenic capability and characteristics of JL/170408 to SPF chicks. ResultThe complete genome sequencing showed that there were the highest identity between the isolate and the FAdV-8a TR59 strain. They showed high identity in the genomic structure and the encoding gene. Phylogenetic analysis based on the complete genome sequence, the isolate JL/170408 was in the cluster of FAdV-E, further was grouped into the branch of serotype 8a based on the hexon gene. Consequently, the isolate JL/170408 was determined as FAdV-8a serotype within the species of FAdV-E. The clinical peak was observed from 3 to 13d.p.i. without death. The virus shedding and viremia was detected as early as 3d.p.i. and last for a long period at least 51d, the antibody was not positive conversion completely and the mean ELISA titer S/P<1, which didn’t provide enough neutralizing ability to eliminate the virus in the blood and the intestinal tract. At 54d.p.i., the ELISA titer reached a peak with a mean S/P>2, with the consequence of viremia disappearing and a sudden drop of virus shedding. Postmortem examination and histopathological detection at 5d.p.i. showed no obvious pathologic change. While the viral load was detected in 15 tissues, suggested that the isolate propagated in multiple tissues and exhibited higher tropism to gizzard. By monitoring the circulating antibody, the infected birds showed later antibody positive conversion until 15d post infection. Not all birds showed positive seroconversion even at 51d post infection, and the antibody level was stable. At 54d post infection, the antibody titer reached peak, suggesting that birds may suffer a second infection. Virus neutralization test based on the antiserum of 63d.p.i. showed that there was no obvious correlation between the circulating antibody and the neutralization antibody. ConclusionThe FAdV-8a strain JL/170408 causes disease to 10-day-old chicks as single pathogen without leading to death, the isolate is determined as low pathogenic strain. JL/170408 propagates in multiple tissues with higher tropism to gizzard. The infected chicks show a long duration of virus shedding with a repetitive characteristics.


Key words: Fowl adenovirus, serotype 8a, genomic characteristic, pathogenicity