Search

Fulminant viral hepatitis (FH) represents a significant clinical challenge, with its pathogenesis not yet fully elucidated. Heat shock protein (HSP)70, a molecular chaperone protein with a broad range of cytoprotective functions, is upregulated in response to stress. However, the role of HSP70 in FH remains to be investigated. Notably, HSP70 expression is upregulated in the livers of coronavirus-infected mice and patients. Therefore, we investigated the mechanistic role of HSP70 in coronavirus-associated FH pathogenesis. FH was induced in HSP70-deficient (HSP70 KO) mice or in WT mice treated with the HSP70 inhibitor VER155008 when infected with the mouse hepatitis virus strain A59 (MHV-A59). MHV-A59-infected HSP70 KO mice exhibited significantly reduced liver damage and mortality. This effect was attributed to decreased infiltration of monocyte-macrophages and neutrophils in the liver of HSP70 KO mice, resulting in lower levels of inflammatory cytokines such as IL-1β, TNFα, and IL-6, and a reduced viral load. Moreover, treatment with the HSP70 inhibitor VER155008 protected mice from MHV-A59-induced liver damage and FH mortality. In summary, HSP70 promotes coronavirus-induced FH pathogenesis by enhancing the infiltration of monocyte-macrophages and neutrophils and promoting the secretion of inflammatory cytokines. Therefore, HSP70 is a potential therapeutic target in viral FH intervention., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Avian hepatitis E virus (HEV) has resulted in significant economic losses in the poultry industry. There is currently no commercial vaccination available to prevent avian HEV infection. Previously, a novel epitope ( 601 TFPS 604 ) was discovered in the ORF2 protein of avian HEV. In this study, peptides were synthesized and assessed for their ability to provide immunoprotecting against avian HEV infection in poultry. Twenty-five Hy-Line Variety Brown laying hens were randomly divided into five groups; groups 1 to 3 respectively immunized with RLLDRLSRTFPS, PETRRLLDRLSR (irrelevant peptide control), or truncated avian HEV ORF2 protein (aa 339-606), while group 4 (negative control) was mock-immunized with PBS and group 5 (normal control) was not immunized or challenged. After the challenge, all hens in groups 2 and 4 showed seroconversion, fecal virus shedding, viremia, alanine aminotransferase (ALT) level increasing, liver lesions and HEV antigen in the liver. There were no pathogenic effects in other groups. Collectively, all of these findings showed that hens were completely protected against avian HEV infection when they were immunized with the peptide containing TFPS of the avian HEV ORF2 protein., Competing Interests: Declaration of competing interest The authors declare that they have no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier B.V. All rights reserved.)

Duck hepatitis A virus 1 (DHAV-1) is the primary cause of duck viral hepatitis, leading to sudden mortality in ducklings and significant economic losses in the duck industry. However, little is known about how DHAV-1 affects duckling liver at the molecular level. We conducted an analysis comparing the expression patterns of mRNAs and miRNAs in DHAV-1-infected duckling livers to understand the underlying mechanisms and dynamic changes. We identified 6,818 differentially expressed mRNAs (DEGs) and 144 differentially expressed microRNAs (DEMs) during DHAV-1 infection. Functional enrichment analysis of DEGs and miRNA target genes using gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) revealed their potential involvement in innate antiviral immunity, mitophagy, and pyroptosis. We constructed coexpression networks of mRNA-miRNA interactions and confirmed key DEMs (novel-mir333, novel-mir288, novel-mir197, and novel-mir71) using RT-qPCR. Further investigation demonstrated that DHAV-1 activates the RLRs signaling pathway, disrupts mitophagy, and induces pyroptosis. In conclusion, DHAV-1-induced antiviral immunity is closely linked to mitophagy, suggesting it could be a promising therapeutic target., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Duck hepatitis A virus 1 (DHAV-1) is a highly contagious etiological agent that causes acute hepatitis in young ducklings. MicroRNAs (miRNAs) play important regulatory roles in response to pathogens. However, the interplay between DHAV-1 infection and miRNAs remains ambiguous. We characterized and compared miRNA and mRNA expression profiles in duck embryo fibroblasts cells (DEFs) infected with DHAV-1. In total, 36 and 96 differentially expressed (DE) miRNAs, and 4110 and 2595 DE mRNAs, were identified at 12 and 24 h after infection. In particular, 126 and 275 miRNA-mRNA pairs with a negative correlation were chosen to construct an interaction network. Subsequently, we identified the functional annotation of DE mRNAs and target genes of DE miRNAs enriched in diverse Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways, which may be important for virus resistance, cell proliferation, and metabolism. Moreover, upregulated miR-222a could negatively regulate DHAV-1 replication in DEFs and downregulate integrin subunit beta 3 (ITGB3) expression by targeting the 3' untranslated region (3'UTR), indicating that miR-222a may modulate DHAV-1 replication via interaction with ITGB3. In conclusion, the results reveal changes of mRNAs and miRNAs during DHAV-1 infection and suggest miR-222a as an antiviral factor against DHAV-1., Competing Interests: Author JH was employed by the company Shandong GreenBlue Biotechnology Co. Ltd. Economic development zone. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Sui, Zhang, Jiang, Yu, Xu, Wang, Zhu, Xie, Hu and Jiang.)

Specificity Protein 3 (SP3) is a newly identified regulator of tumor growth and invasiveness in humans. In this study, we identified and characterized the function of duck SP3 ( duSP3 ). The full-length cDNA sequence of the duSP3 gene was cloned via rapid amplification of cDNA ends. It contained 2468 nucleotides, including a 111 base pair (bp) 5'-untranslated region (UTR), 215 bp 3'-UTR, and 2142 bp open reading frame (ORF), which encoded a 713 amino acid (AA) strongly conserved with Avian SP3 . Tissue specificity analysis demonstrated that duSP3 was constitutively expressed in the eight tissues tested: liver, spleen, lung, heart, kidney, thymus, breast, and leg; and low expression levels were observed in all tissues, except the spleen and thymus. Quantitative real-time polymerase chain reaction (qRT-PCR) analysis revealed that duSP3 expression rapidly increased in vitro after stimulation with both the hepatitis virus (DHV-1) and polyriboinosinic polyribocytidylic acid (poly(I:C)). However, the expression under these treatments varied in kidney and liver tissues; in the liver, duSP3 increased significantly at 36 h after the DHV-1 treatment and peaked at 72 h after poly(I:C) stimulation. These results suggested that SP3 may play a positive role in immune responses against viral infections in ducks.

Background and Aims: Equine hepacivirus (EqHV) is phylogenetically the closest relative of HCV and shares genome organization, hepatotropism, transient or persistent infection outcome, and the ability to cause hepatitis. Thus, EqHV studies are important to understand equine liver disease and further as an outbred surrogate animal model for HCV pathogenesis and protective immune responses. Here, we aimed to characterize the course of EqHV infection and associated protective immune responses., Approach and Results: Seven horses were experimentally inoculated with EqHV, monitored for 6 months, and rechallenged with the same and, subsequently, a heterologous EqHV. Clearance was the primary outcome (6 of 7) and was associated with subclinical hepatitis characterized by lymphocytic infiltrate and individual hepatocyte necrosis. Seroconversion was delayed and antibody titers waned slowly. Clearance of primary infection conferred nonsterilizing immunity, resulting in shortened duration of viremia after rechallenge. Peripheral blood mononuclear cell responses in horses were minimal, although EqHV-specific T cells were identified. Additionally, an interferon-stimulated gene signature was detected in the liver during EqHV infection, similar to acute HCV in humans. EqHV, as HCV, is stimulated by direct binding of the liver-specific microRNA (miR), miR-122. Interestingly, we found that EqHV infection sequesters enough miR-122 to functionally affect gene regulation in the liver. This RNA-based mechanism thus could have consequences for pathology., Conclusions: EqHV infection in horses typically has an acute resolving course, and the protective immune response lasts for at least a year and broadly attenuates subsequent infections. This could have important implications to achieve the primary goal of an HCV vaccine; to prevent chronicity while accepting acute resolving infection after virus exposure., (© 2021 The Authors. Hepatology published by Wiley Periodicals LLC on behalf of American Association for the Study of Liver Diseases.)

Metformin is not only the first-line medication for the treatment of type 2 diabetes, but it is also effective as an anti-inflammatory, anti-oxidative and anti-tumor agent. However, the effect of metformin during viral hepatitis remains elusive. Using an adenovirus (Ad)-induced viral hepatitis mouse model, we found that metformin treatment significantly attenuated liver injury, with reduced serum aspartate transaminase (AST) and alanine transaminase (ALT) levels and liver histological changes, presumably via decreased effector T cell responses. We then demonstrated that metformin reduced mTORC1 activity in T cells from infected mice, as evidenced by decreased phosphorylation of ribosome protein S6 (p-S6). The inhibitory effects on the mTORC1 signaling by metformin was dependent on the tuberous sclerosis complex 1 (TSC1). Mechanistically, metformin treatment modulated the phosphorylation of dynamin-related protein 1 (Drp-1) and mitochondrial fission 1 protein (FIS1), resulting in increased mass in effector T cells. Moreover, metformin treatment promoted mitochondrial superoxide production, which can inhibit excessive T cell activation in viral hepatitis. Together, our results revealed a protective role and therapeutic potential of metformin against liver injury in acute viral hepatitis via modulating effector T cell activation via regulating the mTORC1 pathway and mitochondrial functions., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Xu, Wang, Chen, Soong, Chen, Cai, Liang and Sun.)

The liver is a central regulator of metabolic homeostasis and serum metabolite levels. Hepatocytes are the functional units of the liver parenchyma and not only responsible for turnover of biomolecules but also act as central immune signaling platforms. Hepatotropic viruses infect liver tissue, resulting in inflammatory responses, tissue damage and hepatitis. Combining well-established in vitro and in vivo model systems with transcriptomic analyses, we show that type I interferon signaling initiates a robust antiviral immune response in hepatocytes. Strikingly, we also identify IFN-I as both, sufficient and necessary, to induce wide-spread metabolic reprogramming in hepatocytes. IFN-I specifically rewired tryptophan metabolism and induced hepatic tryptophan oxidation to kynurenine via Tdo2, correlating with altered concentrations of serum metabolites upon viral infection. Infected Tdo2-deficient animals displayed elevated serum levels of tryptophan and, unexpectedly, also vast increases in the downstream immune-suppressive metabolite kynurenine. Thus, Tdo2-deficiency did not result in altered serum homeostasis of the tryptophan to kynurenine ratio during infection, which seemed to be independent of hepatocyte-intrinsic compensation via the IDO-axis. These data highlight that inflammation-induced reprogramming of systemic tryptophan metabolism is tightly regulated in viral hepatitis., Competing Interests: The authors have declared that no competing interests exist.

Duck hepatitis A virus genotypes 3 (DHAV-3) has become the most prevalent pathogen of duck viral hepatitis (DVH) in Asian duck industry in recent years. Previous studies on the pathogenic mechanism of DHAV-3 mainly focused on examine host gene expression levels. However, the study about host protein expression levels has not been reported. For this, proteomics analysis on livers of infected 7-day-old Pekin ducks with DHAV-3 112803 strain was performed to screen differentially expressed proteins. A total of 3,385 proteins were identified, and we found 39 proteins in the challenged group (CH) were significantly up-regulated and 15 proteins were significantly down-regulated in comparison with control group (CON). GO results showed that 9 of the top 20 GO terms were involved in type I interferon regulation, and the KEGG pathway enrichment results showed that innate immune responses were significantly enriched, such as RIG-1-like, Toll-like and NOD-like receptor signaling pathways. Notably, interaction between 11 up-regulation proteins promoted interferon-induced protein synthesis and supported viral genome replication, which could aggravate inflammatory response and liver damage. These findings, together with RT-qPCR verification of related genes, support the view that the type I interferon may play an extremely important role in the pathogenic mechanism of DHAV-3., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Various human disorders are cured by the use of licorice, a key ingredient of herbal remedies. Glycyrrhizic acid (GL), a triterpenoid glycoside, is the aqueous extract from licorice root. Glycyrrhetinic acid (GA) has been reported to be a major bioactive hydrolysis product of GL and has been regarded as an anti-inflammatory agent for the treatment of a variety of inflammatory diseases, including hepatitis. However, the mechanism by which GA inhibits viral hepatic inflammatory injury is not completely understood. In this study, we found that, by consecutively treating mice with a traditional herbal recipe, licorice plays an important role in the detoxification of mice. We also employed a murine hepatitis virus (MHV) infection model to illustrate that GA treatment inhibited activation of hepatic inflammatory responses by blocking high-mobility group box 1 (HMGB1) cytokine activity. Furthermore, decreased HMGB1 levels and downstream signaling triggered by injection of a neutralizing HMGB1 antibody or TLR4 gene deficiency, also significantly protected against MHV-induced severe hepatic injury. Thus, our findings characterize GA as a hepatoprotective therapy agent in hepatic infectious disease not only by suppressing HMGB1 release and blocking HMGB1 cytokine activity, but also via an underlying viral-induced HMGB1-TLR4 immunological regulation axis that occurs during the cytokine storm. The present study provides a new therapy strategy for the treatment of acute viral hepatitis in the clinical setting., Competing Interests: Declaration of Competing Interest All authors have no conflict of interest to disclose., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Objective: The Fragile X mental retardation (FMR) syndrome is a frequently inherited intellectual disability caused by decreased or absent expression of the FMR protein (FMRP). Lack of FMRP is associated with neuronal degradation and cognitive dysfunction but its role outside the central nervous system is insufficiently studied. Here, we identify a role of FMRP in liver disease., Design: Mice lacking Fmr1 gene expression were used to study the role of FMRP during tumour necrosis factor (TNF)-induced liver damage in disease model systems. Liver damage and mechanistic studies were performed using real-time PCR, Western Blot, staining of tissue sections and clinical chemistry., Results: Fmr1 null mice exhibited increased liver damage during virus-mediated hepatitis following infection with the lymphocytic choriomeningitis virus. Exposure to TNF resulted in severe liver damage due to increased hepatocyte cell death. Consistently, we found increased caspase-8 and caspase-3 activation following TNF stimulation. Furthermore, we demonstrate FMRP to be critically important for regulating key molecules in TNF receptor 1 (TNFR1)-dependent apoptosis and necroptosis including CYLD, c-FLIP S and JNK, which contribute to prolonged RIPK1 expression. Accordingly, the RIPK1 inhibitor Necrostatin-1s could reduce liver cell death and alleviate liver damage in Fmr1 null mice following TNF exposure. Consistently, FMRP-deficient mice developed increased pathology during acute cholestasis following bile duct ligation, which coincided with increased hepatic expression of RIPK1, RIPK3 and phosphorylation of MLKL., Conclusions: We show that FMRP plays a central role in the inhibition of TNF-mediated cell death during infection and liver disease., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2020. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Fowl adenovirus (FAdV) is the causative agent of inclusion body hepatitis (IBH) in chickens with significant economic losses due to high mortality and poor production. It was objectives of the study to attenuate and determine the molecular characteristic of FAdV isolate (UPM1137) of Malaysia passages in primary chicken embryo liver (CEL) cells. The cytopathic effect (CPE) was recorded and the present of the virus was detected by polymerase chain reaction (PCR). Nucleotide and amino acid changes were determined and a phylogenetic tree was constructed. The pathogenicity and immunogenicity of the virus at passage 35 (CEL35) with virus titre of 106.7TCID50/mL was determined in day old specific pathogen free (SPF) chicks via oral or subcutaneous route of inoculation. The study demonstrated that the FAdV isolate was successfully propagated and attenuated in CEL cells up to 35th consecutive passages (CEL35) with delayed of CPE formation within 48 to 72 post inoculation (pi) from CEL20 onwards. The virus caused typical CPE with basophilic intranuclear inclusion bodies, refractile and clumping of cells. The virus is belong to serotype 8b with substitution of amino acid at position 44, 133 and 185 in L1 loop of hexon gene and in knob of fiber gene at position 348 and 360 at CEL35. It is non-pathogenic, but immunogenic in SPF chickens. It was concluded that the FAdV isolate was successfully attenuated in CEL cells with molecular changes in major capsid proteins which affect its infectivity in cell culture and SPF chickens., Competing Interests: The authors have declared that no competing interests exist.

Ethnopharmacological Relevance: Formula Le-Cao-Shi (LCS) is a traditional Chinese medicine (TCM), which has long been used as a folk remedy against hepatitis B in China. The present study was conducted to evaluate the anti-hepatitis B effects of aqueous extract of LCS in vivo and in vitro., Materials and Method: we investigated the anti-HBV effects of LCS in vivo and in vitro with duck hepatitis B model and HepG2.2.15 cell line model, respectively. The serologic and cellular biomarkers and the histopathological changes were examined., Results: By a duck hepatitis B model, the extract of LCS was found to restrain the expressions of duck hepatitis B surface antigen (DHBsAg), hepatitis B e antigen (DHBeAg), and HBV-DNA (DHBV-DNA). Moreover, LCS could decrease the levels of aspartate and alanine aminotransferases (AST and ALT) and ameliorate duck liver histological lesions. Correspondingly, in a HepG2.2.15 cellular model, LCS could also significantly inhibit the secretions of HBsAg and HBeAg., Conclusion: LCS exerted potent anti-hepatitis effects against the infection of HBV. The above results demonstrated the first-hand experimental evidences for the anti-hepatitis B efficiency of LCS. Our study provides a basis for further exploration and development of this promising compound prescription to treat hepatitis B disease., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Duck hepatitis A virus (DHAV) causes an infectious disease that mainly affects 1- to 4-week-old ducklings, resulting in considerable loss to the duck industry. Although there have been many studies on DHAV in recent years, the effects on host infection and pathogenesis of DHAV-1 remain largely unknown. This study investigated the effects of the DHAV-1 structural protein VP3 on DHAV-1 virus adsorption and apoptosis to explore the role of VP3 in the viral life cycle. The effects of DHAV-1 VP3 and an antibody against the protein on virion adsorption was analyzed by qRT-PCR. The results showed that the virus copy number for the rabbit anti-VP3 IgG-treated group was significantly lower than that for the negative control group but higher than that for the rabbit anti-DHAV-1 IgG-treated group. This result indicates that VP3 mediates DHAV-1 virus adsorption but that it is not the only protein that involved in this process. In addition, a eukaryotic recombinant plasmid, pCAGGS/VP3, was transfected into duck embryo fibroblasts (DEFs), and the apoptotic rate was determined by DAPI staining, the TUNEL assay and flow cytometry. DAPI staining showed nucleus fragmentation and nuclear edge shifting. TUNEL assay results revealed yellow nuclei, and flow cytometry indicated a significant increase in the apoptotic rate. In addition, qRT-PCR revealed increased in the transcriptional levels of the apoptotic caspase-3, -8 and -9, with the largest increase for caspase-3, followed by caspase-9 and caspase-8. Enzyme activity analysis confirmed these results. Furthermore, the VP3 protein decreased the mitochondrial membrane potential, and the transcriptional levels of the proapoptotic factors Bak, Cyt c and Apaf-1 in the mitochondrial apoptotic pathway were significantly upregulated. These data suggest that expression of VP3 in DEFs induces apoptosis and may primarily activate caspase-3-induced apoptosis through mitochondrion-mediated intrinsic pathways. The findings provide scientific data to clarify DHAV-1 infection and pathogenesis.

Hepatitis E is an important global disease, causing outbreaks of acute hepatitis in many developing countries and sporadic cases in industrialized countries. Hepatitis E virus (HEV) infection typically causes self-limiting acute hepatitis but can also progress to chronic disease in immunocompromised individuals. The immune response necessary for the prevention of chronic infection is T cell-dependent; however, the arm of cellular immunity responsible for this protection is not currently known. To investigate the contribution of humoral immunity in control of HEV infection and prevention of chronicity, we experimentally infected 20 wild-type (WT) and 18 immunoglobulin knockout (JH-KO) chickens with a chicken strain of HEV (avian HEV). Four weeks postinfection (wpi) with avian HEV, JH-KO chickens were unable to elicit anti-HEV antibody but had statistically significantly lower liver lesion scores than the WT chickens. At 16 wpi, viral RNA in fecal material and liver, and severe liver lesions were undetectable in both groups. To determine the role of cytotoxic lymphocytes in the prevention of chronicity, we infected 20 WT and 20 cyclosporine and CD8 + antibody-treated chickens with the same strain of avian HEV. The CD8 + lymphocyte-depleted, HEV-infected chickens had higher incidences of prolonged fecal viral shedding and statistically significantly higher liver lesion scores than the untreated, HEV-infected birds at 16 wpi. The results indicate that CD8 + lymphocytes are required for viral clearance and reduction of liver lesions in HEV infection while antibodies are not necessary for viral clearance but may contribute to the development of liver lesions in acute HEV infection., (© 2019 Wiley Periodicals, Inc.)

Calcium (Ca 2+ ) is a vital secondary messenger in T lymphocytes regulating a vast array of important events including maturation, homeostasis, activation, and apoptosis and can enter the cell through CRAC, TRP, and Ca V channels. Here we describe a mutation in the L-type Ca 2+ channel Ca V 1.4 leading to T lymphocyte dysfunction, including several hallmarks of immunological exhaustion. Ca V 1.4-deficient mice exhibited an expansion of central and effector memory T lymphocytes, and an upregulation of inhibitory receptors on several T cell subsets. Moreover, the sustained elevated levels of activation markers on B lymphocytes suggest that they are in a chronic state of activation. Functionally, T lymphocytes exhibited a reduced store-operated Ca 2+ flux compared to wild-type controls. Finally, modifying environmental conditions by herpes virus infection exacerbated the dysfunctional immune phenotype of the Ca V 1.4-deficient mice. This is the first example where the mutation of a Ca V channel leads to T lymphocyte dysfunction, including the upregulation of several inhibitory receptors, hallmarks of T cell exhaustion, and establishes the physiological importance of Ca V channel signaling in maintaining a nimble immune system., (Copyright © 2019 Fenninger, Han, Stanwood, Nohara, Arora, Choi, Munro, Pfeifer, Shanina, Horwitz and Jefferies.)

Interferon-induced proteins with tetratricopeptide repeats (IFITs) are a family of proteins strongly induced downstream of type I interferon signaling. The function of IFITs has been investigated extensively in mammals. IFIT5 is the sole protein in this family found in birds and little information is available about the function of avian IFIT5. In this study, duck IFIT5 was cloned from peripheral blood mononuclear cells. Multiple amino acid sequence alignment and phylogenetic analysis showed that duck IFIT5 is highly homologous to chicken IFIT5. Tissue specificity analysis demonstrated that duck IFIT5 was ubiquitously expressed in all examined tissues of five-day-old ducklings, with the highest expression levels in heart, followed by thymus, cerebrum, liver, and lung; kidney expressed the lowest. Quantitative real-time PCR (qRT-PCR) analysis revealed that duck IFIT5 expression rapidly increased both in vitro and in vivo after stimulation with polyinosinic:polycytidylic acid [poly (I:C)] and infection with virulent duck hepatitis A virus type 3 (DHAV-3), respectively. Altogether, these results indicate that the expression of duck IFIT5 is positively correlated with viral load and may play an important role in the immune response to DHAV-3 infection. This study lays a foundation for further research into the innate antiviral immune responses of ducklings., (Copyright © 2019. Published by Elsevier Ltd.)

To develop an indirect enzyme-linked immunosorbent assay(I-ELISA) method based on 3A protein of duck hepatitis A virus type 1(DHAV-1) for detection of DHAV-1 antibody, the recombinant protein 3A of DHAV-1 was expressed in E.coli and detected by Western blotting with DHAV-1 infected duck serum. A 3A-ELISA method using the expressed 3A protein as coating antigen for the detection of antibodies to DHAV-1 was developed. The optimal antigen, serum and enzyme-labeled antibody dilutions were 1:200(6.185 μg/ml), 1:20 and 1:2000, respectively. The optimal blocking buffer was 5% BSA. The cutoff value was determined to be 0.274, and the analytical sensitivity was 1:1280. There was no cross reaction between DHAV-1 infected duck serum and other common pathogenic duck serum, indicating that I-ELISA could be used to detect DHAV-1 infected duck serum. The coefficients of variation(CVs) were lower than 10%. The concordance between the I-ELISA based on the 3A subunit of DHAV-1 and that based on the whole DHAV-1 particle was 92.7%. Taken together, the 3A-ELISA method is a highly sensitive and specific test that could be used for screening for DHAV-1 infection and monitoring DHAV-1 antibody., (Copyright © 2019 Elsevier B.V. All rights reserved.)

We previously showed that potassium channel tetramerization domain containing 9 (KCTD9) is aberrantly expressed in natural killer (NK) cells in patients with hepatitis B virus-associated acute-on-chronic liver failure and mice with experimental fulminant hepatitis. However, the mechanism underlying the regulation of NK cell function and fulminant hepatitis progression by KCTD9 is unknown. Here, we investigated the role of Kctd9 in regulation of early development, maturation, and function of NK cells using Kctd9- knockout mice. Compared to wild-type mice, Kctd9 -deficient mice exhibited impaired NK cell lineage commitment, as evidenced by selective reduction in the refined NK progenitors, and incomplete NK cell maturation, as manifested by a higher proportion of CD11b - NK cells and a lower percentage of CD11b + NK cells with high proliferative potential. Moreover, Kctd9 -depleted NK cells displayed insufficient IFN-γ production, degranulation, and granzyme B production in response to cytokine stimulation, and attenuated cytotoxicity to tumor cells in vitro . The defect in NK cells was further supported by ameliorated liver damage and improved survival in Kctd9 -deficient mice following murine hepatitis virus strain-3 (MHV-3) infection, which otherwise leads to immune-mediated fulminant hepatitis, a phenotype homologous to that caused by NK cell depletion in wild-type mice. Further investigation to identify the underlying mechanism revealed that Kctd9 deficiency hindered the expression of transcription factors, including Ets1, Nfil3, Eomes, and Id2 in NK cells. Collectively, our data reveal that Kctd9 acts as a novel regulator for NK cell commitment, maturation, and effector function.

Duck hepatitis A virus type 1 (DHAV-1) is one of the most lethal pathogens in the duck industry. The attenuated vaccine (the CH60 strain) is cultivated through serial passage in chicken embryos and is widely used for the prevention and control of the disease. However, the specific mechanism underlying its adaptation in chicken embryos has not been fully elucidated. In this study, we first infected chicken embryo fibroblasts (CEFs) with the DHAV-1 CH60 strain. The peak of viral proliferation occurred within 36-48 h post-infection. The different DHAV-1 strains significantly induced the expression of IFNα, IFNγ, and Suppressor of cytokine signaling 3 (SOCS3) in CEFs, and we found that SOCS3 overexpression significantly promoted viral replication. Furthermore, SOCS3 overexpression significantly inhibited the expression of IFNα but promoted the expression of IFNγ. In addition, SOCS3 overexpression clearly decreased the mRNA levels of STAT1 and STAT3 in the Janus kinase (JAK)-STAT signaling pathway and inhibited the expression of the antiviral proteins MX1 and OASL. Immune-precipitation assays indicated that SOCS3 and IFNα do not physically interact. Subcellular localization of SOCS3 and IFNα revealed that SOCS3 was mainly located in the nucleus and cytoplasm, while IFNα was located only in the cytoplasm. Co-localization of these two proteins was not observed in the cytoplasm. In conclusion, the DHAV-1 CH60 strain may inhibit the expression of IFNα by increasing the SOCS3 protein and SOCS3 can in turn decrease STAT1 and STAT3 mRNA levels, thereby inhibiting the antiviral protein MX1 and ultimately promoting viral proliferation, indirectly assisting in viral adaptation in chicken embryos.

Duck virus hepatitis (DVH) caused by duck hepatitis A virus type 1 (DHAV-1) is an acute and highly contagious disease affecting young ducklings. The VP1 protein is one of the major structural proteins of DHAV-1 carries critical epitopes responsible for the induction of neutralizing antibodies. In this study, we have successfully constructed an immune phage display VHHs library against DHAV-1 with the size of 6 × 10 6 colonies. A nanobody (Nb) against VP1 protein of DHAV-1, named Nb25, was identified from the immunized phage display library. Nb25 could react with the conserved linear B-cell epitope of 174 PAPTST 179 in DHAV-1 VP1, even though Nb25 showed no neutralizing activity to DHAV-1. To the best of our knowledge, this is the first report about preparation of anti-DHAV-1 Nbs and identification of the specific conserved linear B-cell epitope of DHAV-1 with Nb, which will facilitate the serologic diagnosis of DHAV-1 infection., (Copyright © 2018 Elsevier Inc. All rights reserved.)

The avian adeno-associated virus (AAAV) has been proved to be an efficient gene transfer vector for human gene therapy and vaccine research. In this experiment, an AAAV-based vaccine was evaluated for the development of a vaccine against duck hepatitis a virus type 1 (DHAV-1). The major capsid VP1 gene was amplified and subcloned into pFBGFP containing the inverted terminal repeats of AAAV, and then the recombinant baculovirus rBac-VP1 was generated. The recombinant AAAV expressing the VP1 protein (rAAAV-VP1) was produced by co-infecting Sf9 cells with rBac-VP1 and the other 2 baculoviruses containing AAAV functional genes and structural genes respectively, and confirmed by electron microscopy, Western blotting and immunofluorescence assays. Quantitative real-time PCR revealed that the titer of rAAAV-VP1 was about 9 × 1012 VG/mL. Immunogenicity was studied in ducklings. One day ducklings were injected intramuscularly once with rAAAV-VP1. Serum from rAAAV-VP1-vaccinated ducklings showed a systemic immune response evidenced by VP1-specific enzyme-linked immunosorbent assay and virus neutralization test. Furthermore, all ducklings inoculated with rAAAV-VP1 were protected against DHAV-1 challenge. The data of quantitative real-time RT-PCR from livers of challenged ducklings also showed that the level of virus copies in rAAAV-VP1 group was significantly lower than that of the PBS group. Collectively, these results demonstrate that the AAAV-based vaccine is a potential vaccine candidate for the control of duck viral hepatitis.

Background: Fulminant hepatitis (FH) is a serious threat to human life, accompanied by massive and rapid necroinflammation. Kupffer cells, the major immune cell population involved in innate immune responses, are considered to be central for FH. Fibrinogen-like protein 2 (Fgl2) is a pro-coagulant protein that is substantially induced in macrophages upon viral infection, and Fgl2 depletion represses murine hepatitis virus strain 3 (MHV-3) infection. Clara cell 10 kDa (CC10) protein is a secretory protein with anti-inflammatory properties in allergic rhinitis and asthma. However, its mechanisms of action and pathogenic roles in other disease are still unclear. In this study, we aimed to determine the role of CC10 in FH and the regulation of Fgl2 by CC10. Methods: A mouse FH model was established by peritoneal injection of MHV-3. The mice received CC10 protein through tail vein injection before viral infection. Survival rate, liver function, liver histology, fibrin deposition, and necrosis were examined. The regulatory effect of CC10 on Fgl2 expression was investigated using THP-1 cells and mouse peritoneal macrophages in vitro . Results: In the mouse FH model induced by MHV-3, the survival rate increased from 0 to 12.5% in the CC10 group compared to that in the saline-only control group. Meanwhile, the levels of ALT and AST in serum were significantly decreased and liver damage was reduced. Furthermore, hepatic Fgl2, TNF-α, and IL-1β expression was obviously downregulated together with fibrin deposition, and hepatocyte apoptosis was reduced after administration of CC10 protein. In vitro , CC10 was found to significantly inhibit the expression of Fgl2 in IFN-γ-treated THP-1 cells and MHV-3-infected mouse peritoneal macrophages by western blot and real-time PCR. However, there was no direct interaction between CC10 and Fgl2 as shown by co-immunoprecipitation. Microarray investigations suggested that HMG-box transcription factor 1 (HBP1) was significantly low in CC10-treated and IFN-γ-primed THP-1 cells. HBP1-siRNA treatment abrogated the inhibitory effect of CC10 on Fgl2 expression in Human Umbilical Vein Endothelial cells (HUVECs). Conclusion: CC10 protects against MHV-3-induced FH via suppression of Fgl2 expression in macrophages. Such effects may be mediated by the transcription factor HBP1.

The liver bears unique immune properties that support both immune tolerance and immunity, but the mechanisms responsible for clearance versus persistence of virus-infected hepatocytes remain unclear. Here, we dissect the factors determining the outcome of antiviral immunity using recombinant adenoviruses that reflect the hepatropism and hepatrophism of hepatitis viruses. We generated replication-deficient adenoviruses with equimolar expression of ovalbumin, luciferase, and green fluorescent protein driven by a strong ubiquitous cytomegalovirus (CMV) promoter (Ad-CMV-GOL) or by 100-fold weaker, yet hepatocyte-specific, transthyretin (TTR) promoter (Ad-TTR-GOL). Using in vivo bioluminescence to quantitatively and dynamically image luciferase activity, we demonstrated that Ad-TTR-GOL infection always persists, whereas Ad-CMV-GOL infection is always cleared, independent of the number of infected hepatocytes. Failure to clear Ad-TTR-GOL infection involved mechanisms acting during initiation as well as execution of antigen-specific immunity. First, hepatocyte-restricted antigen expression led to delayed and curtailed T-cell expansion-10,000-fold after Ad-CMV-GOL versus 150-fold after Ad-TTR-GOL-infection. Second, CD8 T-cells primed toward antigens selectively expressed by hepatocytes showed high PD-1/Tim-3/LAG-3/CTLA-4/CD160 expression levels similar to that seen in chronic hepatitis B. Third, Ad-TTR-GOL but not Ad-CMV-GOL-infected hepatocytes escaped being killed by effector T-cells while still inducing high PD-1/Tim-3/LAG-3/CTLA-4/CD160 expression, indicating different thresholds of T-cell receptor signaling relevant for triggering effector functions compared with exhaustion. Conclusion: Our study identifies deficits in the generation of CD8 T-cell immunity toward hepatocyte-expressed antigens and escape of infected hepatocytes expressing low viral antigen levels from effector T-cell killing as independent factors promoting viral persistence. This highlights the importance of addressing both the restauration of CD8 T-cell dysfunction and overcoming local hurdles of effector T-cell function to eliminate virus-infected hepatocytes., (© 2018 The Authors. HEPATOLOGY published by Wiley Periodicals, Inc., on behalf of the American Association for the Study of Liver Diseases.)

CD8 T cells protect the liver against viral infection, but can also cause severe liver damage that may even lead to organ failure. Given the lack of mechanistic insights and specific treatment options in patients with acute fulminant hepatitis, we develop a mouse model reflecting a severe acute virus-induced CD8 T cell-mediated hepatitis. Here we show that antigen-specific CD8 T cells induce liver damage in a perforin-dependent manner, yet liver failure is not caused by effector responses targeting virus-infected hepatocytes alone. Additionally, CD8 T cell mediated elimination of cross-presenting liver sinusoidal endothelial cells causes endothelial damage that leads to a dramatically impaired sinusoidal perfusion and indirectly to hepatocyte death. With the identification of perforin-mediated killing as a critical pathophysiologic mechanism of liver failure and the protective function of a new class of perforin inhibitor, our study opens new potential therapeutic angles for fulminant viral hepatitis.

Historically, fowl adenovirus (FAdV) associated inclusion body hepatitis (IBH) was considered a secondary disease in broiler chickens associated with immunosuppression. However, we previously reported the occurrence of IBH as a primary disease in the broiler chicken industry in Canada as a result of infections with various FAdV serotypes. Therefore, the objectives of this study were to develop an immunization strategy in broiler breeders using live FAdV 11-1047 and FAdV8a-TR59 to confer homologous and heterologous protection in broiler progeny against IBH and to study the efficacy of natural exposure of naïve broiler breeders to a vaccine virus from live FAdV vaccinated birds as an immunization technique. Broiler breeders vaccinated orally with FAdV8a-TR59 (1 × 10 4 TCID 50 /bird) and FAdV11-1047 (1 × 10 4 TCID 50 /bird), FAdV8a-TR59 (1 × 10 6 TCID 50 /bird) and FAdV11-1047 (1 × 10 6 TCID 50 /bird) or FAdV8b (1 × 10 6 TCID 50 /bird) transferred substantial levels of neutralizing antibodies to their progeny. The efficacy of maternal antibodies was studied by challenging 14-day old broiler chickens with 1 × 10 7 TCID 50 of FAdV2-685, FAdV7-x11a like, FAdV8a-TR59, FAdV8b-SK or FAdV11-1047 which are the dominant serotypes causing IBH outbreaks in Canada. Broiler chickens from the low and high dose vaccinated breeders were significantly protected against all serotypes of FAdV (P < 0.05). Comingling of unvaccinated broiler breeders with FAdV-vaccinated broiler breeders was an effective immunization technique for in-contact naïve birds. This study confirms that IBH can be effectively controlled in Canada by vaccination of broiler breeder parents with a bivalent vaccine containing live FAdV8a-TR59 and FAdV11-1047., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Duck viral hepatitis type I is a rapidly spreading infection lethal in young ducklings, caused by the duck hepatitis A virus (DHAV). Vaccination of breeder ducks is a common practice to control DHAV. However, maintaining proper maternal antibody levels in large flocks is difficult. Therefore, a simple vaccination strategy that can induces stable high antibody levels through mass vaccination is desirable. We evaluated a DHAV vaccination strategy for breeder ducks involving oral administration under field conditions, and examined the kinetics of antibody response in the ducks and their progeny. The strategy included a primary intramuscular vaccination, followed by secondary and tertiary oral vaccinations. Five weeks after the primary vaccination, virus-neutralizing antibody titers increased by 8.4 ± 1.3 log 2 . The titers remained stable at around 9.0 ± 1.1 log 2 for up to 36 weeks. None of the progeny died when challenged with virulent DHAV at 1, 7 or 14 days of age. The transfer percentage of antibodies from the breeder ducks to their progeny was 12.8 ± 3.0%. When antibody levels of the progeny were measured from the day of hatching to 20 days of age, the levels steadily declined, reaching a mean titer of 0 log 2 at 20 days. The half-life of the maternally derived antibodies against DHAV was 3.4 ± 1.1 days. Our vaccination strategy might be effective in breeder ducks because it can be easily applied and induced strong immunity. Moreover, our results might provide a foundation for the mechanistic study of maternally derived antibodies in passive protection., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Background & Aims: The macrophage scavenger receptor 1 (Msr1, also called SRA) is a pattern recognition receptor primarily expressed on myeloid cells, which plays an important role in the maintenance of immune homeostasis. Since MSR1 expression was upregulated in the livers of patients with fulminant hepatitis (FH), we investigated the functional mechanism of Msr1 in FH pathogenesis., Methods: Msr1-deficient (Msr1 -/- ) mice and their wild-type (WT) littermates were infected with mouse hepatitis virus strain-A59 (MHV-A59) to induce FH, and the levels of tissue damage, serum alanine aminotransferase, inflammatory cytokines and complement component 5a (C5a) were measured and compared. Liver injury was studied after MHV infection with or without neutrophil depletion., Results: Our results showed that Msr1 -/- mice were resistant to MHV-induced hepatitis. Treatment with the C5a receptor antagonist (C5aRa) diminished the differences in inflammatory responses and liver injury between MHV-infected wild-type and Msr1 -/- mice, suggesting that C5a-induced pro-inflammatory response plays a critical role in the Msr1-mediated regulation of FH pathogenesis. We demonstrated that Msr1 efficiently enhanced transforming growth factor-activated kinase-1 phosphorylation in neutrophils upon MHV-A59 stimulation, thereby promoting the activation of the extracellular signal-regulated kinase pathway and subsequent NETosis formation. Moreover, we provided evidence that blockage of Msr1 attenuated the liver damage caused by MHV-A59 infection., Conclusions: Msr1 promotes the pathogenesis of virus-induced FH by enhancing induction of neutrophil NETosis and subsequent complement activation. Targeting Msr1 may be employed as a new immunotherapeutic strategy for FH., Lay Summary: Virus-induced fulminant hepatitis (FH) is a disease with a high mortality worldwide. Enhanced levels of macrophage scavenger receptor 1 (Msr1) in the liver of patients with FH and of murine experimental FH indicated Msr1 plays a role in the pathogenesis of FH. Herein, we demonstrate that mice deficient in Msr1 are resistant to FH induced by MHV-A59, and the Msr1 inhibitor fucoidan suppresses the progression of FH in mice. Our study suggests that use of drugs inhibiting MSR1 function could be beneficial to patients with FH., (Copyright © 2017 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.)

Duck virus hepatitis caused by duck hepatitis A virus (DHAV) is an acute and contagious disease. To better understand the pathogenic mechanism of DHAV-3 in ducklings, an infection experiment was performed. Our results showed that typical symptoms were observed in the infected ducklings. DHAV-3 could infect many tissues, leading to pathological lesions, especially on the livers and spleen, and the host immune responses are activated in infection. Real-time quantitative PCR demonstrated that expression of many innate immune-related genes was mostly up-regulated in the livers and spleen, and antiviral innate immune response was established, but not sufficient to restrict the virus replication of lethal dose. Many major pattern recognition receptors (PRRs) (RIG-1, MDA5, and TLR7) are involved in the host immune response to DHAV-3, and the expression of interferon (IFNα, IFNβ and IFNγ) and antiviral proteins (MX, OAS and PKR) are also up-regulated in the liver and spleen. The expression of most cytokines (IL-1β, IL-2 and IL-6) was also up-regulated to different degrees and was various; the expression of IL-2 increased most significantly in liver. Our data provide a foundation for further study of the pathogenicity of duck virus hepatitis and extend our understanding of the immune responses of ducklings to DHAV-3 infection., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Ferritin heavy polypeptide 1 (FTH1) plays a pivotal role in response to viral infections. FTH1 expression is modulated by various pathogens, but the regulatory mechanisms are unknown. We firstly construct duck hepatitis virus 1 (DHV-1) infection model, including morbid ducklings, non-morbid ducklings and control ducklings. Then the mRNA expression of duck FTH1 (duFTH1) was measured mRNA expression of duck FTH1 (duFTH1) in the liver and spleen after duck hepatitis virus 1 (DHV-1) infection using quantitative polymerase chain reaction (qPCR) and found that duFTH1 mRNA was down-regulated significantly in morbid ducklings (liver, P < 0.01; spleen, P < 0.05) compared with the control ducklings. We also found that duFTH1 expression was significantly higher in the spleen (P < 0.01) and liver (P < 0.05) of non-morbid ducklings than in morbid ducklings. Moreover, DNA methylation of the duFTH1 promoter was examined by bisulfite sequencing (BSP) and we found that the duFTH1 promoter was hypomethylated, the relative methylation was only 5.9% and 2.0% in the morbid ducklings and non-morbid ducklings, respectively. The promoter contained a -55 C/T mutation in 75% of non-morbid ducklings, and this polymorphism affected promoter activity. Further analysis suggested that this mutation altered the binding site of the transcription factor NRF1. Binding of NRF1 to the FTH1 promoter was confirmed by electrophoretic mobility shift assay (EMSA) analysis. Thus, our findings revealed the NRF1 was a negative regulator, and lossed of binding of NRF1 to duFTH1 promoter due to -55C/T mutation enhances duFTH1 expression in non-morbid ducks, which provided molecular insights into the effect of duFTH1 expression via promoter polymorphisms, but not DNA methylation, in response to DHV-1 challenge., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Duck hepatitis A virus (DHAV) infection is characterized by an acute, rapidly spreading that affects young ducklings. DHAV-1 or DHAV-3 infection is prevalent, and simultaneous co-infection with both viruses has recently become increasingly frequent in the domestic duck farms. In this study, we developed a bivalent live attenuated vaccine (DHV-HSBP100 and AP-04203P100) for DHAV-1 and DHAV-3 and reported the protective efficacy and safety of the vaccine. At 1-day-old, the ducklings received a bivalent vaccine via intramuscular injection. The immunized ducklings showed effective and rapid protection against virulent DHAV-1 and DHAV-3 at 2 or 3 days post vaccination. Moreover, the ducklings showed a potent humoral immune response that peaked at 3 weeks and were maintained at 6 weeks after vaccination. The bivalent vaccine was safe; ducklings administered 10 doses of bivalent vaccines showed no clinical signs, mortality, gross lesions, and body weight changes compared with those observed in the negative controls. Ducklings vaccinated with a bivalent vaccine were evaluated for tissue tropism and viral replication of vaccine strains. Both bivalent vaccine strains were detected in various organs, and the highest virus replication was detected in the kidneys, among the tested organs. No interference occurred during the replication of both vaccine strains. Thus, these experiments suggest that bivalent vaccines would be useful as a promising and practical strategy for control DHAV outbreaks caused by DHAV-1 and DHAV-3 in duck farms., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Fowl adenovirus (FAdV) is comprised of five species (A to E) and 12 serotypes (1-7, 8a, 8b, 9-11). Inclusion body hepatitis (IBH) is caused by FAdV-7, 8a, 8b (species E) and FAdV-2 and 11 (species D). Commercial vaccines against IBH are not available in Canada. Autogenous FAdV broiler breeder vaccines are now used in some areas where outbreaks of IBH are occurring. The objective of this study was to evaluate the efficacy of a bivalent (species D and E) live and an inactivated FAdV broiler breeder vaccine in protecting broiler chicks against IBH through maternal antibody (MtAb) transfer. FAdV seronegative broiler breeders (n = 300/group) received either a live or inactivated bivalent (FAdV-8b-SK + FAdV-11-1047) vaccine. The live vaccine (1 × 10 4 TCID 50 of each virus/bird) was given orally once at 16 weeks of age and the inactivated vaccine (1 × 10 6 TCID 50 of each virus + 20% Emulsigen D) was given intramuscularly at 16 and 19 weeks of age. Controls (n = 150) were given saline orally. The inactivated vaccine group was boosted 3 weeks later with the same vaccine. Neutralizing antibodies (NAb) in sera (n = 10) were detected at 19, 22, 30 and 48 weeks of age. NAb were able to neutralize various FAdV serotypes within species D and E. Mean NAb were similar in the both live and killed vaccine groups at 19, 30 and 48 weeks and ranged from 2.4 to 3.7 log 10 . Approximately 26 ± 7% of MtAbs were passively transferred through eggs to day-old chicks. Progeny challenged with a lethal dose (1 × 10 7 TCID 50 /bird intramuscularly) of FAdV-8b-SK, FAdV-11-1047, or FAdV-2-685 (n = 90/group) at 14 days post-hatch (dph) showed 98-100% protection in broiler chicks to homologous or heterologous FAdV challenges. Our data suggests that a bivalent live and an inactivated FAdV vaccine are equally effective and have the potential for the control of IBH., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Despite drug advances for Hepatitis C virus (HCV), re-infections remain prevalent in high-risk populations. Unfortunately, the role of preexisting viral immunity and how it modulates re-infection is unclear. GBV-B infection of common marmosets is a useful model to study tissue immune responses in hepacivirus infections, and in this study we re-challenged 4 animals after clearance of primary viremia. Although only low-to-absent viremia was observed following re-challenge, GBV-B viral RNA was detectable in liver, confirming re-infection. Microscopic hepatic lesions indicated severe-to-mild lymphocyte infiltration and fibrosis in 3 out of 4 animals. Further, GBV-B-specific T cells were elevated in animals with moderate-to-severe hepatopathology, and up to 3-fold increases in myeloid dendritic and activated natural killer cells were observed after infection. Our data indicate that occult hepacivirus re-infections occur and that new liver pathology is possible even in the presence of anti-hepacivirus T cells and in the absence of high viremia., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Rift Valley fever virus (RVFV) leads to varied clinical manifestations in animals and in humans that range from moderate fever to fatal illness, suggesting that host immune responses are important determinants of the disease severity. We investigated the immune basis for the extreme susceptibility of MBT/Pas mice that die with mild to acute hepatitis by day 3 post-infection compared to more resistant BALB/cByJ mice that survive up to a week longer. Lower levels of neutrophils observed in the bone marrow and blood of infected MBT/Pas mice are unlikely to be causative of increased RVFV susceptibility as constitutive neutropenia in specific mutant mice did not change survival outcome. However, whereas MBT/Pas mice mounted an earlier inflammatory response accompanied by higher amounts of interferon (IFN)-α in the serum compared to BALB/cByJ mice, they failed to prevent high viral antigen load. Several immunological alterations were uncovered in infected MBT/Pas mice compared to BALB/cByJ mice, including low levels of leukocytes that expressed type I IFN receptor subunit 1 (IFNAR1) in the blood, spleen and liver, delayed leukocyte activation and decreased percentage of IFN-γ-producing leukocytes in the blood. These observations are consistent with the complex mode of inheritance of RVFV susceptibility in genetic studies.

Because of their better immunogenicity and the improved protection they afford, live attenuated vaccines derived from serial passaging in an abnormal host are widely used to protect humans or animals from certain pathogens. Here, we used a virulent and a chicken embryo-attenuated duck hepatitis A virus genotype 1 to compare the different regulated immune responses induced by viruses with differing virulence. In this study, the attenuated strains had lower protein expression levels than the virulent strains as identified by immunohistochemistry. This may be caused by apparent codon usage bias selected during passage. Furthermore, lower translation efficiency led to decreased viral replication, which is highly dependent on non-structural viral protein expression. Although the two strains had differing levels of virulence, both could induce strong innate immune responses and robust Tc or Th cell populations during the early stages of the immune response. However, due to fixed single nucleotide polymorphisms (SNPs) selected by passage, the virulent and attenuated strains may induce differing immune responses, with stronger Tc cell immunity induced by the attenuated strain in the spleen and thymus and stronger Tc cell immunity induced by the virulent strain in the liver, lung, bursa of Fabricius and Harderian gland. Four immune related genes (RIG-1, MDA5, IFN-β, and IL-6) were highly differentially expressed in the Harderian gland, bursa of Fabricius and thymus. This study has provided further information about differences in virus-host interactions between duck hepatitis A viruses of differing virulence.

Background: Liver inflammation or hepatitis is a result of pluripotent interactions of cell death molecules, cytokines, chemokines and the resident immune cells collectively called as microenvironment. The interplay of these inflammatory mediators and switching of immune responses during hepatotoxic, viral, drug-induced and immune cell-mediated hepatitis decide the fate of liver pathology. The present review aimed to describe the mechanisms of liver injury, its relevance to human liver pathology and insights for the future therapeutic interventions., Data Sources: The data of mouse hepatic models and relevant human liver diseases presented in this review are systematically collected from PubMed, ScienceDirect and the Web of Science databases published in English., Results: The hepatotoxic liver injury in mice induced by the metabolites of CCl 4 , acetaminophen or alcohol represent necrotic cell death with activation of cytochrome pathway, formation of reactive oxygen species (ROS) and mitochondrial damage. The Fas or TNF-alpha induced apoptotic liver injury was dependent on activation of caspases, release of cytochrome c and apoptosome formation. The ConA-hepatitis demonstrated the involvement of TRAIL-dependent necrotic/necroptotic cell death with activation of RIPK1/3. The alpha-GalCer-induced liver injury was mediated by TNF-alpha. The LPS-induced hepatitis involved TNF-alpha, Fas/FasL, and perforin/granzyme cell death pathways. The MHV3 or Poly(I:C) induced liver injury was mediated by natural killer cells and TNF-alpha signaling. The necrotic ischemia-reperfusion liver injury was mediated by hypoxia, ROS, and pro-inflammatory cytokines; however, necroptotic cell death was found in partial hepatectomy. The crucial role of immune cells and cell death mediators in viral hepatitis (HBV, HCV), drug-induced liver injury, non-alcoholic fatty liver disease and alcoholic liver disease in human were discussed., Conclusions: The mouse animal models of hepatitis provide a parallel approach for the study of human liver pathology. Blocking or stimulating the pathways associated with liver cell death could unveil the novel therapeutic strategies in the management of liver diseases.

Our view on liver macrophages in the context of health and disease has been reformed by the recognition of a remarkable heterogeneity of phagocytes in the liver. Liver macrophages consist of ontogenically distinct populations termed Kupffer cells and monocyte-derived macrophages. Kupffer cells are self-renewing, resident and principally non-migratory phagocytes, serving as sentinels for liver homeostasis. Liver injury triggers Kupffer cell activation, leading to inflammatory cytokine and chemokine release. This fosters the infiltration of monocytes into the liver, which give rise to large numbers of inflammatory monocyte-derived macrophages. Liver macrophages are very plastic and adapt their phenotype according to signals derived from the hepatic microenvironment (e.g. danger signals, fatty acids, phagocytosis of cellular debris), which explains their manifold and even opposing functions during disease. These central functions include the perpetuation of inflammation and hepatocyte injury, activation of hepatic stellate cells with subsequent fibrogenesis, and support of tumor development by angiogenesis and T cell suppression. If liver injury ceases, specific molecular signals trigger hepatic macrophages to switch their phenotype towards reparative phagocytes that promote tissue repair and regression of fibrosis. Novel strategies to treat liver disease aim at targeting macrophages. These interventions modulate Kupffer cell activation (e.g. via gut-liver axis or inflammasome formation), monocyte recruitment (e.g. via inhibiting chemokine pathways like CCR2 or CCL2) or macrophage polarization and differentiation (e.g. by nanoparticles). Evidence from mouse models and early clinical studies in patients with non-alcoholic steatohepatitis and fibrosis support the notion that pathogenic macrophage subsets can be successfully translated into novel treatment options for patients with liver disease., Lay Summary: Macrophages (Greek for "big eaters") are a frequent non-parenchymal cell type of the liver that ensures homeostasis, antimicrobial defense and proper metabolism. However, liver macrophages consist of different subtypes regarding their ontogeny (developmental origin), differentiation and function. Understanding this heterogeneity and the critical regulation of inflammation, fibrosis and cancer by macrophage subsets opens promising new options for treating liver diseases., (Copyright © 2017 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.)

In recent years, a critical role for T cell immunoglobulin mucin domain 3 (Tim-3) and its ligand Galectin-9 (Gal-9) has emerged in infectious disease, autoimmunity and cancer. Manipulating this immune checkpoint may have immunotherapeutic potential and could represent an alternative approach for improving immune responses to viral infections and cancer. The woodchuck (Marmot monax) infected by woodchuck hepatitis virus (WHV) represents an informative animal model to study HBV infection and HCC. In the current study, the cDNA sequences of woodchuck Tim-3 and Gal-9 were cloned, sequenced and characterized. The extracellular domain of Tim-3 cDNA sequence consisted of 576bp coding sequence (CDS) that encoded 192 amino acids. The 1076bp full-length Gal-9 cDNA sequence consisted of 1059bp coding sequence (CDS) that encoded 352 amino acids with a molecular weight of 39.7kDa. The phylogenetic tree analysis revealed that the woodchuck Tim-3 and Gal-9 had the closest genetic relationship with Ictidomys tridecemlineatus. The result of quantification PCR analysis showed that ubiquitous expression of Gal-9 but not Tim-3 in different tissues of naive woodchucks. Elevated liver Gal-9 expression was observed in woodchucks with chronic WHV infection. Moreover, a polyclonal antibody against the extracellular domain of woodchuck Tim-3 were generated and identified by flow cytometry. Our results serve as a foundation for further insight into the role of Tim-3/Galectin-9 signaling pathway in viral hepatitis and HCC in the woodchuck model., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

The VP1 protein of the foot-and-mouth disease virus (FMDV) is a major target of neutralizing antibodies and is responsible for viral attachment to permissive cells via an RGD motif. VP1 of duck hepatitis A type 1 virus (DHAV-1) does not contain any RGD motif. To investigate the antibody and receptor-binding properties of DHAV-1, VP1 has been expressed as a His fusion protein (His-VP1) in baculovirus system. Sera against His-VP1 raised in rabbits effectively neutralized DHAV-1 infection in vitro and in vivo. A flow cytometry binding assay indicated that His-VP1 bound to duck embryo fibroblast cell (DEF) surface receptors. This binding was reduced in a dose-dependent manner by the addition of purified DHAV-1 virions, demonstrating the specificity of this interaction. A separate cell-binding assay also implicated His-VP1 in receptor binding. Importantly, anti-His-VP1 antibodies inhibited the binding of DHAV-1 virions to DEF cells, suggesting that these antibodies exert their neutralizing activity by blocking viral attachment. Similar to the counterpart of FMDV, DHAV-1 VP1 appears to be involved in receptor binding activity and a target of protective antibodies. This study confirms the potential of recombinant VP1 protein to serve as vaccine and diagnostic reagents for the control of DHAV-1 infections., (Copyright © 2016 Elsevier B.V. All rights reserved.)

An estimated 350 million people are chronically infected with hepatitis B virus (HBV), and over one million people die each year due to HBV-associated liver diseases, such as cirrhosis and liver cancer. Current therapeutics for chronic HBV infection are limited to nucleos(t)ide analogs and interferon. These anti-HBV drugs in general reduce viral load and improve the long-term outcome of infection but very rarely lead to a cure. Thus, new therapies for chronic HBV infection need to be developed by utilizing liver cell lines and primary cultures and small laboratory animals capable of replicating HBV or surrogate hepadnaviruses for antiviral testing. Natural infection with woodchuck hepatitis virus (WHV), a hepadnavirus closely related to HBV, occurs in woodchucks. Chronic WHV infection has been established over decades as a suitable model for evaluating direct-acting antivirals as well as vaccines, vaccine adjuvants, and immunotherapeutics because animals are fully immunocompetent. Before HBV-specific compounds are applied to woodchucks, they are usually tested in primary woodchuck hepatocytes (PWHs) replicating WHV at high levels for confirming drug specificity against viral or host targets. Here we describe a protocol for the isolation of PWHs from liver of WHV-infected woodchucks, maintenance in culture, and use in assays for determining antiviral efficacy, safety, and associated host innate immune response of new experimental drugs. Exemplary assays were performed with the nucleoside analog, lamivudine, and the immunomodulator, interferon-alpha.

Viral hepatitis remains a global health challenge despite recent progress in the development of more effective therapies. Although virus-specific CD8 + and CD4 + T cell responses are essential for viral clearance, it remains largely unknown what regulates T cell-mediated viral clearance. Thus, a better understanding of the regulation of anti-viral T cell immunity would be critical for the design of more effective therapies for viral hepatitis. Using a model of adenovirus-induced hepatitis, here we showed that adenoviral infection induced recruitment of Ly6C hi monocytes to the liver in a CCR2-dependent manner. These recruited Ly6C hi monocytes suppressed CD8 + and CD4 + T cell responses to adenoviral infection, leading to a delay in viral clearance. In vivo depletion of Ly6C hi monocytes markedly enhanced anti-viral T cell responses and promoted viral clearance. Mechanistically, we showed that induction of iNOS and the production of NO by Ly6C hi monocytes are critical for the suppression of T cell responses. In addition, a contact-dependent mechanism mediated by PD-1 and PD-L1 interaction is also required for T cell suppression by Ly6C hi monocytes. These findings suggest a critical role for Ly6C hi monocytes in the regulation of T cell immunity in viral hepatitis and may provide new insights into development of more effective therapies for treating viral hepatitis based on targeting the immunosuppressing monocytes.

Viral replication in the liver is generally detected by cellular endosomal Toll-like receptors (TLRs) and cytosolic helicase sensors that trigger antiviral inflammatory responses. Recent evidence suggests that surface TLR2 may also contribute to viral detection through recognition of viral coat proteins but its role in the outcome of acute viral infection remains elusive. In this study, we examined in vivo the role of TLR2 in acute infections induced by the highly hepatotrophic mouse hepatitis virus (MHV) type 3 and weakly hepatotrophic MHV-A59 serotype. To address this, C57BL/6 (wild-type; WT) and TLR2 knockout (KO) groups of mice were intraperitoneally infected with MHV3 or MHV-A59. MHV3 infection provoked a fulminant hepatitis in WT mice, characterized by early mortality and high alanine and aspartate transaminase levels, histopathological lesions and viral replication whereas infection of TLR2 KO mice was markedly less severe. MHV-A59 provoked a comparable mild and subclinical hepatitis in WT and TLR2 KO mice. MHV3-induced fulminant hepatitis in WT mice correlated with higher hepatic expression of interferon-β, interleukin-6, tumour necrosis factor-α, CXCL1, CCL2, CXCL10 and alarmin (interleukin-33) than in MHV-A59-infected WT mice and in MHV3-infected TLR2 KO mice. Intrahepatic recruited neutrophils, natural killer cells, natural killer T cells or macrophages rapidly decreased in MHV3-infected WT mice whereas they were sustained in MHV-A59-infected WT mice and MHV3-infected TLR2 KO. MHV3 in vitro infection of macrophagic cells induced rapid and higher viral replication and/or interleukin-6 induction in comparison to MHV-A59, and depended on viral activation of TLR2 and p38 mitogen-activated protein kinase. Taken together, these results support a new aggravating inflammatory role for TLR2 in MHV3-induced acute fulminant hepatitis., (© 2016 John Wiley & Sons Ltd.)

Unlabelled: Despite its importance in shaping adaptive immune responses, viral clearance, and immune-based inflammation, tissue-specific innate immunity remains poorly characterized for hepatitis C virus (HCV) infection due to the lack of access to acutely infected tissues. In this study, we evaluated the impact of natural killer (NK) cells and myeloid (mDCs) and plasmacytoid (pDCs) dendritic cells on control of virus replication and virus-induced pathology caused by another, more rapidly resolving hepacivirus, GB virus B (GBV-B), in infections of common marmosets. High plasma and liver viral loads and robust hepatitis characterized acute GBV-B infection, and while viremia was generally cleared by 2 to 3 months postinfection, hepatitis and liver fibrosis persisted after clearance. Coinciding with peak viral loads and liver pathology, the levels of NK cells, mDCs, and pDCs in the liver increased up to 3-fold. Although no obvious numerical changes in peripheral innate cells occurred, circulating NK cells exhibited increased perforin and Ki67 expression levels and increased surface expression of CXCR3. These data suggested that increased NK cell arming and proliferation as well as tissue trafficking may be associated with influx into the liver during acute infection. Indeed, NK cell frequencies in the liver positively correlated with plasma (R = 0.698; P = 0.015) and liver (R = 0.567; P = 0.057) viral loads. Finally, soluble factors associated with NK cells and DCs, including gamma interferon (IFN-γ) and RANTES, were increased in acute infection and also were associated with viral loads and hepatitis. Collectively, the findings showed that mobilization of local and circulating innate immune responses was linked to acute virus-induced hepatitis, and potentially to resolution of GBV-B infection, and our results may provide insight into similar mechanisms in HCV infection., Importance: Hepatitis C virus (HCV) infection has created a global health crisis, and despite new effective antivirals, it is still a leading cause of liver disease and death worldwide. Recent evidence suggests that innate immunity may be a potential therapeutic target for HCV, but it may also be a correlate of increased disease. Due to a lack of access to human tissues with acute HCV infection, in this study we evaluated the role of innate immunity in resolving infection with a hepacivirus, GBV-B, in common marmosets. Collectively, our data suggest that NK cell and DC mobilization in acute hepacivirus infection can dampen virus replication but also regulate acute and chronic liver damage. How these two opposing effects on the host may be modulated in future therapeutic and vaccine approaches warrants further study., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

IFI16 and AIM2 are important DNA sensors in antiviral immunity. To characterize these two molecules in a woodchuck model, which is widely used to study hepatitis B virus (HBV) infection, we cloned and analyzed the complete coding sequences (CDSs) of woodchuck IFI16 and AIM2, and found that AIM2 was highly conserved in mammals, whereas the degree of sequence identity between woodchuck IFI16 and its mammalian orthologues was low. IFI16 and IFN-β were upregulated following VACV ds 70 mer transfection, while AIM2 and IL-1β were upregulated following poly (dA:dT) transfection, both in vitro and in vivo; IFI16-targeted siRNA decreased the transcription of IFI16 and IFN-β stimulated by VACV ds 70 mer, and AIM2 siRNA interference downregulated AIM2 and IL-1β transcripts stimulated by poly (dA:dT), in vitro, suggesting that woodchuck IFI16 and AIM2 may play pivotal roles in the DNA-mediated induction of IFN-β and IL-1β, respectively. IFI16 and AIM2 transcripts were upregulated in the liver and spleen following acute WHV infection, while IFI16 was downregulated in the liver following chronic infection, implying that IFI16 and AIM2 may be involved in WHV infection. These data provide the basis for the study of IFI16- and AIM2-mediated innate immunity using the woodchuck model.

TREM1 (Triggering Receptor Expressed on Myeloid Cells 1) is a pro-inflammatory receptor expressed by phagocytes, which can also be released as a soluble molecule (sTREM1). The roles of TREM1 and sTREM1 in liver infection and inflammation are not clear. Here we show that patients with hepatitis B virus (HBV) or hepatitis C virus (HCV) infection manifest elevated serum levels of sTREM1. In mice, experimental viral hepatitis induced by infection with Lymphocytic Choriomeningitis Virus (LCMV)-WE was likewise associated with increased sTREM1 in serum and urine, and with increased TREM1 and its associated adapter molecule DAP12 in the liver. Trem1-/- mice showed accelerated clearance of LCMV-WE and manifested attenuated liver inflammation and injury. TREM1 expression in the liver of wild-type mice was mostly confined to infiltrating neutrophils, which responded to LCMV by secretion of CCL2 and TNF-α, and release of sTREM1. Accordingly, the production of CCL2 and TNF-α was decreased in the livers of LCMV-infected Trem1-/- mice, as compared to LCMV-infected wildtype mice. These findings indicate that TREM1 plays a role in viral hepatitis, in which it seems to aggravate the immunopathology associated with viral clearance, mainly by increasing the inflammatory activity of neutrophils.

We have recently described sustained clinical recovery associated with dampened neuroinflammation and remyelination following transplantation of neural precursor cells (NPCs) derived from human embryonic stem cells (hESCs) in a viral model of the human demyelinating disease multiple sclerosis. The hNPCs used in that study were derived by a novel direct differentiation method (direct differentiation, DD-NPCs) that resulted in a unique gene expression pattern when compared to hNPCs derived by conventional methods. Since the therapeutic potential of human NPCs may differ greatly depending on the method of derivation and culture, we wanted to determine whether NPCs differentiated using conventional methods would be similarly effective in improving clinical outcome under neuroinflammatory demyelinating conditions. For the current study, we utilized hNPCs differentiated from a human induced pluripotent cell line via an embryoid body intermediate stage (EB-NPCs). Intraspinal transplantation of EB-NPCs into mice infected with the neurotropic JHM strain of mouse hepatitis virus (JHMV) resulted in decreased accumulation of CD4+ T cells in the central nervous system that was concomitant with reduced demyelination at the site of injection. Dampened neuroinflammation and remyelination was correlated with a transient increase in CD4+FOXP3+ regulatory T cells (Tregs) concentrated within the peripheral lymphatics. However, compared to our earlier study, pathological improvements were modest and did not result in significant clinical recovery. We conclude that the genetic signature of NPCs is critical to their effectiveness in this model of viral-induced neurologic disease. These comparisons will be useful for understanding what factors are critical for the sustained clinical improvement.

Forty-eight fowl aviadenoviruses (FAdVs) isolated from recent IBH outbreaks across Europe were investigated, by utilizing for the first time the two major adenoviral antigenic domains, hexon loop-1 and fiber, for compound molecular characterization of IBH-associated FAdVs. Successful target gene amplification, following virus isolation in cell culture or from FTA-card samples, demonstrated presence of FAdVs in all cases indicative for IBH. Based on hexon loop-1 analysis, 31 European field isolates exhibited highest nucleotide identity (>97.2%) to reference strains FAdV-2 or -11 representing FAdV-D, while 16 and one European isolates shared >96.0% nucleotide identity with FAdV-8a and -8b, or FAdV-7, the prototype strains representing FAdV-E. These results extend recognition of specific FAdV-D and FAdV-E affiliate genotypes as causative agents of IBH to the European continent. In all isolates, species specificity determined by fiber gene analysis correlated with hexon-based typing. A threshold of 72.0% intraspecies nucleotide identity between fibers from investigated prototype and field strains corresponded with demarcation criteria proposed for hexon, suggesting fiber-based analysis as a complementary tool for molecular FAdV typing. A limited number of strains exhibited inconsistencies between hexon and fiber subclustering, indicating potential constraints for single-gene based typing of those FAdVs. Within FAdV-D, field isolate fibers shared a high degree of nucleotide (>96.7%) and aa (>95.8%) identity, while FAdV-E field isolate fibers displayed greater nucleotide divergence of up to 22.6%, resulting in lower aa identities of >81.7%. Furthermore, comparison with FAdVs from IBH outbreaks outside Europe revealed close genetic relationship in the fiber, independent of the strains' geographic origin., (Copyright © 2016 Elsevier B.V. All rights reserved.)

The underlying immune-mediated mechanisms involved in virus-induced severe hepatitis have not been well elucidated. In this study, we investigated the role of CD3(+)CD4(-)CD8(-) double-negative T (DN T) cells in the pathogenesis of fulminant viral hepatitis (FVH) induced by murine hepatitis virus strain 3 (MHV-3). After MHV-3 infection, the proportions of DN T cells increased significantly in BALB/cJ mice, and splenic DN T cells expressing high levels of CD69 were recruited by MHV-3-infected hepatocytes to the liver. Serum levels of alanine aminotransferase, aspartate aminotransferase and total bilirubin increased, accompanied by massive hepatocyte necrosis. These DN T cells were predominantly consisted of a TCRαβ(+) subset expressing high levels of CD44 and did not produce cytokine except IL-2. Adoptive transfer of this subset of DN T cells to the MHV-3-infected mice resulted in an increase in murine fibrinogen-like protein 2 (mfgl2) expressions in association with massive fibrin deposition in the liver. Following MHV-3 infection, membrane mfgl2 expression and functional procoagulant activity increased remarkably in the DN T cells. Introduction of a recombinant adenovirus which encoded a microRNA specifically targeting mfgl2 gene (Ad-mfgl2-miRNA) in vivo significantly inhibited the hepatic expression of mfgl2 and improved survival in mice. However, under this condition, adoptive transfer of the DN T cells accelerated the disease progression and reversed the benefit from mfgl2 gene silence, leading to a 100 % death rate. Our results demonstrate that DN T cells contribute to the outcome of MHV-3-induced FVH via an important effector molecule mfgl2.

Background . Parvovirus B19 (B19V) is a common finding in endomyocardial biopsy specimens from myocarditis and dilated cardiomyopathy patients. However, current understanding of how B19V is contributing to cardiac damage is rather limited due to the lack of appropriate mice models. In this work we demonstrate that immunization of BALB/c mice with the major immunogenic determinant of B19V located in the unique sequence of capsid protein VP1 (VP1u) is an adequate model to study B19V associated heart damage. Methods and Results . We immunized mice in the experimental group with recombinant VP1u; immunization with cardiac myosin derived peptide served as a positive reference and phosphate buffered saline served as negative control. Cardiac function and dimensions were followed echocardiographically 69 days after immunization. Progressive dilatation of left ventricle and decline of ejection fraction were observed in VP1u- and myosin-immunized mice. Histologically, severe cardiac fibrosis and accumulation of heart failure cells in lungs were observed 69 days after immunization. Transcriptomic profiling revealed ongoing cardiac remodeling and immune process in VP1u- and myosin-immunized mice. Conclusions . Immunization of BALB/c mice with VP1u induces dilated cardiomyopathy in BALB/c mice and it could be used as a model to study clinically relevant B19V associated cardiac damage.