The outcome of experimentally induced inclusion body hepatitis (IBH) by fowl aviadenoviruses (FAdVs) is crucially influenced by the genetic background of the host.

In the present study, inclusion body hepatitis (IBH) was experimentally induced by oral inoculation of two groups of specific pathogen-free (SPF) broilers and two groups of SPF layers at day-old with either a fowl aviadenovirus (FAdV)-D or a FAdV-E strain. A substantial variation in the degree of susceptibility was observed with mortalities of 100 and 96% in the FAdV-E and D infected SPF broiler groups, respectively, whereas in the groups of infected SPF layers mortalities of only 20 and 8% were noticed. Significant changes in clinical chemistry analytes of all infected birds together with histopathological lesions indicated impairment of liver and pancreas integrity and functions. Furthermore, significantly lower blood glucose concentrations were recorded at peak of infection in both inoculated SPF broiler groups, in comparison to the control group, corresponding to a hypoglycaemic status. High viral loads were determined in liver and pancreas of SPF broilers already at 4 days post-infection (dpi), in comparison to SPF layers, indicating a somewhat faster viral replication in the target organs. Overall, highest values were noticed in the pancreas of SPF broilers independent of the virus used for infection. The actual study provides new insights into the pathogenesis of IBH, a disease evolving to a metabolic disorder, to which SPF broilers were highly susceptible. Hence, this is the first study to report a significant higher susceptibility of SPF broiler chickens to experimentally induced IBH in direct comparison to SPF layers.

Identification and characterization of avian hepatitis E virus in 2013 outbreaks of hepatitis-splenomegaly syndrome in two US layer operations.

Two commercial Midwestern egg-type chicken flocks experienced significant increases in mortality rates in April 2013 with clinical signs appearing in 17-week-old pullets on Farm A and in 46-week-old hens on Farm B. Average weekly mortality was 0.44% over a 4-week period on Farm A and 0.17% over an 8-week period on Farm B. On Farm A, flocks in the affected house had a 45% decrease in daily egg production from weeks 19 to 27 when compared with standard egg production curves (P < 0.01) while no decrease in egg production was noticed on Farm B. Post-mortem examination revealed changes consistent with hepatitis-splenomegaly syndrome, including hepatomegaly with serosanguineous fluid in the coelomic cavity and hepatic subcapsular haemorrhages. Microscopic lesions were characterized by multifocal necrotizing hepatitis and intrahepatic haemorrhage. No significant bacteria were recovered from liver samples, but 72 to 100% of the liver samples from affected chickens on Farm A (8/11) and Farm B (7/7) contained detectable amounts of avian hepatitis E virus (aHEV) RNA as determined by polymerase chain reaction. Sequencing and phylogenetic analysis of a 361-base-pair fragment of the helicase gene demonstrated 98.6 to 100% nucleotide identity between the aHEV genomes from Farm A and Farm B, whereas identities ranged from 74.6 to 90.5% when compared with other representative sequences. Sequences from this study clustered within aHEV genotype 2 previously recognized in the USA. In contrast to other reported aHEV outbreaks that occurred in 30-week-old to 80-week-old chickens, in the present investigation clinical aHEV was identified in 17-week-old chickens on one of the farms.

Programmed death (PD)-1-deficient mice are extremely sensitive to murine hepatitis virus strain-3 (MHV-3) infection.

The inhibitory receptor programmed death-1 (PD-1) has the capacity to maintain peripheral tolerance and limit immunopathological damage; however, its precise role in fulminant viral hepatitis (FH) has yet to be described. Here, we investigated the functional mechanisms of PD-1 as related to FH pathogenesis induced by the murine hepatitis virus strain-3 (MHV-3). High levels of PD-1-positive CD4(+), CD8(+) T cells, NK cells and macrophages were observed in liver, spleen, lymph node and thymus tissues following MHV-3 infection. PD-1-deficient mice exhibited significantly higher expression of the effector molecule which initiates fibrinogen deposition, fibrinogen-like protein 2 (FGL2), than did their wild-type (WT) littermates. As a result, more severe tissue damage was produced and mortality rates were higher. Fluorescence double-staining revealed that FGL2 and PD-1 were not co-expressed on the same cells, while quantitative RT-PCR demonstrated that higher levels of IFN-γ and TNF-α mRNA transcription occurred in PD-1-deficient mice in response to MHV-3 infection. Conversely, in vivo blockade of IFN-γ and TNF-α led to efficient inhibition of FGL2 expression, greatly attenuated the development of tissue lesions, and ultimately reduced mortality. Thus, the up-regulation of FGL2 in PD-1-deficient mice was determined to be mediated by IFN-γ and TNF-α. Taken together, our results suggest that PD-1 signaling plays an essential role in decreasing the immunopathological damage induced by MHV-3 and that manipulation of this signal might be a useful strategy for FH immunotherapy.

Cardiotrophin-1 promotes a high survival rate in rabbits with lethal fulminant hepatitis of viral origin.

Rabbit hemorrhagic disease virus (RHDV) causes lethal fulminant hepatitis closely resembling acute liver failure (ALF) in humans. In this study, we investigated whether cardiotrophin-1 (CT-1), a cytokine with hepatoprotective properties, could attenuate liver damage and prolong survival in virus-induced ALF. Twenty-four rabbits were infected with 2 × 10(4) hemagglutination units of RHDV. Twelve received five doses of CT-1 (100 µg/kg) starting at 12 h postinfection (hpi) (the first three doses every 6 h and then two additional doses at 48 and 72 hpi), while the rest received saline. The animals were analyzed for survival, serum biochemistry, and viral load. Another cohort (n = 22) was infected and treated similarly, but animals were sacrificed at 30 and 36 hpi to analyze liver histology, viral load, and the expression of factors implicated in liver damage and repair. All infected rabbits that received saline died by 60 hpi, while 67% of the CT-1-treated animals survived until the end of the study. Treated animals showed improved liver function and histology, while the viral loads were similar. In the livers of CT-1-treated rabbits we observed reduction of oxidative stress, diminished PARP1/2 and JNK activation, and decreased inflammatory reaction, as reflected by reduced expression of tumor necrosis factor alpha, interleukin-1ß, Toll-like receptor 4, VCAM-1, and MMP-9. In addition, CT-1-treated rabbits exhibited marked upregulation of TIMP-1 and increased expression of cytoprotective and proregenerative growth factors, including platelet-derived growth factor B, epidermal growth factor, platelet-derived growth factor receptor ß, and c-Met. In conclusion, in a lethal form of acute viral hepatitis, CT-1 increases animal survival by attenuating inflammation and activating cytoprotective mechanisms, thus representing a promising therapy for ALF of viral origin.

Pathotypic and molecular characterization of a fowl adenovirus associated with inclusion body hepatitis in Saskatchewan chickens.

Inclusion body hepatitis (IBH) is one of the major global disease problems, causing significant economic losses to poultry industry of the United States and Canada. The disease is characterized by its sudden onset and high mortalities. Amongst different serotypes of fowl adenoviruses (FAdVs) associated with IBH, serotype 8 of group I FAdV has been isolated from majority of IBH cases. In present studies, we isolated a FAdV from morbid liver of a 17-day-old broiler from a Saskatchewan broiler farm. This newly isolated virus was designated as IBHV(SK). However, based on the sequence analysis of the L1 region of the hexon gene, the IBHV(SK) may be classified as FAdV 8b strain 764. These studies describe for the first time the complete hexon gene sequence of FAdV serotype 8b. Experimental infection of 2-day-old (n = 48) and 2-wk-old (n = 56) chicks caused 83% and 43% mortalities, respectively. Determination of the complete hexon gene sequence of IBHV(SK) with establishment of a disease model in chickens will facilitate the development of type-specific diagnostic reagents and assays for the evaluation of potential experimental vaccines against pathogenic FAdV infections.

Type I IFN-mediated protection of macrophages and dendritic cells secures control of murine coronavirus infection.

The swift production of type I IFNs is one of the fundamental aspects of innate immune responses against viruses. Plasmacytoid dendritic cell-derived type I IFNs are of prime importance for the initial control of highly cytopathic viruses such as the mouse hepatitis virus (MHV). The aim of this study was to determine the major target cell populations of this first wave of type I IFNs. Generation of bone marrow-chimeric mice expressing the type I IFN receptor (IFNAR) on either hemopoietic or non-bone marrow-derived cells revealed that the early control of MHV depended mainly on IFNAR expression on hemopoietic cells. To establish which cell population responds most efficiently to type I IFNs, mice conditionally deficient for the IFNAR on different leukocyte subsets were infected with MHV. This genetic analysis revealed that IFNAR expression on LysM+ macrophages and CD11c+ dendritic cells was most important for the early containment of MHV within secondary lymphoid organs and to prevent lethal liver disease. This study identifies type I IFN-mediated cross-talk between plasmacytoid dendritic cells on one side and macrophages and conventional dendritic cells on the other, as an essential cellular pathway for the control of fatal cytopathic virus infection.

Role of TNF-alpha produced by nonantigen-specific cells in a fulminant hepatitis mouse model.

In previous studies, the mechanisms of acute liver injury and virus exclusion have been examined using a model wherein HBsAg-specific CTL are injected into HBsAg transgenic (Tg) mice. The importance of the role of TNF-alpha in virus exclusion was shown, but its role in liver injury was unclear. We crossed the TNF-alpha knockout mouse and HBsAg-Tg mouse to establish the HBsAg-Tg/TNF-alpha KO mouse, and examined the influence of TNF-alpha on liver injury. The severity of liver damage, as determined by serum alanine aminotransferase activity, was approximately 100 times greater in HBsAg-Tg/TNF-alpha(+/+) than in HBsAg-Tg/TNF-alpha(-/-) mice after i.v. administration of 5 x 10(6) CTLs. This liver damage reached the peak of its severity within 24-48 h, and was restored 7 days later. Histopathological examination showed hepatocellular necrosis and inflammatory cell infiltrate 24 h after the CTL injection in HBsAg-Tg/TNF-alpha(+/+) mice but not in HBsAg-Tg/TNF-alpha(-/-) mice. The liver damage was fatal for all HBsAg-Tg/TNF-alpha(+/+) mice that received 1.5 x 10(7) CTLs. In contrast, 1.5 x 10(7) CTLs could not kill the HBsAg-Tg/TNF-alpha(-/-) mice. The TNF-alpha production level was enhanced after the CTL injection in not only intrahepatic macrophages but also other types of mononuclear cells from non-HBsAg-Tg/TNF-alpha(+/+) mice. An adoptive transfer examination revealed that severe liver damage occurred in HBsAg-Tg/TNF-alpha(-/-) mice that had received mononuclear cells from TNF-alpha(+/+) mice. In conclusion, the present study provides evidence that TNF-alpha produced by intrahepatic non-Ag-specific inflammatory cells is critical in the development of lethal necroinflammatory liver disease.

Monoclonal antiprothrombinase (3D4.3) prevents mortality from murine hepatitis virus (MHV-3) infection.

The induction of monocyte/macrophage procoagulant activity (PCA) has been implicated in the pathogenesis of murine hepatitis virus strain 3 (MHV-3) infection and disease. Previously, we have shown that induction of PCA by MHV-3 correlated with resistance/susceptibility to infection in different mouse strains. In this study, all BALB/cJ mice that were infected with 10(3) plaque-forming units of MHV-3 developed severe liver disease and died within 96-120 h. Examination of the livers of these animals showed marked hepatic necrosis, deposition of fibrin, and cellular expression of PCA by direct immunofluorescence staining in areas of necrosis as well as in hepatic sinusoids. Splenic mononuclear cells recovered from these mice expressed high concentrations of PCA with time after infection. Infusion into mice of a high-titered monoclonal antibody that neutralized PCA (3D4.3) attenuated the development of hepatic necrosis and enhanced survival in a dose-dependent manner. All of the animals receiving 100 micrograms, and 44% and 22% of the animals that received 50 and 25 micrograms per day, respectively, survived for 10 d and made a full recovery. Administration of the antibody resulted in a dose-dependent reduction in fibrin deposition, PCA expression as detected by direct immunofluorescence staining and by a functional assay. In animals treated with high concentrations of antibody, titers of antibody to PCA fell from 87 +/- 15 micrograms/ml to 100 +/- 7 ng/ml during the active phase of the disease, consistent with sequestration due to binding of the immunoglobulin to cells expressing PCA. Surviving animals, when rechallenged with MHV-3, had a 40% mortality, consistent with the known rates of metabolism of immunoglobulin. This further suggested that protection was by a passive mechanism. The results reported here demonstrate that a neutralizing antibody to PCA protects animals from fulminant hepatitis and death associated with MHV-3 infection, and supports the notion that PCA is a potent inflammatory mediator that plays a pivotal role in the pathogenesis of liver injury resulting from MHV-3 infection.

The association of Equine Parvovirus-Hepatitis (EqPV-H) with cases of non-biologic-associated Theiler's disease on a farm in Ontario, Canada.

Theiler's disease was confirmed within a group horses located on a farm in southwestern Ontario during the summer and autumn of 2005. Five sudden deaths occurred between 3 July and 21 August, 2005, none of which were necropsied, however two of the horses showed clinical signs compatible with hepatic encephalopathy prior to death. No horse on the farm had received a biologic product of equine blood origin in the preceding six months. The only biologics used on the property were the administration of killed vaccines for rabies, tetanus and West Nile Virus to all horses 30 days prior to the onset of the first sudden death. Between 22 August, 2005 and 21 October, 2005, a further four horses died suddenly or were euthanized with all having a confirmed histopathologic diagnosis of acute hepatic necrosis. Serum was collected from all horses on the farm on 30 September, 2005 and this was repeated on 29 October, 2005. Equine parvovirus-hepatitis (EqPV-H) DNA was detected by quantitative-PCR in the serum of 61.8% (34/55) of the horses on the farm on either one or both sampling dates with viral loads ranging from <3.75 × 103 copies/mL to 3.64 × 107 copies/mL. EqPV-H DNA was present in serum samples of three horses with a confirmed diagnosis of Theiler's disease, five horses with subclinical liver disease, and in clinically normal in-contact horses. Subsequent phylogenetic analysis based on partial NS1 of EqPV-H revealed not only high similarity on nucleotide level within the sequenced samples but also within other previously published sequences.

Glucan-induced modification of murine viral hepatitis.

Glucan, a macrophage stimulant, was evaluated for its ability to alter survival and phagocytic dysfunction in mice challenged with mouse hepatitis virus strain MHV-A59. Administration of glucan before the mice were challenged with the virus significantly prolonged median survival time but did not modify overall mortality compared with control mice given dextrose. Maximal effectiveness was achieved when glucan was administered both before and after the viral challenge. In contrast to the marked hepatic parenchymal cell necrosis observed in the control mice, glucan-treated mice exhibited reduced pathology. Intraperitoneal administration of MHV-A59 resulted in a significant depression of phagocytic activity compared with controls that were not exposed to the virus. The enhancement in phagocytic function in glucan-treated control mice was unaltered in virus-challenged, glucan-treated mice. Thus glucan is capable of increasing survival, inhibiting hepatic necrosis, and maintaining an activated state of phagocytic activity in mice challenged with MHV-A59. Macrophage stimulants may have a significant role in the modification of virally induced hepatic lesions.

Assessment of the Effect of Baicalin on Duck Virus Hepatitis.

BACKGROUND: Duck virus hepatitis (DVH) caused by duck hepatitis A virus type 1 (DHAV-1) is a malignant disease in ducklings, causing economic losses in the duck industry. However, there is still no antiviral drug against DHAV-1 in the clinic. OBJECTIVE: Our aim is to investigate the anti-DHAV-1 effect of baicalin, which is a flavonoid derived from the Chinese medicinal herb huangqin (Scutellaria baicalensis Georgi). METHODS: Here, we first detected its anti-DHAV-1 ability in vitro and in vivo. At the same time, the inhibition of baicalin on DHAV-1 reproduction was determined. Finally, we tested and verified the anti-oxidative and immuno-enhancing roles of baicalin on its curative effect on DVH. RESULTS: Baicalin possessed anti-DHAV-1 effect. It improved the cytoactive of DEH which was infected by DHAV-1 as well as reduced the DHAV-1 reproduction in DEH. Under baicalin treatment, mortality of ducklings infected by DHAV-1 decreased, additionally the DHAV-1 level and liver injury in such ducklings were significantly reduced or alleviated. The in vitro mechanism study indicated baicalin inhibited DHAV-1 reproduction via interfering the viral replication and release. Furthermore, the in vivo mechanism study manifested both the anti-oxidative and immuno-enhancing abilities of baicalin, which played crucial roles in its curative effect on DVH. CONCLUSION: This study may provide a scientific basis for developing baicalin as one or a part of the anti-DHAV-1 drugs.

Inactivated and live bivalent fowl adenovirus (FAdV8b + FAdV11) breeder vaccines provide broad-spectrum protection in chicks against inclusion body hepatitis (IBH).

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 × 104 TCID50 of each virus/bird) was given orally once at 16 weeks of age and the inactivated vaccine (1 × 106TCID50 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 log10. Approximately 26 ±â€¯7% of MtAbs were passively transferred through eggs to day-old chicks. Progeny challenged with a lethal dose (1 × 107 TCID50/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.

Cytokine storms are primarily responsible for the rapid death of ducklings infected with duck hepatitis A virus type 1.

Duck hepatitis A virus type 1 (DHAV-1) is one of the most harmful pathogens in the duck industry. The infection of adult ducks with DHAV-1 was previously shown to result in transient cytokine storms in their kidneys. To understand how DHAV-1 infection impacts the host liver, we conducted animal experiments with the virulent CH DHAV-1 strain and the attenuated CH60 commercial vaccine strain. Visual observation and standard hematoxylin and eosin staining were performed to detect pathological damage in the liver, and viral copy numbers and cytokine expression in the liver were evaluated by quantitative PCR. The CH strain (108.4 copies/mg) had higher viral titers than the CH60 strain (104.9 copies/mg) in the liver and caused ecchymotic hemorrhaging on the liver surface. Additionally, livers from ducklings inoculated with the CH strain were significantly infiltrated by numerous red blood cells, accompanied by severe cytokine storms, but similar signs were not observed in the livers of ducklings inoculated with the CH60 strain. In conclusion, the severe cytokine storm caused by the CH strain apparently induces hemorrhagic lesions in the liver, which might be a key factor in the rapid death of ducklings.

Pathogenicity of duck hepatitis A virus type 3 and innate immune responses of the ducklings to virulent DHAV-3.

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, IFNß and IFNγ) and antiviral proteins (MX, OAS and PKR) are also up-regulated in the liver and spleen. The expression of most cytokines (IL-1ß, 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.

Clara Cell 10 kDa Protein Alleviates Murine Hepatitis Virus Strain 3-Induced Fulminant Hepatitis by Inhibiting Fibrinogen-Like Protein 2 Expression.

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-1ß 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.

Synergism between hepatic injuries and a nonhepatotropic reovirus in mice. Enhanced hepatic infection and death.

Reovirus type 1, after intravenous inoculation in the adult mouse, is secreted via bile into the intestine in an infectious form. Although reovirus type 1 is rapidly removed from systemic circulation by the liver and the lung, very few hepatocytes express reovirus antigen during infection. In intestinal cells, reovirus replicates selectively in the crypts. This site preference may be due to active cell proliferation in the crypts. We hypothesized that the state of the cell may affect virus replication and tested this hypothesis by using chemical and surgical means to increase hepatic mitotic activity. Adult mice were treated with carbon tetrachloride or surgical trauma, inoculated with reovirus type 1 intravenously, and subsequently killed. Virus antigen was identified using a highly specific immunohistochemical technique. Liver sections were stained using immunoperoxidase with specific rabbit antireovirus antibody. Hepatotoxin and surgical trauma increase reovirus antigen detection in both Kupffer cells and hepatocytes. Only the sequential administration of CCl4 and virus caused mortality at doses sublethal for each alone. These data demonstrate a synergism between hepatic injury and reovirus which results in a significant increase in the magnitude of viral infection and contributes to mortality. Such synergism may be important in idiopathic liver disease.

A flavone-polysaccharide based prescription attenuates the mitochondrial dysfunction induced by duck hepatitis A virus type 1.

The principal target organ of duck hepatitis A virus type 1 (DHAV-1) is duckling liver, which is an energy-intensive organ and plays important roles in body's energy metabolism and conversion. As the "power house" of the hepatocytes, mitochondria provide more than 90% of the energy. However, mitochondria are much vulnerable to the oxidative stress for their rich in polyunsaturated fatty acids. Although previous researches have demonstrated that DHAV-1 could induce the oxidative stress in the serum of the infected ducklings, no related study on the mitochondria during the pathological process of DVH has been reported by far. To address this issue, we examined the HE stained tissue pathological slices, detected the hepatic SOD, CAT and GPX activities and MDA contents and analyzed the ATP content, mitochondrial ultrastructure and the mitochondrial SOD, GPX activities and MDA content in the liver tissues. The results showed that the hepatic redox status was significantly disturbed so that causing the mitochondrial dysfunction, ATP depletion and mitochondrial oxidative stress during the process of the DHAV-1 infection, and a prescription formulated with Hypericum japonicum flavone, Radix Rehmanniae Recens polysaccharide and Salvia plebeia flavone (HRS), which had been demonstrated with good anti-oxidative activity in serum, could effectively alleviate the hepatic injury and the oxidative stress in liver tissue induced by DHAV-1 thus alleviating the mitochondrial injury and oxidative stress. In a word, this research discovers the oxidative stress induced mitochondrial dysfunction and oxidative stress during the DVH pathological process and demonstrates HRS exerts good anti-oxidative activity in liver tissue to protect mitochondria against reactive oxygen species (ROS).

Synergistic hepatocarcinogenic effect of hepadnaviral infection and dietary aflatoxin B1 in woodchucks.

Interactive hepadnaviral and chemical hepatocarcinogenesis was studied in woodchucks inoculated as newborns with woodchuck hepatitis virus (WHV), which is closely related to the human hepatitis B virus. When the woodchucks reached 12 months of age, aflatoxin B1 (AFB1) was administered in the diet at dose levels of 40 micrograms/kg body weight/day for 4 months and subsequently 20 micrograms/kg body weight/day (5 days/week) for lifetime. WHV DNA was demonstrated by Southern blot hybridization in the serum and by PCR in the serum and/or liver tissue. The histo- and cytomorphology of the liver were investigated by light and electron microscopy. WHV carriers with and without AFB1 treatment developed a high incidence of preneoplastic foci of altered hepatocytes, hepatocellular adenomas, and hepatocellular carcinomas that appeared 6-26 months after the beginning of the combination experiment. Administration of AFB1 to WHV carriers resulted in a significantly earlier appearance of hepatocellular neoplasms and a higher incidence of hepatocellular carcinomas compared to WHV carriers not treated with AFB1. Neither hepatocellular adenomas nor carcinomas (but preneoplastic foci of altered hepatocytes) were detected in woodchucks receiving AFB1 alone, and no preneoplastic or neoplastic lesions were found in untreated controls. These results provide conclusive evidence of a synergistic hepatocarcinogenic effect of hepadnaviral infection and dietary AFB1. Except for the frequent presence of ground glass cells containing surface antigen filaments in the infected woodchucks, the phenotype of preneoplastic foci of altered hepatocytes was similar in WHV carriers with and without exposure to AFB1 and in animals treated with AFB1 alone. Clear cell foci excessively storing glycogen and/or fat, amphophilic cell foci crowded with mitochondria and peroxisomes, and mixed cell foci composed of various cell types including basophilic cells rich in ribosomes predominated. The cellular phenotype in neoplastic lesions varied from clear, amphophilic, and mixed cell populations in highly differentiated adenomas and carcinomas to basophilic cell populations prevailing in poorly differentiated carcinomas. The striking similarities in altered cellular phenotypes of preneoplastic hepatic foci emerging after both hepadnaviral infection and exposure to AFB1 suggest closely related underlying molecular mechanisms that may be mainly responsible for the synergistic hepatocarcinogenic effect of these oncogenic agents.

A disparate subset of double-negative T cells contributes to the outcome of murine fulminant viral hepatitis via effector molecule fibrinogen-like protein 2.

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 TCRαß(+) 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.

Chemokine expression and viral infection of the central nervous system: regulation of host defense and neuropathology.

An effective host response against viral infection of the central nervous system (CNS) is the principal factor dictating the outcome of infection. It is the responsibility of the immune response to contain and control viral replication. Paradoxically, it is the immune response that may also contribute to the development of neuropathology. We have used mouse hepatitis virus (MHV), apositive-strand RNA virus, infection of the CNS to understand the dynamic interaction between viral replication, protection, and pathology with an emphasis on understanding how chemokines participate in these interrelated processes. Herein, we demonstrate the complexity of the chemokine response to MHV infection of the CNS and the delicate balance that exists between host defense and development of disease.