Wild rabies virus detection by plaque assay from naturally infected brains in different species

A simple, sensitive and specific plaque assay protocol for the detection of wild type rabies virus in different species is described using confluent monolayers of chicken embryo cells in 6-well plates. Plaques are produced after application of either agarose or Sephadex G-100 overlay onto cell monolayers and incubation for 96 h after virus infection at 37 degreesC. The parameters affecting plaque appearance include cell seeding concentration, overlay composition and time of incubation after infection. Optimal conditions are seeding at a concentration of 4 x 10(6) cell/cm(3), incubation at 37 degreesC in 5% CO2 atmosphere during 96 h, using either 1% agarose or 2% Sephadex G-100 overlays. The described plaque assay would be a new valuable too] in conducting various quantitative investigations, since the chicken embryo cells are susceptible to rabies virus infection from all species studied. (C) 2004 Elsevier B.V. All rights reserved.

In vitro and in vivo studies of the Interferon-alpha action on distinct Orthobunyavirus

Oropouche, Caraparu, Guama, Guaroa and Tacaiuma viruses (Orthobunyavirus genus) cause human febrile illnesses and/or encephalitis. To achieve a therapeutical agent to prevent and/or treat these diseases we evaluated the antiviral action of Interferon-alpha (IFN-alpha) on these orthobunyaviruses. In vitro results showed that all the studied orthobunyaviruses are susceptible to antiviral action of IFN-alpha, but this susceptibility is limited and dependent on both concentration of drug and treatment period. In vivo results demonstrated that IFN-alpha present antiviral action on Oropouche and Guaroa viruses when used as a prophylactic treatment. Moreover, a treatment initiated 3 It after infection prevented the death of Guaroa virus infected-mice. Additionally, mortality of mice was related to the migration and replication of viruses in their brains. Our results suggest that IFN-alpha could be potentially useful in the prevention of diseases caused by Oropouche virus and in the prevention and/or treatment of diseases caused by Guaroa virus. (C) 2007 Elsevier B.V. All rights reserved.

Antiviral effect of Guazuma ulmifolia and Stryphnodendron adstringens on poliovirus and bovine herpesvirus

Crude extract (CE) and aqueous (AqF) and ethyl acetate (EtOAcF) fractions of Guazuma ulmifolia LAM., Sterculiaceae and the corresponding AqF, EtOAcF of Stryphnodendron adstringens (MART.) COVILLE, Leguminosae were tested for their antiviral activity against poliovirus 1 (P-1) and bovine herpesvirus 1 (BHV-1) in HEp2 cultured cells. The antiviral activity was monitored by plaque assay and immunolluorescence assay (IFA) under virucidal and therapeutic protocols. The therapeutic protocol demonstrated statistically significant positive results with both plants and for both virus strains. The highest percentages of viral inhibition were found for G. ulmifolia EtOAcF which inhibited BHV-1 and P-1 replication by 100% and 99%, respectively (p < 0.05, Student's t-test). For S. adstringens, AqF was the most efficient, inhibiting BHV-1 and P-1 by 97% and 93%, respectively (p < 0.05). In the virucidal protocol, G. ulmifolia CE inhibited the replication of BHV-1 and P-1 by 60% and 26%, respectively (p < 0.05), while, for S. adstringens, inhibition of 62% (p < 0.05) was demonstrated only with EtOAcF for P-1. IFA demonstrated that the greatest reduction in fluorescent cell number occurred with G. ulmifolia, under the therapeutic protocol for both virus strains. However, AqF and EtOAcF of S. adstringens were most efficient with the virucidal protocol for P-1 In conclusion, we demonstrated that G. ulmifolia and S. adstringens inhibited BHV-1 and P-1 replication, as well as, blocked the synthesis of viral antigens in infected cell cultures.

In vitro study of antiviral activity of mycophenolic acid on Brazilian orthobunyaviruses

Objective: Oropouche, Caraparu, Guama, Guaroa and Tacaiuma are ssRNA viruses that belong to the genus Orthobunyavirus and have been associated with human febrile illnesses and/or encephalitis. In this study, we evaluated the antiviral action of mycophenolic acid (MPA) on these orthobunyaviruses to achieve a therapeutic agent to treat the diseases caused by these viruses. Methods: the in vitro antiviral evaluation to MPA was done by using plaque assay at different periods of treatment. Results: Results showed that MPA at a concentration of 10 mu g/ml has significant antiviral activity on Tacaiuma virus when treatment was initiated either 24 h before or 2 h after viral infection. Moreover, MPA has an inhibitory effect on Guama virus replication, but only when treatment was initiated before cell infection. Addition of guanosine in the culture reverted the inhibitory effect of MPA on Tacaiuma and Guama viruses, suggesting that the antiviral activity of this substance was via depletion of the intracellular guanosine pool. Conclusion: Our results suggest that MPA would not be a good therapeutic agent to treat the diseases caused by Oropouche, Caraparu, Guama, Guaroa, and Tacaiuma viruses.

The in vitro antiviral activity of an aliphatic nitro compound from Heteropteris aphrodisiaca

We investigated the antiviral activity of an aliphatic nitro compound (NC) isolated from Heteropteris aphrodisiaca O. Mach. (Malpighiaceae), a Brazilian medicinal ptant. The NC was tested for its antiviral activity against poliovirus type 1 (PV-1) and bovine herpes virus type 1 (BHV-1) by plaque reduction assay in cell culture. The NC showed a moderate antiviral activity against PV-1 and BHV-1 in HEp-2 celts, and the 50% inhibitory concentration (IC50) were 22.01 mu g/ml (selectivity index (SI) = 2.83) and 21.10 mu g/mi (SI = 2.95), respectivety. At the highest concentration of the drug (40 mu g/ml) a reduction of approximately 80% in plaque assay was observed for both viruses. The treatment of cells or virus prior to infection did not inhibit the replication of virus strains. (C) 2006 Elsevier GmbH. All rights reserved.

Association between polyclonal B cell activation and the presence of autoantibodies in mice infected with Yersinia enterocolitica O:3

Eight-week old conventional female Swiss mice were inoculated intravenously with Yersinia enterocolitica O:3. A second group of normal mice was used as control. Five mice from each group were bled by heart puncture and their spleens were removed for spleen cell collection on the 3rd, 5th, 7th, 10th, 14th and 21st day after infection. Immunoglobulin-secreting spleen cells were detected by the isotype-specific protein A plaque assay. Total immunoglobulin levels were determined in mouse serum by single radial immunodiffusion and the presence of autoantibodies was determined by ELISA. We observed a marked increase in the total number of cells secreting immunoglobulins of all isotypes as early as on the 3rd day post-infection and the peak of secretion occurred on the 7th day. At the peak of the immunoglobulin response, the total number of secreting cells was 19 times higher than that of control mice and most immunoglobulin-secreting cells were of the IgG2a isotype. On the 10th day post-infection, total serum immunoglobul in values were 2 times higher in infected animals when compared to the control group, and continued at this level up to the 21st day post-infection. Serum absorption with viable Y. enterocolitica cells had little effect on antibody levels detected by single radial immunodiffusion. Analysis of serum autoantibody levels revealed that Y. enterocolitica infection induced an increase of anti-myosin and anti-myelin immunoglobulins. The sera did not react with collagen. The present study demonstrates that Y. enterocolitica O:3 infection induces polyclonal activation of murine B cells which is correlated with the activation of some autoreactive lymphocyte clones.

Invasin of Yersinia pseudotuberculosis is not a polyclonal activator of mouse B lymphocytes

It is known that the invasin molecule of Yersinia pseudotuberculosis stimulates human peripheral B cells in vitro. In this work we evaluated the in vivo role of invasin as polyclonal activator of B lymphocytes in the mouse experimental model, by comparing strains of Y. pseudotuberculosis expressing invasin and isogenic inv mutants. Swiss mice were infected intravenously with two strains expressing invasin (YpIII pIB1 and an isogenic virulence plasmid-cured strain, YpIII) and with two invasin mutant strains (Yp100 pIB1 and Yp100, plasmid-cured). Spleen cells were sampled on days 7, 14, 21 and 28 after infection. Immunoglobulin (Ig)-secreting spleen cells were detected by protein A plaque assay and specific antibodies were detected in sera by ELISA. The virulent strain YPIII pIB1 (wild type) did not provoke polyclonal activation of B lymphocytes in vivo. In general, fewer Ig-secreting spleen cells of all isotypes were found in the infected animals than in the control animals. Specific IgG antibodies were detected in the sera of animals infected with all strains. The peak response occurred on the 21 st day post-infection, and the Yp100 strain provoked the highest level of these antibodies. We concluded that invasin is not a polyclonal activator of murine B cells.

Effects of Yersinia enterocolitica O:3 derivatives on B lymphocyte activation in vivo

The potential sequelae of intestinal infection with Yersinia enterocolitica include reactive arthritis, erythema nodosum, Reiter's syndrome and other autoimmune diseases. The role of the immune response in the pathogenesis of these diseases has not been fully defined, but autoimmune manifestations may be a consequence of the increase in autoantibodies as a result of polyclonal B-cell activation induced by Yersinia. We investigated the effects of Y enterocolitica 0:3 derivatives on B lymphocyte activation in vivo. Groups of five specific pathogen free (SPF) Swiss mice were inoculated with bacterial cell extract, Yersinia outermembrane proteins (Yops) or lipopolysaccharide (LPS) obtained from Y enterocolitica 0:3 and their immunoglobulin-secreting spleen cells were detected by isotype-specific protein A plaque assay. The presence of specific anti-Yersinia antibodies and autoantibodies was determined in mouse sera by ELISA. In all experiments a marked increase in the number of secretory cells of different isotypes was observed as early as the third day after inoculation. IgG and IgM anti-Yersinia antibodies were detected in the sera of all inoculated mice, and autoantibodies against myosin in the sera of those inoculated with bacterial cell extract. The sera from animals stimulated with LPS reacted with myelin, actin and laminin, while the sera from mice inoculated with Yops reacted with myelin, thyroglobulin and cardiolipin. These results suggest that SPF Swiss mice inoculated with any one of the Y enterocolitica derivatives tested exhibited polyclonal activation of B lymphocytes as a result of stimulation by various bacterial components and not only LPS stimulation.

The Effect of a Supragingival Plaque-Control Regimen on the Subgingival Microbiota in Smokers and Never-Smokers: Evaluation by Real-Time Polymerase Chain Reaction

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Development and application of a Saccharomyces cerevisiae-expressed nucleocapsid protein-based enzyme-linked immunosorbent assay for detection of antibodies against infectious bronchitis virus

A Saccharomyces cerevisiae-expressed nucleocapsid (N) polypeptide of the M41 strain of infectious bronchitis virus (IBV) was used as antigen in a recombinant yeast-expressed N protein-based enzyme-linked immunosorbent assay (Y-N-ELISA). The Y-N-ELISA was rapid, sensitive, and specific for detecting chicken serum antibodies to IBV, and it compared favorably with a commercial ELISA.

An enzyme-linked immunosorbent assay for the detection of IgG antibodies against Babesia equi in horses

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Sensitivity evaluation of a single-step PCR assay using Ehrlichia canis p28 gene as a target and its application in diagnosis of canine ehrlichiosis

O objetivo deste estudo foi aperfeiçoar um ensaio de PCR que amplificasse um fragmento de 843 pares de bases do gene p28 da Ehrlichia canis e compará-lo com outros dois métodos de PCR utilizados para amplificar partes do gene 16S rRNA e dsb do gênero Ehrlichia. Amostras sanguíneas foram colhidas de cães com diagnóstico clínico de erliquiose. A amplificação do gene p28 pela PCR produziu um fragmento de 843pb e esse ensaio permitiu a detecção do DNA de um parasita dentre 1 bilhão de células. Todas as amostras positivas detectadas pela PCR baseada no gene p28 foram também positivas pela nested PCR para detecção do gene 16S rRNA e também pela PCR dsb. Dentre as amostras negativas para a PCR p28, 55,3% foram co-negativas, mas 27,6% foram positivas pela PCR baseada nos genes 16S rRNA e dsb. A PCR p28 parece ser um teste útil para detecção molecular de E. canis, entretanto otimizações na sensibilidade nesta PCR são necessárias, para que esta técnica se torne uma importante alternativa no diagnóstico da erliquiose canina.

Detection of anti-Toxoplasma gondii antibodies in experimentally and naturally infected non-human primates by Indirect Fluorescence Assay (IFA) and indirect ELISA

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

An enzyme-linked immunosorbent assay (ELISA) for the detection of IgM antibodies against Leishmania chagasi in dogs

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

A nested-PCR assay for detection of Xylella fastidiosa in citrus plants and sharpshooter leafhoppers

Aims: Detection of Xylella fastidiosa in citrus plants and insect vectors.Methods and Results: Chelex 100 resin matrix was successfully standardized allowing a fast DNA extraction of X. fastidiosa. An amplicon of 500 bp was observed in samples of citrus leaf and citrus xylem extract, with and without symptoms of citrus variegated chlorosis, using PCR with a specific primer set indicating the presence of X. fastidiosa. The addition of insoluble acid-washed polyvinylpyrrolidone (PVPP) prior to DNA extraction of insect samples using Chelex 100 resin together with nested-PCR permitted the detection of X. fastidiosa within sharpshooter heads with great sensitivity. It was possible to detect up to two bacteria per reaction. From 250 sharpshooter samples comprising four species (Dilobopterus costalimai, Oncometopia facialis, Bucephalogonia xanthopis and Acrogonia sp.), 87 individuals showed positive results for X. fastidiosa in a nested-PCR assay.Conclusions: the use of Chelex 100 resin allowed a fast and efficient DNA extraction to be used in the detection of X. fastidiosa in citrus plants and insect vectors by PCR and nested-PCR assays, respectively.Significance and Impact of the study: the employment of efficient and sensitive methods to detect X. fastidiosa in citrus plants and insect vectors will greatly assist epidemiological studies.