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.

Core transcription restores in vitro inhibition of protein synthesis induced by vaccinia virus.

When Ehrlich acistes tumor cell lysate is incubated in the presence of vaccinia core, protein synthesis is impaired. However, when the same system is coupled with viral transcription, protein synthesis is restored. The reversal of endogenous protein synthesis is inhibited by actinomycin D, suggesting that de novo RNA synthesis is required for the reversal of total protein synthesis. When the in vitro products of synthesis are analysed by polyacrylamide gel electrophoresis, two newly synthesized peptides which are not present in the noncoupled transcription-translation system are observed. These two peptides have molecular weights of 31 000 and 25 000, similar to viral early proteins.

Changes in cell shape and desmin intermediate filament distribution are associated with down-regulation of desmin expression in C2C12 myoblasts grown in the absence of extracellular Ca2+.

Desmin is the main intermediate filament (IF) protein of muscle cells. In skeletal muscle, desmin IFs form a scaffold that interconnects the entire contractile apparatus with the subsarcolemmal cytoskeleton and cytoplasmic organelles. The interaction between desmin and the sarcolemma is mediated by a number of membrane proteins, many of which are Ca2+-sensitive. In the present study, we analyzed the effects of the Ca2+ chelator EGTA (1.75 mM) on the expression and distribution of desmin in C2C12 myoblasts grown in culture. We used indirect immunofluorescence microscopy and reverse transcription polymerase chain reaction (RT-PCR) to analyze desmin distribution and expression in C2C12 cells grown in the presence or absence of EGTA. Control C2C12 myoblasts showed a well-spread morphology after a few hours in culture and became bipolar when grown for 24 h in the presence of EGTA. Control C2C12 cells showed a dense network of desmin from the perinuclear region to the cell periphery, whereas EGTA-treated cells showed desmin aggregates in the cytoplasm. RT-PCR analysis revealed a down-regulation of desmin expression in EGTA-treated C2C12 cells compared to untreated cells. The present results suggest that extracellular Ca2+ availability plays a role in the regulation of desmin expression and in the spatial distribution of desmin IFs in myoblasts, and is involved in the generation and maintenance of myoblast cell shape.

Integrated radiation hybrid and yeast artificial chromosome map of chromosome 9p.

A panel of 93 radiation-reduced hybrids have been screened using PCR amplification and oligonucleotide primers for sequence-tagged sites (STSs) specific for 114 single-copy loci mapping to the short arm of chromosome 9. An x-ray dose of 6,000 rads gave an average retention frequency of approximately 23%. We have constructed a framework map containing 31 markers ordered by analyzing coretention patterns, with support for the order greater than 1,000:1. In addition, we have placed the remaining markers which could not be mapped to a single interval with this support to a range of intervals on the framework map. The STS oligonucleotide primers used in the construction of the radiation hybrid (RH) map have been used to isolate and order yeast artificial chromosomes (YACs) assigned to 9p identified from the CEPH megaYAC library. Eighty-nine STS markers have screened positive with at least one YAC. A total of 88 individual YACs (with an average size of 0.9 MB) have been placed on the map in a series of contigs and in some cases mapped cytogenetically by fluorescence in situ hybridization. Additionally, the YAC information has been used in conjunction with the RH framework placements to generate an integrated map containing 65 loci including 51 uniquely positioned markers, with an average resolution of 0.79 Mb.

The surface charge of L-A9 cells and Aedes albopictus cells infected with Marituba (Bunyaviridae) virus.

The surface charge of Marituba virus infected L-A9 cells and Aedes albopictus cells was estimated by direct measurement of their electrophoretic mobilities. Uninfected L-A9 cells and A. albopictus cells have mean electrophoretic mobilities of -1.083 microns/s X cm/V and -1.019 microns/s X cm/V, respectively. In Marituba virus infected L-A9 cells a progressive decline in the electrophoretic mobility was observed. In contrast, in Marituba virus infected A. albopictus cells the electrophoretic mobility of the cell surface was unaltered.

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.

Selective inhibition of protein synthesis by hypertonic medium in Marituba (Bunyaviridae) virus-infected L-A9 cells.

Elevation of the NaCl concentration in the growth medium of L-A9 cells caused an inhibition of the protein synthesis accompanied by a complete breakdown of polyribosomes. However, a complete recovery of the rate of protein synthesis was observed when isotonicity was restored. In Marituba virus infected cells, protein synthesis became resistant to hypertonic treatment. Under hypertonic conditions cellular protein synthesis was selectively suppressed and an enhancement of virus proteins was observed. Analysis of the virus specific proteins by polyacrylamide gel electrophoresis revealed that the synthesis of G1 was unalterable, and N was stimulated.

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.

A computer-based register for inherited retinal dystrophies in Southern Africa.

A genetic register for inherited retinal degenerative disorders (RDDs) has been established at the Division of Human Genetics, UCT Medical School, Cape Town, South Africa. The primary role of the register is to monitor the progress of molecular research and to facilitate the efficient delivery of services, including genetic counselling, to respective family members and new patients. The database currently holds information on 1829 subjects. The RDD-specific breakdown of the data are presented.

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.

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.

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.

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.

Interferon induction in mouse fibroblast L-A9 cells.

Mouse L-A9 cell interferon was induced by infection with Newcastle disease virus. Interferon production was 1.5 X 10(5) IU/10(7) cells. Interferon was partially purified by precipitation with ammonium sulphate, chromatography on CM-Sephadex and hydrophobic chromatography on octyl-agarose. The specific activity of the final preparation was 1.7 X 10(7) IU/mg protein. Treatment of L-A9 cells with 20 IU/ml interferon prior to viral infection inhibited the intracellular accumulation of reovirus-specific double-stranded RNA. Dose-response studies of the cells to interferon indicated that L-A9 cells require 10, 13 and 15 IU/ml to obtain 50% viral plaque reduction for Marituba virus, vesicular stomatitis virus and reovirus, respectively. The present results demonstrate the potential of mouse L-A9 cells as an interferon-producing system and also as a model for the study of the effect of cellular response to exogenous interferon treatment on the replication of RNA viruses.

Effect of high temperature on Aedes albopictus cells infected with Mayaro virus.

The multiplication of Mayaro virus in Aedes albopictus cells was drastically inhibited after incubation at 37 degrees C. The effect of short-term exposure of infected cells to high temperatures (heat shock) produced a preferential translation of the heat shock messengers when compared to the viral mRNAs. When cells were shifted back to 28 degrees C (the optimum growth temperature for Aedes albopictus cells), preferential translation of viral mRNA occurred. Although the infected cells were programmed for preferential translation of viral messengers, the thermal treatment was able to shift the translational machinery towards synthesis of heat shock proteins.

Changes in cell shape, cytoskeletal proteins and adhesion sites of cultured cells after extracellular Ca2+ chelation.

Although much is known about the molecules involved in extracellular Ca2+ regulation, the relationship of the ion with overall cell morphology is not understood. The objective of the present study was to determine the effect of the Ca2+ chelator EGTA on the major cytoskeleton components, at integrin-containing adhesion sites, and their consequences on cell shape. Control mouse cell line C2C12 has a well-spread morphology with long stress fibers running in many different directions, as detected by fluorescence microscopy using rhodamine-phalloidin. In contrast, cells treated with EGTA (1.75 mM in culture medium) for 24 h became bipolar and showed less stress fibers running in one major direction. The adhesion plaque protein alpha 5-integrin was detected by immunofluorescence microscopy at fibrillar adhesion sites in both control and treated cells, whereas a dense labeling was seen only inside treated cells. Microtubules shifted from a radial arrangement in control cells to a longitudinal distribution in EGTA-treated cells, as analyzed by immunofluorescence microscopy. Desmin intermediate filaments were detected by immunofluorescence microscopy in a fragmented network dispersed within the entire cytoplasm in EGTA-treated cells, whereas a dense network was seen in the whole cytoplasm of control cells. The present results suggest that the role of extracellular Ca2+ in the regulation of C2C12 cell shape can be mediated by actin-containing stress fibers and microtubules and by intermediate filament reorganization, which may involve integrin adhesion sites.

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.

Interferon action on Mayaro virus replication.

Treatment of TC7 cells with interferon (IFN) drastically reduced the yield of infectious Mayaro virus under experimental conditions that virus attachment and penetration into the cells were not affected. In IFN-treated cells, synthesis of Mayaro virus proteins was inhibited and cellular protein synthesis was restored. This phenomenon is dependent on IFN concentration and multiplicity of infection. Electron microscopy of these cells revealed normal and anomalous viral particles inside cytoplasmic vacuoles. This suggests that IFN also interferes with Mayaro virus morphogenesis and inhibits the release of virions from cells.

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.