Complete Genome Sequence Analysis And Pathogenecity Study Of Fowl Adenovirus Group I Serotype 4 GX-1 Strain

Fowl adenovirus group I belongs to the family Adenoviridae and the genus Avian Adenoviruses.According to restriction endonuclease maps and molecular criteria,Avian adenovirus I can be divided into five subgroups（FAdV-A～FAdV-E）,and 12 serotypes（FAdV-1～FAdV-8a,FAdV-8b～FAdV-11）according to cross neutralization test.All 12serotypes of group I avian adenovirus can cause inclusion body hepatitis（IBH）and gizzard erosion（GE）.Currently,only serotype 4（FAdV-4）which belonging to subgroup C can cause hydropericardium-hepatitis syndrome（HHS）.this disease has already caused a large amount of economic losses in Chinese poultry industry.In this study,The GX-1 strain of FAdV-4 causing HHS was isolated from a commercial broiler farm in Guangxi Province.After the FAdV was successfully propagated in chicken embryo liver cell.The harvested virus was successfully reproduced on chicken liver hepatocellular carcinoma cell line（LMH）,and this virus was purified by plaque purification experiment using this cell line.The virus was identified as group I avian adenovirus by PCR.Then the complete genome sequence of the virus was obtained by high throughput sequencing and it is name as GX-1.The complete genome sequence is 43721 bp,and contains 38 open reading frames,of which the content of G+C is 54.87%.Phylogenetic analysis showed that GX-1 belonged to subgroup C and serotype 4 of group I avian adenovirus.The complete genome sequence similarity of GX-1 was 99%to 100%compared with other nine FAdV-4 isolates from China,and 98%to 99%similarity compared with B1 isolates from India and MX-SHP95 isolates from Mexico.Phylogenetic analysis using hexon gene also showed the same result.Transmission electron microscopy（TEM）observation confirmed that the virus particles were icosahedral with sizes of 60～90nm.Hemagglutination test showed that the virus had no hemagglutination effect on chicken blood.Physical and chemical experiments showed that the virus had high temperature tolerance and could resist weak acid.Organic solvents such as ether and chloroform could not inactivate it.Ultraviolet irradiation and formaldehyde solution treatment can inactivate the virus.To explore the pathogenicity of FAdV-4 GX-1 strain to SPF chicken and SPF chicken embryo.The 8-day-old chicken embryos were inoculated with 0.2 m L×10³ TCID₅₀ FAdV-4 GX-1 virus solution.The mortality rate of all chicken embryos within 6 days was 100%.Compared with the control group,the development of infected chicken embryos was retarded and their body size was significantly smaller than that of the control group.In the pathogenicity test of SPF chickens,different doses of virus were used to attack 10-day-old SPF chickens.The mortality rate of 2 dpi（day post infection,dpi）to 4 dpi was 100%after inoculated 0.2 m L×10⁴ TCID₅₀ dose virus solution,84%within 9 dpi after using 0.2m L×10³ TCID₅₀ dose,and 20%after using 0.2 m L×10² TCID₅₀ dose.Liver enlargement,punctate hemorrhage and necrosis,severe pericardial effusion and renal congestion were found during autopsy diseased chickens.Pathological examination revealed a rupture of cardiac muscle fibers accompanied by extensive congestion and bleeding.There were eosinop Hilic intranuclear inclusions,coagulative necrosis and edema degeneration of hepatocytes in the liver.Renal edema is accompanied by tubular degeneration and necrosis.Lymp Hocyte degeneration and necrosis,thymic endothelial cell degeneration and necrosis and lymp Hocytopenia in Bursa of fabricius.A small amount of lymp Hocyte degeneration and endothelial cell necrosis were found in the spleen.The inactivated vaccine of GX-1 strain was prepared by formaldehyde inactivation method.25 SPF chickens immunized once at the age of 10 days and other 25 SPF chickens immunized twice at the age of 10 days and 24 days,respectively.At the 14th day after the end of immunization each group inoculated 0.4 m L×10² TCID₅₀ FAdV-4 GX-1 virus solution.The results showed that the specific antibody of FAdV was detected on the 14th day after the enhanced immunization.The antibody level of the primary immunization group reached the peak around the 35th day after the vaccination,while the antibody level of the enhanced immunization group reached the peak around the 42nd day after the vaccination.The mortality rate of the challenge control group was 100%,the protection rate of the primary immunization group was 90%,and the protection rate of the enhanced immunization group was 100%.Detection of virus by cloacal swab showed that the inactivated vaccine could effectively prevent detoxification.Through the whole gene sequence analysis and pathogenicity study,we can draw the conclusion that the GX-1 strain belongs to group I avian adenovirus serotype 4 with a total genome length of 43721 bp.The inactivated vaccine prepared by formaldehyde inactivation method can provide 90%～100%protection rate and effectively prevent the detoxification of 10-day-old SPF chickens.It can provide reference for the development of commercial vaccines in the future.