Antiviral properties of polysaccharides from Agaricus brasiliensis in the replication of bovine herpesvirus 1.

Natural products are an inexhaustible source of compounds with promising pharmacological activities, including antiviral action. In the present study, the antiviral potential of polysaccharide-peptide (PLS) and an extracted ß-glucan from Agaricus brasiliensis were investigated in the replication of bovine herpesvirus 1 (BoHV-1) in HEp-2 cell cultures. The cytotoxicity (CC50) was assayed by the MTT method and the antiviral activity (IC50) was estimated by the plaque reduction assay. To study the possible mode of action of PLS and ß-glucan, the following protocols were performed: the virucidal assay, adsorption assay and the time-of-addition assay. The PLS presented a selectivity index (SI) higher than 12.50 and ß-glucan 9.19. The antiviral inhibition (67.9%) in cells treated with PLS during virus infection was higher than that in cells treated prior to or post infection. The ß-glucan presented high inhibition of virus replication by plaque assay (83.2%) and by immunofluorescence assay (63.8%). Although the mechanism has yet to be defined, we suggest that PLS and ß-glucan inhibited BoHV-1 replication by interfering with the early events of viral penetration. Additional studies are required for a better understanding of the mechanism of action of PLS and ß-glucan.

Effects of chlorophyllin on replication of poliovirus and bovine herpesvirus in vitro.

AIMS: Chlorophyllin (CHLN), a synthetic derivative of chlorophyll, was assayed in the replication of poliovirus (PV-1) and bovine herpesvirus (BoHV-1) in HEp-2 cell cultures. METHODS AND RESULTS: Virucidal activity of CHLN was evaluated and the time-of-addition assay was performed as follows: before the infection (-1 and -2 h), at the time of the infection (0 h) and after the infection (1 and 2 h). Plaque reduction assay (PRA) showed that CHLN inhibited BoHV-1 and PV-1 infection and the 50% inhibitory concentrations (IC(50)) against BoHV-1 and PV-1 infection were 8.6 and 19.8 microg ml(-1), respectively. The time-of-addition study demonstrated that the CHLN was effective inhibiting viral replication in 51% and 66.5% for PV-1 and BoHV-1, respectively, at the highest concentration of 20.0 microg ml(-1), when added during the infection. The directed effect of CHLN on viral strains demonstrated an inhibition of 62% and 66.4% for PV-1 and BoHV-1, respectively, by PRA. CONCLUSIONS: These results demonstrated that CHLN could be used as an antiviral suggesting directed activity on virus particles and on virus-receptor sites to BoHV. For poliovirus, CHLN also demonstrated virucide activity, moreover, showed to inhibit early steps of the replication cycle. SIGNIFICANCE AND IMPACT OF THE STUDY: CHLN demonstrated promising selectivity index for both virus strains; therefore, it can be used for the development of an antiviral agent.

Antiviral activity of aqueous and ethanol extracts and of an isolated polysaccharide from Agaricus brasiliensis against poliovirus type 1.

AIMS: Agaricus brasiliensis (previously named Agaricus blazei ss. Heinem), also known as the sun mushroom is native of Southeast Brazil, and is widely consumed, mainly in the form of tea, due to its nutritional and pharmacological properties. In this study, we tested aqueous (AqE) and ethanol (EtOHE) extracts and an isolated polysaccharide (PLS) from the fruiting body of A. brasiliensis, for antiviral activity against poliovirus type 1 in HEp-2 cells. METHODS AND RESULTS: The evaluation of the time of addition by plaque assay showed that when AqE, PLS and EtOHE were added, just after the virus inoculation (time 0 h), there was a concentration-dependent reduction in the number of plaques up to 50%, 67% and 88%, respectively, with a selectivity index (SI) of 5.4, 9.9 and 12.3, respectively. CONCLUSIONS: The test substances showed antiviral activity and were more effective when added during the poliovirus infection. As they had little effect on reducing viral adsorption and did not show any virucidal effect, we suggest that they act at the initial stage of the replication of poliovirus. SIGNIFICANCE AND IMPACT OF THE STUDY: These results corroborate that basidiomycetes can be a rich source of potential antiviral compounds.

Chlorophyllin protects HEp-2 cells from nuclear fragmentation induced by poliovirus.

AIMS: Chlorophyllin (CHLN) is a synthetic derivative of chlorophyll that possesses antimutagenic activity against several environmental contaminants. In the present study, CHLN was assayed for its capacity to prevent nuclear fragmentation (NF) in HEp-2 cells infected with poliovirus. METHODS AND RESULTS: CHLN was assayed at concentrations of 0.5 and 2.5 microg ml(-1), and NF was monitored using the comet assay and acridine orange staining. We demonstrated that CHLN reduced the percentage of NF in poliovirus-infected HEp-2 cells, when cells were treated with drug before infection or exposed continuously to drug. However, the highest degree of protection was achieved when the virus was exposed to CHLN before infection followed by protocol where infected cultures were continuously exposed to the drug after infection. CONCLUSIONS: It is suggested that CHLN primarily binds to the virus which inhibits cell penetration, thereby maintaining nuclear integrity. SIGNIFICANCE AND IMPACT OF THE STUDY: Considering that CHLN has several beneficial properties and no significant toxic effects in humans and animals, it would be an ideal candidate drug to test for antiviral activity.

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 stability of porcine rotavirus in feces.

Rotaviruses are known as major causal agents of diarrhea in humans and animals. They affect young animals in intensive rearing and cause great economic losses. This study evaluated the infectivity of porcine rotavirus maintained for 32 months at approximately 10 degrees C in the original stool specimens. Thirty stool specimens of 1-4-week-old piglets from breeding farms located in the southwest of the State of Parana were selected for this study. They were randomly chosen from stool samples positive for rotavirus RNA by polyacrylamide gel electrophoresis (PAGE) at the time of collection. The thirty stool samples maintained for 32 months were re-tested by PAGE and 11 out of 30 were still positive showing physical integrity of the eleven segments of viral RNA. In order to demonstrate the maintenance of viral infectivity processed fecal homogenates were inoculated in MA-104 cell cultures. After an average of three blind passages 5 out of 11 samples demonstrated cytopathic effect similar to that of a simian rotavirus (SA-11) used as positive control. To confirm these findings an immunofluorescence test was performed and typical cytoplasmatic granular fluorescence was observed. Electron microscopy of stool samples showed that most of the virus particles were single-shelled and some were found to be in advanced state of degradation. The viral nucleic acid extracted from six fecal specimens out of those that showed physical integrity of rotavirus RNA by PAGE were also amplified when submitted to RT-PCR demonstrating stability of viral RNA. We therefore concluded that porcine rotavirus infectivity is maintained for a long period of time in stool specimens at low temperature.

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.

Experimental infection of newborn mice with murine EDIM rotavirus.

All four-day old Swiss mice whose dams were seronegative to rotavirus developed diarrhea 72 h after oral infection with EDIM rotavirus (HP strain). Electron microscopy of infected small intestine showed rotavirus particles of different sizes and morphology in the intestinal absorptive cells. In these cells rotavirus particles of 30 to 60 nm representing the various steps of virus maturation were visualized in viroplasm near the rough endoplasmic reticulum (RER). Mature rotavirus particles of 70 nm approximately were observed inside the RER of the intestinal absorptive cells. In the lamina propria rotavirus particles of approximately 30 nm and tubules were observed in phagosome-like structures of subepithelial phagocytes and were probably destroyed by lysosomal enzymes. The absence of signs of viral replication in phagocytes of lamina propria suggests that this may be the mechanism for viral antigen presentation to the host immunological system and may be one of the reasons for the fact that rotavirus infection is limited to the intestine.

Experimental infection of newborn mice with murine EDIM rotavirus

All four-day old Swiss mice whose dams were seronegative to rotavirus developed diarrhea 72 h after oral infection with EDIM rotavirus (HP strain). Electron microscopy of infected small intestine showed rotavirus particles of different sizes and morphology in the intestinal absorptive cells. In these cells rotavirus particles of 30 to 60 nm representing the various steps of virus maturation were visualized in viroplasm near the rough endoplasmic reticulum (RER). Mature rotavirus particles of 70 nm approximately were observed inside the RER of the intestinal absorptive cells. In the lamina propria rotavirus particles of approximately 30 nm and tubules were observed in phagosome-like structures of subepithelial phagocytes and were probably destroyed by lysosomal enzymes. The absence of signs of viral replication in phagocytes of lamina propria suggests that this may be the mechanism for viral antigen presentation to the host immunological system and may be one of the reasons for the fact that rotavirus infection is limited to the intestine

Contaminaçäo ambiental pelo rotavírus / Environmental contamination by bovine rotavirus

Com o objetivo de se isolar o rotavírus humano a partir de material fecal diarréico, inoculou-se o material fecal na forma de homogenato previamente descontaminado, em cultura de células MA-104. O material clínico foi estudado pela microscopia eletrônica (ME) e eletroforese em gel de poliacrilamida (EGPA), sendo que, em ambas as técnicas, detectou-se a presença do vírus, de padräo eletroforético humano. Na terceira passagem em cultivo celular foi observado efeito citopático (ECP). O monitoramento da replicaçäo viral foi positivo pelo teste de imunofluorescência (IF). A identificaçäo do isolado através da reaçäo de neutralizaçäo de focos imunofluorescentes (NFIF) e da EGPA revelou a presença de rotavírus bovino. Este trabalho discute as eventuais fontes de contaminaçäo cruzada em nosso laboratório, onde o vírus bovino também é rotineiramente manipulado. A hipótese de o vírus bovino já estar presente no material fecal humano näo pode ser descartada, porquanto näo foi observada qualquer situaçäo que sugerisse presença das duas espécies virais

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