Isolation And Identification Of Avian Adenovirus 8a And Preliminary Study On The Mechanism Of Liver Necrotizing Apoptosis

Fowl adenovirus（FAdV）is a type of cyste-free double-stranded DNA viruses with typical icosahedral symmetry in structure and was di-vided into three groups（Fadv-Ⅰ～Ⅲ）according to their antigenicity and gene sequence.Among them,the avian adenovirus is divided into 5 sub-groups（A～E）and 12 serotypes（1～8a,8b～11）in pairs.ⅠFAdV can cause Hydropericardium syndrome（HPS）,Inclusion bodyhepatitis（IBH）and Gizzard erosions（GE）in chickens.In recent years,IBH is endemic and the main infection is 3-5 week old broiler chickens.Large area outbreaks of IBH occur in many places of our country.Clinical symptoms and mor-tality reach a peak in 3-4 days.FAdV-8a,8b and D11 have gradually be-come the main serotypes causing IBH,which have caused huge economic losses and severe epidemic prevention challenges to the poultry industry in China.In 2022,only chickens from 20 broiler farms in Nanping area of Fu-jian Province showed hemorrhagic hepatitis,liver enlargement and asci-tes.Liver disease samples from chickens suspected of inclusion body hepatitis were collected from broiler farms in this area,and the test results were positive for avian adenovirus serum type 8a（FADV-8a）nucleic acid.In order to understand the molecular characteristics of FAdV-8a Fujian strain and its pathogenicity to chickens,a strain of FAdV-8a was isolated from liver tissue by inoculation of chicken embryos,named FJNP strain,and part of its hexon gene was amplified by PCR and analyzed by se-quencing.The results showed that the size of the amplified fragment was847bp,and the nucleotides sequence comparison showed that the homol-ogy of the hexon gene of TR59（KT862810.1）of the Australian strain was99.5%with no significant variation,which further proved that the FJNP strain was FADV-8a.The virulence of FJNP strain was subsequently de-tected,and the results showed that the ELD₅₀ was 10^-3.645/0.2 m L,and the TCID₅₀ was 10^-6.76/0.1 m L.In order to study the pathogenicity of FJNP strain on SPF chicks,1-day-old SPF chickens were artificially infected with 2.5×10^3.65ELD₅₀virus by injection into the leg muscle,and a blank control group was set up.Clinical symptoms,histopathological changes and viral load of in-fected chickens were observed at 3,5,7,14 and 21 days after challenge,respectively.The pathogenicity of serum biochemical level was analyzed.The results showed that three days after infection,SPF chickens showed symptoms of listless spirit and decreased appetite,and three to seven days after infection,they died suddenly,with a mortality rate of 42.3%.Com-pared with the blank group,the weight of infected chickens decreased significantly.The virus was widely distributed in SPF chickens,and the viral load in liver tissue was the highest.Autopsy results showed that the liver lesions were the most obvious in the dead chickens,manifested as liver swelling and bleeding,the color was yellow,white necrotic point,crisp quality,kidney swelling and bleeding,spleen swelling and bleeding.The histopathological results of the liver showed that the liver was bleed-ing,a large number of hepatocyte necrosis,intranuclear inclusion bodies,nucleus fragmentation and dissolution,a small amount of watery degen-eration of hepatocytes,cell swelling,loose and light staining of cytoplasm were observed in the visual field.More lymphocyte focal infiltration is seen.In conclusion,the isolated strain of FAdV-8a was highly pathogenic to SPF chicks,resulting in slow growth and pathological damage to mul-tiple organs in the body,with liver function being the most serious injury.In vivo infection test of FAdV-8a showed that the virus was the most susceptible to infecting hepatocytes and caused serious pathological changes rapidly.Besides acute degeneration and necrosis of hepatocytes and the formation of intranuclear inclusion bodies,serum biochemical results showed that 3-21 days after infection,The contents of alanine aminotransferase（ALT）,aspartate aminotransferase（AST）and lactate dehydrogenase（LDH）in serum were significantly increased,and the dif-ferences were significant compared with blank control group（P<0.05）.The contents of total protein（TP）,albumin（ALB）and blood glucose（GLU）in serum were significantly decreased compared with blank con-trol group（P<0.05）.RT-q PCR was used to detect the m RNA relative expression levels of four inflammatory factors（IL-1β,IL-6,IL-8 and TNF-α）genes in the liver of dead chickens.The results showed that the m RNA relative expression levels of the four inflammatory factors in the liver were up-regulated at different time points from 3 to 21 days after infection.Compared with the control group,the difference was signifi-cant（P<0.05）.The m RNA relative expression levels of necrotic apoptot-ic genes RIP1,RIP3,MLKL and caspase 8 in liver of infected chickens were detected by RT-q PCR.The results showed that the m RNA relative expression levels of necrotic apoptotic genes RIP1,RIP3 and MLKL sig-nificantly increased from 3 to 7 days after infection.The relative expres-sion level of caspase 8 m RNA gene was significantly decreased,and the difference was significant compared with the control group（P<0.05）.In vitro experiment,infected LMH cells（MOI=2）,the results showed that the m RNA relative expression levels of four inflammatory factors IL-1β,IL-6,IL-8 and TNF-αgenes were significantly increased at 24 and 36 h after infection,and the differences were significant compared with the control group（P<0.05）.24 h after infection,the m RNA relative expres-sion levels of RIP1 and RIP3 genes were significantly increased com-pared with the control group（P<0.05）,showing an increase at first and then a decrease.At 12 h after infection,the m RNA relative expression of MLKL gene was significantly increased,and the difference was signifi-cant compared with the control group（P<0.05）,showing a decreasing trend.24 h after infection,the m RNA relative expression level of caspase8 gene was significantly decreased,and the difference was significant compared with the control group（P<0.05）,showing a trend of first de-creasing and then increasing.The results showed that FADV-8a infection of SPF chickens induced cell necrotic apoptosis,leading to severe liver injury.In this process,the necrotizing apoptotic signaling pathway RIP1/RIP3/MLKL is activated.IL-1β,IL-6,IL-8 and TNF-αreleased by cells are involved in liver injury.