Biological Characterization And Pathogenicity Of The Recombinant Avian Adenoviruses JS-eGFP

Avian adenoviruses can be subdivided into three groups: I, II and III. Group I inclides the conventional group I adenoviruses, or fowl adenovirus (FAV-1~12) isolates from chickens, turkeys, geese, and other avian species, which share a common group antigen. The role of group I avian adenoviruses as pathogens is not well defined. It has been found that birds were reinfected with the same strain after 8 week, eliciting a secondary response of both neutralizing and precipitating antibodies.We demonstrated the isolation and identification of a strain of fowl adenovirus group I virus (FAVI-JS) from clinical healthy chickens and selection of non-essential region of FAVI-JS. A recombinant virus rFAVI-eGFP expressing the enhanced green fluorescence protein (rFAVI-eGFP) was developed before. Here, we do some research about replicability and pathogenicity of it in vivo and vitro.1, Biological characterization and pathogenicity of the recombinant virus rFAVI-eGFPHA experiments showed that rFAVI-eGFP virus could agglutinate rat cells. From electron microscope it had the same configuration as wt-FAVI-JS. Still, rFAVI-eGFP after 40 passages in CEK was stable. When it was inoculated into CEK or chicken embryo, the kinetics of rFAVI-eGFP and wt-FAVI-JS in CEK were similar.To evaluate the safety of rFAVI-eGFP, SPF chickens were inoculated intramuscularly with l×l0~10 TCID50 of the rFAVI-eGFP and FAVI-JS at 1-day-old respectively. Five birds were euthanized by every 5 days from 1 to 20 days post-inoculation. Clinical signs, gross lesions were examined. Meanwhile, isolation of the recombinant virus was performed in CEK. The results showed that the inoculated chickens were all healthy and no gross lesions. No effects on SPF chickens body weight can be found after inoculated with the viruses. Most of samples of tissues had no histopathological changes, and virus isolation in blood was negative. Only one sample from gut of inoculated birds waspositive for isolation. These results indicated that the rFAVI-eGFP and wt-FAVI-JS showed good safety and could replicated in the inoculated chickens.二, Immunogenicity of recombinant rFAVI-eGFP in chickensThe recombinant rFAVI-eGFP was characterized in vivo. Firstly, groups of 10-day-old SPF chickens and commercial chickens without maternal antibodies were exposed to recombinant or wild type FAVI-JS respectively with dosage of 10~7 10~8, 10~9 TCID50 by intramuscular injection, through the feed or drinking water and one group served as a negative control. Commercial chickens with maternal antibodies were exposed to recombinant or wild type FAVI-JS dosage with 10~8 TCID50 by intramuscular injection.Indirect immunofluorescence assay (IFA) and ELISA detection demonstrated that antibody response was similar between group with recombinant and group with wild type FAVI-JS. The titres of antibodies in all of kinds were still very high at 60 days post-infection. The antibodies response depended on the dosage and the route of inoculation. The antibodies could be detected by ELISA or IFA as early as 1 week after infection. The highest levels of anti-viral antibodies were detected in chickens inoculated intramuscularly (i.m.). The antibodies titer was still very high after 8 weeks of immunization in all groups. The antibodies titre in commercial chickens with maternal antibodies after being inoculated with rFAVI-eGFP was high for long time. This indicated the existing of maternal antibodies in chickens didn't influence virus replication in vivo. The neutralization result showed that the antibody could neutralize rFAVI-eGFP'replication. All results above suggested that the virus could replicate in the inoculated chickens. The rFAVI-eGFP is a avirulent virus.