ß-catenin facilitates fowl adenovirus serotype 4 replication through enhancing virus-induced autophagy.

ß-catenin is a key component of the Wnt/ß-catenin signal transduction cascade which is a highly conserved signaling pathway in eukaryotes. Increasing evidence suggests that the Wnt/ß-catenin signaling pathway is involved in the infection of many viruses. However, its role in fowl adenovirus serotype 4 (FAdV-4) replication remains unclear. In the present study, we showed that FAdV-4 infection increased the expression of ß-catenin and promoted the nuclear translocation of ß-catenin. Overexpression of ß-catenin and LiCl treatment stimulated the accumulation of ß-catenin in the nucleus, and then facilitated FAdV-4 replication. Conversely, repression of ß-catenin by inhibitors and siRNA significantly inhibited FAdV-4 replication. Furthermore, inhibition of autophagy by 3-Methyladenine (3-MA) suppressed the FAdV-4 replication, and repression of ß-catenin inhibited the FAdV-4-triggered autophagy. In conclusion, the nuclear translocation of ß-catenin benefits FAdV-4 replication, and suppression of ß-catenin limits FAdV-4 production by inhibiting FAdV-4-induced autophagy. These findings indicated that ß-catenin is an important regulator of FAdV-4 replication which can serve as a potential target for anti-FAdV-4 agents.

FAdV-4 without Fiber-2 Is a Highly Attenuated and Protective Vaccine Candidate.

Hepatitis-hydropericardium syndrome (HHS) caused by the highly pathogenic fowl adenovirus serotype 4 (FAdV-4) has resulted in huge economic losses to the poultry industry globally. The fiber-2 gene, as a major virulence determiner, is also an important vaccine target against FAdV-4. In this study, we used a CRISPR/Cas9-based homology-dependent recombinant technique to replace the fiber-2 gene with egfp and generate a novel recombinant virus, designated FAdV4-EGFP-rF2. Although FAdV4-EGFP-rF2 showed low replication ability compared to the wild-type FAdV-4 in LMH cells, FAdV4-EGFP-rF2 could effectively replicate in LMH-F2 cells with the expression of Fiber-2. Moreover, FAdV4-EGFP-rF2 was not only highly attenuated in chickens, but also could provide efficient protection against a lethal challenge of FAdV-4. Moreover, FAdV4-EGFP-rF2 without fiber-2 could induce neutralizing antibodies at the same level as FA4-EGFP with fiber-2. These results clearly demonstrate that although fiber-2 affects the viral replication and pathogenesis of FAdV-4, it is not necessary for virus replication and induction of neutralizing antibodies; these findings provide novel insights into the roles of fiber-2 and highlight fiber-2 as an insertion site for generating live-attenuated FAdV-4 vaccines against FAdV-4 and other pathogens. IMPORTANCE Among all serotypes of fowl adenovirus, serotypes FAdV-1, FAdV-4, and FAdV-10 are unique members with two fiber genes (fiber-1 and fiber-2). Recent studies reveal that Fiber-1, not Fiber-2, directly triggers viral infection of FAdV-4, whereas Fiber-2, but not Fiber-1, has been identified as the major virulence determiner and an efficient protective immunogen for subunit vaccines. Here, we replaced fiber-2 with egfp to generate a novel recombinant virus, designated FAdV4-EGFP-rF2. In vitro and in vivo studies on FAdV4-EGFP-rF2 revealed that fiber-2 was not necessary for either virus replication or efficient protection for FAdV-4; these results not only provide a novel live-attenuated vaccine candidate against HHS, but also give new ideas for generating a FAdV-4 based vaccine vector against other pathogens.

An Outbreak of Fowl Aviadenovirus A-Associated Gizzard Erosion and Ulceration in Captive Bobwhite Quail (Colinus virginianus).

A flock of captive bobwhite quail (Colinus virginianus) experienced loose droppings, depression, and increased mortality starting at 3 wk of age. Necropsy of the affected birds revealed intestines dilated with frothy and tan fluid. Irregular dark brown fissures within the koilin layer of the gizzard were found in 20%-30% of the birds. Histologically, gizzards showed multifocal koilin degeneration or fragmentation, degeneration and necrosis of the subjacent epithelial cells, and infiltration of macrophages, lymphocytes, and heterophils. Necrotic epithelial cells occasionally contained large, smudgy, basophilic intranuclear inclusion bodies with marginated nuclear chromatin. Adenoviral paracrystalline arrays composed of icosahedral virions (60-70 nm diameter) were seen on transmission electron microscopy in the nuclei of epithelial cells in the gizzard mucosa. Adenovirus was isolated from gizzard, liver, intestine, and trachea by inoculation of specific-pathogen-free embryonated chicken eggs. Homogenates of the gizzard, liver, and intestine were positive for the adenovirus hexon gene by PCR. Sequencing of PCR amplicons confirmed the virus as fowl aviadenovirus A. The study isolates showed more than 99% and 97% nucleotide identity with quail bronchitis virus and with aviadenoviruses from gizzard erosion and ulceration (GEU) in broilers, respectively. The viral isolates showed six substitutions (G1T, C174A, A229G, C513A, T579A, and G621C) of which two were nonsynonymous (G1T and A229G), resulting in a change in the translated amino acid as A1S and S77G, respectively. These results indicate that adenoviruses of the same type or species can cause different clinical presentations in quails, e.g., bronchitis or GEU.

Artículo regular­Brote de erosiones y ulceraciones de la molleja asociadas con Aviadenovirus A del pollo en codornices de Virginia en cautiverio (Colinus virginianus). Una parvada de codornices de Virginia en cautiverio (Colinus virginianus) mostró heces acuosas, depresión y aumento de la mortalidad a partir de las tres semanas de edad. La necropsia de las aves afectadas reveló intestinos dilatados con líquido espumoso y marrón. Se encontraron fisuras irregulares de color marrón oscuro dentro de la capa de koilin de la molleja en el 20% al 30% de las aves. Histológicamente, las mollejas mostraron degeneración o fragmentación multifocal de la capa de koilin, degeneración y necrosis de las células epiteliales subyacentes e infiltración de macrófagos, linfocitos y heterófilos. Las células epiteliales necróticas contenían ocasionalmente cuerpos de inclusión intranucleares basófilos grandes, con cromatina nuclear marginada. Se observaron matrices paracristalinas adenovirales compuestas de viriones icosaédricos (60-70 nm de diámetro) en el microscopio electrónico de transmisión en los núcleos de las células epiteliales de la mucosa de la molleja. Se aisló adenovirus de molleja, hígado, intestino y tráquea mediante la inoculación de huevos embrionados de pollo libres de patógenos específicos. Los homogeneizados de la molleja, el hígado y el intestino fueron positivos para el gene del hexon del adenovirus por PCR. La secuenciación de amplicones de PCR confirmó la presencia de Aviadenovirus A del pollo. Los aislamientos del estudio mostraron una identidad mayor del 99% y 97% en la secuencia de nucleótidos con el virus de la bronquitis de codorniz y con aviadenovirus asociado con erosión y ulceración de mollejas (con las siglas en inglés GEU) en pollos de engorde, respectivamente. Los aislados virales mostraron seis sustituciones (G1T, C174A, A229G, C513A, T579A y G621C) de las cuales dos eran no-sinónimas (G1T y A229G), lo que resultó en un cambio en el aminoácido traducido como A1S y S77G, respectivamente. Estos resultados indican que los adenovirus del mismo tipo o especie pueden causar diferentes presentaciones clínicas en codornices, por ejemplo, bronquitis o erosión y ulceración de mollejas.

Experimental co-infection of variant infectious bursal disease virus and fowl adenovirus serotype 4 increases mortality and reduces immune response in chickens.

Infectious bursal disease virus (IBDV) and fowl adenovirus serotype 4 (FAdV-4) cause infectious bursal disease (IBD) and hydropericardium-hepatitis syndrome, respectively. Recently, studies have reported co-infections of poultry with IBDV and FAdV-4, which is an important problem in the poultry industry. Here, the variant IBDV strain ZD-2018-1 and FAdV-4 isolate HB1501 were used to assess the pathogenicity of co-infection in 1-day-old specific pathogen-free (SPF) chickens. Compared with chickens infected with only FAdV-4, those coinfected with IBDV and FAdV-4 showed enhanced clinical symptoms, higher mortality, more severe tissue lesions, and higher biochemical index levels. Furthermore, the expression of interleukin (IL)-6, IL-1ß, and interferon-γ mRNAs in the IBDV-FAdV-4 coinfected chickens was delayed, and the antibody response levels were significantly lower in those birds compared with the FAdV-4-infected chickens. These results indicate that co-infection with variant IBDV ZD-2018-1 and FAdV-4 HB1501 could significantly promote the pathogenicity of FAdV-4 and reduce the immune response in chickens. This study provides the foundation for further investigation of the interaction mechanism in IBDV and FAdV-4 co-infection.

A tandem mass tag-based quantitative proteomic analysis of fowl adenovirus serotype 4-infected LMH cells.

Fowl adenovirus serotype 4 (FAdV-4) is recognized as an economically important pathogen for the poultry industry worldwide. FAdV-4 infection causes a metabolic disturbance of hepatocytes, leading to hydropericardium-hepatitis syndrome (HHS) in poultry. However, the metabolic response of hepatocytes to FAdV-4 infection remains poorly investigated. Here, a tandem mass tag (TMT)-based approach was first used to quantitatively identify differentially expressed proteins (DEPs) in leghorn male hepatoma (LMH) cells infected with the virulent FAdV-4 strain GY. We identified 666 DEPs associated with many biological processes and pathways, according to Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway analyses. Functional enrichment analysis revealed that three pathways, including metabolism-related signaling pathways, apoptosis, and autophagy responses, were enriched during FAdV-4 infection. Moreover, excessive induction of metabolism-related signaling pathways by FAdV-4 infection might be associated with HHS induced by the virus. Meanwhile, among the proteins in these pathways, RRM2, SAE1, AEN, and RAD50 were verified through western blotting to be markedly altered in FAdV-4-infected LMH cells. Notably, overexpression of SAE1 inhibited the replication of FAdV-4 in vitro, whereas silencing of SAE1 expression promoted the replication of the virus. Collectively, our findings show for the first time that SAE1 is a host cellular protein that plays roles in regulating the life cycle of FAdV-4.

A novel fiber-2-edited live attenuated vaccine candidate against the highly pathogenic serotype 4 fowl adenovirus.

Recently, the outbreaks of hydropericardium-hepatitis syndrome (HHS) caused by the highly pathogenic fowl adenovirus serotype 4 (FAdV-4) have resulted in huge economic losses to the poultry industry globally. Although several inactivated or subunit vaccines have been developed against FAdV-4, live-attenuated vaccines for FAdV-4 are rarely reported. In this study, a recombinant virus FA4-EGFP expressing EGFP-Fiber-2 fusion protein was generated by the CRISPR/Cas9 technique. Although FA4-EGFP shows slightly lower replication ability than the wild type (WT) FAdV-4, FA4-EGFP was significantly attenuated in vivo compared with the WT FAdV-4. Chickens infected with FA4-EGFP did not show any clinical signs, and all survived to 14 day post-infection (dpi), whereas those infected with FAdV-4 showed severe clinical signs with HHS and all died at 4 dpi. Besides, the inoculation of FA4-EGFP in chickens provided efficient protection against lethal challenge with FAdV-4. Compared with an inactivated vaccine, FA4-EGFP induced neutralizing antibodies with higher titers earlier. All these data not only provide a live-attenuated vaccine candidate against the highly pathogenic FAdV-4 but also give a potential insertion site for developing FAdV-4-based vaccine vectors for delivering foreign antigens.

Fowl Adenovirus Serotype 4 Induces Hepatic Steatosis via Activation of Liver X Receptor-α.

Fowl adenovirus serotype 4 (FAdV-4) is a hepatotropic virus that causes severe hepatic damage characterized by basophilic intranuclear inclusion bodies, vacuolar degeneration, and multifocal necrosis in hepatocytes. Many aspects of FAdV-4 infection and pathogenesis, however, remain unknown. Here, we found that FAdV-4-induced hepatic injury is accompanied by the accumulation of oil droplets (triglycerides) in the cytoplasm of hepatocytes, a typical indicator of steatosis, in FAdV-4-infected chickens. Significant upregulation of adipose synthesis-related genes, such as liver X receptor-α (LXR-α), peroxisome proliferator-activated receptor gamma (PPAR-γ), and sterol regulatory element-binding protein-1c (SREBP-1c), and significant downregulation of low-density lipoprotein secretion-related genes and lipid oxidation- and lipid decomposition-related genes were observed in the infected chickens. FAdV-4 infection in cultured leghorn male hepatoma (LMH) cells caused similar signs of steatosis, with alterations in various lipogenesis-related genes. We eliminated the effect of LXR-α activation on FAdV-4-induced steatosis and found that treatment with an LXR-α antagonist (SR9243) and RNA interference (small interfering RNA targeting LXR-α [Si-LXR-α]) decreased the number of oil droplets and the accumulation of lipogenic genes, but treatment with an LXR-α agonist (T0901317) increased the number of oil droplets and the accumulation of lipogenic genes in the cells. Additionally, SR9243 treatment or Si-LXR-α transfection led to significant reductions in viral DNA level, protein expression, and virus production, whereas T0901317 treatment caused significant increases in viral DNA level, protein expression, and virus production. However, inhibition of SREBP-1c activity had no significant effect on virus production. Collectively, these results indicated that FAdV-4-induced steatosis involves activation of the LXR-α signaling pathway, which might be a molecular mechanism underlying the hepatic injury associated with FAdV-4 infection.IMPORTANCE Fowl adenovirus serotype 4 (FAdV-4) is an important hepatotropic adenovirus in chicken, but the underlying mechanism of FAdV-4-induced hepatic injury remains unclear. We report here that infection with FAdV-4 induced the accumulation of oil droplets (triglycerides) in the cytoplasm of hepatocytes, a typical indicator of steatosis, in the livers of chickens. FAdV-4-induced steatosis might be caused by a disrupted balance of fat metabolism, as evidenced by differential regulation of various lipase genes. The significant upregulation of liver X receptor-α (LXR-α) prompted us to investigate the interplay between LXR-α activation and FAdV-4-induced steatosis. Treatment with an agonist, an antagonist, or RNA interference targeting LXR-α in cultured leghorn male hepatoma (LMH) cells indicated that FAdV-4-induced steatosis was dependent upon LXR-α activation, which contributed to virus replication. These results provide important mechanistic insights, revealing that FAdV-4 induces hepatic steatosis by activating the LXR-α signaling pathway and highlighting the therapeutic potential of strategies targeting the LXR-α pathway for the treatment of FAdV-4 infection.

Emerging of a novel natural recombinant fowl adenovirus in China.

Outbreaks of hydropericardium syndrome and inclusion body hepatitis caused by fowl adenovirus (FAdV) have occurred in China since June 2015, resulting in significant economic loss to poultry industry. In this study, a novel FAdV, designated as AH720, with recombination among serotype FAdV-8a and FAdV-8b was isolated and characterized in China. Full genome analysis revealed that the AH720 has the genome backbone from FAdV-8b and the fibre gene from FAdV-8a. In an infection study, although AH720 was not lethal to chickens, AH720 did cause characteristic lesions of inclusion body hepatitis in the infected chickens. All these data not only provide strong evidences for the recombination among different serotype FAdVs, but also highlight the necessary for monitoring the molecular epidemiology of such recombinant FAdV to develop efficient strategies against FAdV.

Pathogenicity and Immune Responses in Specific-Pathogen-Free Chickens During Fowl Adenovirus Serotype 4 Infection.

Hydropericardium-hepatitis syndrome, a recently emerged disease of chickens, is caused by some strains of fowl adenovirus serotype 4 (FAdV-4). However, the relationship between the immune response and cytokine expression during FAdV-4 infection is largely unknown. In this study, our data showed that all chickens exhibited typical clinical signs and lesions and that the viral load was significantly increased in both the liver and thymus following FAdV-4 infection. We also found that the appearance of tissue lesions in the liver and thymus was consistent with the viral copy numbers, indicating that virus replication in systemic organs closely correlated with disease progression. In addition, the effects of FAdV-4 infection on the transcription of some avian cytokines were studied in vivo. In general, expression of the proinflammatory cytokines interleukin (IL)-2 and interferon (IFN)-α and IFN-ß in the liver and thymus was strongly upregulated. Interestingly, the expression of IL-2 was the most highly upregulated. Expression of the anti-inflammatory cytokines IL-4, IL-10, and transforming growth factor (TGF)-ß1 and TGF-ß2, were also upregulated. Moreover, we investigated both the humoral and cellular immune responses in chickens infected with FAdV-4. Compared to those in the noninfected chickens, the antibody levels in chickens infected with FAdV-4 were significantly increased within 30 days postinfection. In addition, the ratio of CD4+/CD8+ T cells was decreased in FAdV-4-infected chickens. Taken together, these findings increase our understanding of the pathogenesis of FAdV-4 in chickens and provide a foundation for additional pathogenesis studies.

Fowl Adenoviruses Type 8b Isolated from Chickens with Inclusion Body Hepatitis in Japan.

Fowl adenovirus (FAdV) type 8b isolated from chickens with inclusion body hepatitis (IBH) in Japan from 2018 to 2019 were characterized serologically and genetically. Serologically, all isolates were well neutralized by antisera against the FAdV-8b strain, but they were not neutralized by antisera against the FAdV-8a strain. Phylogenetic analysis of the part of the hexon protein gene that includes the L1 region revealed that these isolates were all identical. They were also identical to foreign strains such as the SD1356 strain isolated in China and belonged to FAdV-8b. Furthermore, the 2018-19 Japanese IBH 8b isolates were genetically identical to the SD1356 strain by phylogenetic analysis of fiber genes, but they were different from previous Japanese 8b strains. These findings suggest that the 2018-19 Japanese IBH isolates might have been introduced from other countries.

Research Note: Molecular and pathologic characterization of avian adenovirus isolated from the oviducts of laying hens in eastern Japan.

Cases of poor egg production were investigated in 2 layer farms from Ibaraki Prefecture in eastern Japan. To identify any microbial agents that may have caused the problem, necropsy, bacterial isolation, histopathology, and virus detection were performed. Members of the avian adenoviruses was detected by PCR in oviduct samples from both farms; chicken anemia virus coinfection was also confirmed in one of the farms. Avian adenovirus was isolated from the oviducts of the affected chickens on each farm. Inoculation into chick embryos showed tropism for the chorio-allantoic membrane. Stunting and hemorrhaging was observed in all infected embryos, as well as death in a few. Inoculation of 1-day-old specific pathogen-free chicks, and 400-day-old commercial hens, did not result in any significant findings. The isolated viruses were analyzed by sequencing of the hexon gene and were confirmed as fowl adenovirus type-c serotype-4 (FAdV-4). The 2 virus strains were found to be 99.29% similar to each other. One of the strains, Japan/Ibaraki/Y-H6/2016, was 99.15% similar to the KR5 strain. The other, Japan/Ibaraki/M-HB2/2016, was 99.57% similar to the KR5 strain. Fiber-2 gene analysis confirmed the identity as FAdV-4 that is closely related to nonpathogenic strains. Although nonpathogenic to chicks and laying hens, this infection can possibly cause economic damage. Perhaps the bigger concern is the effect on infected breeder operations. Because the virus is fatal to 9.09% of infected embryos, this could translate to a considerable loss in chick production owing to embryonic death. This is the first report of detection and isolation of FAdV-4 from the chicken oviduct; however, further studies are needed to elucidate its impact on both layer and breeder flocks. Indeed, FAdV-4 has negative effects on the avian reproductive tract as well.

Two novel monoclonal antibodies against fiber-1 protein of FAdV-4 and their application in detection of FAdV-4/10.

BACKGROUND: Recently, serotype 4 fowl adenovirus (FAdV-4) has spread widely and caused huge economic loss to poultry industry. However, little is known about the molecular pathogenesis of FAdV-4. Fiber protein is thought to be vital for its infection and pathogenesis. RESULTS: Two novel monoclonal antibodies (mAbs) targeting the fiber-1 protein of FAdV-4 were generated, designated as mAb 3B5 and 6H9 respectively. Indirect immunofluorescence assay (IFA) showed that both mAbs only reacted with the FAdV-4 and FAdV-10, not with other serotypes including FAdV-1, FAdV-5, FAdV-6, FAdV-7, FAdV-8 and FAdV-9 tested. Although both mAbs did not recognize the linear epitopes, they could efficiently immunoprecipitate the fiber-1 protein in LMH cells either infected with FAdV-4 or transfected with pcDNA3.1-Fiber-1. Moreover, mAb 3B5 as a capture antibody and HRP-conjugated mAb 6H9 as a detection antibody, a novel sandwich ELISA for efficient detection of FAdV-4 was generated. The limit of detection of the ELISA could reach to 1000 TCID50/ml of FAdV-4 and the ELISA could be efficiently applied to detect FAdV-4 in the clinical samples. CONCLUSION: The two mAbs specific targeting fiber-1 generated here would pave the way for further studying on the role of fiber-1 in the infection and pathogenesis of FAdV-4, and the established mAb based sandwich ELISA would provide an efficient diagnostics tool for detection of FAdV-4/10.

Molecular detection and characterization of fowl adenovirus associated with inclusion body hepatitis from broiler chickens in Egypt.

A case-control study was performed to assess prescence of inclusion body hepatitis (IBH) caused by fowl adenoviruses (FAdVs) at Kafr EL-Shiekh Governorate, Egypt, during spring, 2017. The case group consisted of 100 liver and spleen samples collected from 10 broiler chickens flocks (10 samples from each flock) suspected to be infected with IBH depending on clinical manefestations and necropsy examination. Controls were randamly selected from chickens without clinical sings or evidence of the disease on postmortem examination. Molecular screening of the disease disease in collected samples based on the DNA polymerase gene of FAdVs was carried out. Furthermore, the DNA polymerase gene sequence was determined and analyzed with published reference sequences on GeneBank. Respectively, enzyme-linked immunosorbent assay (ELISA) and reverse transcription-polymerase chain reaction (RT-PCR) were used to confirm existence of co-infection with chicken infectious anemia virus (CIAV) and/or infectious bursal disease virus (IBDV in flocks involved in the study. Using PCR, FAdV genome was detected in seven flocks in the case group and one in the control group. FAdV identified in this study revealed close genetic relationship with FAdVs-D previously identified in UK and Canada, suggesting potential virus transmission from these countries. All tested serum samples from diseased chickens were positive for CIAV infection via ELISA while none of the collected bursa of Fabricius samples tested IBDV positive by RT-PCR. Therefore, results obtained from the current study highlighted the importance of implementation of control measures against FAdV and CIAV in Egyptian poultry flocks. This study opens the door for future work toward specific identification of FAdV serotypes circulating in Egyptian poultry farms and molecular characterization of the virus based on hexon gene or full genome sequencing for better understanding of genetic diversity among FAdVs in Egypt at higher reolution.

Fowl adenovirus 9 ORF19, a lipase homolog, is nonessential for virus replication and is suitable for foreign gene expression.

Fowl adenovirus 9 (FAdV-9) has one of the largest genomes (45 kb) so far sequenced from all adenoviruses studied. Genus-specific genes located within the early (E) regions at the right and left ends of the viral genome have unknown functions except for ORF8 (Gam-1 gene), ORF22 and ORF1 (dUTPase gene). ORF19, located at the right end of the genome (nts 34,220-36,443), is predicted to encode a lipase protein and its homologs are also found in all FAdV genomes so far sequenced. The role of ORF19 in virus replication and virulence is unknown. To study ORF19 and explore its potential as a locus for foreign gene insertion, we generated one ORF19-deleted mutant virus (rFAdV-9Δ19-SwaI) and three FAdV-9Δ19-based recombinant viruses replacing ORF19 as follows: rFAdV-9Δ19-CAT and enhanced-green fluorescent protein (EGFP) cassette (CMV promoter-EGFP-poly A) in a rightward (rFAdV-9Δ19-EGFP-R) and leftward orientation (rFAdV-9Δ19-EGFP-L). All recombinant viruses were stable after three passages. In chicken hepatoma cells, rFAdV-9Δ19-SwaI, rFAdV-9Δ19-CAT and rFAdV-9Δ19-EGFP-R replicated at titers similar to that of the wild-type virus, whilst rFAdV-9Δ19-EGFP-L replicated at a much lower titer. Interestingly, FAdV-9Δ19-SwaI replicated at higher titers in cells and in embryonated eggs, respectively than those of wild-type and recombinant viruses. These observations suggest ORF19 is nonessential for replication and can be used as a novel cloning site for engineering FAdV-9-based recombinant viruses and rFAdV-9Δ19-SwaI could be used to determine its role for virus replication in vivo.

Inhibition of fowl adenovirus serotype 4 replication in Leghorn male hepatoma cells by SP600125 via blocking JNK MAPK pathway.

Fowl Adenoviruses (FAdVs) are widely-distributed pathogens across the globe. Fowl adenovirus serotype 4 (FAdV-4), the causative agent for chicken hydropericardium syndrome (HPS) with a high mortality in the infected flocks, has caused severe economic losses to the poultry industry of China in the past few years. Although vaccination against FAdV-4 has been implemented, the prevention and control of FAdV-4 infection is still not very successful. Here, we report that FAdV-4 markedly inhibits Leghorn male hepatoma (LMH) cell growth and this inhibition could be abolished by a small molecule SP600125, a JNK MAPK specific inhibitor. Furthermore, SP600125 considerably suppressed FAdV-4-induced phosphorylation of p38 and JNK MAPK. Importantly, SP600125 promoted type I interferon production associated with inhibition of FAdV-4 replication. Thus, FAdV-4 might employ the JNK MAPK pathway for the benefit of its replication, and SP600125 may have the potential of being used as an anti-virus drug for the control FAdV-4 infection.

Mechanism of fowl adenovirus serotype 4-induced heart damage and formation of pericardial effusion.

Fowl adenovirus serotype 4 (FAdV-4) is the causative agent of hydropericardium syndrome (HPS), which is characterized by the accumulation of a clear, straw-colored fluid in the pericardial sac, and high mortality rates. In order to explore the mechanism of FAdV-4-induced cardiac damage, dynamic pathology, apoptosis, and inflammatory reactions were analyzed in vivo. Moreover, we detected viral proliferation, and ultrastructure, inflammation and apoptosis of cardiomyocytes (CM) after FAdV-4 infection in vitro. The results showed that FAdV-4 impaired cardiac integrity and function by causing apoptosis and inflammation in vivo. Flow cytometry showed that CM infected with FAdV-4 did not show apoptosis in vitro. In addition, the mRNA expression of four inflammatory cytokines (interleukin (il)1B, il6, il8, and tumor necrosis factor), and activity of three myocardial enzymes were significantly different between FAdV-4 and control groups. However, in vitro, these indexes showed no significant difference between the groups. These observations collectively indicated that the heart was not the target organ of FAdV-4, and the virus may not directly lead to the occurrence of CM apoptosis and inflammation. To explore the source of pericardial effusion, we measured total protein, albumin, aspartate aminotransferase, creatine kinase isoenzyme, lactate dehydrogenase, potassium, sodium, and chloride ions in serum and pericardial effusion. Pericardial effusion was derived from vascular exudation rather than CM degeneration. Further studies are needed to investigate the exudation mechanism of vascular endothelial cells in FAdV-4 infection then weakened or eliminated pericardial effusion to minimize heart injury and/or restore damaged CM.

Fowl adenovirus serotype 4-induced apoptosis, autophagy, and a severe inflammatory response in liver.

Fowl adenovirus serotype 4 (FAdV-4) is a hepatotrophic virus that causes severe liver diseases. Upon histological examination, the most remarkable findings in the liver are small multifocal areas of necrosis and mononuclear cell infiltration, including basophilic intranuclear inclusion bodies in hepatocytes surrounded by a clear halo or which fill the entire nucleus. Here, we examined the mechanism responsible for FAdV-4-mediated hepatocyte damage in vivo and in vitro. The results showed that FAdV-4 impaired liver integrity and function, which decreased albumin and blood glucose concentrations and increased the plasma activity of aspartate aminotransferase and lactate dehydrogenase, compared with a non-infected control group (P<0.05). FAdV-4 induced hepatocyte apoptosis in a time-dependent manner in vivo and in vitro. Additionally, we found that FAdV-4 also induced the autophagy of hepatocytes, which promoted the conversion of microtubule-associated protein light chain 3 (LC3-I) to LC3-II, which is a hallmarks of autophagy. Furthermore, the mRNA expressions of interleukin (IL)-1ß, IL-6, IL-8, and tumor necrosis factor (TNF)-α in vivo and in vitro showed a statistically significant increase (P<0.05) compared to that of the control group. However, the molecular mechanisms underlying the FAdV-4-induced apoptotic and autophagic cell death remain unclear. In summation, our observations suggested that FAdV-4 induced liver injury via apoptosis, autophagy, and a severe inflammatory response.

Pancreatitis Is an Important Feature of Broilers Suffering from Inclusion Body Hepatitis Leading to Dysmetabolic Conditions with Consequences for Zootechnical Performance.

The aim of the present study was to further unravel the pathophysiologic mechanisms of inclusion body hepatitis (IBH). In a first trial, the susceptibility of specific-pathogen-free (SPF) broilers to fowl aviadenovirus (FAdV) infections was investigated. Regardless of viral dose, route of infection, and susceptibility to disease on day 1, the 3-week-old SPF broilers showed resistance to IBH, with no mortality being recorded throughout the experiment. In a second trial, SPF broilers were orally infected at 3 weeks of age with a FAdV-E strain, and their digestive and metabolic processes were monitored. The birds' performance decreased from 7 days postinfection (dpi) onward, and hepato- and pancreatomegaly were found at necropsy at 4, 7, and 10 dpi and at 7 dpi, respectively. Clinical chemistry revealed transient hyperlipasemia at 4 dpi and hyperglycemia from 4 dpi onwards, with 25% of infected birds showing glycemia levels suggestive of diabetes mellitus. Histopathology findings included typical adenoviral hepatitis in the liver, while in the pancreas, inflammation characterized by multifocal infiltrations of lymphocytes, together with shrinkage of acinar cells, loss of acinar arrangement, and hyperplasia of islet cells, was noticed. Additionally, the pancreatic tissue had tendentiously lower levels of enzyme activity, and in the ileum, the digestibility of fat was significantly impaired. Hence, our data reinforce the concept of age-related resistance to experimentally induced IBH. Additionally, we demonstrated that FAdV-induced pancreatitis in broilers interferes with the digestive process and evolves into a dysmetabolic condition that resembles diabetes, affecting the health and zootechnical performance of birds, and therefore providing an important component of IBH pathogenesis.

Genetic Characterization of Fowl Adenovirus Strains Isolated from Poultry in China.

Fowl adenoviruses (FAdVs) infect chickens worldwide, resulting in global economic losses in the poultry industry. We examined the strains present in chickens in regions of China where infections are particularly prevalent. Fifteen FAdV strains were successfully isolated in the field. The L1 loop region of the hexon gene was sequenced to genetically identify the FAdV isolates. By comparing these sequences to adenovirus reference strain sequences using phylogenetics, 15 adenovirus strains were found to cluster into two distinct species. One cluster containing 12 strains belonged to the fowl adenoviruses C species and serotyped as FAdV-4. The other cluster containing three strains belonged to the fowl adenoviruses E species and serotyped as FAdV-10. To our knowledge, this is the first report of the existence of fowl adenoviruses E in China. Furthermore, at least two types of fowl adenovirus strains are predominant among poultry in China. Cumulatively, this study helps lays the groundwork for future research on the pathogenicity and potential treatment measures for FAdV infections in chickens.

Phylogenetic Analyses of Fowl Adenoviruses (FAdV) Isolated in China and Pathogenicity of a FAdV-8 Isolate.

Fowl adenoviruses (FAdVs) have a worldwide distribution and are associated with a variety of diseases, causing considerable economic losses to the poultry industry. We characterized 10 FAdVs isolated from China in 2015-2016 and assessed the pathogenicity of a FAdV-8 strain in specific-pathogen-free (SPF) chickens. Phylogenetic analysis of a hexon gene revealed that only 1 of the 10 isolates belonged to FAdV-8, whereas others belonged to FAdV-4, indicating that Chinese FAdVs were mainly FAdV-4 in recent years. The pathogenicity experiment of the FAdV-8 strain CH/SD/2015/09 showed that no clinical signs were observed in infected chickens. Necropsy displayed mild necrotic foci and petechial hemorrhage of livers collected at 5 days postinfection (dpi). Histopathologic examination identified the presence of intranuclear inclusion bodies in hepatocytes. No virus was detected in oral and cloacal swabs at 5 dpi, and only viral DNA could be measured in kidneys collected at the same time. The results revealed that CH/SD/2015/09 had no obvious pathogenicity in 5-wk-old SPF chickens, which could provide a better understanding about the pathogenicity of the FAdV-8 serotype.