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 microg/ml. Treatment of rotavirus-infected cells with 2.5 microg/ml Act D 48 h post-infection reduced the cytoplasmic metachromasia after staining with acridine orange by 25%. 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%. 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.

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

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 alterações 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 relação ao vírus símio, demonstrando maior capacidade de disseminação célula-célula, quase oito vezes mais, a julgar pelo diâmetro dos plaques de ECP. Os elementos do citoesqueleto das células infectadas revelaram uma reorganização semelhante para ambas as estirpes, não sendo possível observar nenhuma diferença, embora o ECP do vírus porcino tenha sido mais acentuado.

Detection of porcine rotavirus type G9 and of a mixture of types G1 and G5 associated with Wa-like VP4 specificity: evidence for natural human-porcine genetic reassortment.

Rotavirus type G5 is a primarily porcine pathogen that has caused frequent and widespread diarrhea in children in Brazil and in piglets elsewhere. Initial results on the rotavirus types circulating in diarrheic piglets in Brazil disclosed a high diversity of strains with distinct G types including G1, G4, G5, and G9 and the novelty of P[8], the predominant human P specificity type. Those results add strong evidence for the emergence of new strains through natural reassortment between rotaviruses of human and porcine origins.

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

Rotavirus and reovirus interaction with mouse peritoneal resident phagocytic cells

Rotaviruses and reoviruses are involved in human and animal diseases. It is known that both viruses penetrate the gastrointestinal tract but their interaction with phagocytic cells is unknown. To study this interaction, peritoneal resident phagocytic cells were used and rotavirus and reovirus replication in peritoneal phagocytic cells was observed. However, rotavirus replication in these cells led to the production of defective particles since MA-104 cells inoculated with rotavirus phagocytic cell lysate did not show any evidence of virus replication. On the basis of these results, we suggest that, althought reovirus dissemination may be helped by these phagocytic cells, these cells may control rotavirus infection and probably contribute to the prevention of its dissemination.

Rotavirus and reovirus interaction with mouse peritoneal resident phagocytic cells.

Rotaviruses and reoviruses are involved in human and animal diseases. It is known that both viruses penetrate the gastrointestinal tract but their interaction with phagocytic cells is unknown. To study this interaction, peritoneal resident phagocytic cells were used and rotavirus and reovirus replication in peritoneal phagocytic cells was observed. However, rotavirus replication in these cells led to the production of defective particles since MA-104 cells inoculated with rotavirus phagocytic cell lysate did not show any evidence of virus replication. On the basis of these results, we suggest that, although reovirus dissemination may be helped by these phagocytic cells, these cells may control rotavirus infection and probably contribute to the prevention of its dissemination.

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.

Comparaçao das tecnicas de microscopia eletronica (ME) eletroforese em gel de poliacrilamida (EPGA), ensaio imunoenzimatico (EI) e aglutinaçao do latex (AL) no diagnostico / Comparative study of the electron microscopy (EM),polyacrylamide gel electrophoresis (PAGE),enzyme immunoassay (EIA) and latex agglutination (LA) for the rotativiruses diagnosis

Duzentos e trinta e uma amostras fecais,coletadas durante o periodo de julho de 1984 a julho de 1988, originarias de crianças de um mes a cinco anos de idade, acometidas de diarreia aguda, foram analisadas atraves de microscopia eletronica (ME), ensaio imunoenzimatico (IE), eletroforese em gel de poliacrilamida (EGPA) do RNA viral e aglutinaçao do latex (AL), a fim de se comparar a sensibilidade e a importancia das tecnicas usadas. Constatou-se que do total, 47 amostras (47/231, 20,3%)foram consideradas positivas para rotavirus por pelo menos uma tecnica, 32 amostras detectadas por ME (32/47,68,0%); 31 amostras por EGPA (31/47, 65,9%), 31 por IE (31/47,65,9%) e 30 amostras por AL (30/47,63,8%). Dezoito amostras (18/47,38,2%) foram positivas pelas quatro tecnicas, nove amostras (9/47,19,1%) positivas por tres tecnicas, sete amostras (7/47, 14,8%) positivas por duas tecnicas e 184 amostras (l84/231,79,6%) negativas pelas quatro tecnicas. A concordancia entre as quatro tecnicas foi de 87,4% (202/231). A analise estatistica (Teste de McNemar) entre os resultados dos testes em que se detectou maior diferença de positividade,i.e., ME x AL, mostrou nao haver diferença significativa na sensibilidade dos metodos utilizados (P>0,5).

Acridine orange metachromasia in the cytoplasm of simian rotavirus (SA-11)-infected MA-104 cell cultures.

1. Acridine orange metachromasia was used to determine the distribution of simian rotavirus double-stranded RNA in cultured MA-104 cells 0 to 72 h post-infection. Correlations were made among time of detection and amount of viral antigens, virus yield and the ultrastructural aspects of infected cells. 2. RNAase-resistant cytoplasmic metachromasia appeared 48 h post-infection, 36 h after the initial detection of viral antigens or infectious virions and 24 h after the appearance of the cytopathic effect. 3. Acridine orange staining is thus useful for monitoring the progress of rotaviral infection in cell cultures due to its simplicity and low cost, in spite of its lower sensitivity compared to other techniques evaluated.

Acridine orange metachromasia in the cytoplasm of simian rotavirus (SA-11)-infected MA-104 cell cultures

Acridine orange metachromasia was used to determine the distribution of simian rotavirus double-stranded RNA in cultured MA-104 cells 0 to 72 h post-infection. Correlations were made among time of detection and amount of viral antigens, virus yield and the ultrastructural aspects of infected cells. RNAase-resistant cytoplasmic metachromasia appeared 48 h post-infection, 36 h after the initial detection of viral antigens or infectious virions and 24 h after the appearance of the cytopathic effect. Acridine orange staining is thus useful for monitoring the progress of rotaviral infection in cell cultures due to its simplicity and low cost, in spite of its lower sensitivity compared to other techniques evaluated

Human rotavirus detection in infantile acute diarrhoea: I. A comparative study of inmunodiffusion in agarose gel (ID) and electron microscopy (EM)

Com o intuito de detectar rotavírus em fezes diarréicas, as técnicas de imunodifusäo em gel de agarose (ID) e microscopia electrônica (ME) foram usadas comaprativamente. O estudo foi realizado com 89 amostras fecais diarréicas colhidas em hospital local, originárias de crianças com até dois anos de idade acometidas de diarréia aguda. O teste de ID foi realizado utilizando-se soro de coelho anti-rotavírus. As observaçöes ao ME foram feitas após ultracentrifugaçäo ou imunoconcentraçäo (IEM) dos homogenatos fecais. Das 89 amostras fecais o antígeno de rotavírus foi detectado em 55% (49% ) dos materiais por ID e em 45% (40) pela ME. Os resultados de ME consistiram no estudo de 71 amostras fecais analisadas após ultracentrifugaçäo com uma positividade de 45% (32) e de 18 amostras estudadas por IEM com positividade de 44% (8). Todas as amostras (40) positivas pela ME foram positivas po ID, entretanto ID detectou antígeno em nove materiais consistemente negativos pela ME. A análise estatística (teste de McNemar) do resultado é favorável à ID em relaçäo à ME (0.01 > 0.0027)

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

1. The antiviral effect of isoprinosine on simian rotavirus (SA-11) replication was studied using MA-104 cell cultures from Rhesus monkey fetal kidney. 2. 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 micrograms/ml to 1 mg/ml. Viral inhibition activity was dependent on drug concentration. No antiviral effect was observed when isoprinosine was tested without replacement (200-500 micrograms/ml). 3. When isoprinosine (1 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. 4. Isoprinosine inhibited synthesis of both viral antigen (protein) and viral double-stranded nucleic acid, as monitored by immunofluorescence and acridine orange staining, respectively. Inhibition of synthesis of viral macromolecules increased with drug concentration.

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

Immunofluorescent antibody to rotavirus and antigen excretion in infantile acute diarrhoea

Realizamos este estudo para avaliar a relaçäo causal diarréia aguda e rotavírus em crianças abaixo de cinco anos de idade. A infecçäo viral foi caracterizada pela reaçäo de imunofluorescência indireta (IF) nos soros dos pacientes. Utilizamos como substrato para a reaçäo células MA-104 infectadas com rotavírus bovino cepa UK. De 80 amostras de soros pareados, verificamos que 23 amostras (28,75%) apresentaram soroconversäo, 19 amostras (23,75%) tiveram elevaçäo no título de 2 vezes e em 38 amostras (47,5%) näo observamos elevaçäo no título de anticorpos anti-rotavírus. Estes resultados säo confrontados com os resultados previamente obtidos na detecçäo do antígeno de rotavírus nas fezes, dos mesmos pacientes, pela técnica de contraimunoeletroforese (CIEOP)