Isolation,Identification And Pathogenic Mechanism Of Fowl Adenovirus

Fowl adenoviruses(FAdVs)are double-stranded non-enveloped DNA viruses with diameters of 60–90 nm,belonging to the family Adenoviridae,genus Aviadenovirus.FAdV-I could be divided into five species(FAdV A,B,C,D,and E)with 12 serotypes(FAdV-1 to 11,FAdV-8 includes FAdV-8a and FAdV-8b),as determined by restriction enzyme digest patterns and serum cross-neutralization tests.FAdVs have been implicated in a wide range of avian diseases,including inclusion body hepatitis(IBH),hydropericardium syndrome(HPS)and gizzard erosion.The infection of IBH in China was first reported in Taiwan at 1976,and the epidemic of HPS was also sporadic subsequently.The diseases have spread all over the country,but it only sporadically occurred in broilers.Therefore,its harm had not caused great concern.However,since the fall of 2014,the number of HPS cases gradually increased,especially involving in the broilers,free range chickens and commercial laying hens in Shandong.Since June 2015,outbreaks of HPS with high mortality rates have occurred in most areas in China,resulting in tremendous economic losses to the poultry industry.In order to investigate the infection of FAdV in the current chicken flocks,we detected and analyzed105 flocks with positive FAdV infection.The aim of this study is to provide a theoretical basis for clarifying epidemic characteristic and prevention of FAdV in the future.Moreover,we investigated the pathogenicity of FAdV-4 in specific-pathogen-free(SPF)chickens,hepatocytes and cardiomyocytes to elucidate mechanism of FAdV-4-induced liver and heart damage and the formation of pericardial effusion.Viral pathogens,including reticuloendotheliosis virus(REV),Marek's disease virus(MDV)and chicken infectious anemia virus(CIAV),were examined directly in the tested tissues via polymerase chain reaction(PCR)or reverse transcription–PCR.To decipher the disease etiology,the isolates full-length sequences of the Hexon gene were cloned and sequenced to construct phylogenetic trees and sequence alignments.Moreover,the natural cases were also analyzed by observation of autopsy and histopathology.The results showed that a variety of types of chicken with multiple days of age could be infected by FAdV.A survey of the mixed infection of FAdV showed that 68 flocks,5 flocks and 2 flocks were infected by FAdV species C,FAdV species D and FAdV species E respectively.30 flocks contained two or more different FAdV strains.Among them,FAdV-4 was dominant(93%of the strains).Through the homology analysis of Hexon gene,we found that the isolated strains were in same branch with those isolated from other countries and had no serious variation.The co-infections with other immunosuppression pathogens results showed that 50%of the FAdV cases were co-infected with CIAV,while 33%were co-infected with MDV,and 16%were co-infected with REV.The autopsy lesions were characterized by hemorrhagic necrotizing hepatitis and pericardial effusion.The pathological histology was characterized by hepatocyte focal necrosis accompanied with intra-nuclear inclusion bodies in nuclear and cardiocytes granular degeneration.By electron microscopy,we found viral particles with about 70 nanometers in diameter accumulated in the liver nucleus formed a lattice pattern.In order to clarify the pathogenicity of FAdV-4 SDDM-4/15 isolates and the occurrence regularity of HPS,SPF chickens were inoculated by subcutaneous injection.Then the clinical symptoms,histopathological changes,organ edema,and function of liver and heart were observed.The results showed that chickens appeared minor depression symptoms at 2 day-post-infection(dpi);the death commenced at 3 dpi and continued for 6 days;the survival rate was only 50%;the weight of chicken infection group was significantly lower than the control group;the death of the chickens showed severe hepatitis and hemorrhagic pericardial effusion symptoms;the occurrence of different degrees of edema was observed in heart,lung,kidney and spleen.Histopathology showed that the degeneration and necrosis of the pancreatic epithelial cells and the basophilic inclusion bodies in the nucleus,lymphocytes of the spleen,bursa of Fabricius and thymus with necrotic lesions,and brain and digestive organs also with pathological damage.The detection of liver and cardiac function enzymes in serum showed that the contents were significantly increased and the structure of the tissues also appeared severely denatured at 3 dpi.To sum,FAdV-4 has highly pathogenicity to SPF chicks,resulting in slow growth,multiple organ damage,especially the serious damage to the structure and function in liver and heart.FAdV-4 is a hepatotrophic virus that causes severe liver damage,which is characteristic by the longer the infection,the more serious pathological changes of the hepatocytes.Therefore,we want to reveal the mechanism of FAdV-4-induced hepatocytes injury in vitro and in vivo.The results showed that FAdV-4 can damage the structure and function of the liver and there were decreases in the serum albumin(ALB)and blood glucose(GLU)concentration,an increase in plasma aspartate aminotransferase(ALT)and lactate dehydrogenase(LDH)content,which was significantly different from the control group(P<0.05).TUNEL staining showed that FAdV-4 could induce hepatocyte apoptosis.By q-PCR,the mRNA expressions of interleukin(Il)1b,Il6,Il8,and tumor necrosis factor(Tnf)a in infected liver cells and LMH showed a statistically significant increase(P<0.05)compared to that of the control group.Through western-blot detection,FAdV-4 induced the autophagy of hepatocytes,which promoted the conversion of microtubule-associated protein light chain3(LC3-I)to LC3-II,which is a hallmark of autophagy.Conversion of cytoplasm-diffuse GFP to membrane-associated GFP puncta was observed in transfected GFP-LC3 LMH by fluorescence microscopy,which confirmed that FAdV-4 infection can induce hepatocytes autophagy.By using autophagy inducer rapamycin inhibitor 3-methyl adenine(3-MA)to change the state of cell autophagy to analyze FAdV-4 replication,the results showed that after the autophagic inducer or inhibitor,the intracellular FAdV-4 content increased or decreased.It indicated that the degree of autophagy was positively correlated with the proliferation of the virus.Chickens infected by FAdV-4 showed severe pericardial effusion.We explored the formation of pericardial effusion and the mechanism of FAdV-4-induced cardiac damage in vitro and in vivo.In order to analyze the formation of pericardial effusion,total protein exudate(TP),albumin(ALB)and aspartate aminotransferase(AST),creatine kinase isoenzyme(CKMB),lactate dehydrogenase(LDH),potassium(K~+),sodium chloride(Na~+),(Cl~-)and virus content was measured.By analyzing the serum cardiac function enzyme and the histopathological changes of the heart,it is found that FAdV-4 can cause structural damage and dysfunction of the heart.To further analyze the cause of the injury,cardiac cell apoptosis assay was carried out.It was found that the apoptosis of cardiac cells could occur when pericardial effusion formed,however the cardiomyocytes(CM)in vitro did not appear apoptosis by infected with FAdV-4.The results found that there was no significant difference in the composition and content of pericardial effusion and serum.The transcription level of four inflammatory factors in hearts increased significantly at different times(P<0.05)compared with those in control group post FAdV-4 infection.However,the mRNA levels of four inflammatory cytokines and activity of three myocardial enzymes did not differ significantly from the controls.In addition,secretion of IL-6,IL-8 and TNF-?in cell supernatants was consistent with mRNA expression and did not differ significantly from those in control group.TUNEL staining showed that CMs infected with FAdV-4 did not show apoptosis compared with the control group.These observations collectively indicated that pericardial effusion was derived from vascular exudation rather than CM degeneration,and the virus could not directly lead to the occurrence of CM apoptosis.Our results clearly demonstrated that the current epidemic of chicken adenovirus disease is mainly caused by FAdV-4,and there was a mixed infection of other serotype FAdV and high proportion of immunosuppressive viruses.FAdV-4 infection mainly caused the degeneration and necrosis of hepatocytes characterized by the inclusion of the inclusion body in the nucleus,and induced apoptosis and autophagy of the liver cells.FAdV-4 did not directly infect cardiac myocytes,which could not induce cardiomyocyte apoptosis directly.The effusion of cardiac pericardium was from blood exudation,and similar edema lesions also occured in other organs such as lung and kidney.