Fowl Adenovirus Serotype 4 Infection Research Articles

Hypervirulent fowl adenovirus serotype 4 elicits early innate immune response and promotes virus-induced cellular autophagy in the spleen

The recent emergence of hepatitis-hydropericardium syndrome caused by highly pathogenic fowl adenovirus serotype 4 (FAdV-4) has resulted in significant economic losses to the poultry industry. However, the early innate immune response of immune organs within 24 hpi and the induction of autophagy in vivo after FAdV-4 infection have not been fully elucidated. In this study, 35-day-old specific pathogen-free (SPF) chickens were artificially infected with hypervirulent FAdV-4, which resulted in a mortality rate of up to 90%. The results showed that FAdV-4 infection rapidly triggered the innate immune response in vivo of chickens, with the spleen eliciting a stronger innate immune response than the thymus and bursa. During the early stage of viral infection within 24 hpi, the main receptors TLR3/7/21, MDA5, and cGAS were activated via the NF-κB and TBK1/IRF7-dependent signaling pathways, which up-regulated production of inflammatory cytokines and type I interferons. Additionally, the expression levels of the autophagy-related molecules LC3B, Beclin1, and ATG5 were significantly up-regulated at 24 hpi, while degradation of SQSTM1/p62 was observed, suggesting that FAdV-4 infection elicits a complete autophagy response in the spleen. Besides, the colocalization of Fiber2 and LC3B suggested that FAdV-4 infection induced autophagy which benefits FAdV-4 replication in vivo. This study provides new insights into the immunoregulation signal pathways of the early innate immunity in response to hypervirulent FAdV-4 infection in vivo within 24 hpi and the close relationship between viral replication and autophagy.

Differential innate immune responses to fowl adenovirus serotype 4 infection in Leghorn male hepatocellular and chicken embryo fibroblast cells

Fowl adenovirus serotype 4 (FAdV-4) infections result in substantial economic losses in the poultry industry. Recent findings have revealed that FAdV-4 significantly suppresses the host immune response upon infection; however, the specific viral and host factors contributing to this immunomodulatory activity remain poorly characterized. Moreover, diverse cell types exhibit differential immune responses to FAdV-4 infection. To elucidate cell-specific host responses, we performed transcriptomic analysis of FAdV-4 infected leghorn male hepatocellular (LMH) and chicken embryo fibroblast (CEF) cells. Although FAdV-4 replicated more efficiently in LMH cells, it provoked limited interferon-stimulated gene induction. In contrast, FAdV-4 infection triggered robust antiviral responses in CEF cells, including upregulation of cytosolic DNA sensing and interferon-stimulated genes. Knockdown of key cytosolic DNA sensing molecules enhanced FAdV-4 replication in LMH cells while reducing interferon-stimulated gene expression. Our findings reveal cell-specific virus-host interactions that provide insight into FAdV-4 pathogenesis while identifying factors that mediate antiviral immunity against FAdV-4.

FAdV-4-induced ferroptosis affects fat metabolism in LMH cells

Ferroptosis is a form of controlled cell death that was first described relatively recently and that is dependent on the formation and accumulation of lipid free radicals through an iron-mediated mechanism. A growing body of evidence supports the close relationship between pathogenic infections and ferroptotic cell death, particularly for viral infections. Ferroptosis is also closely tied to the pathogenic development of hepatic steatosis and other forms of liver disease. Fowl adenovirus serotype 4 (FAdV-4) is a hepatotropic aviadenovirus causing hydropericardium syndrome (HPS) that is capable of impacting fat metabolism. However, it remains uncertain as to what role, if any, ferroptotic death plays in the context of FAdV-4 infection. Here, FAdV-4 was found to promote ferroptosis via the p53-SLC7A11-GPX4 axis, while ferrostain-1 was capable of inhibiting this FAdV-4-mediated ferroptotic death through marked reductions in lipid peroxidation. The incidence of FAdV-4-induced fatty liver was also found to be associated with the activation of ferroptotic activity. Together, these results offer novel insights regarding potential approaches to treating HPS.

Identification of ORF1B as a unique nonstructural protein for fowl adenovirus serotype 4

Fowl adenovirus serotype 4 (FAdV-4), the causative agent of hepatitis-hydropericardium syndrome (HHS), is a double-stranded DNA virus. Although many structural proteins have been deeply studied, the coding potential of some other open reading frames (ORFs) and the biological functions of their products during virus infection have not been fully elucidated. Here, a unique nonstructural protein ORF1B of FAdV-4 was identified and its expression kinetics along infection was analyzed. Except that of FAdV-10, a member of the same genus as FAdV-4, FAdV-4 ORF1B shared as low homologous identity as 29.2% in amino acid sequence with the other ten counterparts. Structurally, ORF1B was mapped on the N-terminal region of the genome between 1485 nt to 1808 nt and predicted to only contain two α-helix. Confocal immunofluorescence assay with homemade rabbit polyclonal antibody demonstrated that ORF1B could be simultaneously observed with structural protein Fiber 1 in FAdV-4-infected cells. Western blot further showed that ORF1B could only be detected in the infected cells but not mature virions, suggesting ORF1B was a nonstructural protein. Subsequently, the expression level of ORF1B detected by qRT-PCR and IFA was gradually decreased along with FAdV-4 infection, suggesting ORF1B was an early gene transcript. These results will lay a solid foundation to further study the biological effect of ORF1B on the replication and pathogenicity of FAdV-4.

An efficient and convenient Fiber -2- based latex agglutination test for the detection of antibodies against fowl adenovirus serotype 4 in clinical samples

To detect the antibody against fowl adenovirus serotype 4 (FAdV-4) in clinical practice, the latex agglutination test (LAT) was developed by using the Fiber-2 protein of FAdV-4 as an antigen bound to sensitized latex microspheres. The concentration, time, and temperature of sensitization latex microspheres by the Fiber-2 protein were studied and optimized; the specificity, sensitivity, and repeatability of LAT were tested; and the method developed in the study was applied. The results showed that the optimum sensitization concentration of Fiber-2 protein was 0.8 mg/mL, the time was 120 min, and the temperature was 37 ℃. Except for antiserum against FAdV-4 and FAdV-10, LAT developed in the study could not agglutinate antisera against FAdV-1, FAdV-2, FAdV-3, FAdV-4, FAdV-5, FAdV-6, FAdV-8a, FAdV-8b, FAdV-11, Newcastle disease virus, infectious bronchitis virus, egg drop syndrome virus and Clostridium perfringens. Compared with the commercial FAdV-4 ELISA Kit, the titers in 21 clinical samples were low when tested by the developed LAT method, but there was no significant difference. The coefficients of variation among different batches and the same batch of latex-sensitized particles were between 0 % and 13.3 % and 0–8.7 %, respectively. The critical value of immune protective antibody against FAdV-4 was 25, and the titers in 40.9 % of clinical samples were higher than the immune critical point. The results showed that the Fiber-2-based LAT developed in the study has the characteristics of high specificity, sensitivity and repeatability, has the advantages of free equipment, long shelf life, and fast and easy operation, and is an effective and convenient method for serological diagnosis of FAdV-4 infection and evaluating the efficacy of vaccines.

Design and Identification of a Novel Antiviral Affinity Peptide against Fowl Adenovirus Serotype 4 (FAdV-4) by Targeting Fiber2 Protein.

Outbreaks of hydropericardium hepatitis syndrome caused by fowl adenovirus serotype 4 (FAdV-4) with a novel genotype have been reported in China since 2015, with significant economic losses to the poultry industry. Fiber2 is one of the important structural proteins on FAdV-4 virions. In this study, the C-terminal knob domain of the FAdV-4 Fiber2 protein was expressed and purified, and its trimer structure (PDB ID: 7W83) was determined for the first time. A series of affinity peptides targeting the knob domain of the Fiber2 protein were designed and synthesized on the basis of the crystal structure using computer virtual screening technology. A total of eight peptides were screened using an immunoperoxidase monolayer assay and RT-qPCR, and they exhibited strong binding affinities to the knob domain of the FAdV-4 Fiber2 protein in a surface plasmon resonance assay. Treatment with peptide number 15 (P15; WWHEKE) at different concentrations (10, 25, and 50 μM) significantly reduced the expression level of the Fiber2 protein and the viral titer during FAdV-4 infection. P15 was found to be an optimal peptide with antiviral activity against FAdV-4 in vitro with no cytotoxic effect on LMH cells up to 200 μM. This study led to the identification of a class of affinity peptides designed using computer virtual screening technology that targeted the knob domain of the FAdV-4 Fiber2 protein and may be developed as a novel potential and effective antiviral strategy in the prevention and control of FAdV-4.

Fowl adenovirus serotype 4 enters leghorn male hepatocellular cells via the clathrin-mediated endocytosis pathway

Hepatitis-hydropericardium syndrome (HHS) induced by fowl adenovirus serotype-4 (FAdV-4) has caused large economic losses to the world poultry industry in recent years. HHS is characterized by pericardial effusion and hepatitis, manifesting as a swollen liver with focal necroses and petechial haemorrhage. However, the process of FAdV-4 entry into hepatic cells remains largely unknown. In this paper, we present a comprehensive study on the entry mechanism of FAdV-4 into leghorn male hepatocellular (LMH) cells. We first observed that FAdV-4 internalization was inhibited by chlorpromazine and clathrin heavy chain (CHC) knockdown, suggesting that FAdV-4 entry into LMH cells depended on clathrin. By using the inhibitor dynasore, we showed that dynamin was required for FAdV-4 entry. In addition, we found that FAdV-4 entry was dependent on membrane cholesterol, while neither the knockdown of caveolin nor the inhibition of a tyrosine kinase-based signalling cascade affected FAdV-4 infection. These results suggested that FAdV-4 entry required cholesterol but not caveolae. We also found that macropinocytosis played a role, and phosphatidylinositol 3-kinase (PI3K) was required for FAdV-4 internalization. However, inhibitors of endosomal acidification did not prevent FAdV-4 entry. Taken together, our findings demonstrate that FAdV-4 enters LMH cells through dynamin- and cholesterol-dependent clathrin-mediated endocytosis, accompanied by the involvement of macropinocytosis requiring PI3K. Our work potentially provides insight into the entry mechanisms of other avian adenoviruses.

Multiantigen epitope fusion recombinant proteins from capsids of serotype 4 fowl adenovirus induce chicken immunity against avian Angara disease

Avian Angara disease caused by fowl adenovirus serotype 4 (FAdV-4) has spread widely and brought economic losses to the poultry industry in some countries. Effective vaccines for Angara disease control are currently lacking. In this study, four capsid proteins (hexon, penton, fiber1 and fiber2) from FAdV-4 were selected, and their optimal efficient antigenic epitopes predicted by bioinformatics software were tandemly linked with the flexible linker GGGGS. Based on their amino acid sequences, the DNA sequences for the genes encoding the multiantigen epitope tandem proteins (MAETPs) FAdV4:F1, FAdV4:P, FAdV4:F2 and FAdV4:H were chemosynthesized and then ligated by T4 ligases at the cleavage sites of restriction endonucleases to construct DNAs encoding the multilinked fusion recombinant proteins (MLFRPs) used as protective antigens from avian Angara disease. These genes ligated into the expression vector pET-28a were successfully expressed using the Escherichia coli prokaryotic expression system to prepare five kinds of MLFRPs (FAdV4:F1-P-F2-H, FAdV4:F1-F2-P-H, FAdV4:F1-F2-H-P, FAdV4:F1-P-H-F2 and FAdV4:F1-H-F2-P) for use to immunize chicks. FAdV-4 was injected into MLFRP-immunized chickens, and the challenge protection rate was evaluated. FAdV4:F1-P-F2-H produced the best protection against FAdV-4, with a single immunization resulting in a 100 % protection rate, followed by FAdV4:F1-F2-P-H (83.33 %) and FAdV4:F1-F2-H-P (66.67 %). FAdV4:F1-P-H-F2 and FAdV4:F1-H-F2-P were not able to induce a good immune protection effect after one immunization. However, all of the MLFRPs were capable of protecting the host from FAdV-4 infection after two immunizations. In conclusion, these MLFRPs generated based on capsid proteins of FAdV-4 are promising candidate subunit vaccines against Angara disease.

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

β-catenin is a key component of the Wnt/β-catenin signal transduction cascade which is a highly conserved signaling pathway in eukaryotes. Increasing evidence suggests that the Wnt/β-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 β-catenin and promoted the nuclear translocation of β-catenin. Overexpression of β-catenin and LiCl treatment stimulated the accumulation of β-catenin in the nucleus, and then facilitated FAdV-4 replication. Conversely, repression of β-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 β-catenin inhibited the FAdV-4-triggered autophagy. In conclusion, the nuclear translocation of β-catenin benefits FAdV-4 replication, and suppression of β-catenin limits FAdV-4 production by inhibiting FAdV-4-induced autophagy. These findings indicated that β-catenin is an important regulator of FAdV-4 replication which can serve as a potential target for anti-FAdV-4 agents.

TMT-based quantitative proteomics analysis reveals the role of Notch signaling in FAdV-4-infected LMH cell.

Fowl adenovirus serotype 4 (FAdV-4) is recognized as a pathogen that causes hydropericardium syndrome. Irrespective of the pathway used by the virus to invade the chicken, the pathological characteristics of the disease include degeneration and necrosis of hepatocytes, formation of intranuclear inclusions, as well as inflammatory cell infiltration. Liver dysfunction constitutes one of the critical factors leading to death. Therefore, it is vital to investigate the virus-mediated severe pathological liver damage to further understand the pathogenesis of FAdV-4. Here, proteomics, a tandem mass tag (TMT)-based approach to directly analyze protein expression, was used to determine the protein expression during FAdV-4 proliferation in leghorn male hepatoma (LMH) cells. We identified 177 differentially expressed proteins associated with various biological processes and pathways. The functional enrichment analysis revealed that FAdV-4 could downregulate some signaling pathways in LMH cells, including NOD-like receptor signaling, RIG-I-like receptor signaling, NF-κB signaling, TNF signaling pathway, and Notch signaling, FoxO signaling, PI3K-Akt signaling, and autophagy. The results of proteomics screening suggested an association between FAdV-4 infection and Notch signaling in LMH in vitro, indicating that Notch signaling regulated the expression of inflammatory cytokines and interferons but not viral replication in LMH cells. These data contributed to the understanding of the immunopathogenesis and inflammopathogenesis of FAdV-4 infection and also provided valuable information for the further analysis of the molecular mechanisms underlying viral pathogenesis.

Transcriptome analysis reveals critical factors for survival after adenovirus serotype 4 infection

Fowl adenovirus serotype-4 (FAdV-4) is highly lethal to poultry, making it one of the leading causes of economic losses in the poultry industry. However, a small proportion of poultry can survive after FAdV-4 infection. It is unclear whether there are genetic factors that protect chickens from FAdV-4 infection. Therefore, the livers from chickens uninfected with FAdV-4 (Normal), dead after FAdV-4 infection (Dead) or surviving after FAdV-4 infection (Survivor) were collected for RNA-seq, and 2,649 differentially expressed genes (DEGs) were identified. Among these, many immune-related cytokines and chemokines were significantly upregulated in the Dead group compared with the Survivor group, which might indicate that death is related to an excessive inflammatory immune response (cytokine storm). Subsequently, the KEGG results for DEGs specifically expressed in each comparison group indicated that cell cycle and apoptosis-related DEGs were upregulated and metabolism-related DEGs were downregulated in the Dead group, which also validated the reliability of the samples. Furthermore, GO and KEGG results showed DEGs expressed in all three groups were mainly associated with cell cycle. Among them, BRCA1, CDK1, ODC1, and MCM3 were screened as factors that might influence FAdV-4 infection. The qPCR results demonstrated that these 4 factors were not only upregulated in the Dead group but also significantly upregulated in the LMH cells after 24 h infection by FAdV-4. Moreover, interfering with BRCA1, CDK1, ODC1, and MCM3 significantly attenuated viral replication of FAdV-4. And interfering of BRCA1, CDK1, and MCM3 had more substantial hindering effects. These results provided novel insights into the molecular changes following FAdV-4 infection but also shed light on potential factors driving the survival of FAdV-4 infection in chickens.

Hsp70 Inhibits the Replication of Fowl Adenovirus Serotype 4 by Suppressing Viral Hexon with the Assistance of DnaJC7.

Fowl adenovirus serotype 4 (FAdV-4) infection results in serious hepatitis-hydropericardium syndrome (HHS) in broilers, which has caused great economic losses to the poultry industry; however, the specific host responses to FAdV-4 remain unknown. In this study, we identified 141 high-confidence protein-protein interactions (PPIs) between the main viral proteins (Hexon, Fiber 1, Fiber 2, and Penton bases) and host proteins via a liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay. We found that heat shock protein 70 (Hsp70), the protein with the highest score, and its cofactor DnaJ heat shock protein 40 family member C7 (DnaJC7) could negatively regulate the replication of FAdV-4. Furthermore, the nucleotide binding domain (NBD) of Hsp70 and the J domain of DnaJC7 were necessary for inhibiting FAdV-4 replication. We verified that DnaJC7 as a bridge could bind to Hsp70 and Hexon, assisting the indirect interaction between Hsp70 and Hexon. In addition, we found that FAdV-4 infection strongly induced the expression of autophagy proteins and cellular Hsp70 in a dose-dependent manner. Blockage of Hexon by Hsp70 overexpression was significantly reduced when the autophagy pathway was blocked by the specific inhibitor chloroquine (CQ). Our results showed that Hsp70 was co-opted by DnaJC7 to interact with viral Hexon and inhibited Hexon through the autophagy pathway, leading to a considerable restriction of FAdV-4 replication. IMPORTANCE FAdV-4, as the main cause of HHS, has quickly spread all over the world in recent years, seriously threatening the poultry industry. The aim of this study was to identify the important host proteins that have the potential to regulate the life cycle of FAdV-4. We found that Hsp70 and DnaJC7 played crucial roles in regulating the amount of viral Hexon and extracellular viral titers. Moreover, we demonstrated that Hsp70 interacted with viral Hexon with the assistance of DnaJC7, followed by suppressing Hexon protein through the autophagy pathway. These results provide new insight into the role of the molecular chaperone complex Hsp70-DnaJC7 in FAdV-4 infection and suggest a novel strategy for anti-FAdV-4 drug development by targeting the specific interactions among Hsp70, DnaJC7 and Hexon.

Transcriptome Analysis of Genes Responding to Infection of Leghorn Male Hepatocellular Cells With Fowl Adenovirus Serotype 4.

Fowl adenovirus serotype 4 (FAdV-4) is a highly pathogenic virus with a broad host range that causes huge economic losses for the poultry industry worldwide. RNA sequencing has provided valuable and important mechanistic clues regarding FAdV-4–host interactions. However, the pathogenic mechanism and host's responses after FAdV-4 infection remains limited. In this study, we used transcriptome analysis to identify dynamic changes in differentially expressed genes (DEGs) at five characteristic stages (12, 24, 36, 48, and 60 h) post infection (hpi) with FAdV-4. A total of 8,242 DEGs were identified based on comparison of five infection stages: 0 and 12, 12 and 24, 24 and 36, 36 and 48, and 48 and 60 hpi. In addition, at these five important time points, we found 37 common upregulated or downregulated DEGs, suggesting a common role for these genes in host response to viral infection. The predicted function of these DEGs using Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analyses revealed that these DEGs were associated with viral invasion, host metabolic pathways and host immunosuppression. Interestingly, genes involved in viral invasion, probably EGR1, SOCS3, and THBS1, were related to FAdV-4 infection. Validation of nine randomly selected DEGs using quantitative reverse-transcription PCR produced results that were highly consistent with those of RNA sequencing. This transcriptomic profiling provides valuable information for investigating the molecular mechanisms underlying host–FAdV-4 interactions. These data support the current molecular knowledge regarding FAdV-4 infection and chicken defense mechanisms.

The Cellular and Viral circRNAs Induced by Fowl Adenovirus Serotype 4 Infection.

Circular RNAs (circRNAs) are a new class of noncoding RNAs that play vital roles in many biological processes. Virus infection induces modifications in cellular circRNA transcriptomes and expresses viral circRNAs. The outbreaks of Hydropericardium-hepatitis syndrome (HHS) caused by fowl adenovirus serotype 4 (FAdV-4) have resulted in huge economic losses to the poultry industry worldwide. To investigate the expression of circRNAs during FAdV-4 infection, we performed transcriptome analysis of FAdV-4-infected leghorn male hepatoma (LMH) cells. In total, 19,154 cellular circRNAs and 135 differentially expressed (DE) cellular circRNAs were identified. The characteristics of the DE cellular circRNAs were analyzed and most of them were related to multiple biological processes according to GO and KEGG enrichment analysis. The accuracy of 10 cellular circRNAs were verified by semiquantitative RT-PCR and sequencing. The change trend was consistent with the RNA sequencing results. Moreover, 2014 viral circRNAs were identified and 10 circRNAs were verified by the same methods. Our analysis showed that seven circRNAs with the same 3′ terminal and variable 5′ terminal regions were located at pTP protein and DNA pol protein of FAdV-4, which may be generated via alternative splicing events. Moreover, the expression level of viral circRNAs was closely related to the replication efficiency of the virus and partial of the viral circRNAs promoted the replication of FAdV-4. Competing endogenous RNA analysis further showed that the effects of cellular and viral circRNAs on host or viral genes may act via miRNAs. Collectively, our findings first indicate that FAdV-4 infection induced the differential expression of cellular circRNAs and FAdV-4 also expressed viral circRNAs, some of which affected FAdV-4 replication. These findings will provide new clues for further understanding FAdV-4 and provide a basis for investigating host-virus interactions.

Gga-miR-30c-5p Enhances Apoptosis in Fowl Adenovirus Serotype 4-Infected Leghorn Male Hepatocellular Cells and Facilitates Viral Replication through Myeloid Cell Leukemia-1.

Fowl adenovirus serotype 4 (FAdV-4) is the primary causative agent responsible for the hepatitis-hydropericardium syndrome (HHS) in chickens, leading to considerable economic losses to stakeholders. Although the pathogenesis of FAdV-4 infection has gained attention, the underlying molecular mechanism is still unknown. Here, we showed that the ectopic expression of gga-miR-30c-5p in leghorn male hepatocellular (LMH) cells enhanced apoptosis in FAdV-4-infected LMH cells by directly targeting the myeloid cell leukemia-1 (Mcl-1), facilitating viral replication. On the contrary, the inhibition of endogenous gga-miR-30c-5p markedly suppressed apoptosis and viral replication in LMH cells. Importantly, the overexpression of Mcl-1 inhibited gga-miR-30c-5p or FAdV-4-induced apoptosis in LMH cells, reducing FAdV-4 replication, while the knockdown of Mcl-1 by RNAi enhanced apoptosis in LMH cells. Furthermore, transfection of LMH cells with gga-miR-30c-5p mimics enhanced FAdV-4-induced apoptosis associated with increased cytochrome c release and caspase-3 activation. Thus, gga-miR-30c-5p enhances FAdV-4-induced apoptosis by directly targeting Mcl-1, a cellular anti-apoptotic protein, facilitating FAdV-4 replication in host cells. These findings could help to unravel the mechanism of how a host responds against FAdV-4 infection at an RNA level.

Cellular protein HSC70 promotes fowl adenovirus serotype 4 replication in LMH cells via interacting with viral 100K protein

Fowl adenovirus serotype 4 (FAdV-4), the predominant causative agent of hepatitis-hydropericardium syndrome (HHS), has caused severe economic losses to poultry industry since 2015. Although fiber2 and hexon have been confirmed to be the virulence-related factors, the roles of nonstructural viral proteins in pathogenicity of FAdV-4 remain poorly understood. Here, a tandem mass spectrometry (MS) was used to identify host factors interacted with 100K protein of hypervirulent FAdV-4 isolate (CH/HNJZ/2015), and 2595 cellular proteins associated with many biological processes and pathways were identified according to Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway analyses. Among the proteins, HSC70 was verified to interact with 100K through co-immunoprecipitation assay. Notably, overexpression of HSC70 promoted the replication of FAdV-4 in LMH cells, whereas blocking HSC70 with inhibitor ver-155008 markedly suppressed viral replication. Collectively, these findings suggested that many cellular proteins involved in FAdV-4 infection through interacting with 100K and HSC70 positively regulated virus replication.

Antiviral and Virucidal Activities of Camptothecin on Fowl Adenovirus Serotype 4 by Blocking Virus Replication.

Fowl adenovirus serotype 4 (FAdV-4) caused hepatitis–hydropericardium syndrome in poultry and caused huge economic losses to the poultry industry. At present, antiviral drugs have not been reported to be effective against this virus, and new treatment methods are urgently needed to treat FAdV-4. Camptothecin has been shown to have antiviral activity against various viruses; however, whether it can inhibit FAdV-4 infection remains unclear. This study aimed to explore the anti-FAdV-4 effects and mechanisms of camptothecin in vitro and in vivo. Several camptothecin treatments were used to study the antiviral activity of camptothecin on FAdV-4-infected Leghorn male hepatocellular (LMH) cells. The FAdV-4 titers of mock and camptothecin-treated infected cell cultures were determined using tissue culture infective dose assay, and the FAdV-4 copy number was determined using quantitative real-time polymerase chain reaction. In addition, the therapeutic effect of camptothecin on FAdV-4-infected chickens was also evaluated. The results showed that camptothecin significantly reduced the viral replication in LMH cells in a dose-dependent manner, resulting in a reduction in viral titer, viral copy number, and viral Hexon protein expression. Camptothecin was also found to have a significant inhibitory effect on the viral replication step. Finally, camptothecin showed anti-FAdV-4 efficacy in the chicken infection model, and the survival rate was improved. This study was novel in proving that camptothecin had a protective effect against FAdV-4, indicating its potential as an antiviral drug against FAdV-4 infection.

FAdV-4 induce autophagy via the endoplasmic reticulum stress-related unfolded protein response

Hydropericardium syndrome caused by the fowl adenovirus serotype 4 (FAdV-4) is prevalent disease in China with a high mortality rate. Many studies have demonstrated some viral infections to induce stress in the endoplasmic reticulum (ER). When the ER stress exceeds or persists, it activates autophagy, eventually triggering the onset of diseases. However, no report has ever stated FAdV-4 infection to induce ER stress-mediated autophagy. Previous studies have identified FAdV-4 infection in triggering autophagy in the hepatocytes; however, the underlying mechanism of this induction remains unknown. This study investigated the mechanism of ER stress-mediated autophagy induced by FAdV-4 infection. Here, ER stress was found to be triggered by FAdV-4 infection, as evident from the increased expression of the ER stress marker glucose-regulated protein 78, and the dilated morphology of the ER. Three pathways linked with the unfolded protein response (UPR) were found to be triggered in the hepatocellular carcinoma cell line, which included the PKR-like ER protein kinase (PERK), transcription factor 6, and inositol-requiring enzyme 1 (IRE1) pathways, respectively. Additionally, our results demonstrated that autophagy is involved in the PERK-eukaryotic initiation factor 2 subunit - C/EBP homologous protein and IRE1-c-Jun-N-terminal kinase pathways. Furthermore, treatment with the small interfering RNAs, or specific chemical inhibitors for the two pathways were found to reduce the interfering activity and could suppress the FAdV-4 replication. Collectively, these results developed new insight into the mechanisms of FAdV-4-induced autophagy by activating the ER stress-related UPR pathway and provided the experimental bases and novel ideas for developing antiviral drugs.

Metabolomic Profiling Reveals New Insight of Fowl Adenovirus Serotype 4 Infection.

Highly pathogenic fowl adenovirus serotype 4 (FAdV-4) is the causative agent of hydropericardium syndrome (HPS), which is characterized by pericardial effusion and hepatitis, and is one of the foremost causes of economic losses to the poultry industry over the last 30 years. However, the metabolic changes in cells in response to FAdV-4 infection remain unclear. In order to understand the metabolic interactions between the host cell and virus, we utilized ultra-high-performance liquid chromatography/quadrupole time-of-flight tandem mass spectrometry to analyze the metabolic profiles with hepatocellular carcinoma cell line (LMH) infected with FAdV-4. The results showed that FAdV-4 could restore metabolic networks in LMH cells and tricarboxylic acid cycle, glycolysis, and metabolism of purines, pyrimidines, alanine, aspartate, glutamate, and amino sugar and nucleotide sugar moieties. Moreover, FAdV-4 production was significantly reduced in LMH cells cultured in glucose or glutamine-deficient medium. These observations highlighted the importance of host cell metabolism in virus replication. Therefore, similarities and disparities in FAdV-4-regulation of the metabolism of host cells could help improve targeted drug and reduce infection.

Hypervirulent FAdV-4 infection induces activation of the NLRP3 inflammasome in chicken macrophages

Fowl adenovirus serotype 4 (FAdV-4) is the primary causative agent of hepatitis–hydropericardium syndrome (HHS) causing great economic losses to the world poultry industry. The exact factors responsible for the pathogenesis of hypervirulent FAdV-4 have not been completely elucidated. Hypervirulent FAdV-4 infection induces inflammatory damages in accompany with a high level of proinflammatory interleukin-1 beta (IL-1β) secretion in a variety of organs. Investigation of the mechanisms underlying hypervirulent FAdV-4-induced IL-1β secretion would contribute to understanding of the pathogenesis of FAdV-4. Here, we investigated whether FAdV-4 infection activates NLRP3 inflammasome in chicken macrophage cell line HD11. The results showed that stimulation of HD11 with hypervirulent FAdV-4 induced NLRP3- and Caspase-1-dependent secretion of IL-1β. Genetic knockdown of NLRP3 or Caspase-1 expression, a critical component of inflammasome, significantly downregulated IL-1β expression, indicating that activation of the NLRP3 inflammasome contributed to the FAdV-4-induced IL-1β secretion. Moreover, ATP signaling and potassium efflux were involved in the process of NLRP3 inflammasome activation. Our data indicated that hypervirulent FAdV-4 infection induces the activation of NLRP3 inflammasome and followed by massive secretion of IL-1β of macrophages, which thereby contribute to the inflamed lesion of tissues.