| Since July 2015,hydropericardium-hepatitis syndrome(HPS)has been widely spread in most intensive poultry farming areas in China,which has brought huge economic losses.In this study,a highly pathogenic HLJFAdl5 strain was isolated and identified as FAdV-4 from liver tissue of layers with severe HPS.Complete genome sequence analysis of HLJFAd15 showed a natural large genomic deletion of ORF19 and ORF27,which was consistent with the highly pathogenic strains recently isolated in China.The emerging FAdV-4 has been defined as a novel genotype FAdV-4 based on the natural deletion.We have successfully developed the infection model in SPF chickens of the novel FAdV-4 using HLJFAd15 strain.The lethal rates of oral inoculation and intramuscular injection were 76.9%and 100%,respectively.Meanwhile,the clinical symptoms in natural infection,such as pericardial effusion and inclusion body hepatitis,were replicated in infected SPF chickens.The SPF chicken infection model successfully established in this study showed great significance for the development of FAdV-4 vaccine and the pathogenic mechanism study.There is no complete genome sequence of duck-origin FAdV-4 until now,we isolated and identified a duck-origin FAdV-4 strain HLJDAdl5 in this study.The whole genome of HLJDAdl5 was sequenced and submitted to GenBank with an accession number KX538980,which enriched the diversity of FAdV-4 and provided reference diagnostic targets for the novel FAdV-4.The pathogenicity of HLJFAd15 showed that the novel FAdV-4 was more susceptible to SPF chickens with considerable mortality and rapid replication,while no obvious pathological changes were observed in SPF ducks,although FAdV-4 could continuously replicate in SPF ducks.The replication patterns of the novel FAdV-4 in SPF chickens and SPF ducks were completely different,suggesting that ducks may play as an intermediate host in the transmission of the novel FAdV-4 in China.The cross-transmission of the novel FAdV-4 further increased the difficulty in the prevention and control of HPS,and the transmission mechanism needs to be further studied.A natural 1966del deletion was prevalent in the novel FAdV-4 strains in China through the sequence alignment of 17 clinical FAdV-4 isolates,but the effect of the deletion on the virulence has not been identified.In order to investigate the relationship between the natural 1966del deletion and the increased virulence of the novel FAdV-4,CRISPR/cas9 technology was used to establish a reverse genetic operation system of.the novel FAdV-4 for the first time.The developed system was used to rescue the Re 1966 strain containing the 1966del of a non-pathogenic strain KR5.The natural 1966del was proved to be non-essential on the enhancement virulence of the novel FAdV-4 emerged in China based on the biological characterization in vitro and the pathogenicity investigation in vivo.In this study,the reverse genetic operating system based on CRISPR/cas9 technology is capable to efficiently edit FAdV-4,which provides a powerful tool for further screening the virulence genes of the novel FAdV-4 emerged in China.In order to further study the pathogenic mechanism of the novel FAdV-4,we focused on the process of virus adsorption into host cells.The binding of virus fiber protein and cell receptor is the first step of virus infection,but so far,the cellular receptor of FAdV-4 has not been identified.FAdV-4 has a unique fiber antigen pattern,the virus particles have two independent fiber molecules(fiber 1 and fiber 2).Therefore,FAdV-4 has an identifiable molecular mechanism in the process of binding the host cells.In this study,we identified the chicken CAR homology molecule as the cellular receptor for FAdV-4 infection for the first time,and FAdV-4 utilized its short fiber 1 to attach the D2 domain of CAR to complete the binding to host cells.This study revealed a novel adenovirus-receptor binding mechanism,and provided important information for developing FAdV-4 vaccine and screening drug targets. |
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