The Subunit Vaccine Of FAdV-4

Fowl adenovirus?FAdV?serotype-4 is highly pathogenic for chickens,especially for broilers aged 3 to 5 wk,and it has emerged as one of the foremost causes of economic losses to the poultry industry in the last 30 years.Its mortality rate ranges from 30 to 70%.The liver is a major target organ of FAdV-4 infections,and virus-infected chickens usually show symptoms of hydropericardium syndrome?HPS?which is characterized by the accumulation of a clear,straw-colored fluid in the pericardial sac,as well as nephritis and hepatitis.The virus is very contagious,and it is spread both vertically and horizontally.It can be isolated from infected liver homogenates and detected by several laboratory diagnostic methods?including an agar gel immunodiffusion test,indirect immunofluorescence assays,enzyme-linked immunosorbent assays,restriction endonuclease analyses,polymerase chain reaction?PCR?,real-time PCR,and high-resolution melting-curve analyses?.Although inactivated tissue vaccines have been deployed widely to control the disease,new vaccines are more attractive vaccine candidates.The different sources of subunit vaccine from FAdV-4were investigated,and the main results of this paper are as follows.1.The isolation and identification of FAdV-4HPS suspected materials collected from a poultry farm in Dali,xianyang,shaanxi province,in October 2015 were homogenated and inoculated to the chick embryo chorioallantoic membrane,CEL cells,and LMH cells.PCR amplification,indirect immunofluorescence,transmission electron microscopy and animal regression experiment were used to detect the virus.The results showed that the virus were isolated from the LMH cells.Phylogenetic analysis showed that the strain was located in the same branch with JSJ13and ON1 which were the FAdV-4 reference strains and belonged to FAdV-C.The results of animal regression test showed that the mobility and mortality of chicks were 80%and 80%.A typical HPS was found in the dead chickens by pathological examination,characterized by the accumulation of a clear,straw-colored fluid in the pericardial sac,enlargement,yellow brown coloring and hemorrhagic necrotic foci in the liver,hyperemia,swelling and uric acid salt deposition in the kidney,and no pathological changes were found in the control group.A FAdV-4strainwassuccessfullyisolatedfromthisstudy,named FAdV-4/Chicken/Shaanxi/China/2015.2.Immune protection efficacy of FAdV-4 surface proteins fiber-1,fiber-2,hexon and penton baseThe immune protection induced by Escherichia coli-expressed capsid proteins of fowl adenovirus serotype 4,including fiber-1,fiber-2,penton base and hexon?loop-1 region?were compared in chickens at different inoculation amounts.According to challenge mortalities and tissue gross/micro lesion results,fiber-2 induced the best protection,followed by fiber-1and hexon.Fiber-1 and fiber-2 provided complete protection against 10^5.5 TCID₅₀ viral load challenge with 100 or 50?g doses per chicken,respectively.Penton could induce effective protection only at the high dosage of 200?g per chicken.The immunoprotective characteristics of these FAdV-4 capsid proteins may prove useful for developing subunit vaccines to control hydropericardium syndrome.3.The production and immune efficacy evaluation of virus like particles?VLP?FAdV-4 capsid protein fiber-1,fiber-2 and penton base were expressed respectively by using human type 5 replication deficient adenovirus expression system and identified by Western blot.The negative staining indicated that there was no virus like particles in the rhAd-fiber-1 cell culture media,purified virus like particles can be found in the co-transduction of HEK293T cell culture media.Confocal microscopy showed that a large number of FAdV-4 VLPs could internalize into HeLa cells.After immunizing with FAdV-4VLP,rhAd-fiber-1,rhAd-fiber-2,rhAd-penton base and inactivated FAdV-4 vaccine,the humoral immune response,cell immune response,challenge mortalities and tissue gross/micro lesion results were used to evaluate the immune protection of the vaccines.FAdV-4VLP induced 100%protection and the cellular immune response showed that the immuno related cytokines,IFN-?,IL-4 and MHC-?,of FAdV-4 VLP group were significantly higher than that of the control group.