Isolation And Identification Of Fowl Adenovirus Serotype 4 And Preliminary Study On Inactivated Vaccine

Hydropcricardium hepatitis syndrome is mainly caused by Fowl adenovirus serotype 4.In 2015,the disease broken out in many provinces in China and became a regional epidemic.At present,there are no effective prevention and control measures in China,therefore,it has caused huge economic losses to China's Poultry industry.In this study,69 FAdV-4 strains were isolated from 92 suspected diseases were collected,the separation rate is 75%.Diseases were from several chicken farms in Henan,Shandong,Jiangxi and Guangdong from 2015 to 2017,which Shown that FAdV-4 is widespread in our country.After identifying,sequencing,and phylogenetic analysis of the hexon gene of the isolate strains,which shown that the isolates were closely related to the representative strains and the identification was 100%.Compared with foreign strains,the identification is between 98.9%⁹9.1%.Total of 9 amino acid mutation sites in the isolate compared with the hexon protein of a foreign representative strain,which are 188 I^R,193 Q^R,195 E^Q,238 N^D,240 A^T,243 E^N,410 T^A,797 A^P,842 G^A,these mutation sites are mainly distributed in the anterior and middle parts of the hexon protein,while the anterior and middle parts are the main antigenic sites of the hexon protein.Therefore,it is suggested that there may be a change in the epitope of the FAdV-4 which has erupted in the country,which shown this isolate has a certain degree of representation.This study selected the pathogenicity of the isolate HZ-06-01 as a representative virus based on the separation time and clinical characteristics.To study the pathogenicity of the virus,this strain was purified through plaque purification in LMH cells and the whole genome of the virus was sequenced and analyzed,finally the pathogenicity of the virus was studied by animal experiments.Genome-wide phylogenetic analysis showed that HZ-06-01 isolate belonged to the same branch of the Chinese representative strains HB1510 and JSJ13.Sequence alignment revealed that the HZ-06-01 isolate had a deletion of the ORF19 and ORF27 genes compared with the representative strains MX-SHP95 and NC₀15323.1?unnamed?.The challenge experiments showed that there were significant differences in pathogenicity between different routes of challenge.Among them,the intramuscular injection group had a mortality rate of 100%?10/10?and virus could be detected in multiple organs of diseased chickens;however,the mortality rate of the fed group was 0%?0/10?and the antibody was positive in the second week.Histopathological examination showed that the liver had obvious nucleus inclusion bodies,severe reduction and necrosis of lymphocytes showed in spleen.After isolation,identification,screening and pathogenicity analysis of FAdV-4,the HZ-06-01 strain is homologe to the domestic representative strains of FAdV-4,and can cause a typical HHS,therefore,the preliminary study on the HZ-06-01 F7 as the seed strain for the inactivation of FAdV-4 vaccine.The results of genetic stability analysis show that,the strains of HZ-06-01 F7,F50,and F100 are conserved in the structural protein genes of hexon,penton,fiber1,and fiber2,and the nucleotide sequence identity is 100%,however,the sequence mutations of the non-structural protein genes such as dUTPpase,PTP,33 KDa,100KDa and ITR occurred.The results of the vaccine quality test showed that the finished vaccine was nonpolluting and there was no adverse reactions after inoculation of 3-week-old SPFchickens,and the inoculation site was well absorbed.Immunoprotective experiment results show that,after twice immunization of 3-week-old SPF chickens with 25 ?L finished vaccine,and in the eighth week,chickens were challenged and can effectively prevent the HZ-06-01 F7 virus attack,the protection rate is 100%?5/5?.The experimental results of antibody lengthening show that,after immunization of 3-week-old SPF chickens with 250 ?L of finished vaccine,in the seventh week it still have a higher serum antibody levels to FAdV-4?? 4461.478?.Therefore,the HZ-06-01 strain can be used as a candidate strain for further study of the inactivated FAdV-4 vaccine.