Evaluation of developmental changes in bovine in vitro produced embryos following exposure to bovine Herpesvirus type 5

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

Susceptibility of neuron-like cells derived from bovine Wharton's jelly to bovine herpesvirus type 5 infections

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

Astrocyte intermediate filaments are important markers in olfactory bulb of bovine herpesvirus type 5 natural infections

Astroglial cells are the most abundant cells in the mammalian central nervous system, yet our knowledge about their function in bovine Herpesvirus type 5 (BoHV-5) has been limited. The aim of this study was to detect by immunohistochemistry assay the reactive astrocytes for glial fibrilary acidic protein (GFAP) and vimentin (VIM), considered intermediate filaments of the cytoskeleton, localized in olfactory bulb from natural acute cases of BoHV-5 infection. All samples were submitted to virus isolation, real-time polymerase chain reaction (RT-PCR) and in situ hybridization (ISH) technique to confirm the virus transcription and respective genome. Samples were classified into four groups according to the severity of histological lesions. Groups III and IV, which histological lesions were classified as alacia, gliosis, satellitosis, neuronophagia and neuronal necrosis, 35% (± 1.8-2.1) of the inflammatory mononuclear cells, corresponded to CD3 positive lymphocytes. In the same group, 35% (± 1.8) of astrocytes were described as reactive to GFAP and VIM proteins. An agreement of r = 1.0 (P<0.0001) was found between histological lesions, intermediate filaments expression, viral DNA and transcription and CD3 lymphocytes. However, samples with mild histological lesions, 10.8 to 14.2% of astrocytes were classified as reactive to GFAP and VIM filaments. Our findings suggest that GFAP and VIM reactive astrocytes, in primary site of virus replication, seems to play an important role in neurovirulence, in spite of many questions concerning the virus immunopathology remains unclear.

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.

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.

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.

Bovine herpesvirus type 5 infection regulates Bax/BCL-2 ratio

Bovine herpesvirus 5 (BoHV-5) is an α-herpesvirus that causes neurological disease in young cattle and is also occasionally involved in reproductive disorders. Although there have been many studies of the apoptotic pathways induced by viruses belonging to the family Herpesviridae, there is little information about the intrinsic programmed cell death pathway in host-BoHV-5 interactions. We found that BoHV-5 is able to replicate in both mesenchymal and epithelial cell lines, provoking cytopathology that is characterized by cellular swelling and cell fusion. Viral antigens were detected in infected cells by immunofluorescence assay at 48 to 96 h post-infection (p.i.). At 48 to 72 h p.i., anti-apoptotic BCL-2 antigens were found at higher levels than Bax antigens; the latter is considered a pro-apoptotic protein. Infected cells had increased BCL-2 phenotype cells from 48 to 96 h p.i., based on flow cytometric analysis. At 48 to 96 h p.i., Bax mRNA was not expressed in any of the infected cell monolayers. In contrast, BCL-2 mRNA was found at high levels at all p.i. in both types of cells. BoHV-5 replication apparently modulates BCL-2 expression and gene transcription, enhancing production of virus progeny. © FUNPEC-RP.

Avaliação histopatológica, imunoistoquímica e detecção molecular do DNA viral no sistema nervoso central de bovinos inoculados experimentalmente com herpesvirus bovino 5

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

Ocorrência e diversidade molecular de rotavírus em rebanhos bovinos nas regiões Sudeste e Centro-Oeste do Brasil

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

Ocorrência de patógenos de origem bacteriana e viral e marcadores de virulência de Escherichia coli e Rhodococcus equi isolados das fezes de aves silvestres de cativiero da fauna brasileira

Pós-graduação em Medicina Veterinária - FMVZ

Molecular detection and phylogenetic analysis of bovine astrovirus in Brazil

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

Immunohistochemical approach to the pathogenesis of clinical cases of bovine Herpesvirus type 5 infections

Meningoencephalitis by Herpesvirus type 5 (BoHV-5) in cattle has some features that are similar to those of herpetic encephalitis in humans and other animal species. Human Herpesvirus 3 (commonly known as Varicella-zoster virus 1), herpes simplex viruses (HSV), and equid Herpesvirus 1 (EHV-1) induce an intense inflammatory, vascular and cellular response. In spite of the many reports describing the histological lesions associated with natural and experimental infections, the immunopathological mechanisms for the development of neurological disorder have not been established. A total of twenty calf brains were selected from the Veterinary School, University of São Paulo State, Araçatuba, Brazil, after confirmation of BoHV-5 infection by virus isolation as well as by a molecular approach. The first part of the study characterized the microscopic lesions associated with the brain areas in the central nervous system (CNS) that tested positive in a viral US9 gene hybridization assay. The frontal cortex (Fc), parietal cortex (Pc), thalamus (T) and mesencephalon (M) were studied. Secondly, distinct pathogenesis mechanisms that take place in acute cases were investigated by an immunohistochemistry assay. This study found the frontal cortex to be the main region where intense oxidative stress phenomena (AOP-1) and synaptic protein expression (SNAP-25) were closely related to inflammatory cuffs, satellitosis and gliosis, which represent the most frequently observed neurological lesions. Moreover, MMP-9 expression was shown to be localized in the leptomeninges, in the parenchyma and around mononuclear infiltrates (p < 0.0001). These data open a new perspective in understanding the role of the AOP-1, MMP-9 and SNAP-25 proteins in mediating BoHV-5 pathogenesis and the strategies of host-virus interaction in order to invade the CNS.

Estimation of the diagnostic accuracy of the glyco-C and US9 gene-based polymerase chain reaction technique for the detection of bovine Herpesvirus type 5 DNA in decomposed brain suspension from a slaughter house using Bayesian analysis, Brazil

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

Comparative analysis of the replication of bovine herpesvirus 1 (BHV1) and BHV5 in bovine derived-neuron-like cells

Members of the subfamily Alphaherpesvirinae use the epithelium of the upper respiratory and/or genital tract as preferential sites for primary replication. However, bovine herpesvirus 5 (BoHV5) is neurotropic and neuroinvasive and responsible for meningoencephalitis in cattle and in animal models. A related virus, BoHV1 has also been occasionally implicated in natural cases of neurological infection and disease in cattle. The aim of the present study was to assess the in vitro effects of BoHV1 and BoHV5 replication in neuron-like cells. Overall, cytopathic effects, consisting of floating rounded cells, giant cells and monolayer lysis, induced by both viruses at 48 h postinfection (p.i.) resulted in a loss of cell viability and high virus titres (r = 0.978). The BoHV1 Cooper strain produced the lowest titres in neuron-like cells, although viral DNA was detected in infected cells during all experiments. Virus replication in infected cells was demonstrated by immunocytochemistry, flow cytometry and qPCR assays. BoHV antigens were better visualized at 48 h p.i. and flow cytometry analysis showed that SV56/90 and Los Angeles antigens were present at higher levels. In spite of the fact that BoHV titres dropped at 48 h p.i, viral DNA remained detectable until 120 h p.i. Sensitive TUNEL (terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling) and annexin V assays were used to identify apoptosis. BoHV5 induced death in approximately 50 % of cells within 24 h p.i., similar to what has been observed for BoHV1 Los Angeles. Infection with the BoHV1 Cooper strain resulted in 26.37 % of cells being in the early stages of apoptosis; 63.69 % of infected cells were considered viable. Modulation of mitochondrial function, as measured by mitochondrial membrane depolarization, was synchronous with the virus replication cycle, cell viability and virus titres at 48 h p.i. Our results indicate that apoptosis plays an important role in preventing neuronal death and provides a bovine-derived in vitro system to study herpesvirus-neuron interactions.