Development and characterization of a bovine serum evaluation panel as a standard for immunoassays based on detection of antibodies against foot-and-mouth disease viral non-capsid proteins.

The widespread perception of the effectiveness of applying tests based on the detection of antibodies against foot-and-mouth disease (FMD) viral non-capsid proteins (NCPs) to assess virus circulation irrespective of vaccination triggered the demand for international standards to evaluate the comparative performance of the upcoming assays against the OIE Index test developed at the Pan American Foot-and-Mouth Disease Center, PAHO/WHO. To this end, a panel was developed composed of 34 cattle sera from animals with an unambiguous exposed/infected status, covering serotypes O, A and C, obtained either under experimental conditions or from the field in regions with different epidemiological situations. Reference values in the Index test and their reproducibility in other laboratories, data on stability as well as results in four other commercial kits and one in house test were obtained. The characteristics of the panel which comprise adequate preparation following international guidelines, a broad range of antibody reactivity, proper stability and the ability to assess comparative diagnostic sensitivity, make it suitable as a reference standard to evaluate if tests equivalent to the OIE Index method are used in support of FMD control programs and by trading partners, and also whether they maintain their standards of diagnostic performance.

Effect of monensin on Mayaro virus replication in monkey kidney and Aedes albopictus cells.

The effect of a cationic ionophore, monensin, on the replication of Mayaro virus in monkey kidney TC7 and Aedes albopictus cells has been studied. Treatment of these cells with 1 micromol/l monensin during infection did not affect the virus protein synthesis but inhibited severely the virus replication. Electron microscopy of the cells infected with Mayaro virus and treated with monensin revealed that the morphogenesis of Mayaro virus was impaired in TC7 but not in A. albopictus cells.

Inactivation of classical swine fever virus: association of hydrostatic pressure and ultraviolet irradiation.

Reversible pressure-induced disassembly of several viruses has suggested the idea of using hydrostatic pressure to suppress virus infectivity. In this study, the effects of high hydrostatic pressure and ultraviolet (UV) irradiation were investigated on classical swine fever virus (CSFV) in an attempt to eliminate residual infectivity. The structural modifications were followed by intrinsic fluorescence and biological activity assays. The kinetics of CSFV inactivation showed that pressure-induced inactivation was not enough to eliminate viral infectivity. However, when pressure was applied in association with UV irradiation no infectious focus was observed. The application of these two methods against CSFV can be an attractive inactivation strategy for the development of a vaccine.

Effect of prostaglandin A1, arsenite and aspirin on stress proteins response in mosquito cells.

The stress response of eukaryotic cells is characterized by changes in the metabolism of responding cells, most notably by increased synthesis of a group of proteins known as heat shock (HSP) proteins In this paper the effect of prostaglandin A1 (PGA1), arsenite and aspirin in Aedes albopictus cells was investigated. In cells treated with PGA1 (10 microg/ml) we observed the induction of several polypeptides with molecular masses of 87, 80, 70, 57, 29 and 23 kDa. Immunoblot analysis revealed that arsenite induces a marked synthesis of HSP70, and aspirin administered during the hyperthermic treatment caused a small increase of HSP70 synthesized.

The metastable state of nucleocapsids of enveloped viruses as probed by high hydrostatic pressure.

Enveloped viruses fuse their membranes with cellular membranes to transfer their genomes into cells at the beginning of infection. What is not clear, however, is the role of the envelope (lipid bilayer and glycoproteins) in the stability of the viral particle. To address this question, we compared the stability between enveloped and nucleocapsid particles of the alphavirus Mayaro using hydrostatic pressure and urea. The effects were monitored by intrinsic fluorescence, light scattering, and binding of fluorescent dyes, including bis(8-anilinonaphthalene-1-sulfonate) and ethidium bromide. Pressure caused a drastic dissociation of the nucleocapsids as determined by tryptophan fluorescence, light scattering, and gel filtration chromatography. Pressure-induced dissociation of the nucleocapsids was poorly reversible. In contrast, when the envelope was present, pressure effects were much less marked and were highly reversible. Binding of ethidium bromide occurred when nucleocapsids were dissociated under pressure, indicating exposure of the nucleic acid, whereas enveloped particles underwent no changes. Overall, our results demonstrate that removal of the envelope with the glycoproteins leads the particle to a metastable state and, during infection, may serve as the trigger for disassembly and delivery of the genome. The envelope acts as a "Trojan horse," gaining entry into the host cell to allow release of a metastable nucleocapsid prone to disassembly.

Inhibition of Mayaro virus replication by prostaglandin A(1) in Vero cells.

Prostaglandins exhibit antiviral activity against a wide variety of RNA and DNA viruses. In the present report, we describe the effect of cyclopentenone prostaglandin A(1) (PGA(1)) on Mayaro virus replication in Vero cells. Virus yield was significantly reduced at nontoxic concentrations which did not suppress DNA, RNA or protein synthesis in uninfected or infected cells. Antiviral action decreased if PGA(1) was added at later times after infection. In Mayaro virus-infected cells, PGA(1) inhibited the synthesis of virus proteins. This effect is accompanied by the induction of heat shock proteins (HSPs). Actinomycin D treatment not only inhibited the induction of HSPs but also partially prevented PGA(1) antiviral activity.

Weak bases affect late stages of Mayaro virus replication cycle in vertebrate cells.

This paper describes the effect of two weak bases (ammonium chloride and chloroquine) on the morphogenesis of Mayaro virus. When Mayaro virus-infected TC7 (monkey kidney) cells were treated with these agents it was observed that weak bases caused a significant reduction in virus yield. Also, cellular protein synthesis, which is inhibited by Mayaro virus infection, recovered to nearly normal levels. However, the synthesis of Mayaro virus proteins was affected. These phenomena were dose-dependent. The process of Mayaro virus infection in vertebrate cells is very rapid. Virus precursors are not observed in cell cytoplasm and budding through the plasma membrane seems to be the only way of virus release. Electron microscopy of cells infected with Mayaro virus and treated with weak bases revealed an accumulation of virus structures in cell cytoplasm. The study also noted an inhibition of budding through the plasma membrane and the appearance of virus particles inside intracytoplasmic vacuoles. These observations indicate an impairment at the final stages of the virus replication cycle.

Effect of brefeldin A on Mayaro virus replication in Aedes albopictus and Vero cells.

Brefeldin A (BFA), a fungal metabolite that blocks transport of newly synthesized proteins from the endoplasmic reticulum, was found to inhibit Mayaro virus replication. At the concentration of 0.05 microgram/ml, the yield of the virus was inhibited by 94% in Aedes albopictus cells and by 99.5% in Vero cells. Treatment of A. albopictus cells with BFA did not inhibit the virus protein synthesis. However, this compound drastically reduced viral protein synthesis in Vero cells. The inhibitory effect progressively declined when BFA was added at late times post infection (p.i.). The effect of BFA on protein glycosylation is discussed.

Inhibition of Mayaro virus replication by prostaglandin A1 and B2 in Vero cells

The effect of prostaglandins (PGA1 and PGB2) on the replication of Mayaro virus was studied in Vero cells. PGA1 and PGB2 antiviral activity was found to be dose-dependent. However, while 10 µg/ml PGB2 inhibited virus yield by 60 percent, at the same dose PGA1 suppressed virus replication by more than 90 percent. SDS-PAGE analysis of [35S]-methionine-labelled proteins showed that PGA1 did not alter cellular protein synthesis. In infected cells, PGA1 slightly inhibited the synthesis of protein C, while drastically inhibiting the synthesis of glycoproteins E1 and E2

Effect of prostaglandin A1 in the induction of stress proteins in Aedes albopictus cells.

Prostaglandins are natural fatty acid derivatives with diverse physiological effects, including immune function and the control of cell growth. While the action of prostaglandins in the induction of stress proteins in vertebrate cells is well documented, their functions in invertebrate cells have been poorly investigated. The purpose of the present study was to investigate the effect of prostaglandin A1 (PGA1; 0.25, 1.25 and 12.5 micrograms/ml) on protein synthesis during the growth of Aedes albopictus cells. We found that PGA1 stimulates the synthesis of several polypeptides with molecular masses of 87, 80, 70, 57, 29, 27 and 23 kDa in Aedes albopictus cells. When the proteins induced by PGA1 and those induced by heat treatment were compared by polyacrylamide gel electrophoresis, PGA1 was found to induce the stress proteins. The HSP70 family and the low-molecular weight polypeptides (29 and 27 kDa, respectively) were induced by PGA1 in the lag phase. We also observed that PGA1 is able to induce a 23-kDa polypeptide independently of the growth phase of the cell.

Effect of prostaglandin A1 in the induction of stress proteins in Aedes albopictus cells

Prostaglandin are natural fatty acid derivatives with diverse physiological effects, including immune function and the control of cell growth. While the action of prostaglandins in the induction of stress proteins in vertebrate cells in well documented, their functions in invertebrate cells have been poorly investigated. The purpose of the present study was to investigate the effect of prostaglandin A1 (PGA1; 0.25, 1.25 and 12.5 mug/ml) on protein synthesis during the growth of Aedes albopictus cells. We found that PGA1 stimulates the synthesis of several polypeptides with molecular masses of 87,80,70,57,29,27 and 23 kDa in Aedes albopictus cells. When the protein induced by PGA1 and those induced by heat treatment were compared by polyacrylamide gel electrophoresis, PGA1 was found to induce the stress proteins. The HSP70 family and the low-molecular weight polypeptides (29 and 27 kDa, respectively) were induced by PGA1 in the lag phase. We lso observed that PGA1 is able to induce a 23-kDa polypeptide independently of the growth phase of the cell.

Prostaglandin A1 inhibits replication of classical swine fever virus.

Prostaglandins (Pgs) have been shown to inhibit the replication of several DNA and RNA viruses. Here we report the effect of prostaglandin (PgA1) on the multiplication of a positive strand RNA virus, Classical Swine Fever Virus (CSFV) in PK15 cells. PgA1 was found to inhibit the multiplication of CSFV. At a concentration of 5 micrograms/ml, which was nontoxic to the cells, PgA1 inhibitis virus production in 99%. In PgA1 treated cells the size and number of characteristic Classical Swine Fever focus decreased in amount.

Inhibition of Mayaro virus replication by prostaglandin A1 and B2 in Vero cells.

The effect of prostaglandins (PGA1 and PGB2) on the replication of Mayaro virus was studied in Vero cells. PGA1 and PGB2 antiviral activity was found to be dose-dependent. However, while 10 micrograms/ml PGB2 inhibited virus yield by 60%, at the same dose PGA1 suppressed virus replication by more than 90%. SDS-PAGE analysis of [35S]-methionine-labelled proteins showed that PGA1 did not alter cellular protein synthesis. In infected cells, PGA1 slightly inhibited the synthesis of protein C, while drastically inhibiting the synthesis of glycoproteins E1 and E2.

Inhibition of Mayaro virus replication by cerulenin in Aedes albopictus cells.

The antibiotic cerulenin, an inhibitor of lipid synthesis, was shown to suppress Mayaro virus replication in Aedes albopictus cells at non-cytotoxic doses. Cerulenin blocked the incorporation of [3H]glycerol into lipids when present at any time post infection (p.i.). Cerulenin added at the beginning of infection inhibited the synthesis of virus proteins. However, when this antibiotic was added at later stages of infection, it had only a mild effect on the virus protein synthesis. The possibility that cerulenin acts by blocking an initial step in the Mayaro virus replication after virus entry and before late viral translation is discussed.

In situ relationship between sucrose exposure and the composition of dental plaque.

The aim of this study was to analyze the composition of dental plaque according to sucrose exposure. Twelve adult volunteers took part in this crossover study done in four phases of 28 days each. For each phase of the study, an acrylic resin appliance containing four human dental enamel blocks was constructed for each volunteer. A 20% sucrose solution was dripped onto the enamel blocks from 0 to 8 times/day. The volunteers were randomly assigned to the treatments. During the experimental period all the subjects used fluoride-free dentifrice, refrained from brushing the enamel blocks and drank water fluoridated at 0.70 ppm F. After each phase the concentrations of fluoride (F), calcium (Ca), phosphorus (P) and total carbohydrate were determined in dental plaque. Statistical analyses showed that frequent sucrose exposure significantly (p < 0.05) reduced the F, Ca and P concentrations in dental plaque, but increased the alkali-soluble carbohydrate concentration. The results suggest that the cariogenicity of dental plaque formed in the presence of sucrose cannot be attributed only to its higher porosity, but the lower inorganic concentration may also be important.

Basic surface properties of Aedes albopictus cells: effect of Mayaro virus infection on electrostatic charge and surface tension.

Aedes albopictus cells possess a negative cell surface charge of -12.7 mV with an isoelectrophoretic point (IEP) located between pH 3.0 and 4.0. Infection with Mayaro virus rendered the surface of A. albopictus cells less negative reaching a zeta-potential value of -9.7 mV after 100 h of infection. Concomitantly, the IEP of the infected cells were also altered from 3.0-4.0 to 4.0-5.0. Furthermore, the contact angle measurements clearly showed qualitative alterations in the cell surface of infected cells.

Effect of isoprinosine on rotavirus replication in vitro.

Isoprinosine (IPS) is a synthetic drug whose antiviral effect on rotavirus replication in vitro has been characterized in terms of the decrease in metachromasia after acridine orange staining. The present study describes the effect of IPS on the synthesis of viral RNA in vitro. MA-104 cell cultures infected with simian rotavirus strain SA-11 were incubated with zero, 250, 500 and 1,000 micrograms/ml IPS and 22, 24, 48, 52, 72 and 76 h after infection the cultures were submitted to a 1-h starvation period, followed by a 2-h pulse with 10 microCi/ml of [3H]-uridine. The homogenates of virus-infected cultures treated or not with IPS were submitted to phenol/chloroform extraction followed by polyacrylamide gel electrophoresis. The amount of radioactivity in viral RNA eluted from the gel strips was determined. Inhibition of viral RNA synthesis was highest at the IPS concentration of 1,000 micrograms/ml at 72 h after infection, corresponding to 78% inhibition. Although the results obtained in vitro suggest that IPS may be useful for the treatment of rotavirus infection, an in vivo demonstration of its efficacy is needed.

Effect of isoprinosine on rotavirus replication in vitro

Isoprinosine (IPS) is a synthetic drug whose antiviral effect on rotavirus replication in vitro has been characterized in terms of the decrease in metachromasia after acridine orange staining. The present study describes the effect of IPS on the synthesis of viral RNA in vitro. MA-104 cell cultures infected with simian rotavirus strain SA-11 were incubated with zero, 250, 500 and 1,000 microg/ml IPS and 22, 24, 48, 52, 72 and 76 h after infection the cultures were submitted to a 1-h starvation period, followed by a 2-h pulse with 10 microCi/ml of [3H]-uridine. The homogenates of virus-infected cultures treated or not with IPS were submitted to phenol/chloroform extraction followed by polyacrylamide gel electrophoresis. The amount of radioactivity in viral RNA eluted from the gel strips was determined. Inhibition of viral RNA synthesis was highest at the IPS concentration of 1,000 microg/ml at 72 h after infection, corresponding to 78 percent inhibition. Although the results obtained in vitro suggest that IPS may be useful for the treatment of rotavirus infection, an in vivo demonstration of its efficacy is needed.

Prostaglandin A1 inhibits replication of Mayaro virus in Aedes albopictus cells.

Prostaglandin A1 (PGA1) reduced Mayaro virus replication in Aedes albopictus (mosquito) cells in culture. The highest nontoxic dose of PGA1, 7.5 microM, decreased virus production by 90%. In Mayaro virus-infected cells, PGA1 inhibited virus-specific protein synthesis. However, in mock-infected cells the presence of PGA1 stimulated the synthesis of several proteins with molecular masses of 70, 57 and 23 kDa, respectively. The data obtained from this study show that PGA1 plays a role in the metabolic regulation of Aedes albopictus cells, blocking the synthesis of Mayaro virus and inducing the synthesis of cellular polypeptides.

Prostaglandin A1, inhibits replication of Mayaro virus in Aedes albopictus cells

Prostaglandin A1 (PGA1) reduced Mayaro virus replication in Aedes albopictus (mosquito) cells in culture. The highest nontoxic dose of PGA1, 7.5µM, decreased virus production by 90 percent. In Mayaro virus-=infected cells, PGA1 inhibited virus-specific protein synthesis. However, in mock-infected cells the presence of PGA, stimulated the synthesis of several proteins with molecular masses of 70, 57 and 23 kDa, respectively. The data obtained from this study show that PGA1 plays a role in the metabolic regulation of Aedes albopictus cells, blocking the synthesis of Mayaro virus and inducing the synthesis of cellular polypeptides