Detection of an untyped strain of bovine respiratory syncytial virus in a dairy herd

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.

Systemic and local antibodies induced by an experimental inactivated vaccine against bovine herpesvirus type 1

Uma vacina experimental inativada contra o herpesvírus bovino tipo 1 (BoHV-1) foi produzida com o objetivo de se avaliar a resposta imune humoral local e sistêmica contra o BoHV-1, em 12 novilhas soronegativas, após a vacinação e a revacinação. Os soros foram submetidos à prova de vírus-neutralização para quantificação do título de anticorpos neutralizantes e a um ELISA para detecção de IgG1 e IgG2. Os swabs nasais também foram submetidos ao ELISA para detecção de IgG1 e IgG2 na secreção nasal. Os resultados demonstraram que títulos de anticorpos neutralizantes foram induzidos após a revacinação, em níveis moderados a altos, permanecendo em níveis significativos no soro sanguíneo e na secreção nasal até o dia 114 pós-vacinação. O IgG2 foi o isótipo predominante na maior parte do período pós-vacinação, tanto na secreção nasal, como no compartimento sistêmico. A vacina experimental inativada contra o BoHV-1 estimulou níveis de anticorpos potencialmente protetores dos isótipos IgG1 e IgG2, tanto no compartimento sistêmico, como nas mucosas.

Detection of bovine herpesvirus 5 (BoHV-5) in formalin-fixed, paraffin-embedded bovine brain by nested PCR in Colombian cattle

Fifteen cases of viral meningoencephalitis in Colombian cattle were tested by nested PCR analysis for the detection of bovine herpesvirus 5 (BoHV-5). All fatal cases had shown severe neurological signs and had occurred following natural outbreaks of the disease. The neurological infection was histologically characterized by mild to moderate inflammatory changes in the brain and cerebellum, including meningitis, mononuclear perivascular cuffing, gliosis, haemorrhage, and the presence of Gitter cells (macrophages) accompanying large areas of malacia. No intranuclear inclusion bodies were seen in any of the cases. Results from BoHV-5 molecular extraction analyses showed there were five positive cases thus confirming the presence of the virus in Colombia.

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.

Detection of bovine Herpesvirus type 5 in formalin-fixed, paraffin-embedded bovine brain by PCR: a useful adjunct to conventional tissue-based diagnostic test of bovine encephalitis

The aim of this study was to evaluate the application of PCR technique for the detection of BoHV-5 in routinely formalin-fixed, paraffin-embedded brain tissues in 20 naturally infected calves affected by fatal meningoencephalitis. Brains were divided into two halves, one kept fresh for virus isolation and PCR assay, targeting the glycoprotein C gene from BoHV-5 genome. The other half brain, corresponding to posterior cortex region, was submitted to formalin fixation and embedded into paraffin blocks for microscopic evaluation and total DNA isolation. Most of the slides showed severe multifocal non-supurative encephalitis with neuronal degeneration, neurophagia, and no acidophilic intranuclear inclusions could be found in neurons and glial. The 20 fresh samples were confirmed, by virus isolation and PCR assay, as having the BoHV-5 virus and, respective glicoprotein C sequence, while 15 of 20 formalin-fixed, paraffin-embedded samples were considered positive for the same analysis. The results revealed the first description of PCR efficiency, applied to formalin-fixed, paraffin-embedded brain collected from naturally infected calves, improving the detection of BoHV-5 from archival samples in South America. (c) 2007 Published by Elsevier B.V.

A novel in situ Polymerase chain reaction hybridisation assay for the direct detection of bovine herpesvirus type 5 in formalin-fixed, paraffin-embedded tissues

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

Optimization of an amperometric biosensor for the detection of hepatitis C virus using fractional factorial designs

Fractional factorial design and factorial with center point design were applied to the development of an amperometric biosensor for the detection of the hepatitis C virus. Biomolecules were immobilized by adsorption on graphite electrodes modified with siloxane-poly(propyleneoxide) hybrid matrix prepared using the sol-gel method. Several parameters were optimized, such as the streptavidin concentration at 0.01 mg mL(-1) and 1.0% bovine serum albumin, the incubation time of the electrodes in the complementary DNA solution for 30 minutes and a 1: 1500 dilution of the avidin-peroxidase conjugate, among others. The application of chemometric studies has been efficient, since the best conditions have been established with a restricted number of experiments, indicating the influence of different factors on the system.

Apoptotic and developmental effects of bovine Herpesvirus type-5 infection on in vitro-produced bovine embryos

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

Bull semen varibles after experimental exposure with Bovine Herpesvirus type 5

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

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)

Effect of capacitation of stallion sperm with polyvinylalcohol or bovine serum albumin on penetration of bovine zona-free or partially zona-removed equine oocytes

Experiments were conducted to study effects of macromolecules on stallion sperm capacitation and fertilization as determined by penetration of bovine zona-free and equine partially zona-removed oocytes. Stallion sperm were capacitated in TYH medium (modified Krebs-Ringer bicarbonate) supplemented with either 1 mg/mL of polyvinylalcohol (PVA) or 4 mg/ mL of BSA. Capacitation was induced with 8 bromoadenosine cyclic monophosphate (8BrcAMP; 0.5 mM) alone or in combination with 0.1 μM of ionomycin. Intraspecies gametes were co-incubated in TYH/PVA or TYH/ BSA for 18 to 20 h. For zona-free bovine oocytes, penetration rate (35%) with the combination of 8BrcAMP and ionomycin in PVA-containing medium was higher (P < 0.05) than any treatment in BSA-containing medium (5 to 6%). A similar study was conducted using equine oocytes with partially removed zonae. Sperm capacitated and used for in vitro fertilization (IVF) in PVA-containing medium had higher penetration rates (P < 0.01) than sperm in BSA-containing medium (54 vs. 11%). The effect of equine preovulatory follicular fluid on bovine oocyte penetration was assessed. Bovine oocytes were matured in tissue culture medium-199 with 0, 20, 50, or 100% equine preovulatory follicular fluid, and 1 IU/mL of equine chorionic gonadotropin. Stallion sperm were treated with 8BrcAMP + ionomycin in PVA- or BSA-containing media. The penetration rates of bovine zona-free oocytes by stallion sperm were again higher with PVA (47%) than BSA (18%; P < 0.01). Penetration rates of oocytes matured in 100% follicular fluid were higher (P < 0.05) than for oocytes matured with 0% follicular fluid. The effects of equine follicular fluid and PVA/BSA during sperm capacitation on standard bovine IVF were examined. Culture of bovine oocytes with equine follicular fluid did not affect oocyte maturation or penetration rates after IVF. Bovine sperm capacitated with heparin in PVA-containing medium yielded lower (P < 0.05) fertilization rates than those capacitated in BSA-containing medium when incubated with both zona-intact and zona-free bovine oocytes. In summary, PVA was superior to BSA for ionophore-induced capacitation of equine sperm for penetration of zona-free bovine oocytes or partially zona-removed equine oocytes, but not for standard bovine IVF with bovine sperm. Zona-free bovine oocytes may be useful for assaying in vitro capacitation and fertilization of stallion sperm. © 2003 American Society of Animal Science. All rights reserved.

Application of a serum/protein-free medium for the growth of mammalian cell lines and high level production of classical, variant and very virulent strain of infectious bursal disease virus (IBDV)

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.