| Since 2015,severe outbreaks of hepatitis-hydropericardium syndrome(HHS)induced by fowl adenovirus serotype 4(FAdV-4)have been reported in several provinces of China,causing up to 80%mortality to the infected chickens and huge economic losses to the Chinese poultry industry.Although basic knowledge was gained for the genetic properties,virulence genes of FAdV-4 and the inflammatory responses induced by FAdV4 infection,there is still a lack of in-depth understanding of the mechanism of FAdV-4 pathogenesis.In the current study,a sequence alignment of the structural protein Hexon of the Chinese epidemic pathogenic FAdV-4 strain CH/HNJZ/2015 and non-pathogenic FAdV-4 strain ON1 was performed first to locate the key amino acids(aa)affecting the pathogenicity of FAdV-4,and recombinant FAdV-4 strains were constructed by exchanging the key amino acid using a reverse genetic technique.The role of Hexon protein in FAdV-4 pathogenesis was investigated by comparing the pathogenicity of the constructed recombinant viruses and parental viruses,the inflammatory responses,and mitophagy induced by viral infection.The present study lays a theoretical foundation for the understanding of FAdV-4 pathogenesis and provides new insights into prevention and control strategies of HHS.To identify the key amino acids determining the three-dimensional(3D)conformation of Hexon protein,an amino acid sequence alignment was performed for Hexon of the pathogenic FAdV-4 strain CH/HNJZ/2015 and the non-pathogenic FAdV-4 strain ON1.It was discovered that Hexon aa188,193 and 195 mutations led to conformational changes of Hexon of the pathogenic FAdV-4 strain CH/HNJZ/2015.HexonR188I mutation altered the theoretical tertiary structure of Loop 1 hypervariable region(HVR)1 of CH/HNJZ/2015 Hexon and was predicted to give Loop 1 HVR 1 the same conformation as ON1 Hexon;HexonQ193R mutation also altered the theoretical tertiary structure of Loop 1 HVR 1 of CH/HNJZ/2015 Hexon,but the predicted structure resulted from HexonQ193R mutation was different from ON1 Hexon;HexonQ195E mutation caused conformational changes at Loop 1 HVR 1 and HVR 2 of CH/HNJZ/2015 Hexon,and the altered structure completely aligned with the same regions of ON1 hexon.To study the role of Hexon aa188 in FAdV-4 pathogenesis,recombinant FAdV-4 H/H/R188I,O/O/I188R and H/O/I188R were constructed using the previously established FAdV-4 reverse genetic technique.The recombinant virus H/H/R188I was constructed by substituting Hexon aa188 in the pathogenic strain CH/HNJZ/2015 with Hexon aa188 in the non-pathogenic strain ON1,and O/O/I188R was constructed vice versa.The recombinant virus H/O/I188R was constructed using the previously constructed virus rHNJZ-hexon/ON1.Hexon of CH/HNJZ/2015 was substituted by Hexon of ON1,and Hexon aa188 was mutated back to that of CH/HNJZ/2015.Sequencing results indicated that the target amino acid was correctly replaced in the recombinant viruses,and the recombinant viruses were genetically stable and shared similar replication kinetics with parental viruses.To investigate the role of Hexon aa188 mutation in the pathogenicity of FAdV-4 and inflammatory responses induced by FAdV-4,the mRNA expression levels of Toll-like receptors(TLRs)and inflammatory cytokines in LMH cells induced by FAdV-4 were measured by real-time fluorescent quantitative polymerase chain reaction(qRT-PCR).The results indicated that HexonR188I mutation in CH/HNJZ/2015 caused significant downregulation of several TLRs and inflammatory cytokines in the infected cells,and HexonI188R mutation in ON1 caused significant up-regulation of IL-1β and IL-18 in the infected cells.The role of Hexon aa188 mutation in the pathogenicity of FAdV-4 was studied by animal tests.The results indicated that the HexonR188I mutation in CH/HNJZ/2015 greatly reduced the virulence of CH/HNJZ/2015:the mortality of chickens infected with H/H/R188I reduced to zero,and the viral loads in different organs reduced significantly.The HexonI188R mutation led to increased virulence of ON1.Chickens exhibited temporary clinical symptoms,but no mortality was induced.Autopsy and histopathological examination indicated that the organs of H/H/R188I infected chickens had no obvious lesion compared to the control group,but O/O/I188R infected chickens showed inclusion hepatitis.Viral load analysis indicated that only a background level of viral loads was detected in organs of O/O/I188R infected chickens.The results of qRTPCR and transcriptome analysis indicated that the HexonR188I mutation significantly down-regulated the inflammatory responses in the infected heart and liver,and the HexonI188R mutation had a milder effect on the in vivo inflammatory responses induced by ON1.Theses results implied that the dominant role of Hexon aa188 in FAdV-4 pathogenicity varies in FAdV-4 strains with different genetic contents.To further elucidate the role of Hexon aa188 in FAdV-4 pathogenesis,in vitro and in vivo mitophagy induced by FAdV-4 was studied by electron microscopy,Western bot and qRT-PCR.The results indicated that FAdV-4 induced different degrees of mitochondrial damage in the infected LMH cells,hepatocytes and cardiomyocytes.Compared to the H/H/R188I and ON1 infected chickens,the protein expression of Parkin increased significantly,P62 reduced significantly and LC3-II/LC3-I level increased significantly at 48 hours post-infection in the hepatocytes of CH/HNJZ/2015 and O/O/I188R infected chickens,suggesting the presence of mitophagy in the infected liver.Pathogenic FAdV-4 infection also induced a certain degree of mitophagy in the infected heart.In summary,the Hexon aa188 mutation affected mitophagy induced by FAdV-4.HexonI188R mutation enhanced mitophagy whereas HexonR188I mutation reduced the degree of mitophagy induced by FAdV-4 infection. |
