Construction And Partial Characteristics Of Recombinant Serotype 4 Fowl Adenovirus Expressing RFP

Currently,Serotype 4 Fowl Adenovirus(FAdV-4)has been widespread in the world.The infection of FAdV-4 mainly causes hapetitis-hydropericardium syndrome(HHS).Hepatitis,nephritis and pericardial effusion are the main clinical symptoms of HHS.The characteristics of HHS caused by FAdV-4 are short incubation period,rapid onset and high mortality.The fatality rate of chickens infected with FAdV-4 is up to 80%.Since 2015,HHS caused by FAdV-4 has swept through many provinces and cities in China,with a large-scale outbreak,causing huge economic losses to domestic poultry industry.However,the only commercial vaccines against the disease in China are the two Newcastle Disease-Avian Influenza(H9N2)-Fowl Adenovirus(Group I,Serotype 4)triple traditional inactivated vaccines,which were approved in 2021,and the live-attenuated vaccine has not been approved.Recently,Xie et al.targeted the virulence factor Fiber-2 of FAdV-4 and constructed three attenuated recombinant viruses,which fused EGFP with FAdV-4,deleted partial sequence of Fiber-2 and completely replaced Fiber-2 with EGFP.In order to explore whether Fiber-1,another Fiber of FAdV-4,could also be edited to construct an attenuated FAdV-4,in this study,we tried to target Fiber-1 of FAdV-4 by CRISPR/Cas9 to construct a novel recombinant FAdV-4 expressing the fusion protein of RFP and Fiber-1 stably,and determine the biological characteristics of virus in vitro and in vivo.1.Construction of the recombinant FAdV-4 virus expressing RFPIn order to test whether Fiber-1 of FAdV-4 could be targeted and edited to generate recombinant viruses,in this study,we intended to insert RFP as an exogenous gene into Fiber-1 to construct a recombinant virus expressing RFP.Firstly,The RFP was inserted into the region between shaft and tail of Fiber-1,and the homologous arm(HA)was designed in 1kb length at both ends to construct donor plasmid for recombinant virus.Based on CRISPR/Cas9,the LMH cells were infected with wild-type FAdV-4 after transfection with sgRNA,and then transfected with the donor plasmid to rescue recombinant virus.The viruses with red fluorescent in LMH cells were purified by limited dilution and plaque assay continuously.Then,the purified recombinant viruses were sequenced and identified by Western Blot.The results showed that recombinant FAdV-4 virus expressing RFP with high purity was successfully obtained in this study,named as FAdV4-RFP_F1.Although the FAdV4-RFP_F1 replicated slower than the wild-type FAdV-4,it still could replicate efficiently and stably in LMH cells.All these data demonstrate that fiber-1 of FAdV-4 can be as a target gene to be edited to generate recombinant FAdV-4 and lay a foundation for developing novel FAdV-4-based genetic engineering vaccine.2.Pathogenicity and protective efficacy of recombinant virus FAdV4-RFP_F1In order to evaluate the replication ability,pathogenicity and protective efficacy of FAdV4-RFP_F1 in vivo,in this study,we carried out infection experiments with SPF chickens.3-week-old SPF chickens were inoculated with the same dose of FAdV4-RFP_F1 and wild-type FAdV-4.Chickens without infection were as negative control at the same time.The clinical signs of the infected chickens were observed and recorded daily.The cloacal swabs and organs of liver,spleen and kidney of chickens were collected at indicated time points,and then titrated for viral titers.The serum of the chickens were also collected at indicated time points for detection of the neutralizing antibody titer.The results showed that the recombinant virus FAdV4-RFP_F1 was highly attenuated in vivo.Chickens infected with FAdV4-RFP_F1 had no any clinical symptoms,without virus shedding in cloaca swabs,and the virus could not be isolated from tissues.However,FAdV4-RFP_F1 could induce high level of neutralizing antibody against FAdV-4.Then,all chickens alive were challenged with the lethal dose of wild-type FAdV-4 at 21 dpi.The results showed that FAdV4-RFP_F1 could block the virus shedding in cloaca and inhibit the virus loading in tissues,and provide 100%protection against the highly pathogenic FAdV-4.All these data demonstrate that the recombinant virus FAdV4-RFP_F1 is highly attenuated and can provide efficient protection against the highly pathogenic FAdV-4,providing the technical support for further development of genetic engineering multiple Fiber-l-edited and live-attenuated recombinant FAdV-4 vaccines.