A TGFß Signaling Inhibitor, SB431542, Inhibits Reovirus-mediated Lysis of Human Hepatocellular Carcinoma Cells in a TGFß-independent Manner.

BACKGROUND/AIM: Oncolytic reovirus, which is a non-enveloped virus possessing a 10-segmented double-stranded RNA genome, has been anticipated as a novel class of antitumor agent. Hepatocellular carcinoma (HCC) is considered to be a target suitable for reovirus-mediated virotherapy. Transforming growth factor (TGF)-ß plays an important role in the pathogenesis of HCC. TGF-ß-signaling inhibitors have proceeded to clinical trials as potential antitumor agents for HCC. On the other hand, TGF-ß is involved in induction of expression of cathepsins B and L, which are important for reovirus infection. It remains to be examined whether TGF-ß signaling inhibitors affect reovirus-mediated lysis of HCC cells. The aim of this study was to evaluate the effects of TGF-ß-signaling inhibitors on tumor cell lysis efficiency of reovirus in human HCC cells. MATERIALS AND METHODS: Reovirus was added to four types of human HCC cell lines pretreated with one of three TGF-ß type I receptor inhibitors: SB431542, A-83-01, or galunisertib (LY2157299). Cell viability, virus genome copy numbers, and virus protein expression were evaluated following reovirus infection. RESULTS: SB431542 significantly inhibited reovirus-mediated killing of human HCC cell lines, while A-83-01 and galunisertib did not inhibit. CONCLUSION: These data indicate that SB431542 inhibited reovirus-mediated lysis of human HCC cells in a TGF-ß signaling-independent manner.

Mammalian orthoreovirus core protein µ2 reorganizes host microtubule-organizing center components.

Filamentous mammalian orthoreovirus (MRV) viral factories (VFs) are membrane-less cytosolic inclusions in which virus transcription, replication of dsRNA genome segments, and packaging of virus progeny into newly synthesized virus cores take place. In infected cells, the MRV µ2 protein forms punctae in the enlarged region of the filamentous VFs that are co-localized with γ-tubulin and resistant to nocodazole treatment, and permitted microtubule (MT)-extension, features common to MT-organizing centers (MTOCs). Using a previously established reconstituted VF model, we addressed the functions of MT-components and MTOCs concerning their roles in the formation of filamentous VFs. Indeed, the MTOC markers γ-tubulin and centrin were redistributed within the VF-like structures (VFLS) in a µ2-dependent manner. Moreover, the MT-nucleation centers significantly increased in numbers, and γ-tubulin was pulled-down in a binding assay when co-expressed with histidine-tagged-µ2 and µNS. Thus, µ2, by interaction with γ-tubulin, can modulate MTOCs localization and function according to viral needs.

Bacillus pumilus ribonuclease rescues mice infected by double-stranded RNA-containing reovirus serotype 1.

Reoviruses (respiratory enteric orphan viruses) are nonenveloped viruses with segmented dsRNA genome. Viruses in the family Reoviridae are quite stable in the environment. Recently, they have been identified with various pathologies and physiologic dysfunctions in a wide range of organs and tissues, including the hepatobiliary system, the myocardium, lungs, and endocrine tissues. Although most cases of reovirus infection are mild or subclinical diseases, the prevention measures are currently needed, especially for young children suffering from dehydrating gastroenteritis. To inhibit viral replication, different RNases targeting viral RNA are proposed. Here, we first have shown that RNase from Bacillus pumilus (binase) acts as an antiviral agent at the level of the whole animal organism infected by Mammalian orthoreovirus 1 strain Lang (TL1). The results obtained on the mice model infected with 10 LD50 and 20 LD50 doses of reovirus indicate the restoration of mice physiological parameters under binase treatment at the dose of 50 µg/mouse. Thus, our research supports the relevance of binase as a promising antiviral agent that affects viral RNA.

The Antibiotic Neomycin Enhances Coxsackievirus Plaque Formation.

Coxsackievirus typically infects humans via the gastrointestinal tract, which has a large number of microorganisms collectively referred to as the microbiota. To study how the intestinal microbiota influences enteric virus infection, several groups have used an antibiotic regimen in mice to deplete bacteria. These studies have shown that bacteria promote infection with several enteric viruses. However, very little is known about whether antibiotics influence viruses in a microbiota-independent manner. In this study, we sought to determine the effects of antibiotics on coxsackievirus B3 (CVB3) using an in vitro cell culture model in the absence of bacteria. We determined that an aminoglycoside antibiotic, neomycin, enhanced the plaque size of CVB3 strain Nancy. Neomycin treatment did not alter viral attachment, translation, or replication. However, we found that the positive charge of neomycin and other positively charged compounds enhanced viral diffusion by overcoming the negative inhibitory effect of sulfated polysaccharides present in agar overlays. Neomycin and the positively charged compound protamine also enhanced plaque formation of reovirus. Overall, these data provide further evidence that antibiotics can play noncanonical roles in viral infections and that this should be considered when studying enteric virus-microbiota interactions.IMPORTANCE Coxsackieviruses primarily infect the gastrointestinal tract of humans, but they can disseminate systemically and cause severe disease. Using antibiotic treatment regimens to deplete intestinal microbes in mice, several groups have shown the bacteria promote infection with a variety of enteric viruses. However, it is possible that antibiotics have microbiota-independent effects on viruses. Here we show that an aminoglycoside antibiotic, neomycin, can influence quantification of coxsackievirus in cultured cells in the absence of bacteria.

A single mutation in the mammalian orthoreovirus S1 gene is responsible for increased interferon sensitivity in a virus mutant selected in Vero cells.

In a previous study, a mammalian orthoreovirus mutant was isolated based on its increased ability to infect interferon-defective Vero cells and was referred to as Vero-cells-adapted virus (VeroAV). This virus exhibits reduced ability to resist the antiviral effect of interferon. In the present study, the complete genome sequence of VeroAV was first determined. Reverse genetics was then used to identify a unique mutation on the S1 gene, overlapping the σ1 and σ1 s reading frame, resulting in increased sensitivity to interferon. A virus lacking σ1 s expression consecutive to mutation of its initiation codon was then shown to exhibit a further increase in sensitivity to interferon, supporting the idea that σ1 s is the viral protein responsible. This identification of a new determinant of reovirus sensitivity to interferon gives credentials to the idea that multiple reovirus genes are responsible for the level of interferon induction and susceptibility to the interferon-induced antiviral activities.

Multiple proteins differing between laboratory stocks of mammalian orthoreoviruses affect both virus sensitivity to interferon and induction of interferon production during infection.

In the course of previous works, it was observed that the virus laboratory stock (T3DS) differs in sequence from the virus encoded by the ten plasmids currently in use in many laboratories (T3DK), and derived from a different original virus stock. Seven proteins are affected by these sequence differences. In the present study, replication of T3DK was shown to be more sensitive to the antiviral effect of interferon. Infection by the T3DK virus was also shown to induce the production of higher amount of ß and α-interferons compared to T3DS. Two proteins, the µ2 and λ2 proteins, were found to be responsible for increased sensitivity to interferon while both µ2 and λ1 are responsible for increased interferon secretion. Altogether this supports the idea that multiple reovirus proteins are involved in the control of induction of interferon and virus sensitivity to the interferon-induced response. While interrelated, interferon induction and sensitivity can be separated by defined gene combinations. While both µ2 and λ2 were previously suspected of a role in the control of the interferon response, other proteins are also likely involved, as first shown here for λ1. This also further stresses that due caution should be exerted when comparing different virus isolates with different genetic background.

Antiviral activity and metal ion-binding properties of some 2-hydroxy-3-methoxyphenyl acylhydrazones.

Here we report on the results obtained from an antiviral screening, including herpes simplex virus, vaccinia virus, vesicular stomatitis virus, Coxsackie B4 virus or respiratory syncytial virus, parainfluenza-3 virus, reovirus-1 and Punta Toro virus, of three 2-hydroxy-3-methoxyphenyl acylhydrazone compounds in three cell lines (i.e. human embryonic lung fibroblast cells, human cervix carcinoma cells, and African Green monkey kidney cells). Interesting antiviral EC50 values are obtained against herpes simplex virus-1 and vaccinia virus. The biological activity of acylhydrazones is often attributed to their metal coordinating abilities, so potentiometric and microcalorimetric studies are here discussed to unravel the behavior of the three 2-hydroxy-3-methoxyphenyl compounds in solution. It is worth of note that the acylhydrazone with the higher affinity for Cu(II) ions shows the best antiviral activity against herpes simplex and vaccinia virus (EC50 ~ 1.5 µM, minimal cytotoxic concentration = 60 µM, selectivity index = 40).

[Exogenous Bacillus pumilus RNase (binase) suppresses the reproduction of reovirus serotype 1].

The experimental study identified the antiviral activity of Bacillus pumilus RNase (binase) against the reovirus of serotype 1/strain Lang. For the first time, it has been found that 50 µg/mL of binase effectively reduced the hemagglutinin and cytocidal activity of reovirus in Vero cell line. The preincubation of the enzyme with reovirus before infection of the cells inhibited the viral replication. To determine the stagedependent effect of reovirus reproduction upon binase inhibition, the infected cells were treated with binase or RNase A at different phases of the infectious cycle. The treatment of virus-infected cells has revealed that both enzymes have a maximal antiviral effect on the reovirus propagation during early phases of the reovirus reproduction cycle, with binase being more effective than RNase A. It has been hypothesized that the combined action of the oncolytic reovirus and binase is promising for the elimination of tumor cells carrying mutated RAS gene.

Novel antiviral effect of lithium chloride on mammalian orthoreoviruses in vitro.

Reovirus not only causes considerable economic loss in the swine industry of the United States and other countries, but also threatens the public health due to its zoonotic potential. According to previous reports, LiCl has antiviral activity against a number of viruses. The inhibitory effects of LiCl on reovirus life cycle in Vero cells were evaluated. The unpaired t-test and one-way ANOVA were used to analyze the differences between experimental groups. We first found that LiCl treatment significantly inhibited reovirus replication in a dose-dependent manner. Furthermore, we found that this antiviral activity of LiCl targets the early stage of viral replication. LiCl could be a potential drug against reovirus infection.

Type I interferon signaling limits reoviral tropism within the brain and prevents lethal systemic infection.

In vivo and ex vivo models of reoviral encephalitis were utilized to delineate the contribution of type I interferon (IFN) to the host's defense against local central nervous system (CNS) viral infection and systemic viral spread. Following intracranial (i.c.) inoculation with either serotype 3 (T3) or serotype 1 (T1) reovirus, increased expression of IFN-α, IFN-ß, and myxovirus-resistance protein (Mx1; a prototypical IFN stimulated gene) was observed in mouse brain tissue. Type I IFN receptor deficient mice (IFNAR(-/-)) had accelerated lethality, compared to wildtype (B6wt) controls, following i.c. T1 or T3 challenge. Although viral titers in the brain and eyes of reovirus infected IFNAR(-/-) mice were significantly increased, these mice did not develop neurologic signs or brain injury. In contrast, increased reovirus titers in peripheral tissues (liver, spleen, kidney, heart, and blood) of IFNAR(-/-) mice were associated with severe intestinal and liver injury. These results suggest that reovirus-infected IFNAR(-/-) mice succumb to peripheral disease rather than encephalitis per se. To investigate the potential role of type I IFN in brain tissue, brain slice cultures (BSCs) were prepared from IFNAR(-/-) mice and B6wt controls for ex vivo T3 reovirus infection. Compared to B6wt controls, reoviral replication and virus-induced apoptosis were enhanced in IFNAR(-/-) BSCs indicating that a type I IFN response, initiated by resident CNS cells, mediates innate viral immunity within the brain. T3 reovirus tropism was extended in IFNAR(-/-) brains to include dentate neurons, ependymal cells, and meningeal cells indicating that reovirus tropism within the CNS is dependent upon type I interferon signaling.

[Examination of biocides for their effectiveness against animal viruses according to European Union Standards with emphasis on the selection of a suitable test virus]. / Prüfung von Bioziden auf Wirksamkeit gegen animale Viren nach EU-Norm im Hinblick auf die Auswahl eines geeigneten Testvirus.

Because of the changes to be expected in the methods for testing disinfectants deemed to be used in the veterinary field, candidate viral species were evaluated for their suitability as test virus. Considered viral species included different non-enveloped viruses [bovine enterovirus type 1 (ECBO (Enteric Cytopathogenic Bovine Orphan) virus), mammalian reovirus type 1, feline calici virus (FCV), and bovine parvovirus (BPV)], as well as enveloped viruses, as equine arteritisvirus (EAV), bovine herpesvirus type 1 (BHV1), Newcastle disease virus (NDV) and vaccinia virus. Viruses were tested for their tenacity against different biocidal agents (formaldehyde, formic acid, peracetic acid, and sodium hypochlorite) in the suspension test at a temperature of 20 degrees C which is given as an optional test temperature according to prEN 14675 "Quantitative suspension test for the evaluation of virucidal activity of chemical disinfectants and antiseptics used in veterinary field--Test method and requirements"elaborated by the "Comite Européen de Normalisation"(CEN) (Anonym, 2004). Of the animal viruses tested for their tenacity highest tenacity against the disinfectants. FCV and the enveloped viruses were of lower resistance. In addition to the tenacity of viruses, other parameters, such as the ability of the virus to replicate in permanent cells, the magnitude of the virus titre that can be obtained from such cultures, as well as the threat a virus poses to humans and animals are to be considered when selecting a suitable test virus. Based on these criteria and despite its tenacity being inferior to that of BPV, the ECBO virus was chosen as the most suitable test virus. The result of the efficacy of disinfectants is not based on the most resistant virus in this case. This circumstance is to be considered when giving recommendations for the practical use of disinfectants.

3,3'-Diindolylmethane stimulates murine immune function in vitro and in vivo.

3,3'-Diindolylmethane (DIM), a major condensation product of indole-3-carbinol, exhibits chemopreventive properties in animal models of cancer. Recent studies have shown that DIM stimulates interferon-gamma (IFN-gamma) production and potentiates the IFN-gamma signaling pathway in human breast cancer cells via a mechanism that includes increased expression of the IFN-gamma receptor. The goal of this study was to test the hypothesis that DIM modulates the murine immune function. Specifically, the effects of DIM were evaluated in a panel of murine immune function tests that included splenocyte proliferation, reactive oxygen species (ROS) generation, cytokine production and resistance to viral infection. DIM was found to induce proliferation of splenocytes as well as augment mitogen- and interleukin (IL)-2-induced splenocyte proliferation. DIM also stimulated the production of ROS by murine peritoneal macrophage cultures. Oral administration of DIM, but not intraperitoneal injection, induced elevation of serum cytokines in mice, including IL-6, granulocyte colony-stimulating factor (G-CSF), IL-12 and IFN-gamma. Finally, in a model of enteric virus infection, oral DIM administration to mice enhanced both clearance of reovirus from the GI tract and the subsequent mucosal IgA response. Thus, DIM is a potent stimulator of immune function. This property might contribute to the cancer inhibitory effects of this indole.

Guanidine hydrochloride inhibits mammalian orthoreovirus growth by reversibly blocking the synthesis of double-stranded RNA.

Millimolar concentrations of guanidine hydrochloride (GuHCl) are known to inhibit the replication of many plant and animal viruses having positive-sense RNA genomes. For example, GuHCl reversibly interacts with the nucleotide-binding region of poliovirus protein 2C(ATPase), resulting in a specific inhibition of viral negative-sense RNA synthesis. The use of GuHCl thereby allows for the spatiotemporal separation of poliovirus gene expression and RNA replication and provides a powerful tool to synchronize the initiation of negative-sense RNA synthesis during in vitro replication reactions. In the present study, we examined the effect of GuHCl on mammalian orthoreovirus (MRV), a double-stranded RNA (dsRNA) virus from the family Reoviridae. MRV growth in murine L929 cells was reversibly inhibited by 15 mM GuHCl. Furthermore, 15 mM GuHCl provided specific inhibition of viral dsRNA synthesis while sparing both positive-sense RNA synthesis and viral mRNA translation. By using GuHCl to provide temporal separation of MRV gene expression and genome replication, we obtained evidence that MRV primary transcripts support sufficient protein synthesis to assemble morphologically normal viral factories containing functional replicase complexes. In addition, the coordinated use of GuHCl and cycloheximide allowed us to demonstrate that MRV dsRNA synthesis can occur in the absence of ongoing protein synthesis, although to only a limited extent. Future studies utilizing the reversible inhibition of MRV dsRNA synthesis will focus on elucidating the target of GuHCl, as well as the components of the MRV replicase complexes.

Correlation between interferon sensitivity of reovirus isolates and ability to discriminate between normal and Ras-transformed cells.

Mammalian reoviruses exhibit a propensity to replicate in transformed cells. It is currently believed that the interferon-inducible RNA-dependent protein kinase (PKR), an intracellular host-cell resistance factor that is inhibited by an activated Ras-dependent pathway in transformed cells, is responsible for this discrimination. In the present study, reovirus isolates differing in their sensitivity to interferon were obtained by chemical mutagenesis, and examined for their replicative properties in parental and Ras-transformed mouse NIH-3T3 cells. It was observed that most isolates can bypass resistance mechanisms of parental cells at high m.o.i., and that there is a correlation between the ability to discriminate between transformed and parental cells, and interferon sensitivity. Most interestingly, an interferon-hypersensitive mutant virus was more dependent on Ras activation than any other viral isolate. Altogether, this suggests that optimal reovirus isolates could be selected to attack tumour cells depending on the nature of the alterations in interferon-inducible pathways found in these cells.

Evaluation of virucidal activity of three commercial disinfectants and formic acid using bovine enterovirus type 1 (ECBO virus), mammalian orthoreovirus type 1 and bovine adenovirus type 1.

A modified version of the test method of the Comité Européen de Normalisation (CEN) was developed using formic acid and three commercial disinfectants to evaluate virucidal activity against three non-enveloped viruses, bovine enterovirus type 1 (ECBO virus), mammalian orthoreovirus type 1 and bovine adenovirus type 1 (BAV 1). Determination of the effects of temperature was carried out at 20 and 10 degrees C. All tests with protein load used bovine serum albumin (BSA) and yeast extract. The investigations were performed in suspension tests and in carrier tests using poplar wood virus carriers. The carrier tests showed that ECBO virus could be inactivated at 20 degrees C with 1% formic acid within a 60 min reaction time. For disinfection of ECBO virus at 10 degrees C within 60 min, a 2% concentration of formic acid was necessary. Formic acid was ineffective against reovirus and bovine adenovirus and cannot be recommended as a reference disinfectant. Inactivation of ECBO virus and adenovirus type 1 using a disinfectant containing aldehydes and alcohols could be achieved, but only at room temperature. The disinfection of reovirus type 1 at room temperature with this product was possible without a protein load. This disinfectant exhibited disinfection ability at 10 degrees C at a concentration of more than 2% or with a longer exposure time. A disinfectant containing aldehydes was effective at room temperature but its effect was reduced in the presence of organic matter. Inactivation at 10 degrees C was found only against adenovirus. The fourth disinfectant, which contained peroxiacetic acid, inactivated all test viruses at a concentration of 0.5% within 15 min independent of temperature and protein load.

Cathepsin L and cathepsin B mediate reovirus disassembly in murine fibroblast cells.

After attachment to receptors, reovirus virions are internalized by endocytosis and exposed to acid-dependent proteases that catalyze viral disassembly. Previous studies using the cysteine protease inhibitor E64 and a mutant cell line that does not support reovirus disassembly suggest a requirement for specific endocytic proteases in reovirus entry. This study identifies the endocytic proteases that mediate reovirus disassembly in murine fibroblast cells. Infection of both L929 cells treated with the cathepsin L inhibitor Z-Phe-Tyr(t-Bu)-diazomethyl ketone and cathepsin L-deficient mouse embryo fibroblasts resulted in inefficient proteolytic disassembly of viral outer-capsid proteins and decreased viral yields. In contrast, both L929 cells treated with the cathepsin B inhibitor CA-074Me and cathepsin B-deficient mouse embryo fibroblasts support reovirus disassembly and growth. However, removal of both cathepsin B and cathepsin L activity completely abrogates disassembly and growth of reovirus. Concordantly, cathepsin L mediates reovirus disassembly more efficiently than cathepsin B in vitro. These results demonstrate that either cathepsin L or cathepsin B is required for reovirus entry into murine fibroblasts and indicate that cathepsin L is the primary mediator of reovirus disassembly. Moreover, these findings suggest that specific endocytic proteases can determine host cell susceptibility to infection by intracellular pathogens.

Thermostability of reovirus disassembly intermediates (ISVPs) correlates with genetic, biochemical, and thermodynamic properties of major surface protein mu1.

Kinetic analyses of infectivity loss during thermal inactivation of reovirus particles revealed substantial differences between virions and infectious subvirion particles (ISVPs), as well as between the ISVPs of reoviruses type 1 Lang (T1L) and type 3 Dearing (T3D). The difference in thermal inactivation of T1L and T3D ISVPs was attributed to the major surface protein mu1 by genetic analyses with reassortant viruses and recoated cores. Irreversible conformational changes in ISVP-bound mu1 were shown to accompany thermal inactivation. The thermal inactivation of ISVPs approximated first-order kinetics over a range of temperatures, permitting the use of Arrhenius plots to estimate activation enthalpies and entropies that account for the different behaviors of T1L and T3D. An effect similar to enthalpy-entropy compensation was additionally noted for the ISVPs of these two isolates. Kinetic analyses with other ISVP-like particles, including ISVPs of a previously reported thermostable mutant, provided further insights into the role of mu1 as a determinant of thermostability. Intact virions, which contain final sigma3 bound to mu1 as their major surface proteins, exhibited greater thermostability than ISVPs and underwent thermal inactivation with kinetics that deviated from first order, suggesting a role for final sigma3 in both these properties. The distinct inactivation behaviors of ISVPs are consistent with their role as an essential intermediate in reovirus entry.