ABSTRACT Propolis is a rich source of bioactive molecules made by bees by collecting an infinite variety of secondary metabolites from plants. This study determined the chromatographic and mass spectrometry profile along with the biological activity of propolis produced by the native Brazilian bee Melipona quadrifasciata. A hydroalcoholic crude extract was prepared and partitioned in solvents of different polarities, generating aqueous, dichloromethane, ethyl acetate, butanol and insoluble fractions. The phenolic and flavonoid content was also determined in crude aqueous and methanolic extracts as well. The antioxidant activity was determined by three different methods and correlated with the phenolic and flavonoid content. Extracts and fractions were tested against the herpes simplex virus type 1, and the cytotoxicity was evaluated in fibroblast L929 cells. The chromatographic and mass spectrometry analysis revealed the presence of catechin, epicatechin, aromadendrin, naringenin, pinocembrin and p-coumaric acid. The methanolic extract, ethyl acetate fraction and insoluble fraction possess higher phenolic and flavonoid content along with better antioxidant activities. The ethyl acetate and butanol fractions, along with the FM14 and FM45 subfractions demonstrated better viral inhibition, with 50% inhibitory concentrations of 90.5, 294, 188 and 58.5 µg ml−1, respectively. Among these, the ones presenting the best selectivity index were the ethyl acetate and FM45 fractions, with a pronounced virucidal effect. The methanolic extract stood out in the antioxidant activities and showed no cytotoxicity bellow 125 µg ml−1, exhibiting the most promising biological activity. Although this propolis seems to act in the early stages of herpetic infections, it is still difficult to correlate this activity with a single constituent of this complex mixture, suggesting a synergism in the extract components. To our knowledge, this is the first study of its kind with this propolis type.

BACKGROUND:Herpes simplex virus (HSV) infection is an endemic disease and it is estimated that 6095% of the adult population are infected with symptoms that are usually self-limiting, though they can be serious, extensive and prolonged in immunocompromised individuals, highlighted by the emergence of drug-resistant strains. The study of the wild-type HSV strains based on the cytopathogenic features and its antiviral sensitivity are important in the establishment of an antivirogram for controlling the infection.OBJECTIVE:This study sought to isolate and examine the cytopathological characteristics of circulating strains of the Herpes simplex virus, from clinical specimens and their sensitivity to commercially available antiherpesvirus drugs, acyclovir, phosphonophormic acid and trifluridine.METHODS:Herpes simplex virus isolation, cytopathological features and antiviral sensitivity assays were performed in cell culture by tissue culture infectious dose or plaque forming unit assay.RESULTS:From twenty-two clinical specimens, we isolated and adapted nine strains. Overall, the cytopathic effect was detected 24 h post-infection (p.i.) and the presence of syncytia was remarkable 48 h p.i., observed after cell staining. Out of eight isolates, four developed plaques of varying sizes. All the isolates were sensitive to acyclovir, phosphonophormic and trifluridine, with the percentage of virus inhibition (%VI) ranging from 49.7-100%.CONCLUSIONS:The methodology for HSV isolation and characterization is a straightforward approach, but the drug sensitivity test, regarded as being of great practical importance, needs to be better understood.

Acute diarrheal disease is still one of the major public health problems worldwide. Rotaviruses (RV) are the most important viral etiologic agents and children under five years of age are the target population. OBJECTIVE: To investigate the rate of RV infection in hospitalized patients due to acute diarrhea in the cities of Ponta Grossa, Londrina and Assai - Paraná. METHODS: Latex agglutination (LA); immunochromatography (ICG); polyacrylamide gel electrophoresis (PAGE) and negative staining electron microscopy (ME) tests were used to detect the virus. For the genotyping, RT-PCR and RT-PCR-ELISA were used, respectively, for NSP4 and VP4/VP7. RESULT: Out of 124 samples there were 69 positive stool samples for RV, for at least one of the used tests, 67 of them being RV group A (RV-A). Overall, most of the RV positive stool samples came from children under thirteen years of age. However, 12 positive cases occurred in patients aged 13 years or above, including an 81-year old patient. CONCLUSION: The data showed similar electropherotypes and genotypes G, P and NSP4 of the inland wild circulating strains of RV.

O trabalho apresenta o desenvolvimento de um ensaio imunoenzimático indireto para a detecção de antígeno de poliovírus. O antígeno viral foi obtido em cultura de células LLC-MK2 e usado para imunização de coelho e cobaia. Os soros hiperimunes foram avaliados por imunodifusão dupla e teste de neutralização. Após padronização, o soro de captura, produzido em cobaia, foi usado na concentração protéica de 15.0 µg/ml para sensibilizar microplacas de poliestireno e o soro de coelho (detector) foi usado na concentração de 7.94 µg/ml. A curva padrão resultante da utilização de oito diferentes concentrações do antígeno padrão definiu um coeficiente de variação de 2.1% a 7.8%. A relação dose-resposta foi determinada por regressão linear simples com o estabelecimento do coeficiente de correlação (R²) igual a 96.4%. O ensaio possibilitou a detecção mínima de 2.3 µg/ml de antígeno de poliovírus.

An indirect solid-phase enzyme immunoassay (EIA) was developed for the detection of poliovirus antigen. Virus antigen was obtained in LLC-MK2 cell cultures and used to prepare antibodies in rabbit and guinea pig. Antibodies were evaluated by double immunodiffusion and neutralization test. Optimal concentrations of guinea pig and rabbit immunoglobulins were determined by checkerboard titration. Microtitre plates were coated with 15.0 µg/ml guinea pig anti-polio immunoglobulin and rabbit anti-polio immunoglobulin at the concentration of 7.94 µg/ml was used as detecting antibody. The standard curve with eight different antigen concentrations in eight replicates resulted in a coefficient of variation (CV) between 2.1% to 7.8%. The dose-response relationship was determined by simple linear regression with a coefficient of correlation (R²) equal to 96.4%. The assay detected a minimum of 2.3 µg/ml poliovirus antigen.

O extrato aquoso de Agaricus blazei Murill ss. Heinem, um basidiomiceto nativo do Brasil, usado na medicina popular, na forma de chá, foi avaliado quanto suas propriedades antivirais contra herpes simplex tipo 1 (HSV-1) e herpes bovino tipo 1 (BoHV-1) em cultura de células HEp-2. A inibição da replicação viral foi monitorada pelos ensaio de placa e reação de imunofluorescência. O extrato apresentou atividade virucida mais efetiva do que terapêutica para ambos os vírus, sendo mais efetivo portanto para HSV-1, inibindo em mais de 70% o número de plaques e em cerca de 47% o número de células apresentando fluorescência específica, nas concentrações de 50 e 100 µg/mL, nas duas técnicas utilizadas. Os resultados obtidos sugerem que o extrato aquoso de A. blazei deve agir principalmente sobre a partícula viral, embora a inibição durante o ciclo replicativo do vírus não deva ser excluída.

The aqueous extract of Agaricus blazei Murill ss. Heinem, a basidiomycete native from Brazil, frequently used by popular medicine, mainly in the form of tea, was assessed to its antiviral action against herpes simplex type 1 (HSV-1) and bovine herpes type 1 (BoHV-1) in HEp-2 cell culture. Viral replication inhibition was evaluated by plaque assay and immunofluorescence test. The extract demonstrated virucide action for both viruses, being more effective against HSV-1, inhibiting its infectivity in 78.4 and 73.9% at the concentrations of 50 and 100 µg/mL, respectively moreover, reduction in 47% the number of fluorescent cells was observed for both concentrations. The extract also showed discrete therapeutic activity. These results suggest that A. blazei extract acts mainly in the viral particle, however, the effect during virus replication can not be ruled out.

Rotaviruses have been implicated as the major causal agents of acute diarrhoea in mammals and fowls. Experimental rotavirus infection have been associated to a series of sub-cellular pathologic alterations leading to cell lysis which may represent key functions in the pathogenesis of the diarrhoeic disease. The current work describes the cytopathic changes in cultured MA-104 cells infected by a simian (SA-11) and a porcine (1154) rotavirus strains. Trypan blue exclusion staining showed increased cell permeability after infection by both strains, as demonstrated by cell viability. This effect was confirmed by the leakage of infected cells evaluated by chromium release. Nuclear fragmentation was observed by acridine orange and Wright staining but specific DNA cleavage was not detected. Ultrastructural changes, such as chromatin condensation, cytoplasm vacuolisation, and loss of intercellular contact were shown in infected cells for both strains. In situ terminal deoxynucleotidyl transferase (Tunel) assay did not show positive result. In conclusion, we demonstrated that both strains of rotavirus induced necrosis as the major degenerative effect.

A infecciosidade do rotavírus cepa SA-11 foi estudada em cultura de células MA-104 em presença de concanavalina A (ConA). Foi demonstrada a presença de receptores para a ConA nestas células através de ensaio de fluorescência e bloqueio específico dos mesmos através de D-manose. A cinética de replicação viral foi realizada pelo método de plaque e o vírus monitorado, pontualmente, pela microscopia eletrônica de transmissão e eletroforese em gel de poliacrilamida. Concluímos que a ConA não afetou consistentemente a replicação viral, entretanto interferiu no efeito citopático.

Rotavirus (RV) strain SA-11 was studied with respect to its infectivity in MA-104 cell cultures and the effect of concanavalin A (ConA). Receptors for ConA at the surface of MA-104 cell were determined by fluorescence assay and specifically inhibited by D-mannose. The kinetics of virus growth was carried out by plaque assay. Electron microscopy and polyacrylamide gel electrophoresis were used for monitoring the experiments. It was concluded that RV replication was not affected consistently by ConA, however it interfered with the development of cytopathic effect (CPE) without altering virus yields.

Os rotavírus são patógenos comuns e causam diarréia aguda em crianças e animais jovens. Neste trabalho avaliamos a participação do vírus na diarréia de populações humanas das áreas urbana e rural da cidade de Londrina, Paraná. Foram analisadas 905 amostras fecais de indivíduos com diarréia aguda, sendo 686 e 219 amostras das zonas urbana e rural, respectivamente. Trinta e oito amostras (4,2%) foram consideradas positivas pelas técnicas de eletroforese em gel de poliacrilamida do RNA viral e aglutinação passiva de látex, das quais 36 da área urbana e dois da área rural. Das amostras positivas, 17 foram genotipadas por RT-PCR tendo sido caracterizadas 16 cepas G1 e uma considerada mistura dos genótipos G1 e G3.

Rotaviruses are common pathogens and the causal agents of acute diarrhea among children and young animals. The involvement of rotavirus in human diarrheal disease among population of urban and rural areas of the city of Londrina, Parana was evaluated. Nine hundred and five fecal specimens from persons with diarrhea were studied, being 686 and 219 from urban and rural areas, respectively. Thirty-eight samples (4,2%) were positive for rotavirus by polyacrylamide gel electrophoresis of viral RNA and latex agglutination test of which 36 were from urban and two from rural areas. Out of the positive specimens, 17 strains were further characterized by RT-PCR typing assay, resulting in 16 strains of G1 genotype while one sample was found to be a mixture of G1 and G3 genotypes.

Quatro linhagens de Lentinula edodes (Le10, 46, K2, ASSAI) foram avaliadas quanto ao seu efeito inibitório sobre quatro espécies de fungos filamentosos de importância agrícola (Helminthosporium euphorbiae, Helminthosporium sp., Fusarium solani, Phomopsis sojae) e sobre o sorotipo Alagoas vírus da estomatite vesicular (VSA). Foi observado que as linhagens de L. edodes estudadas apresentaram variabilidade quanto ao seu efeito, tanto sobre os fungos filamentosos quanto sobre o vírus VSA. As linhagens K2 e Le10 apresentaram-se antagônicas sobre os fungos e as linhagens 46 e K2 foram eficientes na inibição do vírus VSA. Os resultados obtidos permitem ampliar a lista de efeitos benéficos de algumas linhagens de L. edodes no controle e prevenção de vírus patogênicos animais e de fungos filamentosos.

Four Lentinula edodes strains (Le10, 46, K2, Assai) were assessed for their antagonistic effect on four filamentous fungus species of agricultural importance (Helminthosporium euphorbiae, Helminthosporium sp, Fusarium solani and Phomopsis sojae) and on Alagoas serotype of Vesicular Stomatitis Virus (VSA). The L. edodes strains studied had variable effects on the filamentous fungi and on VSA. The K2 and Le10 strains were antagonistic on the fungi assessed and the 46 and K2 strains were efficient on the Vesicular Stomatitis Virus. The results widened the list of beneficial effects of L. edodes on the control and prevention of animal pathogenic virus and filamentous fungi.

Os rotavírus constituem-se nos principais patógenos da diarréia em humanos e animais. Afetam os animais jovens em criações 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 leitões de 1-4 semanas de idade, provenientes de granjas da região 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 manutenção 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 manutenção 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 típicamente 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 degradação. 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 manutenção do vírus viável em condição natural assim como para a transmissão da doença.

Rotaviruses are the major pathogen for both human and animal. They affect young animals in intensive rearing and cause great economic losses. This study intended to evaluate the infectivity of porcine rotavirus maintained for 32 months at approximately 10ºC. in the original stools specimens. Thirty stools specimens of 1-4-week-old piglets originating from breeding farms located in the southwest region of the Paraná State were selected for this study. They were randomly chosen from stools samples positive for rotavirus by polyacrylamide gel electrophoresis (PAGE) at the time of collection. The thirty stools samples maintained for 32 months at approximately 10ºC were re-tested by PAGE and 11 out of 30 were still positive showing the integrity of the eleven bands of viral RNA. To demonstrate the maintenance of viral infectivity, clarified and trypsin-treated stools specimens homogenates were inoculated in MA-104 cell cultures. After an average of 3 blind passages 5 out of 11 samples demonstrated cytophathic effect similar to that of standard simian rotavirus (SA-11). To confirm these findings, a immunofluorescent test was used and demonstrated typical cytoplasmic granular fluorescence. Electron microscopy of stools samples showed that most of the virus particles were single- shelled and some were found to be in an advanced state of degradation. Therefore the conclusion was that porcine rotavirus infectivity is maintained for a long period of time in stool specimens at low temperature. This certainly is an important aspect for the maintenance of viable virus in natural condition as well as for the transmission of the disease.