Preliminary characterization of a reovirus isolated from golden ide Leuciscus idus melanotus

Some characteristics of a reovirus recently isolated from golden ide Leuciscus idus melanotus and tentatively designated as golden ide reovirus (GIRV) were determined. Spherical non-enveloped particles with an outer capsid of about 70 nm and an inner capsid of about 50 nm were observed by electron microscopy. The density of the virus determined in CsCl gradients was 1.36 g ml-1. The genome contained 11 segments of dsRNA. GIRV differed from other aquareoviruses by a slight reduction of infectivity after treatment with chloroform and by the absence of forming syncytia in cell monolayers.     

Inactivation of Reovirus Type 2 by a Combination of Chloroform and Moderate Temperature

The infectivity and hemagglutinins of reovirus type 2 were destroyed at 37 C in the presence of chloroform, whereas no effect of chloroform was noted at 0 C.     

Effect of proteins on reovirus adsorption to clay minerals

Organic matter in sewage, soil, and aquatic systems may enhance or inhibit the infectivity of viruses associated with particulates (e.g., clay minerals, sediments). The purpose of this investigation was to identify the mechanisms whereby organic matter, in the form of defined proteins, affects the adsorption of reovirus to the clay minerals kaolinite and montmorillonite and its subsequent infectivity. Chymotrypsin and ovalbumin reduced the adsorption of reovirus to kaolinite and montmorillonite homoionic to sodium. Lysozyme did not reduce the adsorption of the virus to kaolinite, but it did reduce adsorption to montmorillonite. The proteins apparently competed with the reovirus for sites on the clay. As lysozyme does not adsorb to kaolinite by cation exchange, it did not inhibit the adsorption of reovirus to this clay. The amount of reovirus desorbed from lysozyme-coated montmorillonite was approximately 38% less (compared with the input population) than that from uncoated or chymotrypsin-coated montmorillonite after six washings with sterile distilled water. Chymotrypsin and lysozyme markedly decreased reovirus infectivity in distilled water, whereas infectivity of the virus was enhanced after recovery from an ovalbumin-distilled water-reovirus suspension (i.e., from the immiscible pelleted fraction plus supernatant). The results of these studies indicate that the persistence of reovirus in terrestrial and aquatic environments may vary with the type of organic matter and clay mineral with which the virus comes in contact.     

Effect of proteins on reovirus adsorption to clay minerals.

Organic matter in sewage, soil, and aquatic systems may enhance or inhibit the infectivity of viruses associated with particulates (e.g., clay minerals, sediments). The purpose of this investigation was to identify the mechanisms whereby organic matter, in the form of defined proteins, affects the adsorption of reovirus to the clay minerals kaolinite and montmorillonite and its subsequent infectivity. Chymotrypsin and ovalbumin reduced the adsorption of reovirus to kaolinite and montmorillonite homoionic to sodium. Lysozyme did not reduce the adsorption of the virus to kaolinite, but it did reduce adsorption to montmorillonite. The proteins apparently competed with the reovirus for sites on the clay. As lysozyme does not adsorb to kaolinite by cation exchange, it did not inhibit the adsorption of reovirus to this clay. The amount of reovirus desorbed from lysozyme-coated montmorillonite was approximately 38% less (compared with the input population) than that from uncoated or chymotrypsin-coated montmorillonite after six washings with sterile distilled water. Chymotrypsin and lysozyme markedly decreased reovirus infectivity in distilled water, whereas infectivity of the virus was enhanced after recovery from an ovalbumin-distilled water-reovirus suspension (i.e., from the immiscible pelleted fraction plus supernatant). The results of these studies indicate that the persistence of reovirus in terrestrial and aquatic environments may vary with the type of organic matter and clay mineral with which the virus comes in contact.     

Interaction of reovirus with cell surface receptors. II. Generation of suppressor T cells by the hemagglutinin of reovirus type 3

In studying reovirus interactions with lymphocytes, we have found that reovirus type 3, but not type 1, inhibits the in vitro proliferative response of murine splenic lymphocytes to concanavalin A (Con A). By analyzing recombinant clones containing genes from both reovirus types 1 and 3, we found that the S1 gene, the gene that encodes the viral hemagglutinin, is responsible for the inhibitory effect. In addition we found that type 3, but not type 1, generates suppressor T cells in vitro capable of suppressing Con A proliferation. By analyzing recombinant clones, we also found that the viral hemagglutinin is responsible for the generation of suppressor T cells by reovirus type 3. These effects were observed whether UV-inactivated or live virus was used. Reovirus type 3 inhibition of the proliferative response of murine splenic lymphocytes to Con A was blocked by anti-reovirus type 3 antibody but not by anti-reovirus type 1 antibody. Antiviral antibody had no effect on the ability of reovirus type 3 induced suppressor cells to inhibit Con A proliferation. We have previously demonstrated a receptor on murine lymphocytes for the hemagglutinin of reovirus type 3, and our results suggest that the in vitro suppression of Con A proliferation of murine lymphocytes by reovirus type 3 is secondary to the interaction of the viral hemagglutinin with a receptor on the surface of murine lymphocytes, which results in the generation of functionally active suppressor T cells.     

Reovirus: Effect of Noninfective Viral Components on Cellular Deoxyribonucleic Acid Synthesis

We determined the effects of noninfective reovirus components on cellular deoxyribonucleic acid (DNA) synthesis. Reovirus inactivated by ultraviolet light inhibited cellular DNA synthesis, whereas reovirus cores and empty capsids did not. Both cores and empty capsids were adsorbed to cells. Adenine-rich ribonucleic acid (RNA) from reovirus, adsorbed to cells in the presence of diethyl-aminoethyl-dextran, produced a partial inhibition of DNA synthesis. RNA was synthesized in the presence of actinomycin D after infection with ultraviolet light-irradiated reovirus, and this RNA synthesis was not due to multiplicity reactivation of virus infectivity. These data suggest that viral structural proteins do not inhibit DNA synthesis and that the inhibition produced by ultraviolet-irradiated virus may be mediated in part or in toto by a newly synthesized viral product.     

Absence of Adenine-Rich Ribonucleic Acid from Purified Infectious Reovirus 3

The basic properties of released and cell-associated reovirus are the same. Both contained as their total nucleic acid complement only double-stranded ribonucleic acid (RNA) with an adenine content of 28%. Preparations of purified cell-associated virus, but not released virus, contained adenine-rich RNA which could be separated from the virus with little or no loss of infectivity. These adenine-rich ribonucleic acids were present in the virus preparations either as free RNA or associated with some structures of molecular weight less than 25 x 10(6) daltons. In contrast to our previous report, double-stranded reovirus RNA possessed little or no template activity for the Escherichia coli deoxyribonucleic acid and RNA polymerases.     

Reovirus variants with mutations in genome segments S1 and L2 exhibit enhanced virion infectivity and superior oncolysis

Reovirus preferentially replicates in transformed cells and is being explored as a cancer therapy. Immunological and physical barriers to virotherapy inspired a quest for reovirus variants with enhanced oncolytic potency. Using a classical genetics approach, we isolated two reovirus variants (T3v1 and T3v2) with superior replication relative to wild-type reovirus serotype 3 Dearing (T3wt) on various human and mouse tumorigenic cell lines. Unique mutations in reovirus λ2 vertex protein and σ1 cell attachment protein were associated with the large plaque-forming phenotype of T3v1 and T3v2, respectively. Both T3v1 and T3v2 exhibited higher infectivity (i.e., a higher PFU-to-particle ratio) than T3wt. A detailed analysis of virus replication revealed that virus cell binding and uncoating were equivalent for variant and wild-type reoviruses. However, T3v1 and T3v2 were significantly more efficient than T3wt in initiating productive infection. Thus, when cells were infected with equivalent input virus particles, T3v1 and T3v2 produced significantly higher levels of early viral RNAs relative to T3wt. Subsequent steps of virus replication (viral RNA and protein synthesis, virus assembly, and cell death) were equivalent for all three viruses. In a syngeneic mouse model of melanoma, both T3v1 and T3v2 prolonged mouse survival compared to wild-type reovirus. Our studies reveal that oncolytic potency of reovirus can be improved through distinct mutations that increase the infectivity of reovirus particles.     

Functional and topographical analyses of epitopes on the hemagglutinin (VP4) of the simian rotavirus SA11.

An immunochemical analysis of the hemagglutinin (VP4) of the simian rotavirus SA11 was performed to better understand the structure and function of this molecule. Following immunization of mice with double-shelled virus particles and VP4-enriched fractions from CsCl gradients, a battery of anti-SA11 hybridomas was generated. A total of 13 clones secreting high levels of anti-VP4 monoclonal antibody (MAb) was characterized and compared with two cross-reactive anti-VP4 MAbs generated against heterologous rhesus (RRV) and porcine (OSU) rotavirus strains. These cross-reactive MAbs effectively neutralized SA11 infectivity in vitro. The epitopes recognized by these 15 MAbs were grouped into six antigenic sites on the SA11 hemagglutinin. These sites were identified following analysis of the MAbs by using a simple competitive binding enzyme-linked immunosorbent assay (ELISA) and biological assays. Three of the antigenic sites were involved in neutralization of virus infectivity in vitro. All the MAbs with neutralization activity and two nonneutralizing MAbs were able to inhibit viral hemagglutination of human erythrocytes. Competitive binding ELISA data showed a positive cooperative binding effect with some pairs of the anti-VP4 MAbs, apparently due to a conformational change induced by the binding of the first MAb. Some of the MAbs also bound better to trypsin-treated virus than to non-trypsin-treated virus. A topographic map for VP4 is proposed on the basis of the observed properties of each antigenic site.     

Specificity of virus adsorption to clay minerals

Competitive adsorption studies indicated that reovirus type 3 and coliphage T1 did not share common adsorption sites on kaolinite and montmorillonite. Compounds in the minimal essential medium (e.g., fetal bovine serum, amino acids) in which the reovirus was maintained blocked adsorption of coliphage T1 to kaolinite and partially to montmorillonite in synthetic estuarine water, but they had no effect on coliphage adsorption to montmorillonite in distilled water or on the adsorption of the reovirus to either clay. The blockage of positively charged sites on kaolinite or montmorillonite by treatment of the clays with sodium metaphosphate or with the supernatants from montmorillonite or kaolinite, respectively, had no effect on adsorption of the reovirus. These data indicate that there was a specificity in adsorption sites for mixed populations of reovirus type 3 and coliphage T1 and emphasize the importance of using more than one type of virus, especially in combination, to predict virus behavior (e.g., adsorption, loss of infectivity) in soils and sediments containing clay minerals.     

Removal of carbohydrate from influenza A virus and its hemagglutinin and the effect on biological activities.

Treatment of influenza virus and its purified hemagglutining with glycosidases from Diplococcus pneumoniae, which included beta-galactosidase, beta-N-acetylglucosminidase, and endoglycosidase D, released amino and neutral sugars from the virus and these as well as large oligosaccharides from the purified hemagglutinin. The released glucosamine-containing oligosaccharides were of one discrete size. Large oligosaccharides not removed by the glycosidases were found on the virus as well as the hemagglutinin. Some oligosaccharides on the virus were inaccessible to the enzymes, since they could be removed only from the purified hemagglutinin. Approximately 50% of the hemagglutinin carbohydrates could be removed without effect on hemagglutinating activity. Similarly, removal of 20 to 25% of the carbohydrates from intact virus particles did not alter infectivity.     

Physicochemical properties of Nebraska calf diarrhea virus hemagglutinin.

Highly purified Nebraska calf diarrhea virus (NCDV) was prepared by cesium chloride density gradient centrifugation. The effect of temperature, pH, different concentrations of formaldehyde, chloroform, ether, ethyl alcohol, and methyl alcohol on NCDV hemagglutinin and virus morphology was studied. NCDV hemagglutinin was inactivated by temperature, pH 2.0, chloroform, ethyl alcohol, and methyl alcohol.     

Delayed hypersensitivity in mice infected with reovirus. I. Identification of host and viral gene products responsible for the immune response

Delayed-type hypersensitivity (DTH) can be demonstrated in mice infected with reovirus by challenging primed animals in the footpad with virus. Maximal responses occur 7 days after immunization with as little as 10(5) viral particles. DTH to reovirus is transferable by lymph node cells and is mediated by T cells as the transfer of reactivity can be abrogated by treatment of cells with anti-Thy 1.2 plus complement. DTH to reovirus is serotype specific, animals infected with reovirus type 1 or 3 only develop DTH responses when challenged with the same serotype with which they were infected. Using recombinant viral clones containing genes from both parental serotypes, we have demonstrated that the S1 gene, the gene encoding the viral hemagglutinin, determines serotype specificity. Furthermore, in adoptive transfer experiments between mice of varying histocompatibility backgrounds, it was found that D or K, IA-IB region identity was required for the transfer of reactivity. These studies demonstrate that specific host and viral genes determine the in vivo cellular immune response to reovirus and should allow a more precise definition of the host cellular immune response to viral antigens.     

Characterization of a common high-affinity receptor for reovirus serotypes 1 and 3 on endothelial cells.

During viremia, viruses may be cleared from the bloodstream and taken up by specific organs. The uptake of virus from the bloodstream is dependent on the association of viral particles with endothelial cells that line the luminal surfaces of large and small blood vessels. To understand the nature of this interaction, we have studied the binding of reovirus serotypes 1 and 3 to these cells in vitro. Both serotypes of reovirus productively infected endothelial cells. By using [35S]methionine-biolabeled reovirus as a tracer ligand, we found that both viruses rapidly bind to endothelial cells and that equilibrium is reached after 4 h. The binding of the radiolabeled viruses was saturable and mediated by a homogeneous population of cellular receptors with very high affinity (Kd = 0.5 nM) for the virus ligands. Both serotypes bind to the same receptor, since the attachment of each radiolabeled serotype is inhibited by both the homologous and heterologous unlabeled virus. Exposure of labeled virus to monoclonal antibodies directed against the viral hemagglutinin (sigma 1 protein) inhibited binding, demonstrating that the attachment of reovirus to endothelial cells is mediated by the hemagglutinin for both serotypes. By using a novel ligand-blotting assay, the binding of both viruses to a 54,000-dalton protein could be demonstrated. The binding of each radiolabeled serotype to this protein was inhibited by the homologous and heterologous unlabeled serotype. By using cell fractionation after homogenization, we demonstrated that this 54-kilodalton protein is a membrane protein, in agreement with its proposed role as a cell surface receptor for reovirus serotypes 1 and 3.     

Immune response to a molecularly defined internal image idiotope

A monoclonal anti-idiotope termed 87.92.6 mimics the neutralization/cell-attachment site of the reovirus type 3 hemagglutinin (HA3). The second complementarity determining regions of the VH and VL of 87.92.6 share sequence similarity with a determinant on the HA3. We have used synthetic peptides (termed VH, VL, and Reo peptides, respectively) to probe the immunologic significance of this sequence similarity. Antibodies specific for Reo peptide or VL peptide neutralized reovirus type 3 infectivity. Although Reo peptide was an effective immunogen by itself, free VL peptide or VH peptide were unable to elicit antibodies unless they were linked to each other (VH-VL peptide). Immunization with reo peptide, 87.92.6, or the HA3 elicited a specific lymphocyte proliferative response to VH peptide, indicating that VH peptide may bear an important TH determinant. As found previously for 87.92.6, VL peptide elicited a delayed-type hypersensitivity response specific for reovirus type 3. Reovirus type 3 specific cytolytic lymphocytes specifically lysed targets coated with VH-VL peptide, but not VH or VL peptide alone. These results suggest that immune cross-reactivity between an external Ag and an internal image antibody can be understood at the primary structural level. These observations may have important implications for understanding the development of autoantibodies, network interactions, and the regulation of immune responses.     

Virulence factors of influenza A viruses: WSN virus neuraminidase required for plaque production in MDBK cells.

The genetic basis for the distinctive capacity of influenza A/WSN/33 (H0N1) virus (WSN virus) to produce plaques on bovine kidney (MDBK) cells was found to be related to virus neuraminidase. Recombinant viruses that derived only the neuraminidase of WSN virus were capable of producing plaques, whereas recombinant viruses identical to WSN except for neuraminidase did not produce plaques. With viruses that do not contain WSN neuraminidase, infectivity of virus yields from MDBK cells was increased approximately 1,000-fold after in vitro treatment with trypsin. In contrast, no significant increase in infectivity was observed after trypsin treatment of viruses containing WSN neuraminidase. In addition, polyacrylamide gel analysis of proteins of WSN virus obtained after infection of MDBK cells demonstrated that hemagglutinin was present in the cleaved form (HA1 + HA2), whereas only uncleaved hemagglutinin was obtained with a recombinant virus that derived all of its genes from WSN virus except its neuraminidase. These data are in accord with the hypothesis that neuraminidase may facilitate production of infectious particles by removing sialic acid residues and exposing appropriate cleavage sites on hemagglutinin.     

Effect of kaolinite on the specific infectivity of reovirus

Abstract The infectivity of enteric viruses (e.g., poliovirus, rotavirus, reovirus) is prolonged when these viruses are adsorbed on naturally occurring particulates (sediments, clay minerals) in terrestrial and aquatic environments. Furthermore, in vitro assays of these and other particulate-associated viruses often display infectivity levels (specific infectivity) greater than those of the same concentration of viruses in the absence of particulates. This investigations attempted to identify interactions at the particulate-virus-cell interface and to define the mechanism(s) whereby the apparent infectivity of viruses is enhanced when complexed with particulates. Reovirus type 3 and the clay mineral, kaolinite, were used as the model systems. Scanning electron micrographs after critical point drying showed that kaolinite was not present on the surface of cell monolayers of L-929 mouse fibroblasts 3 h after inoculation with a kaolinite-reovirus complex. However, the virus was observed on the surface of the cells. No change in dispersion of the virus particles was observed nor was the integrity of the cell surface altered by kaolinite. These results indicated that kaolinite enhanced the transport of viral particles, in conjunction with diffusion and Brownian movement, to receptors for the reovirus on the cell surface.     

Isolation and properties of reovirus from cattle in an outbreak of acute respiratory disease.

A cytopathogenic virus was isolated in the primary culture of bovine kidney cells from a nasal swab of affected calves in an outbreak of acute respiratory disease in Japan in 1971. It agglutinated human type O erythrocytes and produced cytoplasmic inclusion bodies. Viral replication was inhibited by 5-iodo-2'-deoxyuridine, indicating that the viral nucleic acid was RNA. The virus was resistant to ether, chloroform, sodium deoxycholate, and acid, and passed readily through Sartorius' membrane filter 100 nm in pore size, but not through the filter 50 nm in pore size. Electron microscopy showed many spherical particles 60 approximately 75 nm in diameter with a double-layered capsid in a sample taken at a buoyant density of 1.34 produced by CaCl equilibrium centrifugation. The virus suspended in 1M MgCl2 solution was stable against heating at 50 degrees C for 30 minutes, but not against freezing at -20 degrees C for 60 minutes. The virus was resistant to, and increased in infectivity after, treatment with 0.063 approximately 1.0% trypsin. These properties were consistent with those established for the reoviruses. Most affected cattle showed a significant rise of antibody titer against reovirus and bovine respiratory syncytial virus, whereas only a few of them presented a serological evidence for recent infection with parainfluenza virus type 3, bovine adenovirus type 7, and bovine parovirus.     

Avian reoviruses cause apoptosis in cultured cells: viral uncoating,but not viral gene expression,is required for apoptosis induction

The cytopathic effect evidenced by cells infected with avian reovirus S1133 suggests that this virus may induce apoptosis in primary cultures of chicken embryo fibroblasts. In this report we present evidence that avian reovirus infection of cultured cells causes activation of the intracellular apoptotic program and that this activation takes place during an early stage of the viral life cycle. The ability of avian reoviruses to induce apoptosis is not restricted to a particular virus strain or to a specific cell type, since different avian reovirus isolates were able to induce apoptosis in several avian and mammalian cell lines. Apoptosis was also provoked in ribavirin-treated avian reovirus-infected cells and in cells infected with UV-irradiated reovirions, indicating that viral mRNA synthesis and subsequent steps in viral replication are not needed for apoptosis induction in avian reovirus-infected cells and that the number of inoculated virus particles, not their infectivity, is the critical factor for apoptosis induction by avian reovirus. Our finding that apoptosis is no longer induced when intracellular viral uncoating is blocked indicates that intraendosomal virion disassembly is required for apoptosis induction and that attachment and uptake of parental reovirions are not sufficient to cause apoptosis. Taken together, our results suggest that apoptosis is triggered from within the infected cell by viral products generated after intraendosomal uncoating of parental reovirions.     

Chikungunya Virus Vaccine Prepared by Tween-Ether Extraction

Chikungunya virus vaccines prepared by Tween 80 and ether inactivation of virus grown in green monkey kidney cell cultures were shown to be as immunogenic as comparable Formalin-inactivated vaccines. Both types of vaccine stimulated hemagglutination-inhibiting, complement-fixing, and neutralizing antibody and afforded protection to mice against a live virus challenge. It was shown after Tween-ether treatment of chikungunya virus that the infectivity of the virus was lost and the hemagglutinin titer was increased. By characterization of the resultant hemagglutinin by sucrose and cesium chloride density gradient centrifugation, it was found that the extracted particle was smaller in size and greater in density than the parent virus particle. Removal of lipid may account for the alterations in the physical characteristics of the infectious virus particle. Conditions for treatment of chikungunya virus with Tween and ether were found that preserved high titers of hemagglutinin as well as the immunogenicity of the virus preparations.