Uso de la inmunohistoquímica como herramienta epidemiológica para el diagnóstico de rabia bovina a partir de casos no conclusivos

Objective. Identify the rabies virus in cases of nervous disease archived in the laboratory with diagnosis of nonspecific encephalitis. Materials and methods. Twelve samples of bovine brain suspected of rabies, were processed by indirect immunoperoxidase technique using polyclonal antibodies against the viral agent. Results. Was demonstrated the presence of viral antigens in three cases in the form of small aggregates in the cytoplasm of neurons, with a pattern of round or oval and a variable number of viral inclusion bodies. We discussed the importance of the results in Colombia, the usefulness of the technique in the difficult conditions for sending samples to the laboratory, plus the possible relationship of the negative cases with bovine herpesvirus 5. Conclusions. The use of immunohistochemical technique to demonstrate rabies virus antigens in formalin fixed bovines tissues can help in the construction of an epidemiological map of rabies disease in Colombia and may reduce the high under-diagnosis of diseases of the nervous system of cattle, reported in some regions.

Herpes Virus Bovino tipo 1 (BoHV-1) como posible causa de encefalitis en bovinos de la región del Magdalena Medio Colombiano. Estudio serológico y análisis epidemiológico

The bovine Herpesvirus type 1 and type 5 (BoHV-1 and BoHV-5), causing diseases and significant economic losses in farms of worldwide. Both affect the nervous system of cattle, although BoHV-5 has been the most associated with this type of pathogenesis. Given the death of animals with nervous symptoms and negative diagnoses for rabies virus in the area of study, this research focused on the detection of positive reactors to bovine herpes virus serum neutralization. We collected 518 blood samples from animals without Herpesvirus vaccine, in the municipalities of Caparrapi, Cimitarra, Honda and Victoria, in the Middle Magdalena River Region. In addition, epidemiological information useful to discuss neurological disease was collected through primary and secondary sources. For the analysis of data was used chi-square test by identification of relationship between evidence of viral infection and the variables recorded. The results revealed that 286 cases were positive for Herpesvirus infection, corresponding to a prevalence of 55.5%, however, there was no statistical relationship (p < 0.05) between the presence of antibodies and the variables analyzed. In conclusion, some cases of neurological disease in cattle in this region could be due to infection with herpes viruses. We discussed about the presence of BoHV-1 and BoHV-5 in the ambient, diagnosis and monitoring plans, as well as economic losses, which may cause in herds in this area.

Faecal contamination (viral and bacteria) detection in groundwater used for drinking purposes in São Paulo, Brazil

This project has been developed to evaluate the possible relationship between the cesspit (pit latrine) in as far as it degrades the quality of underground water. Its importance is due to the fact that in the rural communities in the State of São Paulo (Brazil) this type of cesspit is very common as a means of sewage disposal and these communities use underground water for their supply of drinking water. Rural properties distributed over the rural area in the municipality of São José do Rio Preto were selected. A preliminary study was then set up to determine the social situation and health of the households as well as qualitative evaluations on the type of water supply and sewage disposal of these communities. Campaigns of water sampling then followed and laboratory analyses of water taken from wells were carried out. Parameters were set up to evaluate the potability according to Brazilian legislation (2004) paying attention to microbiologic (coliforms, Crytosporidium sp., and adenovirus). The analyses showed evidence of possible interaction between the wells and the sewage effluents and drainage in these communities. A PCR reaction to detect adenovirus showed a presence in 53.3% of the samples. The tests for the detection of Cryotosporidium sp all showed a negative result.

Chitosan nanoparticles for non-viral gene therapy

The cationic polysaccharide chitosan has been widely used for non-viral transfection in vitro and in vivo and has many advantages over other polycations. Chitosan is biocompatible and biodegradable and protects DNA against DNase degradation. However following administration the ChitosanDNA polyplexes must overcome a series of barriers before DNA is delivered to the cell nucleus. This paper describes the most important parameters involved in the chitosan-DNA interaction and their effects of on the condensation, shape, size and protection of DNA. Strategies developed for chitosanDNA polyplexes to avoid non-specific interaction with blood components and to overcome intracellular obstacles as the crossing of die cell membrane, endosomal escape and nuclear import are presented. © 2006 American Chemical Society.

Inactivation of vesicular stomatitis virus through inhibition of membrane fusion by chemical modification of the viral glycoprotein

Membrane fusion is an essential step in the entry of enveloped viruses into their host cells triggered by conformational changes in viral glycoproteins. We have demonstrated previously that modification of vesicular stomatitis virus (VSV) with diethylpyrocarbonate (DEPC) abolished conformational changes on VSV glycoprotein and the fusion reaction catalyzed by the virus. In the present study, we evaluated whether treatment with DEPC was able to inactivate the virus. Infectivity and viral replication were abolished by viral treatment with 0.5 mM DEPC. Mortality profile and inflammatory response in the central nervous system indicated that G protein modification with DEPC eliminates the ability of the virus to cause disease. In addition, DEPC treatment did not alter the conformational integrity of surface proteins of inactivated VSV as demonstrated by transmission electron microscopy and competitive ELISA. Taken together, our results suggest a potential use of histidine (His) modification to the development of a new process of viral inactivation based on fusion inhibition. © 2006 Elsevier B.V. All rights reserved.

Induction pegylated interferon Alfa-2a and high dose ribavirin do not increase SVR in heavy patients with HCV genotype 1 and high viral loads

Background & Aims Patients infected with hepatitis C virus (HCV) genotype 1, body weight <85 kg, and high baseline viral load respond poorly to standard doses of pegylated interferon (peginterferon) and ribavirin. We evaluated intensified therapy with peginterferon alfa-2a plus ribavirin. Methods This double-blind randomized trial included HCV genotype 1-infected outpatients from hepatology clinics with body weight <85 kg and HCV RNA titer <400,000 IU/mL. Patients were randomized to 180 μg/wk peginterferon alfa-2a for 48 weeks plus 1200 mg/day ribavirin (standard of care) (group A, n = 191) or 1400/1600 mg/day ribavirin (group B, n = 189). Additional groups included 360 μg/wk peginterferon alfa-2a for 12 weeks then 180 μg/wk peginterferon alfa-2a for 36 weeks plus 1200 mg/day ribavirin (group C, n = 382) or 1400/1600 mg/day ribavirin (group D, n = 383). Follow-up lasted 24 weeks after treatment. Results Sustained virologic response rates (HCV RNA level <15 IU/mL at end of follow-up) in groups A, B, C, and D were 38%, 43%, 44%, and 41%, respectively. There were no significant differences among the 4 groups or between pooled peginterferon alfa-2a regimens (A + B vs C + D: odds ratio [OR], 1.08; 95% confidence interval [CI], 0.831.39; P = .584) or pooled ribavirin regimens (A + C vs B + D: OR, 1.00; 95% CI, 0.791.28; P = .974). Conclusions In patients infected with HCV genotype 1 who are difficult to treat (high viral load, body weight <85 kg), a 12-week induction regimen of peginterferon alfa-2a and/or higher-dose ribavirin is not more effective than the standard regimen. © 2010 AGA Institute.

First defection of the equine herpesvirus 1 neuropathogenic variant in Brazil

This report describes the first detection of an equine herpesvirus 1 (EHV-1) neuropathogenic variant (G 2254/D 752) in Brazil from a case of fatal equine herpesvirus myeloencephalopathy (EHM) in a mare. The results of nucleotide sequencing of the EHV-1 ORF30 gene showed that two other Brazilian EHV-1 isolates from EHM cases are representatives of the non-neuropathogenic variant (A 2254/N 752), suggesting that other unidentified factors are probably also involved in the neuropathogenicity of EHV-1 in horses. These findings will contribute to the epidemiological knowledge of EHV-1 infection in Brazil.

Atividade viral do vírus respiratório sincicial bovino (brsv) em bezerros leiteiros

The purpose of the present study was to evaluate the antimicrobial activity of propolis extracts diluted in different solvents against bacteria from Staphylococcus genus. The study was performed in the Immunology and Microbiology Laboratory from Universidade Federal do Vale do São Francisco. The propolis extracts were prepared using brown propolis diluted in different solvents such as chloroform, methanol, ethyl acetate and grain alcohol. In order to determine the antimicrobial potential of extracts, agar well diffusion method was used, with controls for each diluent. After that, Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) methods were used. All tests were performed in triplicate. In the agar well diffusion test, the measurements of the inhibition zone for propolis extract were as follows: grain alcohol and propolis (2.88mm), methanol and propolis (2.41mm), chloroform and propolis (2.40mm) and ethyl acetate and propolis (0.83mm). The MBC of propolis extracts in different solvents were 93.75 μg/mL for grain alcohol, 375 μg/mL for chloroform and methanol and 3,000 μg/ml for ethyl acetate. Statistically significant differences were achieved comparing the inhibition zones of propolis diluted in grain alcohol and ethyl acetate (2.88 and 0.83 mm, respectively). Considering the low cost of therapy and the activity of the propolis against caprine mastitis pathogens, other studies regarding in vivo activity and chemical characterization are necessary, in addition to evaluation of the toxicological aspects of propolis extracts.

Infecção pelo vírus da diarreia viral bovina em vacas gestantes abatidas no Estado de São Paulo-Brasil

The most important pathogens in the bovine livestock nowadays in the virus of the viral diarrhea mainly for triggered clinical manifestations related to the reproductive sphere. The infection in pregnant females, may result in abortions, embryonic resorptions, fetal mummification, birth of weak and malformation of the cattle. Moreover, their birth with persistently infected and immunotolerant virus, which the source of infection and dissemination of their disease. Nowadays, the complexity of the diagnosis and consequently its pathogenesis are tilted in the genotypic differences agent. So, this study aimed to verify the occurrence of the BVDV-1 (SINGER) and BVDV-2 (VS-253) genotypes in cows and their respective fetuses, slaughtered in an abattoir in the State of Sao Paulo. Through blood serum, using virus neutralization technique. All in all, 52,51% (115/219) of the cows which were tested reacted, but no fetus (0/219) reacted, to its virus neutralization. Through this cross-examination we observed that 42% (92/219) of cows reacted for both BVDV-1 and BVDV-2. Furthermore 4,10% (9/219) of them reacted only to the genotype BVDV-1 and 6,39% (14/219) responded only to the genotype 2 of BVDV. Therefore it was noticed that both strains are widespread in the regions studied, which justifies the use of different antigens to avoid false-negative diagnosis. Finally antibodies showed no fetus or fetal abnormalities, it is already developed and can be considered immunocompetent, independent child born to a reagent.

Intensified peginterferon α-2a dosing increases sustained virologic response rates in heavy, high viral load hepatitis c genotype 1 patients with high low-density lipoprotein

BACKGROUND AND GOAL: Patients infected with hepatitis C virus (HCV) with elevated low-density lipoprotein (LDL) levels achieve higher sustained virologic response (SVR) rates after peginterferon (PegIFN)/ribavirin treatment versus patients with lower LDL. Our aim was to determine whether SVR rates in patients with low/elevated LDL can be improved by dose intensification. STUDY: In PROGRESS, genotype 1 patients with baseline HCV RNA≥400,000 IU/mL and body weight ≥85 kg were randomized to 48 weeks of 180 μg/wk PegIFN α-2a (40 kDa) plus ribavirin (A: 1200 mg/d; B: 1400/1600 mg/d) or 12 weeks of 360 μg/wk PegIFN α-2a followed by 36 weeks of 180 μg/wk, plus ribavirin (C: 1200 mg/d; D: 1400/1600 mg/d). This retrospective analysis assessed SVR rates among patients with low (<100 mg/dL) or elevated (≥100 mg/dL) LDL. Patients with high LDL (n=256) had higher baseline HCV RNA (5.86×10 IU/mL) versus patients with low LDL (n=262; 4.02×10 IU/mL; P=0.0003). RESULTS: Multiple logistic regression analysis identified a significant interaction between PegIFN α-2a dose and LDL levels on SVR (P=0.0193). The only treatment-related SVR predictor in the nested multiple logistic regression was PegIFN α-2a dose among patients with elevated LDL (P=0.0074); therefore, data from the standard (A+B) and induction (C+D) dose arms were pooled. Among patients with low LDL, SVR rates were 40% and 35% in the standard and induction-dose groups, respectively; SVR rates in patients with high LDL were 44% and 60% (P=0.014), respectively. CONCLUSIONS: Intensified dosing of PegIFN α-2a increases SVR rates in patients with elevated LDL even with the difficult-to-cure characteristics of genotype 1, high baseline viral load, and high body weight. Copyright © 2013 by Lippincott Williams & Wilkins.

Bovine herpesvirus-5 infection in a rabbit experimental model: Immunohistochemical study of the cellular response in the CNS

Since little information is available regarding cellular antigen mapping and the involvement of non-neuronal cells in the pathogenesis of bovine herpesvirus type 5 (BHV-5) infection, it were determined the BHV-5 distribution, the astrocytic reactivity, the involvement of lymphocytes and the presence of matrix metalloproteinase (MMP)-9 in the brain of rabbits experimentally infected with BHV-5. Twelve New Zealand rabbits that were seronegative for BHV-5 were used for virus inoculation, and five rabbits were used as mock-infected controls. The rabbits were kept in separate areas and were inoculated intranasally with 500 μl of virus suspension (EVI 88 Brazilian isolate) into each nostril (virus titer, 107.5 TCID50). Control rabbits were inoculated with the same volume of minimum essential medium. Five days before virus inoculation, the rabbits were submitted to daily administration of dexamethasone. After virus inoculation, the rabbits were monitored clinically on a daily basis. Seven rabbits showed respiratory symptoms and four animals exhibited neurological symptoms. Tissue sections were collected for histological examination and immunohistochemistry to examine BHV-5 antigens, astrocytes, T and B lymphocytes and MMP-9. By means of immunohistochemical and PCR methods, BHV-5 was detected in the entire brain of the animals which presented with neurological symptoms, especially in the trigeminal ganglion and cerebral cortices. Furthermore, BHV-5 antigens were detected in neurons and/or other non-neural cells. In addition to the neurons, most infiltrating CD3 T lymphocytes observed in these areas were positive for MMP-9 and also for BHV-5 antigen. These infected cells might contribute to the spread of the virus to the rabbit brain along the trigeminal ganglia and olfactory nerve pathways. © 2013 Elsevier Ltd.

Impact of Epstein-Barr virus load, virus genotype, and frequency of the 30bp deletion in the viral BNLF-1 gene in patients harboring the human immunodeficiency virus

Patients infected with the human immunodeficiency virus (HIV) are at higher risk of developing Epstein-Barr Virus (EBV)-associated lymphomas. The usefulness of monitoring EBV in peripheral blood mononuclear cells (PBMCs) of patients infected with HIV has not been established. The aim of this study was to evaluate the EBV viral load in PBMCs, the frequency of viral genotypes, and the presence of the 30-bp deletion in the BNLF-1 gene. DNA samples from 156 patients attending the HIV/AIDS Day Clinic at Botucatu School of Medicine, Sao Paulo State University were evaluated. The EBV viral load was detectable by real time PCR in 123/156 (78.8%) cases and was higher in patients not receiving antiretroviral treatment or under therapeutic failure than in patients under successful highly active antiretroviral therapy (HAART) (P=0.0076). Overall, the profile of patients with high EBV viral load included elevated HIV viremia (P=0.0005), longer time of HIV diagnosis (P=0.0026), and increased levels of T CD8 + lymphocytes (P=0.0159). The successful amplification of the EBNA-2 gene by nested-PCR was achieved in 95 of 123 (77.2%) cases, of which 75.8% were EBV-1, 9.5% EBV-2, and 14.7% were co-infected with both EBV-1 and -2. The analysis of the BNLF-1 gene was possible in 99 of 123 (80.5%) cases, of which 50.5% had the 30-bp deletion. EBV-1 was more common than EBV-2, which may reflect the fact that the cohort was predominantly Caucasian and heterosexual. J. Med. Virol. 85:2110-2118, 2013. © 2013 Wiley Periodicals, Inc.

Modelagem molecular da interação entre a proteína de fusão do vírus sincicial respiratório humano e inibidores da ação viral. -

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Validação de recursos de cargas viral do HIV-1 obtidos para insumos/kids/equipamentos de diferentes procedênias

Pós-graduação em Pesquisa e Desenvolvimento (Biotecnologia Médica) - FMB