Biological Characteristics Of Fowl Adenovirus Type 4 Attenuated Strain ON1 And Preliminary Evaluation Of Its Immunological Efficacy

Hepatitis-Hyperpericardium Syndrome(HHS)is a viral infection caused by Fowl adenovirus 4(FAdV-4)in chicken.Its' typical symptoms are sudden death of chickens aged 3-9 weeks,with pericardial effusion,inclusion body hepatitis,and high morbidity and mortality.The disease was first reported in the Ankara area near Karachi,Pakistan,in 1987,and also in countries such as Mexico,India,Russia,South Korea,and Japan.Since 2015,the HHS caused by the new genotype of FAdV-4,whose mortality rate is as high as 80%,has been endemic in Shandong,Henan and Jiangsu provinces,China.That has resulted in significant economic losses to the poultry industry in China.At present,immunization is still an important means of preventing and controlling poultry adenovirus infections,but a more formalized vaccine to prevent the disease is not yet seen in the market so far.Research on vaccines targeting the new genotype of FAdV-4 will help prevent and control HHS in China.The attenuated live vaccine can simulate the whole process of virus infecting the body and induce the body to produce all levels of immune response including mucosal immune response.In order to develop a live attenuated FAdV-4 vaccine used for prevention and control of HHS,the biological characteristics and in vitro proliferation of FAdV-4 strain ON1 isolated from healthy chickens,as well as challenge against FAdV-4 Chinese epidemic strains were performed in this study,which would lay the foundation for the development of a live attenuated vaccine for prevention and control of HHS.The study includes the following two aspects:1.Biological Characteristics of FAdV-4 Attenuated Strain ON1To understand the biological characteristics of FAdV-4 strain ON1,SYBR Green I real-time fluorescence quantitative PCR and virus titer assays were used to study the growth characteristics of this strain in chicken liver cancer epithelial cell line(LMH).The results showed that the FAdV-4 strain ON1 could proliferate well in LMH and exhibited typical adenovirus-infected cytopathicity.The virus titers at different time points were positively correlated with the viral genome copy number,and the titer reached highest point at 72 h after viral infection in LMH.The culture supernatant also contained a large number of mature virus particles;7-day-old SPF chickens orally were infected with the FAdV-4 strain ON1;all infected chickens did not display clinical symptoms;ON1 virus could be isolated from the cloacae 12 hours after infection,and also shedding virus consistently detected during the observation period of 2 weeks.Histopathological examination showed that there was no significant difference between the tissues and organs of ON1-infected chickens and uninfected control chickens.It was suggested that the strain ON1 is clinically non-pathogenic to chickens.2.Immunogenicity and Immune Efficacy of FAdV-4 Attenuated Strain ON1In order to evaluate the immunogenicity of the FAdV-4 strain ON1,60 7-day-old SPF chickens were randomly divided into two groups,one group were orally inoculated with FAdV-4 strain ON1 cultured in the chicken hepatocellular carcinoma cell line(LMH),and then the specific antibody to FAdV-4 was detected at different time points after immunization.The immunogenicity of the virus was analyzed,and cloacal swabs were also used to detect virus-shedding situation.The results showed that ON1-immunized chickens produced both FAdV-4 specific antibodies and neutralizing antibodies,whose level could reach the peak at 2 weeks post-immunization and persisted for a longer period of time.ON1 immunized chickens continued to shed virus during the observation period of 2 weeks after infection,and the duration of shedding was consistent with the level of induced antibodies.The FAdV-4 Chinese virulent strain was used to challenge the virus 2 weeks after immunization to evaluate the immune efficacy of the attenuated vaccine.The results showed that,2 weeks after the ON1 immunization,75% chicken were protected from challenge of the FAdV-4 Chinese virulent strain,while the control group all died.It was demonstrated that immunization with the FAdV-4 ON1 strain provide partial protection against the FAdV-4 virulence strain.