Carlos Nozawa

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.

Genotyping of enteric adenoviruses by using single-stranded conformation polymorphism analysis and heteroduplex mobility assay.

ABSTRACT Single-stranded conformation polymorphism (SSCP) analysis and heteroduplex mobility assays (HMAs) were used to identify and genotype enteric adenoviruses (EAd). The results were compared to those of restriction endonuclease assays, species-specific PCRs, and direct nucleotide sequence analyses. Of the 31 stool samples tested, 15 isolates were identified as EAd and 7 were identified as nonenteric Ad by all methods. An agreement of 100% was found between the SSCP and HMA results.

The infectivity of pig rotavirus in stools

Os rotavirus constituem-se nos principais patogenos da diarreia em humanos e animais. Afetam os animais jovens em criacoes intensivas e causam grandes perdas economicas. Este estudo avaliou a infecciosidade do rotavirus suino mantido por 32 meses a aproximadamente 10oC nas amostras originais de fezes. Trinta amostras de fezes de leitoes de 1-4 semanas de idade, provenientes de granjas da regiao sudoeste do Parana, foram selecionadas para o estudo. As amostras foram colhidas no periodo de marco a outubro de 1991 e selecionadas ao acaso dentre as positivas para rotavirus pela eletroforese em gel de poliacrilamida (EGPA), a epoca da colheita. Estas foram retestadas por EGPA 32 meses apos manutencao a 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 manutencao da infecciosidade viral, os homogenatos fecais clarificados, previamente tratados com tripsina, foram inoculados em culturas de celulas MA-104. Das 11 amostras, 5 demonstraram efeito citopatico semelhante ao do rotavirus simio (SA-11), apos em media 3 passagens cegas e confirmado pelo teste de imunofluorescencia indireta, demonstrado pela fluorescencia especifica citoplasmatica tipicamente granular. A microscopia eletronica das amostras fecais mostrou que a maioria das particulas virais apresentavam-se sem capsidio externo e outras encontravam-se em adiantado estado de degradacao. Concluiu-se, portanto, que a infecciosidade do rotavirus suino e mantida por longo periodo em amostras fecais em baixa temperatura. Este certamente e um aspecto importante para a manutencao do virus viavel em condicao natural assim como para a transmissao da doenca.

Antiviral Activity of Sulfated Polysaccharide of Adenanthera pavonina against Poliovirus in HEp-2 Cells

Adenanthera pavonina, popularly known as red-bead tree, carolina, pigeons eye, and dragons eye, is a plant traditionally used in Brazil for the treatment of several diseases. The present study aimed at evaluating the activity of sulfated polysaccharide from the Adenanthera pavonina (SPLSAp) seeds against poliovirus type 1 (PV-1) in HEp-2 cell cultures. The SPLSAp presented a cytotoxic concentration (CC50) of 500u2009μg/mL in HEp-2 cell cultures, evaluated by the dimethylthiazolyl-diphenyltetrazolium bromide method (MTT). The SPLSAp exhibited a significant antiviral activity, with a 50% inhibitory concentration (IC50) of 1.18u2009µg/mL, determined by plaque reduction assay and a high selectivity index (SI) of 423. The maximum inhibition (100%) of PV replication was found when the SPLSAp treatment was concomitant with viral infection (time 0u2009h), at all tested concentrations. The maximal inhibition was also found when the SPLSAp was used 1u2009h and 2u2009h postinfection, albeit at 50u2009μg/mL and 100u2009μg/mL. Therefore, we demonstrated that the SPLSAp inhibited PV growth. We also suggested that SPLSAp inhibited PV in more than one step of the replication, as the mechanism of antiviral action. We, therefore, selected the compound as a potential candidate for further development towards the control of the infection.

Characterization and antiherpetic activity of native and chemically sulfated polysaccharide from Adenanthera pavonina.

The herpes simplex virus (HSV) is a widespread human pathogen and for many reasons the development of anti-herpetic drugs from natural products has been encouraged. Adenanthera pavonina (Ap) is a medicinal plant widely used in Brazil, among other uses. Herein, a native Ap seed polysaccharide (PLSAp) and its chemically sulfated derivate (SPLSAp) were studied by Fourier transform IR spectra (FT-IR), gel permeation chromatography (GPC) for molar mass determination and their intrinsic viscosity [η]. Biologically, the compounds were evaluated for anti-HSV activity, in HEp2 cell cultures. The cytotoxic concentrations (CC50) and the inhibitory concentrations (IC(50)) of the polysaccharides were determined by the colorimetric assay (dimethyl-thiazolyl-diphenyltetrazolium bromide) and plaque reduction assay (PRA), respectively. The SPLSAp showed a better antiviral activity when compared to the PLSAp with a CC(50) of 500 μg/ml, the IC(50) equal to 15 μg/ml and the selectivity index (SI) of 33.3. The time-of-addition and the time-of-removal assays demonstrated the highest inhibitory activity between 8-16h after the infection. The inhibition of viral DNA and protein syntheses by SPLSAp monitored by PCR and immunofluorescence assay (IFA), respectively, has also demonstrated. These findings demonstrated that the SPLSAp inhibited HSV-1 infection in different steps of the replication and, therefore, represents a valuable compound for preclinical studies in anti-herpetic therapy.