Ginsenoside Rg3 inhibits grass carp reovirus replication in grass carp ovarian epithelial cells.

Ginseng exhibits multiple medicinal properties, including the improvement of immune function and enhancing disease resistance. In this study, we investigated the inhibitory effects of ginsenoside Rg3 on grass carp reovirus (GCRV) infection of grass carp ovarian (CO) epithelial cells, in order to provide a baseline framework for future high-efficacy antiviral drug screening investigations. Ginsenoside Rg3 was added to GCRV-infected CO cells, and cells were cultured at 27 °C before cell proliferation was measured by MTT assays. Label-free real-time cellular analysis (RTCA) after 72 h of experimentation demonstrated that 100 µg/mL ginsenoside Rg3 treatment had the highest inhibitory effect on GCRV (among 1,10,100 µg/mL treatments). We then measured the capacity for cellular antioxidant ability. Cells treated with 1,10,100 µg/mL ginsenoside Rg3 exhibited increases in Total Antioxidant Capacity activity relative to controls, respectively. Furthermore, Antioxidant assay and reverse transcript quantitative polymerase chain reaction (RT-qPCR) showed that ginsenoside Rg3 were efficient to restrain the replication of GCRV in CO cells. Expression analysis of immune-related genes via RT-qPCR showed that treatment with ginsenoside Rg3 promoted expression of IRF-3 and IRF-7 increases, respectively. Moreover, expression of IFN-1 was induced, which then inhibition the expression of tumor necrosis factor-alpha (TNF-α). In conclusion, we demonstrated that ginsenoside Rg3 promotes CO cell proliferation, inhibits GCRV activity, promotes CO cell immune activities, and thereby enhances the resistance of CO to GCRV infection.

Enteric viruses exploit the microbiota to promote infection.

Enteric viruses infect the mammalian gastrointestinal tract which is home to a diverse community of intestinal bacteria. Accumulating evidence suggests that certain enteric viruses utilize these bacteria to promote infection. While this is not surprising considering their proximity, multiple viruses from different viral families have been shown to bind directly to bacteria or bacterial components to aid in viral replication, pathogenesis, and transmission. These data suggest that the concept of a single virus infecting a single cell, independent of the environment, needs to be reevaluated. In this review, I will discuss the current knowledge of enteric virus-bacterial interactions and discuss the implications for viral pathogenesis and transmission.

Inhibitor analysis revealed that clathrin-mediated endocytosis is involed in cellular entry of type III grass carp reovirus.

BACKGROUND: Grass carp (Ctenopharyngodon idella) hemorrhagic disease is caused by an acute infection with grass carp reovirus (GCRV). The frequent outbreaks of this disease have suppressed development of the grass carp farming industry. GCRV104, the representative strain of genotype III grass carp (Ctenopharyngodon idella) reovirus, belongs to the Spinareovirinae subfamily and serves as a model for studying the strain of GCRV which encodes an outer-fiber protein. There is no commercially available vaccine for this genotype of GCRV. Therefore, the discovery of new inhibitors for genotype III of GCRV will be clinically beneficial. In addition, the mechanism of GCRV with fiber entry into cells remains poorly understood. METHODS: Viral entry was determined by a combination of specific pharmacological inhibitors, transmission electron microscopy, and real-time quantitative PCR. RESULTS: Our results demonstrate that both GCRV-JX01 (genotype I) and GCRV104 (genotype III) of GCRV propagated in the grass carp kidney cell line (CIK) with a typical cytopathic effect (CPE). However, GCRV104 replicated slower than GCRV-JX01 in CIK cells. The titer of GCRV-JX01 was 1000 times higher than GCRV104 at 24 h post-infection. We reveal that ammonium chloride, dynasore, pistop2, chlorpromazine, and rottlerin inhibit viral entrance and infection, but not nystatin, methyl-ß-cyclodextrin, IPA-3, amiloride, bafilomycin A1, nocodazole, and latrunculin B. Furthermore, GCRV104 and GCRV-JX01 infection of CIK cells depended on dynamin and the acidification of the endosome. This was evident by the significant inhibition following prophylactic treatment with the lysosomotropic drug ammonium chloride or dynasore. CONCLUSIONS: Taken together, our data have suggested that GCRV104 enters CIK cells through clathrin-mediated endocytosis in a pH-dependent manner. We also suggest that dynamin is critical for efficient viral entry. Additionally, the phosphatidylinositol 3-kinase inhibitor wortmannin and the protein kinase C inhibitor rottlerin block GCRV104 cell entry and replication.

A mitochondrial membrane protein is a target for rice ragged stunt virus in its insect vector.

Rice ragged stunt virus (RRSV; Reoviridae) is exclusively transmitted by the brown planthopper Nilaparvata lugens in a persistent-propagative manner. It is understood that RNA viral proliferation is associated with the intracellular membranes of the insect host cells. However, the molecular mechanisms of the interaction between the RRSV proliferation and the intracellular membranes remain essentially unknown. It will be of great interest to determine whether RRSV protein(s) directly interact with intracellular membrane components of its host cells. In this study, we identified a RRSV nonstructural protein Pns10 interacting with a host oligomycin-sensitivity conferral protein (OSCP) using yeast two-hybrid system. The interaction between RRSV Pns10 and N. lugens OSCP was verified by a glutathione S-transferase pull-down assay. Confocal miscopy revealed colocalization of these two proteins in the cytoplasm of the salivary gland cells during the viral infection. The virions were further detected in the mitochondria under confocal miscopy and transmission electron microscopy combined with western blotting assay. This is the first observation that RRSV protein has a direct link with mitochondria. Suppressing OSCP gene expression by RNA interference notably decreased the viral loads in RRSV-infected insects. These findings revealed novel aspects of a viral protein in targeting the host mitochondrial membrane and provide insights concerning the mitochondrial membrane protein-based virus proliferation mode in the insect vector.

Development of an Ussuri catfish Pseudobagrus ussuriensis skin cell line displaying differential cytopathic effects to three aquatic animal viruses.

An Ussuri catfish Pseudobagrus ussuriensis skin (UCS) cell line was developed and subcultured for more than 60 passages. UCS cells consisted of mostly epithelial-like cells and multiplied well in TC199 medium supplemented with 10% fetal bovine serum at 25°C. Chromosome analysis revealed that most UCS cells had a normal diploid karyotype with 2n=52. UCS cells showed differential cytopathic effects (CPEs) after inoculation of spring viremia of carp virus (SVCV, a negative-strand RNA virus), grass carp reovirus (GCRV, a multi-segmented double-stranded RNA virus) and Rana grylio virus (RGV, a large double-stranded DNA virus), and were indicative of high sensitivities to these three aquatic animal viruses by a virus titration study. The CPE caused by SVCV appeared as rounded and granular cells, grape-like clusters and small lytic plaques. Characteristic CPE containing plaque-like syncytia was induced by GCRV. RGV-infected cells produced typical CPE characterized by cells shrinkage and aggregation, formation of clear plaques and cell sheet detachment. Furthermore, significant fluorescent signals were observed after UCS cells were transfected with green fluorescent protein reporter plasmids, and the development of CPE induced by a recombinant RGV, ΔTK-RGV, in UCS cells was illustrated using a combination of light and fluorescence microscopy. The data from this study suggested that UCS cell line can potentially serve as a useful tool for the comparison study of different aquatic animal viruses and the isolation of some newly emerging viruses in Ussuri catfish farming.

Histopathology of cotton bollworm midgut infected with Helicoverpa armigera cytoplasmic polyhedrosis virus

This research was carried out to examine cytopathological effects of Helicoverpa armigera Cytoplasmic polyhedrosis virus (HaCPV) on infected midgut cotton bollworm (Helicoverpa armigera) using transmission and scanning electron microscope. The symptoms on infected host larvae of the host, compared with healthy ones, were getting swollen with milky-white and fragile Histopathological examinations showed infection with HaCPV small polyhedral inclusion bodies (PIB) after 1 or 2 days which were observed in columnar cells of midgut. Virions were partially or completely occupied in a polyhedral matrix to form polyhedral inclusion bodies (PIB) at periphery of virogenic stroma. PIBs were measured 0.5 to 3.5 mm and virions about 46 nm in diameter. Microvilli of infected columnar cells were affected and degenerated immediately prior to rupture of the cell. Some infected columnar cells ruptured to release PIB into the gut lumen 3 days after infection. In addition,PIB were found in goblet cells, 5 or 6 days after infection. Infected goblet cells degenerate to such an extent that only a few of the original microvillus-like cytoplasmic projections and cell organells were left. These cytopathic effects caused in the midgut by HaCPV on cotton bollworm larvae are essentially similar to those have been reported for lepidoperan and dipteran infection by CPV.

Apoptosis induced by mammalian reovirus is beta interferon (IFN) independent and enhanced by IFN regulatory factor 3- and NF-κB-dependent expression of Noxa.

A variety of signal transduction pathways are activated in response to viral infection, which dampen viral replication and transmission. These mechanisms involve both the induction of type I interferons (IFNs), which evoke an antiviral state, and the triggering of apoptosis. Mammalian orthoreoviruses are double-stranded RNA viruses that elicit apoptosis in vitro and in vivo. The transcription factors interferon regulatory factor 3 (IRF-3) and nuclear factor kappa light-chain enhancer of activated B cells (NF-κB) are required for the expression of IFN-ß and the efficient induction of apoptosis in reovirus-infected cells. However, it is not known whether IFN-ß induction is required for apoptosis, nor have the genes induced by IRF-3 and NF-κB that are responsible for apoptosis been identified. To determine whether IFN-ß is required for reovirus-induced apoptosis, we used type I IFN receptor-deficient cells, IFN-specific antibodies, and recombinant IFN-ß. We found that IFN synthesis and signaling are dispensable for the apoptosis of reovirus-infected cells. These results indicate that the apoptotic response following reovirus infection is mediated directly by genes responsive to IRF-3 and NF-κB. Noxa is a proapoptotic BH3-domain-only protein of the Bcl-2 family that requires IRF-3 and NF-κB for efficient expression. We found that Noxa is strongly induced at late times (36 to 48 h) following reovirus infection in a manner dependent on IRF-3 and NF-κB. The level of apoptosis induced by reovirus is significantly diminished in cells lacking Noxa, indicating a key prodeath function for this molecule during reovirus infection. These results suggest that prolonged innate immune response signaling induces apoptosis by eliciting Noxa expression in reovirus-infected cells.

Host response to polyomavirus infection is modulated by RNA adenosine deaminase ADAR1 but not by ADAR2.

Adenosine deaminases acting on RNA (ADARs) catalyze the C-6 deamination of adenosine (A) to produce inosine (I), which behaves as guanine (G), thereby altering base pairing in RNAs with double-stranded character. Two genes, adar1 and adar2, are known to encode enzymatically active ADARs in mammalian cells. Furthermore, two size forms of ADAR1 are expressed by alternative promoter usage, a short (p110) nuclear form that is constitutively made and a long (p150) form that is interferon inducible and present in both the cytoplasm and nucleus. ADAR2 is also a constitutively expressed nuclear protein. Extensive A-to-G substitution has been described in mouse polyomavirus (PyV) RNA isolated late times after infection, suggesting modification by ADAR. To test the role of ADAR in PyV infection, we used genetically null mouse embryo fibroblast cells deficient in either ADAR1 or ADAR2. The single-cycle yields and growth kinetics of PyV were comparable between adar1(-/-) and adar2(-/-) genetic null fibroblast cells. While large T antigen was expressed to higher levels in adar1(-/-) cells than adar2(-/-) cells, less difference was seen in VP1 protein expression levels between the two knockout MEFs. However, virus-induced cell killing was greatly enhanced in PyV-infected adar1(-/-) cells compared to that of adar2(-/-) cells. Complementation with p110 protected cells from PyV-induced cytotoxicity. UV-irradiated PyV did not display any enhanced cytopathic effect in adar1(-/-) cells. Reovirus and vesicular stomatitis virus single-cycle yields were comparable between adar1(-/-) and adar2(-/-) cells, and neither reovirus nor VSV showed enhanced cytotoxicity in adar1(-/-)-infected cells. These results suggest that ADAR1 plays a virus-selective role in the host response to infection.

CUG2, a novel oncogene confers reoviral replication through Ras and p38 signaling pathway.

As we have recently found a novel oncogene, the cancer-upregulated gene 2 (CUG2), which was elevated in a variety of tumor tissues such as the ovary, liver, lung and pancreas, we examined whether reovirus could efficiently induce cytolysis in cancer cells expressing CUG2 and thus be used as a potential cancer therapeutic agent. In this study, we describe experiments in which we use reovirus to treat NIH3T3 cells stably expressing either CUG2 (NIH-CUG2) or vector only (NIH-Vec). NIH-CUG2 cells readily support reoviral proliferation and undergo apoptosis, whereas NIH-Vec cells are highly resistant to reoviral infection and virus-induced apoptosis. This notable result may be explained by the observation that CUG2 expression inhibits PKR activation, leading to reoviral proliferation in nonpermissive NIH3T3 cells. Furthermore, reovirus infection results in almost complete regression of tumorgenic NIH-CUG2 cells in transplanted nude mice. As we found that CUG2 enhances activation of MAPK (ERK, JNK and p38), Src kinase and Ras, we examined whether CUG2 confers reoviral replication independent of the Ras or p38 MAPK signaling pathway. From these experiments we found that either inhibition of p38 MAPK or Ras blocks reoviral proliferation even in the presence of CUG2 but inhibition of ERK, JNK and Src kinase does not, indicating that activation of p38 MAPK and Ras has critical roles in reoviral replication in CUG2-expressing tumor cells. Accordingly, we propose that reovirus can be useful in the treatment of transformed cells expressing CUG2, which is commonly detected in various tumor tissues.

Supervivencia y transporte de los virus entéricos humanos a través de los alimentos / Survival and transport of human enteric viruses in foods

Los virus se transmiten a los seres humanos vía los alimentos como resultado de la contaminación directa o indirecta de los mismos con heces; ésta es la esencia del análisis de peligro...

Intracellular ATP and total adenylate concentrations are critical predictors of reovirus productivity from Vero cells.

The productivity of reovirus type-3 Dearing was studied in cultures of Vero cells in serum-free media. Viral productivity was dependent upon the metabolic state of the cells rather than the phase of growth at which the cells were infected. Cells at different energy states were established by 24-h incubation in nutrient-depleted media. This resulted in variable intracellular nucleotide concentrations but high cellular viability was maintained. Of the nucleotides analyzed at the time of infection only the intracellular [ATP] and total adenylate nucleotides were positively correlated with viral productivity. The correlated data followed a sigmoidal plot with an equation defined by polynomial regression analysis. Apparent threshold values of 3.2 fmol/cell and 3.3 fmol/cell were established for ATP and total adenylate, respectively, at which the viral production was 50% the maximal value. Cultures with lower ATP and total adenylate levels at the time of infection resulted in as much as a 95% reduction in overall viral titer compared to the control. The adenylate energy charge (AEC) showed a negative correlation with viral production with an AEC value >0.97 resulting in low virus productivity. Intracellular ATP or total adenylate concentration at the point of infection may be used as a predictor of viral yield in bioprocesses designed for virus/vaccine production.

No role for pepstatin-A-sensitive acidic proteinases in reovirus infections of L or MDCK cells.

Strong evidence indicates that virions of mammalian reoviruses undergo proteolytic processing by acid-dependent cellular proteinases as an essential step in productive infection. Proteolytic processing takes the form of a series of cleavages of outer-capsid proteins final sigma3 and mu1/mu1C. Previous studies showed an effect of both NH4Cl and E-64 on these cleavages, indicating that one or more of the acid-dependent cysteine proteinases in mammalian cells (cathepsins B and L, for example) is required; however, these studies did not address whether acid-dependent aspartic proteinases in those cells (cathepsin D, for example) may also be required. To determine the role of aspartic proteinases in reovirus entry, studies with pepstatin A, a specific inhibitor of aspartic proteinases, were performed. The results showed that pepstatin A neither blocks nor slows reovirus infection of L or MDCK cells. Experiments using ribonuclease A and other proteins as cleavable substrates showed that cathepsin-D-like proteinases from these cells are inhibited within the tested range of pepstatin A concentrations both in vitro and within living cells. In other experiments, virion-bound final sigma3 protein was shown to be a poor substrate for cleavage by cathepsin D in vitro, consistent with the findings with inhibitors. In sum, the data indicate that cathepsin-D-like aspartic proteinases provide little or no activity toward proteolytic events required for infection of L or MDCK cells with reovirus virions.

The molecular basis of viral oncolysis: usurpation of the Ras signaling pathway by reovirus.

NIH-3T3 cells, which are resistant to reovirus infection, became susceptible when transformed with activated Sos or Ras. Restriction of reovirus proliferation in untransformed NIH-3T3 cells was not at the level of viral gene transcription, but rather at the level of viral protein synthesis. An analysis of cell lysates revealed that a 65 kDa protein was phosphorylated in untransformed NIH-3T3 cells, but only after infection with reovirus. This protein was not phosphorylated in infected or uninfected transformed cells. The 65 kDa protein was determined to be the double-stranded RNA-activated protein kinase (PKR), whose phosphorylation leads to translation inhibition. Inhibition of PKR phosphorylation by 2-aminopurine, or deletion of the Pkr gene, led to drastic enhancement of reovirus protein synthesis in untransformed cells. The emerging picture is one in which early viral transcripts trigger PKR phosphorylation in untransformed cells, which in turn leads to inhibition of translation of viral genes; this phosphorylation event is blocked by an element(s) in the Ras pathway in the transformed cells, allowing viral protein synthesis to ensue. The usurpation of the Ras signaling pathway therefore constitutes the basis of reovirus oncolysis.

Lack of restriction of growth for aquareovirus in mammalian cells.

The striped bass (SBR) virus, a member of the recently described aquareoviruses, infected, caused cytopathic effects (CPE), and replicated in mammalian cells. The virus caused CPE in all 7 of the mammalian cell lines investigated. SBR virus functioned best at lower temperatures and it is these lower temperatures that appeared to be restricting factors for growth of some mammalian cells. At 22 degrees C the SBR virus grew to similar titers in both chinook salmon embryo (CHSE) cells and in mammalian cells. Analysis of viral polypeptide and RNA synthesis suggests that the restriction for viral growth at higher temperatures occurs after adsorption but before transcription and translation of viral genes. The fact that SBR virus did not grow at 37 degrees C implies that aquareoviruses are unlikely to be human pathogens.

Characterization of avian reovirus-induced cell fusion: the role of viral structural proteins.

Cell fusion induced by avian reovirus was analyzed using virus strain FC and Vero cells. One-step growth curves showed that cell fusion was directly associated with viral replication. Cell fusion occurred most efficiently at basic pH (8.0-8.5) and fusion from without could not be demonstrated. Actinomycin D, at low concentrations, increased cell fusion, and cycloheximide prevented cell fusion, indicating that viral protein(s) were responsible for the induction of cell fusion. Immunofluorescence tests indicated that viral proteins were present on the infected cell surface. Radioimmuno-precipitation identified structural proteins mu 2C and sigma 2 as predominant viral protein species present on the infected cell surface. Cell fusion was inhibited by virus-specific antisera, suggesting that mu 2C and/or sigma 2 present on the infected cell surface were involved in the induction of cell fusion. Trypsin and chymotrypsin treatment of purified viruses cleaved both mu 2C and sigma 2 proteins, but generated different cleavage products with each protein. The addition of trypsin to the culture media following infection increased cell fusion, whereas chymotrypsin treatment decreased cell fusion. The opposite effects of trypsin and chymotrypsin on the cell fusion, together with the different specificities of these two proteases in cleavage of mu 2C and sigma 2 proteins, further suggest that the cell surface-associated mu 2C and/or sigma 2 are involved in the syncytium formation.

Viral susceptibility of an immortalized human microvascular endothelial cell line.

CDC/EU.HMEC-1 is the first immortalized human microvascular endothelial cell line that retains morphologic, phenotypic, and functional characteristics of a normal human microvascular endothelial cell. This study evaluates a variety of viruses and their effects on this human endothelial cell line. The data indicate that adenoviruses, some herpesviruses, reoviruses and most picornaviruses grow well in HMEC-1, with distinctive cytopathic effects. The paramyxoviruses, however, do not appear to propagate, nor does HIV. The findings indicate that microvascular endothelial cells may act as a reservoir of these viruses; it also suggests the possibility that microvascular endothelium could be involved in the processing and presentation of antigen to immune cells.

Regulation of expression of the reovirus receptor on differentiated HL60 cells.

The reovirus receptor on mammalian cells has not been fully characterized and controversy exists over the nature of this receptor. We report here that the expression of this receptor is dependent on the differentiation status of a human promyelocytic leukaemia cell line (HL60). Phorbol treatment of HL60 cells for 24 h, at a concentration range of 160 nM down to 1 nM, led to differentiation of these cells towards monocytes and a loss of approximately 80% of their ability to bind reovirus in a fluorescence assay. These cells also lost their susceptibility to T1 and T3 reovirus infection. DMSO treatment for 24 h at a concentration of 1.25% (v/v) led to differentiation towards granulocytes. This was accompanied by an increase of approximately 15% in binding of reovirus to these cells. After being infected by T1 or T3 reovirus, the granulocytes produced higher titres of progeny virus than did untreated HL60 cells. Similar differences were noted when virus binding to HL60 cells was assayed using radiolabelled reovirus. These effects were not detected when murine L fibroblasts were treated with DMSO or phorbol. ATCC-derived murine R1.1 cells did not bind reovirus. Competition data indicated that there may be two reovirus receptors on HL60 cells, and that T1 can bind to only one receptor whereas T3 can bind to both receptors. Our data also suggested that the beta-adrenergic receptor was unlikely to act as the reovirus receptor on HL60 cells.

A 2-year survey of the prevalence of enteric viral infections in children compared with contamination in locally-harvested oysters.

We studied, for two years, the prevalence of indigenous human enteric viruses in wild oysters gathered each month from the bottom of Mikawa Bay, Aichi Prefecture, Japan. Viruses were detected periodically in 9 out of 54 oyster pools prepared by the acid or polyethylene glycol precipitation method although all these 9 pools met current national bacteriological safety standards. Since most of the serotypes of the enteric viruses detected in the oysters were identical with those of viruses isolated from sick children living in the area, it is suggested that contamination of enteric viruses in the oysters would depend on the prevalence of enteric viral infections in the local inhabitants.

Proteins of Palyam serogroup viruses.

The replication and polypeptide synthesis of Abadina virus, a member of the Palyam serogroup of orbiviruses, was studied. The first virus-specified proteins could be demonstrated 2 to 4 h post-infection (p.i.) by immunoprecipitation. The rate of synthesis increased rapidly until 12 h p.i. after which it remained fairly constant until 18 h p.i. when it began to decline. Host cell protein synthesis shutoff was incomplete. Twelve virus-induced polypeptides were identified in infected cell lysates, ranging in Mr from 36K to 143K. Three small polypeptides (Mr 15K to 20K) identified in homologous immunoprecipitation studies are also thought to be virus-specified. Nine structural proteins were identified, four being major components of the purified virion. Partial proteolysis was used to demonstrate homology between some proteins. Pulse-chase experiments provided no evidence for a precursor-product relationship between any of the Abadina virus proteins. A non-structural protein was found to be phosphorylated.

Susceptibilities of 14 cell lines to bluetongue virus infection.

The effect of bluetongue virus (BTV) infection was investigated in 14 cell lines. The cell lines included the following vertebrate cells: baby hamster kidney, African green monkey kidney (Vero), rabbit kidney, bovine kidney, canine kidney, bovine turbinate, bovine endothelium (CPAE), bighorn sheep tongue, equine dermis, gekko lung, rainbow trout gonad, and mouse fibroblast (L929); they also included the following invertebrate lines: mosquito and biting midge. Comparisons between the cell lines were made on the basis of time to observed cytopathic effects, titer in 50% tissue culture infectious doses, and titer in plaque-forming units. The CPAE cell line produced the highest BTV 50% tissue culture infectious dose of all cell lines tested. The Vero and L929 cells gave the most discrete plaques in plaque assays. Of the 14 cell lines tested, the CPAE cells were the most susceptible to both cell culture-adapted and animal source BTV. Bovine endothelial cells demonstrate significant potential as a cell culture system for BTV investigations.