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.

Hormonal regulation of visfatin gene in avian Leghorn male hepatoma (LMH) cells.

Visfain has been extensively studied in mammals and has been shown to play an important role in obesity and insulin resistance. However, there is a paucity of information on visfatin regulation in non-mammalian species. After characterization of chicken visfatin gene, we undertook this study to determine its hormonal regulation in avian (non-mammalian) liver cells. Addition of 5 ng/mL TNFα, 100 ng/mL leptin, 1, 3, 10 or 100 ng/mL T3 for 24 h upregulated visfatin gene expression by 1.2, 1.8, 1.95, 1.75, 1.80, and 2.45 folds (P < .05), respectively, compared to untreated LMH cells. Administration of 10 ng/mL of orexin A significantly down regulated visfatin gene expression by 1.35 folds compared to control cells. In contrast, treatment with IL-6 or orexin B for 24 h did not influence visfatin mRNA abundance. These pro-inflammatory cytokines and obesity-related hormones modulate the expression of CRP, INSIG2, and nuclear orphan receptors. Hepatic CRP gene expression was significantly upregulated by IL-6, TNFα, orexin B, and T3 and down regulated by leptin and orexin A. LXR mRNA abundances were increased by orexin A, decreased by orexin B, and T3, and did not affected by IL6, TNFα, or leptin. The expression of FXR gene was induced by IL-6, leptin, and T3, but it was not influenced by TNFα, orexin A or B. CXR gene expression was up regulated by TNFα, leptin, orexin B, and T3, down regulated by 5 ng/mL orexin A, and did not affected by IL-6. INSIG2 mRNA levels were increased by TNFα (5 ng/mL), leptin (100 ng/mL), and T3 (1, 3, 10, and 100 ng/mL), decreased by orexin A, and remained unchanged with IL-6 or orexin B treatment. Together, this is the first report showing hormonal regulation of visfatin in avian hepatocyte cells and suggesting a potential role of CRP, INSIG2, and nuclear orphan receptor LXR, FXR, and CXR in mediating these hormonal effects.

Clinicopathological characterization and genomic sequence differences observed in a highly virulent fowl Aviadenovirus serotype 4.

Highly virulent fowl aviadenoviruses (genus: Aviadenovirus) represent a significant risk in poultry farming that may contribute to increased mortality rates and may adversely affect the growth performance of poultry flocks. In this study, we performed the clinicopathological characterization of a FAdV strain SHP95 isolated from a commercial farm and its whole genome sequencing. The study revealed that the isolated strain is a highly virulent serotype 4 FAdV that can cause 100% mortality in day-old specific pathogen free (SPF) chickens with a dose of 2.5 × 10(5) TCID50. At a lower viral dose (1.5 × 10(4) TCID50), the infection in day-old SPF chickens caused 40% mortality and lesions characteristic for Hepatitis-hydropericardium syndrome (HHS). The viral strain was detectable by real time PCR in chicken organs, including the lymphoid organs until day 28 after infection. The whole genome assembly of strain SHP95 revealed a size of 45,641 bp, which encodes for 42 viral open reading frame (ORF). The comparative analysis in the genome shows 98.1% similarity between strain SHP95 and other FAdV-4 genomes reported. The major differences in the genome sequence between pathogenic and non-pathogenic fowl Adenovirus were identified in the right arm of the genome.

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.

Resveratrol and (-)-Epigallocatechin-3-gallate Regulate Lipid Metabolism by Activating the AMPK Pathway in Hepatocytes.

The purpose of this study was to explore the effects of Res and EGCG on cell growth, cellular antioxidant levels, and cellular lipid metabolism in hepatocytes. In this experiment, leghorn male hepatoma (LMH) cells were used as hepatocytes. The results showed that 6.25-25 µM Res and EGCG had no adverse effects on cell viability and growth. Meanwhile, with the increasing dosage of Res and EGCG, the contents of total cholesterol (TC), total glyceride (TG), and malondialdehyde (MDA) in hepatocytes decreased significantly (p < 0.05), while the contents of glutathione peroxidase (GSH-Px), total superoxide dismutase (T-SOD), and catalase (CAT) increased significantly (p < 0.05). In addition, western blot results showed that Res and EGCG could significantly increase the expression of p-AMPK protein and reduce the expression of 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR) protein in hepatocytes (p < 0.05). Moreover, q-PCR results showed that with the increase in Res and EGCG, the expression of cholesterol- and fatty acid synthesis-related genes decreased significantly (p < 0.05). In conclusion, Res and EGCG can increase the antioxidant capacity of hepatocytes and reduce the synthesis of TC and TG in hepatocytes by activating AMPK, thereby regulating lipid metabolism in hepatocytes.

Oleic acid-induced steatosis model establishment in LMH cells and its effect on lipid metabolism.

Hepatic steatosis is a highly prevalent liver disease, yet research on it is hampered by the lack of tractable cellular models in poultry. To examine the possibility of using organoids to model steatosis and detect it efficiently in leghorn male hepatocellular (LMH) cells, we first established steatosis using different concentrations of oleic acid (OA) (0.05-0.75 mmol/L) for 12 or 24 h. The subsequent detections found that the treatment of LMH cells with OA resulted in a dramatic increase in intracellular triglyceride (TG) concentrations, which was positively associated with the concentration of the inducing OA (R2 > 0.9). Then, the modeled steatosis was detected by flow cytometry after NileRed staining and it was found that the intensity of NileRed-A was positively correlated with the TG concentration (R2 > 0.93), which demonstrates that the flow cytometry is suitable for the detection of steatosis in LMH cells. According to the detection results of the different steatosis models, we confirmed that the optimal induction condition for the establishment of the steatosis model in LMH cells is OA (0.375 mmol/L) incubation for 12 h. Finally, the transcription and protein content of fat metabolism-related genes in steatosis model cells were detected. It was found that OA-induced steatosis could significantly decrease the expression of nuclear receptor PPAR-γ and the synthesis of fatty acids (SREBP-1C, ACC1, FASN), increasing the oxidative decomposition of triglycerides (CPT1A) and the assembly of low-density lipoproteins (MTTP, ApoB). Sterol metabolism in model cells was also significantly enhanced (HMGR, ABCA1, L-BABP). This study established, detected, and analyzed an OA-induced steatosis model in LMH cells, which provides a stable model and detection method for the study of poultry steatosis-related diseases.

Sodium selenite (Na2SeO3) attenuates T-2 toxin-induced iron death in LMH cells through the ROS/PI3K/AKT/Nrf2 pathway.

T-2 toxin, is a monotrichous mycotoxin commonly found in animal feed and agricultural products that can damage tissues and organs through oxidative stress. Selenium is a trace element with favorable antioxidant effects. However, it is unclear whether T-2 toxin-induces ferroptosis in LMH cells and whether Na2SeO3 has a protective role in this process. To investigate the process of hepatic injury by T-2 toxin and its antagonistic effect by Na2SeO3, we used 20 ng/mL T-2 toxin as well as 160 nmol/L Na2SeO3 to treat the LMH cells. The results demonstrated that exposure to the T-2 toxin induced iron death by increasing the quantity of ROS, leading to oxidative damage, decreasing the quantities of SOD, GPx, and T-AOC, and increasing the accumulation of MDA and H2O2, which resulted in the accumulation of Fe2+ and the down-regulation of the manifestation of linked genes and proteins including FTH1, Gpx4, NQO-1, and HO-1. After the addition of Na2SeO3, the PI3K/AKT/Nrf2 pathway is activated by regulating the selenoproteins gene level, and the above abnormal changes are reversed. In summary, Na2SeO3 alleviated T-2 toxin-induced iron death via the PI3K/AKT/Nrf2 pathway. These study not only broaden the cytotoxic knowledge regarding T-2 toxin, but also serve as a foundation for the use of Na2SeO3 in daily life.

Transcriptomic analysis of LMH cells in response to the overexpression of a protein of Eimeria tenella encoded by the locus ETH_00028350.

A protein of Eimeria tenella (encoded by the locus ETH_00028350) homologous to Toxoplasma gondii dense granule protein 9, designated as EtHGRA9 hereafter, was reported to be expressed in all life cycle stages of E. tenella. However, no data are currently available regarding its functional properties. In the present study, a recombinant vector harboring a 741 bp gene segment encoding the mature form of EtHGRA9 was constructed and transfected into leghorn male hepatoma (LMH) cells. Then, transcriptomic analysis of the transfected LMH cells was carried out by using a high-throughput RNA-seq technology. The LMH cells overexpressing EtHGRA9 was validated by means of Western blotting as well as indirect immunofluorescence staining. The results demonstrated that the expression of 547 genes (275 upregulated genes and 272 downregulated genes) was altered by EtHGRA9. The quantitative real-time polymerase chain reaction (qRT-PCR) validation of the ten genes with differential expression between the two groups was consistent with the transcriptome analysis. According to pathway enrichment analysis for the obtained differentially expressed genes, seven pathways were significantly affected by EtHGRA9, such as cytokine-cytokine receptor interaction, MAPK signaling pathway, and protein processing in endoplasmic reticulum. Our data reveal several possible roles of EtHGRA9 in immune or inflammatory responses, which paves the way for a better understanding of the molecular interplay between E. tenella and its host.

Transcriptional changes in LMH cells induced by Eimeria tenella rhoptry kinase family protein 17.

Though a number of Eimeria tenella rhoptry kinase family proteins have been identified, little is known about their molecular functions. In the present study, the gene fragment encoding the matured peptide of E. tenella rhoptry kinase family protein 17 (EtROP17) was used to construct a recombinant vector, followed by transfection into leghorn male hepatoma (LMH) cells. Then, the transcriptional changes in the transfected cells were determined by RNA-seq. The expression of EtROP17 in LMH cells was validated by both Western blot and indirect immunofluorescence analysis. Our analysis showed that EtROP17 altered the expression of 309 genes (114 downregulated genes and 195 upregulated genes) in LMH cells. The quantitative real-time polymerase chain reaction (qRT-PCR) results of the selected differentially expressed genes (DEGs) were consistent with the RNA-seq data. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis showed that DEGs were significantly enriched in nine pathways, such as toll-like receptor signaling pathway, ECM-receptor interaction, intestinal immune network for IgA production and focal adhesion. These findings reveal several potential roles of EtROP17, which contribute to understanding the molecular mechanisms underlying the host-parasite interplay.

Chicken telomerase reverse transcriptase mediates LMH cell pyroptosis by regulating the nuclear factor-kappa B signaling pathway.

The activation of human telomerase reverse transcriptase is regulated by the nuclear factor kappa B (NF-κB) signaling pathway to various degrees to promote the occurrence and development of tumors. However, the regulatory roles of chicken telomerase reverse transcriptase (chTERT) and the NF-κB signaling pathway in chickens are still elusive, particularly in respect to the regulation of cell pyroptosis. In this study, we found that chTERT upregulated the expression of p65 and p50, downregulated the expression of IκBα, promoted the phosphorylation of p65, p50, and IκBα, and significantly increased the transcript levels of the inflammatory cytokines IFNγ, TNFα, and IL-6 in LMH cells. The activity of NF-κB was significantly decreased after siRNA-mediated chTERT silencing. The expression of chTERT and telomerase activity were also significantly decreased when the NF-κB signaling pathway was blocked by p65 siRNA, MG132 or BAY 11-7082. In cells treated with LPS, the activity of NF-κB signaling pathway and the expression of chTERT were significantly upregulated. All of the results suggested that chTERT and the NF-κB pathway could regulate each other, reciprocally. Moreover, the expression of Caspase-1, NLRP3, GSDMA, IL-18, and IL-1ß and caused membrane perforation, suggesting the development of pyroptosis by chTERT in LMH cells. And the expression of caspase-11 did not significantly increased in chTERT overexpression group. Genetic silence of NF-κB p65 or chTERT gene by siRNA suppressed the expression of these proinflammatory cytokines, indicating that chTERT mediates pyroptosis by regulating the NF-κB signaling pathway in LMH cells.

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.

Nano-selenium alleviating the lipid metabolism disorder of LMH cells induced by potassium dichromate via down-regulating ACACA and FASN.

Chromium (Cr) VI is a common environmental contaminant highly toxic to livers. To explore the protective effect of nano-selenium (NANO-Se) on broiler liver damage caused by Cr (VI), this experiment was conducted with chicken hepatocellular carcinoma cell line (LMH) as the research object, using potassium dichromate (PDC) and NANO-Se gel for culturing cells. The results indicated that: (1) in the PDC-exposure group, LMH cells being treated with 20 µmol/L PDC for 24 h, IC50 (median inhibition concentration) = 23.427 could significantly reduce cell activity (p < 0.01) which decreased over time. PDC markedly increased the concentration of triglycerides (TG) and low-density lipoprotein cholesterol (LDL-C) in LMH cells (p < 0.01), which increased over time. In addition, PDC could substantially augment the transcription and protein levels of acetyl-CoA carboxylases alpha (ACACA) and fatty acid synthase (FASN) in LMH cells (p < 0.01). (2) Compared with the PDC-exposure group, the addition of 8 µmol/L NANO-Se after 12 h of PDC treatment could significantly increase the cell viability (p < 0.01) but decreased over time; the levels of TG and LDL-C in LMH cells declined markedly (p < 0.01). In addition, the transcription and protein levels of ACACA and FASN in LMH cells were significantly reduced (p < 0.01). (3) The LMH cells were cultured in advance with 8 µmol/L NANO-Se for 12 h and then with PDC for 24 h. The obtained results were similar to the above. There were no obvious differences in TG and LDL-C levels (p > 0.05). However, significant differences were found in the activity of LMH cells and the expression of genes related to lipid metabolism (p < 0.05).All these results suggest that the exposure to PDC promotes the increase of lipid synthesis in LMH cells and causes disorders in the lipid metabolism. Moreover, NANO-Se can partially attenuate the damage caused by PDC through down-regulating of the lipid metabolism-related genes (ACACA and FASN) in LMH cells.

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.

Protective Effect of Purple Tomato Anthocyanidin on Chromium(VI)-Induced Autophagy in LMH Cells by Inhibiting Endoplasmic Reticulum Stress.

This study aimed to investigate the role of purple tomato anthocyanin (PTA) in autophagy induced by chromium(VI) in a chicken hepatocellular carcinoma cell line (LMH cells). LMH cells were exposed to Cr(VI), PTA, and Cr(VI) + PTA. The changes in endoplasmic reticulum (ER) stress, autophagy, related proteins, and COX-2 were detected. Results showed that the cell viability was reduced after Cr(VI) treatment, and the decrease was also restrained by 3-MA or PTA. Levels of ER stress-related proteins (GRP78/Bip and PERK) and COX-2 increased after Cr(VI) treatment, which resulted in an increase in autophagy-related proteins (Beclin1 and LC3-II), inhibition of autophagy pathway protein mTOR, and degradation of autophagy-related protein p62, leading to excessive autophagy and cell damage. Meanwhile, the changes of these indicators induced by Cr(VI) were alleviated by PTA. In conclusion, our study suggested that Cr(VI) can induce excessive autophagy in LMH cells, while PTA can ameliorate Cr(VI)-induced autophagy by inhibiting ER stress.

Establishment and Characterization of a Novel Tissue-specific DNA Construct and Culture System with Potential for Avian Bioreactor Generation.

Transgenic chickens are of great interest for the production of recombinant proteins in their eggs. However, the use of constitutive strong promoters or the tissue-specific ovalbumin promoter for the generation of the transgenic chickens have different drawbacks that have to be overcome in order to make chicken bioreactor an efficient production system. This prompted us to investigate the use of an alternative tissue-specific promoter, the vitellogenin promoter, which could overcome the difficulties currently found in the generation of chicken bioreactors. In the present work we establish and characterize a DNA construct consisting of a fragment of the 5´-flanking region of the chicken vitellogenin II gene cloned in a reporter vector. This construct is capable of showing the ability of the promoter to drive expression of a reporting gene in a tissue-specific manner and in a way that closely resembles physiologic regulation of vitellogenin, making it an ideal candidate to be used in the future for generation of avian bioreactors. Besides, we validate an in vitro culture system to test the performance of the DNA construct under study that could be used as a practical tool before generating any transgenic chicken. These results are important since they provide the proof of concept for the use of the vitellogenin promoter for future genetic modification of chickens bioreactors with improved characteristics in terms of quality of the recombinant protein produced.

Oleic acid induces the novel apolipoprotein O and reduces mitochondrial membrane potential in chicken and human hepatoma cells.

Non-alcoholic fatty liver disease (NAFLD) is marked by hepatic fat accumulation and reflects a spectrum of chronic liver diseases associated with obesity, impaired insulin sensitivity and dyslipidemia. Apolipoprotein O (ApoO) is a new member of the plasma apolipoprotein family that may play a role in lipid metabolism and electron transport activity of the mitochondrium. However, its physiological functions have not been elucidated yet. Based on our previous data in a non-mammalian experimental system [1], we hypothesized that hepatic expression of ApoO is tightly linked not only to diet-induced hepatosteatosis, but also to increased lipoprotein-production induced by, e.g., hormones and oxidative stress. To gain insight into a mammalian experimental system, we compared the effects of lipid loading on ApoO regulation in chicken hepatoma LMH cells with those in the human hepatoma cell line HepG2. Incubation of the cells with BSA-complexed oleic acid (OA-Alb) induced triglyceride accumulation, but did not affect cell viability. qPCR using specific primer pairs and Western blot analysis with in-house produced rabbit anti-ApoO antisera demonstrated significant increase in ApoO transcript and protein levels in both cell lines. ROS formation due to OA-Alb treatment was only slightly altered in LMH cells, indicating an intact antioxidant defense system of the cells. Oxidative stress applied by addition of H2O2 revealed induction of ApoO transcript and protein level in the same or even higher extent as monitored in the presence of OA-Alb. Upon treatment with estrogen for 24 h quantitative analysis of ApoO transcript and protein revealed increases of ApoO expression supporting the assumption that estrogen affects lipoprotein metabolism at various points. Furthermore, both cell lines showed a significant decrease of the mitochondrial membrane potential upon incubation with OA-Alb. Therefore, we assume that our findings support a role of ApoO as an effector of compromised mitochondrial function that likely accompanies the onset of non-alcoholic fatty liver disease.

Insights into leghorn male hepatocellular cells response to fowl adenovirus serotype 4 infection by transcriptome analysis.

Fowl adenovirus serotype 4 (FAdV-4), a member of the Aviadenovirus genus of the Adenoviridae family, causes hepatitis-hydropericardium syndrome (HHS) in chickens. It causes mortality of up to 80% in 3-6-week-old broilers, posing a substantial threat to the poultry industry. However, the specific host responses to the virus are not well understood. To better understand the interactions between the host and FAdV-4 and to explore the pathogenesis of this virus, a high-throughput RNA-seq technology was utilized with leghorn male hepatocellular (LMH) cells at 12, 24, and 48 h after FAdV-4 infection. We identified a total of 7000 differentially expressed genes (DEGs), which were enriched in a variety of biological processes and pathways using the Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis. Several immune related pathways, including Toll-like receptor (TLR) signaling pathway and cytokine-cytokine receptor interaction pathway, were activated after the FAdV-4 infection. The transcriptional data were validated by quantitative real-time PCR. The expression profiles of 10 genes involved in FAdV-4-infected chicken livers, including TLR2A, TLR3, TLR5, MyD88, IL12B, IL15, IL18, CCL20, TNFRSF21, and CD30, were consistent with RNA-seq profiles. By transfecting small interfering RNA into LMH cells, our results confirmed that MyD88 mediated FAdV-4-induced inflammation. To our knowledge, this was the first study to use transcriptome analysis to investigate host responses to FAdV-4 infection. These findings provide insights into the mechanisms of FAdV-4 pathogenesis and host-FAdV-4 interaction.

Induction of spotty liver disease in layer hens by infection with Campylobacter hepaticus.

Spotty liver disease (SLD) in chickens can present with variable impacts on mortality and production, ranging from sporadic mortalities of individual birds and no notable impact on production to severe reduction in egg output and increased mortality in layer flocks of greater than 1% per day. It was first described over 60 years ago and there have been sporadic reports of the disease throughout the intervening decades, particularly in the US, UK and Germany. Recently it has become of increasing concern as outbreaks of the disease have occurred more frequently, particularly in the Australian poultry industry. An understanding of the causes of the disease has proven elusive. However, recent studies of SLD have strongly implicated a novel Campylobacter species, Campylobacter hepaticus, as the causative agent. Here we demonstrate that C. hepaticus is highly invasive in LMH cells, an immortalised chicken hepatoma cell line, and can induce disease when orally delivered to mature layer birds. Challenged birds developed liver lesions, typical of those seen in field clinical cases, within 5days of challenge. The bacterium used to challenge the birds could be recovered from the diseased liver and from bile, thus demonstrating that C. hepaticus is the causative agent of chicken SLD.

Cell-culture derived fowl adenovirus serotype 4 inactivated vaccine provides complete protection for virus infection on SPF chickens.

Inclusion body hepatitis and hepatitis-hydropericardium syndrome caused by high-pathogenic fowl adenovirus serotype 4 has recently plagued Chinese poultry industry and caused huge economic losses since 2013. So far, there is no commercial vaccine available to control this disease. In this study, we reported the development of both embryo-adapted and cell-culture derived inactivated FAdV-4 vaccines and evaluated their efficacies in chicken. Compared to embryo-adapted vaccine, cell-culture derived vaccine induced significantly earlier and higher serological response measured by AGP and ELISA. After virus challenge, chicken immunized with cell-culture derived vaccine did not showed any gross and histopathological lesions, whereas inclusion body hepatitis was observed in the liver of chicken vaccinated with embryo-adapted vaccine. No mortality was observed in both the vaccinated groups. The above results suggested that cell-culture derived FAdV-4 inactivated vaccine could be a better vaccine candidate than embryo-adapted vaccine to control FADV-4 infections in China.