Inducible Nitric Oxide Synthase (iNOS) Mediates Vascular Endothelial Cell Apoptosis in Grass Carp Reovirus (GCRV)-Induced Hemorrhage.

Hemorrhage is one of the most obvious pathological phenomena in grass carp reovirus (GCRV) infection. The etiology of GCRV-induced hemorrhage is unclear. We found inducible nitric oxide synthase (iNOS) may relate to viral hemorrhage according to the previous studies, which is expressed at high levels after GCRV infection and is related to apoptosis. In this study, we aimed to investigate the mechanism of iNOS on apoptosis and hemorrhage at the cell level and individual level on subjects who were infected with GCRV and treated with S-methylisothiourea sulfate (SMT), an iNOS inhibitor. Cell structure, apoptosis rate, and hemorrhage were evaluated through fluorescence microscopy, Annexin V-FITC staining, and H&E staining, respectively. Cell samples and muscle tissues were collected for Western blotting, NO concentration measure, caspase activity assay, and qRT-PCR. iNOS-induced cell apoptosis and H&E staining showed that the vascular wall was broken after GCRV infection in vivo. When the function of iNOS was inhibited, NO content, apoptosis rate, caspase activity, and hemorrhage were reduced. Collectively, these results suggested iNOS plays a key role in apoptosis of vascular endothelial cells in GCRV-induced hemorrhage. This study is the first to elucidate the relationship between iNOS-induced cell apoptosis and GCRV-induced hemorrhage, which lays the foundation for further mechanistic research of virus-induced hemorrhage.

Avian reovirus σA and σNS proteins activate the phosphatidylinositol 3-kinase-dependent Akt signalling pathway.

The present study was conducted to identify avian reovirus (ARV) proteins that can activate the phosphatidylinositol 3-kinase (PI3K)-dependent Akt pathway. Based on ARV protein amino acid sequence analysis, σA, σNS, µA, µB and µNS were identified as putative proteins capable of mediating PI3K/Akt pathway activation. The recombinant plasmids σA-pcAGEN, σNS-pcAGEN, µA-pcAGEN, µB-pcAGEN and µNS-pcAGEN were constructed and used to transfect Vero cells, and the expression levels of the corresponding genes were quantified by immunofluorescence and Western blot analysis. Phosphorylated Akt (P-Akt) levels in the transfected cells were measured by flow cytometry and Western blot analysis. The results showed that the σA, σNS, µA, µB and µNS genes were expressed in Vero cells. σA-expressing and σNS-expressing cells had higher P-Akt levels than negative control cells, pcAGEN-expressing cells and cells designed to express other proteins (i.e., µA, µB and µNS). Pre-treatment with the PI3K inhibitor LY294002 inhibited Akt phosphorylation in σA- and σNS-expressing cells. These results indicate that the σA and σNS proteins can activate the PI3K/Akt pathway.

Cell entry-associated conformational changes in reovirus particles are controlled by host protease activity.

Membrane penetration by reovirus requires successive formation of two cell entry intermediates, infectious subvirion particles (ISVPs) and ISVP*s. In vitro incubation of reovirus virions with high concentration of chymotrypsin (CHT) results in partial digestion of the viral outer capsid to form ISVPs. When virions are instead digested with low concentrations of chymotrypsin, the outer capsid is completely proteolyzed to form cores. We investigated the basis for the inverse relationship between CHT activity and protease susceptibility of the reovirus outer capsid. We report that core formation following low-concentration CHT digestion proceeds via formation of particles that contain a protease-sensitive form of the µ1C protein, a characteristic of ISVP*s. In addition, we found that both biochemical features and viral genetic requirements for ISVP* formation and core formation following low-concentration CHT digestion are identical, suggesting that core formation proceeds via a particle resembling ISVP*s. Furthermore, we determined that intermediates generated following low-concentration CHT digestion are distinct from ISVPs and convert to ISVP*-like particles much more readily than ISVPs. These results suggest that the activity of host proteases used to generate ISVPs can influence the efficiency with which the next step in reovirus cell entry, namely, ISVP-to-ISVP* conversion, occurs.

Impact of host proteases on reovirus infection in the respiratory tract.

Virion uncoating is an essential early event in reovirus infection. In natural enteric infections, rapid proteolytic uncoating of virions is mediated by pancreatic serine proteases. The proteases that promote reovirus disassembly and cell entry in the respiratory tract remain unknown. In this report, we show that endogenous respiratory and inflammatory proteases can promote reovirus infection in vitro and that preexisting inflammation augments in vivo infection in the murine respiratory tract.

Cooperative roles of fish protein kinase containing Z-DNA binding domains and double-stranded RNA-dependent protein kinase in interferon-mediated antiviral response.

The double-stranded RNA (dsRNA)-dependent protein kinase (PKR) inhibits protein synthesis by phosphorylating eukaryotic translation initiation factor 2α (eIF2α). In fish species, in addition to PKR, there exists a PKR-like protein kinase containing Z-DNA binding domains (PKZ). However, the antiviral role of fish PKZ and the functional relationship between fish PKZ and PKR remain unknown. Here we confirmed the coexpression of fish PKZ and PKR proteins in Carassius auratus blastula embryonic (CAB) cells and identified them as two typical interferon (IFN)-inducible eIF2α kinases, both of which displayed an ability to inhibit virus replication. Strikingly, fish IFN or all kinds of IFN stimuli activated PKZ and PKR to phosphorylated eIF2α. Overexpression of both fish kinases together conferred much more significant inhibition of virus replication than overexpression of either protein, whereas morpholino knockdown of both made fish cells more vulnerable to virus infection than knockdown of either. The antiviral ability of fish PKZ was weaker than fish PKR, which correlated with its lower ability to phosphorylate eIF2α than PKR. Moreover, the independent association of fish PKZ or PKR reveals that each of them formed homodimers and that fish PKZ phosphorylated eIF2α independently on fish PKR and vice versa. These results suggest that fish PKZ and PKR play a nonredundant but cooperative role in IFN antiviral response.

A brain slice culture model of viral encephalitis reveals an innate CNS cytokine response profile and the therapeutic potential of caspase inhibition.

Viral encephalitis is a significant cause of human morbidity and mortality in large part due to suboptimal diagnosis and treatment. Murine reovirus infection serves as a classic experimental model of viral encephalitis. Infection of neonatal mice with T3 reoviruses results in lethal encephalitis associated with neuronal infection, apoptosis, and CNS tissue injury. We have developed an ex vivo brain slice culture (BSC) system that recapitulates the basic pathological features and kinetics of viral replication seen in vivo. We utilize the BSC model to identify an innate, brain-tissue specific inflammatory cytokine response to reoviral infection, which is characterized by the release of IL6, CXCL10, RANTES, and murine IL8 analog (KC). Additionally, we demonstrate the potential utility of this system as a pharmaceutical screening platform by inhibiting reovirus-induced apoptosis and CNS tissue injury with the pan-caspase inhibitor, Q-VD-OPh. Cultured brain slices not only serve to model events occurring during viral encephalitis, but can also be utilized to investigate aspects of pathogenesis and therapy that are not experimentally accessible in vivo.

Caspase-3 activation is required for reovirus-induced encephalitis in vivo.

Reovirus infection of neonatal mice provides a classic experimental system for understanding the molecular pathogenesis of central nervous system (CNS) viral infection. CNS tissue injury, caused by many human neurotropic viruses, including herpes viruses and West Nile virus, is associated with caspase-dependent apoptotic neuronal cell death. We have previously shown that reovirus-induced CNS tissue injury results from apoptosis and is associated with activation of both death-receptor and mitochondrial apoptotic pathways culminating in the activation of the downstream effector caspase, caspase-3. In order to directly investigate the role of caspase-3 in virus-induced neuronal death and CNS tissue injury during encephalitis, we have compared the pathogenesis of reovirus CNS infection in mice lacking the caspase-3 gene (caspase-3 (-/-)) to syngeneic wild-type mice. Prior studies of antiapoptotic treatments for reovirus-infected mice have indicated that protection from reovirus-induced neuronal injury can occur without altering the viral titer in the brains of infected mice. We now show that reovirus infection of caspase-3 (-/-) mice was associated with dramatic reduction in severity of CNS tissue injury, decreased viral antigen and titer in the brain, and enhanced survival of infected mice. Following intracerebral inoculation, the authors also show that virus spread from the brain to the eyes in reovirus-infected caspase-3 (-/-) mice, indicating that viral spread was intact in these mice. Examination of brains of long-term survivors of reovirus infection among caspase-3 (-/-) mice showed that these mice eventually clear their CNS viral infection, and do not manifest residual or delayed CNS tissue injury.

Effect of beta-glucan on activity of antioxidant enzymes and Mx gene expression in virus infected grass carp.

The effects of beta-glucan, an immunostimulatory agent, on the superoxide dismutase (SOD) and catalase (CAT) activities of erythrocytes and Mx gene expression were studied from grass carp that were challenged with grass carp hemorrhage virus (GCHV). The SOD and CAT activities in erythrocytes and Mx gene expression in spleen from the fish were detected by spectrophotometry and RT-PCR, respectively. Negative control fish were injected with PBS; positive control groups were injected with either beta-glucan or GCHV only; and the experimental groups were pre-injected with beta-glucan 15 days prior to injection with GCHV. The results show that the SOD and CAT activities were higher in fish injected with beta-glucan for 15 days than the negative control group injected with PBS. The SOD and CAT activities significantly decreased when the fish were challenged with GCHV, but it was higher in the group pre-treated with beta-glucan than in infected fish not pre-treated, 15 days after GCHV infection. Mx gene expression levels increased during the early stages (at 12 h and 36 h) of GCHV infection, and it remained at higher levels from the 6th till the 10th day in the beta-glucan pre-treated group, but it was falling from the 6th day in the beta-glucan untreated group. The GCHV-infected group pre-treated with beta-glucan had a higher survival rate (60%) than the group not pre-treated with beta-glucan (20%), suggesting that beta-glucan possesses or enhances anti-viral responses.

Novel strategy for treatment of viral central nervous system infection by using a cell-permeating inhibitor of c-Jun N-terminal kinase.

Viral encephalitis is a major cause of morbidity and mortality worldwide, yet there is no proven efficacious therapy for most viral infections of the central nervous system (CNS). Many of the viruses that cause encephalitis induce apoptosis and activate c-Jun N-terminal kinase (JNK) following infection. We have previously shown that reovirus infection of epithelial cell lines activates JNK-dependent apoptosis. We now show that reovirus infection resulted in activation of JNK and caspase-3 in the CNS. Treatment of reovirus-infected mice with a cell-permeating peptide that competitively inhibits JNK activity resulted in significantly prolonged survival of intracerebrally infected mice following an otherwise lethal challenge with T3D (100 x 50% lethal dose). Protection correlated with reduced CNS injury, reduced neuronal apoptosis, and reduced c-Jun activation without altering the viral titer or viral antigen distribution. Given the efficacy of the inhibitor in protecting mice from viral encephalitis, JNK inhibition represents a promising and novel treatment strategy for viral encephalitis.

Viral induction of inflammatory cytokines in human epithelial cells follows a p38 mitogen-activated protein kinase-dependent but NF-kappa B-independent pathway.

The initial step in an immune response toward a viral infection is the induction of inflammatory cytokines. This innate immune response is mediated by expression of a variety of cytokines exemplified by TNF-alpha and IL-1beta. A key signal for the recognition of intracellular viral infections is the presence of dsRNA. Viral infections and dsRNA treatment can activate several signaling pathways including the protein kinase R pathway, mitogen-activated protein kinase (MAPK) pathways, and NF-kappaB, which are important in the expression of inflammatory cytokines. We previously reported that activation of protein kinase R was required for dsRNA induction of TNF-alpha, but not for IL-1beta. In this study, we report that activation of the p38 MAPK pathway by respiratory viral infections is necessary for induction of inflammatory cytokines in human bronchial epithelial cells. Inhibition of p38 MAPK by two different pharmacological inhibitors showed that expression of both TNF-alpha and IL-1beta required activation of this signaling pathway. Interestingly, inhibition of NF-kappaB did not significantly reduce viral induction of either cytokine. Our data show that, during the initial infections of epithelial cells with respiratory viruses, activation of the p38 MAPK pathway is associated with induction of inflammation, and NF-kappaB activation may be less important than previously suggested.

Reovirus-induced apoptosis requires both death receptor- and mitochondrial-mediated caspase-dependent pathways of cell death.

Apoptosis plays an important role in the pathogenesis of many viral infections. Despite this fact, the apoptotic pathways triggered during viral infections are incompletely understood. We now provide the first detailed characterization of the pattern of caspase activation following infection with a cytoplasmically replicating RNA virus. Reovirus infection of HEK293 cells results in the activation of caspase-8 followed by cleavage of the pro-apoptotic protein Bid. This initiates the activation of the mitochondrial apoptotic pathway leading to release of cytochrome c and activation of caspase-9. Combined activation of death receptor and mitochondrial pathways results in downstream activation of effector caspases including caspase-3 and caspase-7 and cleavage of cellular substrates including PARP. Apoptosis is initiated by death receptor pathways but requires mitochondrial amplification producing a biphasic pattern of caspase-8, Bid, and caspase-3 activation.

Calpain inhibition protects against virus-induced apoptotic myocardial injury.

Viral myocarditis is an important cause of human morbidity and mortality for which reliable and effective therapy is lacking. Using reovirus strain 8B infection of neonatal mice, a well-characterized experimental model of direct virus-induced myocarditis, we now demonstrate that myocardial injury results from apoptosis. Proteases play a critical role as effectors of apoptosis. The activity of the cysteine protease calpain increases in reovirus-infected myocardiocytes and can be inhibited by the dipeptide alpha-ketoamide calpain inhibitor Z-Leu-aminobutyric acid-CONH(CH(2))3-morpholine (CX295). Treatment of reovirus-infected neonatal mice with CX295 protects them against reovirus myocarditis as documented by (i) a dramatic reduction in histopathologic evidence of myocardial injury, (ii) complete inhibition of apoptotic myocardial cell death as identified by terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling, (iii) a reduction in serum creatine phosphokinase, and (iv) improved weight gain. These findings are the first evidence for the importance of a calpain-associated pathway of apoptotic cell death in viral disease. Inhibition of apoptotic signaling pathways may be an effective strategy for the treatment of viral disease in general and viral myocarditis in particular.

A glycosyl hydrolase activity of mammalian reovirus sigma1 protein can contribute to viral infection through a mucus layer.

The mammalian reovirus sigma1 protein is responsible for viral attachment to host cells and hemagglutination properties of the virus. In the present study, sequence similarity between sigma1 and chicken-type lysozymes prompted us to investigate additional functions of the sigma1 protein. Expression in Pichia pastoris yeast cells showed that sigma1 can actually cleave lysozyme substrates, including complex sugars found in bacterial cell walls. Replacement by site-directed mutagenesis of acidic amino acid residues in sigma1 by their respective isosteric, uncharged, amino acid residues has allowed us to identify Glu36 and Asp54 as the catalytic pair involved in sigma1-mediated glycosidase activity. The enzyme appears inactive in virions but its activity is unmasked upon generation of infectious subviral particles (ISVPs) by partial proteolytic removal of the outer capsid proteins. Purified sigma1 protein and ISVPs can also hydrolyze mucins, heavily glycosylated glycoproteins that are a major component of the mucus layer overlaying the intestinal epithelium. Furthermore, reovirus infection of epithelial Madin Darby canine kidney cells was inhibited tenfold in cells expressing mucin at their apical surface, while this inhibition was overcome by ISVPs. Unmasking of sigma1 mucinolytic activity in the intestine, consecutive to proteolytic cleavage of virions to ISVPs, thus likely contributes to the known increase in infectivity of reovirus ISVPs compared to complete virions. This work presents the first evidence that some mammalian viruses have evolved mechanisms to facilitate their penetration through the protective barrier of the mucus layer in the intestinal tract.

Small intestinal epithelial brush border enzymatic changes in suckling mice infected with reovirus type 3.

Suckling mice infected with reovirus type 3 were examined for changes in the epithelial brush border of the small intestine. After 3 days of infection with reovirus type 3, no significant changes were found in intestinal morphology or activity of any enzymes tested. After 6 days, villi were shortened and blunted with lymphangiectatic lesions and mild mononuclear infiltration in the lamina propria. In addition, there was a significant decrease in lactase (P < 0.001) and enterokinase activity (P < 0.05). However, there were no significant changes in the activities of alkaline phosphatase. In contrast, maltase (P < 0.001) and leucine aminopeptidase (P < 0.05) activities in the infected mice were significantly increased. These data suggest that brush border enzymes are affected differently by reovirus infection.

Reovirus type 3 infection in a suckling mouse: the effects on pancreatic structure and enzyme content.

Alterations in pancreatic function and structure were examined in suckling mice infected intraperitoneally with reovirus type 3. The results were compared to pancreatic zymogen enzyme activities and histology in adult mice infected with the same virus. No effect of the rovirus type 3 on the adult mice could be elicited. In contrast, the suckling mice infected by the reovirus type 3 revealed a definite change in pancreatic zymogen enzymes. However, the zymogen enzymes were affected in a nonparallel fashion and three groups of enzymes with different responses were noted. Amylase and lipase activities were significantly diminished (P less than 0.001) at 6 days of viral infection. The endopeptidases, trypsin (P less than 0.025) and cymotrypsin (P less than 0.001) activities were increased significantly in the infected group. The exopeptidases, carboxypeptidase A and B in the infected animals were not changed significantly compared to the control. It seems reasonable that the reovirus type 3 infection in the suckling mouse causes diminished lipase and amylase activities that might contribute to the pathogenesis of viral enteritis.

Changes in intestinal structure and function of neonatal calves infected with reovirus-like agent and Eschericia coli.

Measurements of villus/crypt length ratio and mucosal beta-galactosidase activity were made on calves less than 3 weeks of age which had diarrhoea associated with reovirus-like agent and E. coli. In calves with diarrhoea, the villus/crypt length ratios at all sites examined along the small intestine were less than in normal calves of similar age. This was attributed to a reduction in length of vili in calves infected with the reovirus-like agent. The activity of mucosal beta-galactosidase in the intestine of calves with diarrhoea was less than in normal calves, at all sites examined. A relationship existed between beta-galactosidase activity in vitro and lactose hydrolysis in vivo. It was concluded that calves with diarrhoea associated with reovirus-like agent, have a reduced ability to utilize dietary lactose.

Is lactase the receptor and uncoating enzyme for infantile enteritis (rota) viruses?

Rotaviruses are now regarded as important causes of diarrhoea in man, cattle, pigs, mice, and possibly other animals. Characteristically, disease occurs in newborn and young animals, and infection seems limited to the differentiated gut epithelial cells. The major surface polypeptide of the calf scours rotavirus is glycosylated, and highly purified beta-galactosidase (lactase) interacts with the virus in vitro causing removal of the outer shell of the capsid (uncoating). It is suggested that lactase present in the brush border of the intestinal epithelial cell performs a similar function in vivo by acting as a combined receptor and uncoating enzyme for the rotavirus. This hypothesis is consistent with the observations that rotaviruses seem to infect only gut epithelial cells, and that infant animals, whose lactase concentrations are generally higher than those of adult animals, seem more susceptible to rotavirus infections. Implications of the hypothesis include possible new approaches to laboratory cultivation of rotaviruses, which should be more successful in cells selected for surface lactase activity, and the suggestion that the epidemiology of human rotavirus infections may be influenced by the fact that different ethnic groups have different lactase levels (and hence lactose intolerance) in adulthood.