Effect of actinomycin D on simian rotavirus (SA11) replication in cell culture

Rotaviruses are the major cause of viral diarrhea in humans and animals. Actinomycin D (Act D) is an antibiotic that intercalates DNA and therefore inhibits DNA-dependent transcription. The current study was carried out to assess the influence of Act D on the replication of simian rotavirus (SA11) in cell culture. Virus-infected MA-104 cell cultures were studied in the presence of Act D at concentrations of 1.25 and 2.5 æg/ml. Treatment of rotavirus-infected cells with 2.5 æg/ml Act D 48 h post-infection reduced the cytoplasmic metachromasia after staining with acridine orange by 25 percent. Viral RNA labeled with ³H-uridine in the presence of the drug was separated by polyacrylamide gel electrophoresis. Viral RNA replication was not affected by Act D, but increased ³H-uridine uptake was demonstrable by infected cells in the presence of the drug. This possibly was due to the inhibition of cellular RNA synthesis by Act D, which thus enhances incorporation of the radionuclide into the viral RNA. Act D reduced the number of infected cells presenting virus-specific fluorescence 48 h post-infection by more than 50 percent. These data suggest that Act D may have complexed with viral RNA and prevented newly synthesized mRNA from being translated, but may not have prevented early replication

The in vitro comparative cytopathology of a porcine rotavirus and the simian prototype (SA-11)

A citopatologia in vitro de uma cepa de rotavírus porcino adaptado em cultura de células foi comparada à estirpe-protótipo símia (SA-11). O efeito citopático (ECP) produzido pelos vírus foi semelhante embora a estirpe porcina tivesse apresentado algumas alteraçöes diferentes, como o acentuado estreitamento do citoplasma, com grande perda do volume citoplasmático. O vírus porcino apresentou menor número de plaques de ECP porém com diâmetro maior em relaçäo ao vírus símio, demonstrando maior capacidade de disseminaçäo célula-célula, quase oito vezes mais, a julgar pelo diâmetro dos plaques de ECP. Os elementos do citoesqueleto das células infectadas revelaram uma reorganizaçäo semelhante para ambas as estirpes, näo sendo possível observar nenhuma diferença, embora o ECP do vírus porcino tenha sido mais acentuado

Infecciosidade de rotavírus suíno em fezes / The infectivity of pig rotavirus in stools

Os rotavírus constituem-se nos principais patógenos da diarréia em humanos e animais. Afetam os animais jovens em criaçöes intensivas e causam grandes perdas econômicas. Este estudo avaliou a infecciosidade do rotavírus suíno mantido por 32 meses a aproximadamente 10ºC nas amostras originais de fezes. Trinta amostras de fezes de leitöes de 1-4 semanas de idade, provenientes de granjas da regiäo sudoeste do Paraná, foram selecionadas para o estudo. As amostras foram colhidas no período de março a outubro de 1991 e selecionadas ao acaso dentre as positivas para rotavírus pela eletroforese em gel de poliacrilamida (EGPA), à época da colheita. Estas foram retestadas por EGPA 32 meses após manutençäo à temperatura de aproximadamente 10ºC. Onze das 30 amostras ainda foram positivas, mostrando a integridade das 11 bandas de RNA viral. Com o intuito de demonstrar a manutençäo da infecciosidade viral, os homogenatos fecais clarificados, previamente tratados com tripsina, foram inoculados em culturas de células MA-104. Das 11 amostras, 5 demonstraram efeito citopático semelhante ao do rotavírus símio (SA-11), após em média 3 passagens cegas e confirmado pelo teste de imunofluorescência indireta, demonstrado pela fluorescência específica citoplasmática tipicamente granular. A microscopia eletrônica das amostras fecais mostrou que a maioria das partículas virais apresentavam-se sem capsídio externo e outras encontravam-se em adiantado estado de degradaçäo. Concluiu-se, portanto, que a infecciosidade do rotavírus suíno é mantida por longo período em amostras fecais em baixa temperatura. Este certamente é um aspecto importante para a manutençäo do vírus viável em condiçäo natural assim como para a transmissäo da doença

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.

The in vitro antiviral activity of isoprinosine on simian rotavirus (SA-11)

The antiviral effect of isoprinosine on simian rotavirus (SA-11) replication was studied using MA-104 cell cultures from Rhesus monkey fetal kidney. Isoprinosine (N,N-dimethylamino-2-propanol-p-acetamidobenzoate in association with inosine) added after viral infection (therapeutic test) inhibited viral replication by more than 90%. In these experiments, the drug was added to the medium and replaced daily at concentrations varying from 62.5 microng/ml to l mg/ml. Viral inhibition activity was dependent on drug concentration. No antiviral effect was observed when isoprinosine was tested without replacement (200-500 microng/ml). When isoprinosine (l mg/ml) was added to cell cultures only before viral infection (prophylactic test), inhibition of viral replication occurred but was less than 90%. Inhibition by less than 90% is not considered to be significant in this type of test. Isoprinosine inhibited synthesis of both viral antigen (protein) and viral double-stranded nucleic acid, as monitored by immunofluorescence and acridine orange staining, respectively. Inhibiton of synthesis of viral macromolecules increased with drug concentration