A bovine herpesvirus 1 (BoHV-1) defective in glycoprotein E (gE) was constructed from a Brazilian genital BoHV-1 isolate, by replacing the full gE coding region with the green fluorescent protein (GFP) gene for selection. Upon co-transfection of MDBK cells with genomic viral DNA plus the GFP-bearing gE-deletion plasmid, three fluorescent recombinant clones were obtained out of approximately 5000 viral plaques. Deletion of the gE gene and the presence of the GFP marker in the genome of recombinant viruses were confirmed by PCR. Despite forming smaller plaques, the BoHV-1△gE recombinants replicated in MDBK cells with similar kinetics and to similar titers to that of the parental virus (SV56/90), demonstrating that the gE deletion had no deleterious effects on replication efficacy in vitro. Thirteen calves inoculated intramuscularly with BoHV-1△gE developed virus neutralizing antibodies at day 42 post-infection (titers from 2 to 16), demonstrating the ability of the recombinant to replicate and to induce a serological response in vivo. Furthermore, the serological response induced by recombinant BoHV-1△gE could be differentiated from that induced by wild-type BoHV-1 by the use of an anti-gE antibody ELISA kit. Taken together, these results indicated the potential application of recombinant BoHV-1 △gE in vaccine formulations to prevent the losses caused by BoHV-1 infections while allowing for differentiation of vaccinated from naturally infected animals.

Bovine herpesviruses 1 (BoHV-1) and 5 (BoHV-5) share high genetic and antigenic similarities, but exhibit marked differences in tissue tropism and neurovirulence. The amino-terminal region of glycoprotein C (gC), which is markedly different in each of the viruses, is involved in virus binding to cellular receptors and in interactions with the immune system. This study investigated the genetic and antigenic differences of the 5′ region of the gC (5′ gC) gene (amino-terminal) of South American BoHV-1 (n=19) and BoHV-5 (n=25) isolates. Sequence alignments of 374 nucleotides (104 amino acids) revealed mean similarity levels of 97.3 and 94.2% among BoHV-1 gC (gC1), respectively, 96.8 and 95.6% among BoHV-5 gC (gC5), and 62 and 53.3% between gC1 and gC5. Differences included the absence of 40 amino acid residues (27 encompassing predicted linear epitopes) scattered throughout 5′ gC1 compared to 5′ gC5. Virus neutralizing assays testing BoHV-1 and BoHV-5 antisera against each isolate revealed a high degree of cross-neutralization between the viruses, yet some isolates were neutralized at very low titers by heterologous sera, and a few BoHV-5 isolates reacted weakly with either sera. The virus neutralization differences observed within the same viral species, and more pronounced between BoHV-1 and BoHV-5, likely reflect sequence differences in neutralizing epitopes. These results demonstrate that the 5′ gC region is well conserved within each viral species but is divergent between BoHV-1 and BoHV-5, likely contributing to their biological and antigenic differences.

The immunostimulatory properties of inactivated Parapoxvirus ovis (iPPVO) have long been investigated in different animal species and experimental settings. In this study, we investigated the effects of iPPVO on cytokine expression in mice after intraperitoneal inoculation. Spleen and sera collected from iPPVO-treated mice at intervals after inoculation were submitted to cytokine mRNA determination by real-time PCR (qPCR), serum protein concentration by ELISA, and interferon (IFN)-α/β activity by bioassay. The spleen of iPPVO-treated animals showed a significant increase in mRNA expression of all cytokines assayed, with different kinetics and magnitude. Proinflammatory cytokines interleukin (IL)-1β, tumor necrosis factor-alpha (TNF-α), and IL-8 mRNA peaked at 24 hours postinoculation (hpi; 5.4-fold increase) and 48 hpi (3- and 10-fold increases), respectively. A 15-fold increase in IFN-γ and 6-fold IL-12 mRNA increase were detected at 48 and 24 hpi, respectively. Increased expression of autoregulatory cytokines (Th2), mainly IL-10 and IL-4, could be detected at later times (72 and 96 hpi) with peaks of 4.7- and 4.9-fold increases, respectively. IFN-I antiviral activity against encephalomyocarditis virus was demonstrated in sera of treated animals between 6 and 12 hpi, with a >90% reduction in the number of plaques. Measurement of serum proteins by ELISA revealed increased levels of IL-1, TNF-α, IL-12, IFN-γ, and IL-10, with kinetics similar to those observed by qPCR, especially for IL-12 and IFN-γ. These data demonstrate that iPPVO induced a transient and complex cytokine response, initially represented by Th1-related cytokines followed by autoregulatory and Th2 cytokines.

Este artigo descreve uma investigação da virulência/atenuação de recombinantes do herpesvírus bovino tipo 5 (BoHV-5) com deleções nos genes da glicoproteína E (BoHV-5gEΔ), timidina quinase (BoHV-5TKΔ), e ambos gE e TK (BoHV-5gEΔTKΔ). Bezerros soronegativos (80-90 dias de idade) inoculados com o vírus parental SV-507/99 (n=5) excretaram o vírus em secreções nasais por até 15 dias (média de 10,8 dias). Nos animais inoculados com os recombinantes, a duração da excreção viral foi de 11 dias (BoHV-5gEΔ), 9,6 dias (BoHV-5TKΔ) e 6,2 dias (BoHV-5gEΔTKΔ). Os maiores títulos foram observados entre os dias 1 e 6 pós-inoculação (pi), sendo de 10(6,8)TCID50/mL para o SV-507/99, 10(5,1)TCID50/mL (BoHV-5gEΔ), 10(5,9)TCID50/mL (BoHV-5TKΔ) e 10(4,7)TCIΔ50/mL (BoHV-5gEΔTKΔ). Os bezerros inoculados com o vírus parental apresentaram anorexia e apatia; três deles mostraram apatia profunda e perda da condição corporal. Dois bezerros foram eutanasiados in extremis nos dias 10 e 11 pi, respectivamente e o vírus foi isolado de várias regiões do encéfalo. Já os bezerros inoculados com os recombinantes permaneceram saudáveis; alguns apresentaram uma secreção nasal serosa transitória. Administração de dexametasona (Dx) no dia 42 pi resultou em excreção viral por todos os bezerros inoculados com o vírus parental (duração média de 3,7 dias), por 2 de 5 bezerros dos grupos BoHV-5TKΔ (dois dias) e BoHV-5gEΔ (um dia). Os bezerros inoculados com o duplo mutante BoHV-5gEΔTKΔ não excretaram o vírus após o tratamento com Dx. Pesquisa de DNA viral por PCR no dia 30 pós-Dx revelou uma ampla distribuição do DNA do vírus parental no encéfalo; poucas seções (3/30) foram positivas no encéfalo dos animais do grupo BoHV-5gEΔ, e não detectou-se DNA latente no encéfalo dos animais dos grupos BoHV-5TKΔ e BoHV-5gEΔTKΔ. Esses resultados demonstram que os mutantes simples (gE and tk-deletados) são atenuados para bezerros e estabelecem e/ou reativam infecção latente ineficientemente. Já o duplo mutante BoHV-5gEΔTKΔ é atenuado e parece não estabelecer e/ou não reativar eficientemente a infecção latente. Portanto, os vírus recombinantes, e em especial o duplo mutante BoHV-5gEΔTKΔ apresentam um fenótipo compatível com a sua inclusão em vacinas vivas modificadas.

This article describes an investigation on the virulence/attenuation of bovine herpesvirus type 5 (BoHV-5) recombinants deleted in the genes encoding glycoprotein E (BoHV-5gEΔ), thymidine kinase (BoHV-5TKΔ), and both gE and TK (BoHV-5gEΔTKΔ). Seronegative calves (80 to 90 days-old) inoculated with the parental strain (SV-507/99, n=5) shed virus in nasal secretions for up to 15 days (average 10.8 days). Duration of virus shedding was 11 days for BoHV-5gΔ, 9.6 days for BoHV-5TKΔ and 6.2 days for BoHV-5gEΔTKΔ groups. The highest titers were observed between days 1 and 6 post-infection (pi) for SV-507/99 (10(6.8)TCID50/mL), 10(5.1)TCID50/mL (BoHV-5gEΔ), 10(5.9)TCID50/mL (BoHV-5TKΔ) and 10(4.7)TCID50/mL (BoHV-5gEΔTKΔ). Calves inoculated with the parental virus presented anorexia, profound apathy and loss of body condition. Two calves were euthanized in extremis on days 10 and 11 pi; infectious virus was recovered from several areas of the brain. In contrast, calves inoculated with the recombinants remained healthy and a few presented a mild and transient nasal secretion. Dexamethasone (Dx) administration at day 42 pi resulted in virus shedding by all controls calves (mean duration 3.7 days), by 2/5 of BoHV-5TKΔ calves (two days) and 2/5 of BoHV-5gEΔ (one day). No virus shedding was detected in BoHV-5gEΔTKΔ calves upon Dx treatment. PCR examination of brain sections of calves euthanized at day 30 post Dx treatment revealed the presence of latent viral DNA widely distributed in the brain of SV-507/99 calves. Latent viral DNA was detected in a few sections (3/30) of the brains of BoHV-5gEΔ calves and was not detected in the brains of calves inoculated with BoHV-5TKΔ and BoHV-5gEΔTKΔ. These results show that the single BoHV-5 mutants (gE and tk-deleted) are attenuated for calves and establish and/or reactivate latent infection inefficiently. The double mutant BoHV-5gEΔTKΔ is fully attenuated and appears not to establish or not reactivate efficiently from latent infection. Thus, these recombinants, especially the double mutant BoHV-5gEΔTKΔ, display an adequate phenotype for use in modified-live vaccine formulations.

Bovine herpesvirus type 5 (BoHV-5) is an important pathogen of cattle in South America. We describe here the construction and characterization of deletion mutants defective in the glycoprotein E (gE) or thymidine kinase (TK) gene or both (gE/TK) from a highly neurovirulent and well-characterized Brazilian BoHV-5 strain (SV507/99). A gE-deleted recombinant virus (BoHV-5 gE∆) was first generated in which the entire gE open reading frame was replaced with a chimeric green fluorescent protein gene. A TK-deleted recombinant virus (BoHV-5 TK∆) was then generated in which most of the TK open reading frame sequences were deleted and replaced with a chimeric β-galactosidase gene. Subsequently, using the BoHV-5 gE∆ virus as backbone, a double gene-deleted (TK plus gE) BoHV-5 recombinant (BoHV-5 gE/TK∆) was generated. The deletion of the gE and TK genes was confirmed by immunoblotting and PCR, respectively. In Madin Darby bovine kidney (MDBK) cells, the mutants lacking gE (BoHV-5 gE∆) and TK + gE (BoHV-5 gE/TK∆) produced small plaques while the TK-deleted BoHV-5 produced wild-type-sized plaques. The growth kinetics and virus yields in MDBK cells for all three recombinants (BoHV-5 gE∆, BoHV-5 TK∆ and BoHV-5 gE/TK∆) were similar to those of the parental virus. It is our belief that the dual gene-deleted recombinant (BoHV-5 gE/TK∆) produced on the background of a highly neurovirulent Brazilian BoHV-5 strain may have potential application in a vaccine against BoHV-5.

Bovine herpesvirus 5 (BoHV-5), the agent of herpetic meningoencephalitis in cattle, is an important pathogen of cattle in South America and several efforts have been made to produce safer and more effective vaccines. In the present study, we investigated in rabbits the virulence of three recombinant viruses constructed from a neurovirulent Brazilian BoHV-5 strain (SV507/99). The recombinants are defective in glycoprotein E (BoHV-5gEΔ), thymidine kinase (BoHV-5TKΔ) and both proteins (BoHV-5gEΔTKΔ). Rabbits inoculated with the parental virus (N = 8) developed neurological disease and died or were euthanized in extremis between days 7 and 13 post-infection (pi). Infectivity was detected in several areas of their brains. Three of 8 rabbits inoculated with the recombinant BoHV-5gEΔ developed neurological signs between days 10 and 15 pi and were also euthanized. A more restricted virus distribution was detected in the brain of these animals. Rabbits inoculated with the recombinants BoHV-5TKΔ (N = 8) or BoHV-5gEΔTKΔ (N = 8) remained healthy throughout the experiment in spite of variable levels of virus replication in the nose. Dexamethasone (Dx) administration to rabbits inoculated with the three recombinants at day 42 pi did not result in viral reactivation, as demonstrated by absence of virus shedding and/or increase in virus neutralizing titers. Nevertheless, viral DNA was detected in the trigeminal ganglia or olfactory bulbs of all animals at day 28 post-Dx, demonstrating they were latently infected. These results show that recombinants BoHV-5TKΔ and BoHV-5gEΔTKΔ are attenuated for rabbits and constitute potential vaccine candidates upon the confirmation of this phenotype in cattle.

A mamilite causada pelo herpesvírus bovino tipo 2 (BoHV-2) possui importante repercussão sanitária em gado leiteiro de alguns países, mas a sua patogenia permanece pouco conhecida. Este trabalho descreve a reprodução e caracterização da mamilite aguda em ovelhas lactantes inoculadas com o BoHV-2 na pele do úbere e dos tetos. Cinco de oito ovelhas inoculadas desenvolveram extensas placas com necrose focal, pequenas vesículas e formação de crostas nos locais de inoculação. As lesões foram inicialmente observadas no 4º dia pós-inoculação (pi), progrediram em extensão e gravidade até os dias 7-8pi e posteriormente regrediram. O vírus foi isolado das lesões entre os dias 7 e 8 pi. Antígenos virais e partículas típicas de herpesvírus foram demonstrados por microscopia eletrônica em biópsias de lesões coletadas nos dias 5, 6 e 10pi. Os achados histológicos foram caracterizados por necrose epitelial, erosões e úlceras, com formação de células sinciciais e infiltrado inflamatório linfoplasmocitário na derme. Associadas a essas lesões observaram-se inclusões eosinofílicas intranucleares em células epiteliais, sinciciais e inflamatórias. Em um segundo experimento, 7 de 10 cordeiros inoculados na mucosa nasal desenvolveram hiperemia e descarga nasal, bem como vesículas e erosões no focinho. O vírus foi isolado das secreções nasais por até três dias e todos os cordeiros soroconverteram ao BoHV-2. Tentativas de reativar a infecção latente pela administração de dexametasona no 40º dia pi foram infrutíferas, pois não foram observados re-excreção viral, recrudescência clínica ou soroconversão. Esses resultados demonstram que ovelhas são susceptíveis à infecção experimental pelo BoHV-2 e desenvolvem lesões semelhantes às observadas em casos naturais de mamilite herpética. Além disso, esses resultados sugerem o uso dessa espécie animal como modelo para o estudo de vários aspectos da biologia da infecção pelo BoHV-2.

Mammillitis caused by bovine herpesvirus type 2 (BoHV-2) is an important disease in dairy herds yet its pathogenesis remains largely unknown. This report describes the reproduction and characterization of acute mammillitis in lactating ewes inoculated with BoHV-2 in the skin of the udder and teats. Five out of eight inoculated ewes developed large plaques, with focal necrosis, small vesicles and crust formation in the inoculated areas. The lesions were first observed on day 4 post-inoculation (pi), progressed in size and severity up to days 7-8pi and subsided progressively thereafter. Infectious virus was isolated from the lesions at days 7 and 8pi. Viral antigens and herpesvirus-like particles were demonstrated by electron microscopy in lesions examined at days 5, 6 and 10pi. Histological findings included epithelial necrosis, erosions and ulcers, and formation of syncytial cells. Intranuclear inclusions bodies in epithelial, syncytial and inflammatory cells and lymphoplasmacytic inflammatory infiltrate in the dermis were also observed. In a second experiment, seven out of ten lambs inoculated into the nostrils and muzzle developed nasal hyperemia and discharge, vesicles, and erosions in the nose. Infectious virus was isolated from lesions during up to three days and all lambs seroconverted to BoHV-2. Attempts to reactivate the latent infection by dexamethasone administration on day 40pi failed, since virus shedding, clinical recrudescence or seroconversion were not observed. The reproduction of acute infection and mammillitis resembling that occurring in cattle paves the way for the use of sheep to study several aspects of the biology of BoHV-2 infection.

Calves born persistently infected with non-cytopathic bovine viral diarrhea virus (ncpBVDV) frequently develop a fatal gastroenteric illness called mucosal disease. Both the original virus (ncpBVDV) and an antigenically identical but cytopathic virus (cpBVDV) can be isolated from animals affected by mucosal disease. Cytopathic BVDVs originate from their ncp counterparts by diverse genetic mechanisms, all leading to the expression of the non-structural polypeptide NS3 as a discrete protein. In contrast, ncpBVDVs express only the large precursor polypeptide, NS2-3, which contains the NS3 sequence within its carboxy-terminal half. We report here the investigation of the mechanism leading to NS3 expression in 41 cpBVDV isolates. An RT-PCR strategy was employed to detect RNA insertions within the NS2-3 gene and/or duplication of the NS3 gene, two common mechanisms of NS3 expression. RT-PCR amplification revealed insertions in the NS2-3 gene of three cp isolates, with the inserts being similar in size to that present in the cpBVDV NADL strain. Sequencing of one such insert revealed a 296-nucleotide sequence with a central core of 270 nucleotides coding for an amino acid sequence highly homologous (98%) to the NADL insert, a sequence corresponding to part of the cellular J-Domain gene. One cpBVDV isolate contained a duplication of the NS3 gene downstream from the original locus. In contrast, no detectable NS2-3 insertions or NS3 gene duplications were observed in the genome of 37 cp isolates. These results demonstrate that processing of NS2-3 without bulk mRNA insertions or NS3 gene duplications seems to be a frequent mechanism leading to NS3 expression and BVDV cytopathology.

Bovine herpesvirus type 5 (BHV-5) is a major agent of meningoencephalitis in cattle and establishes latent infections mainly in sensory nerve ganglia. The distribution of latent BHV-5 DNA in the brain of rabbits prior to and after virus reactivation was studied using a nested PCR. Fifteen rabbits inoculated intranasally with BHV-5 were euthanized 60 days post-inoculation (group A, N = 8) or submitted to dexamethasone treatment (2.6 mg kg-1 day-1, im, for 5 days) and euthanized 60 days later (group B, N = 7) for tissue examination. Two groups of BHV-1-infected rabbits (C, N = 3 and D, N = 3) submitted to each treatment were used as controls. Viral DNA of group A rabbits was consistently detected in trigeminal ganglia (8/8), frequently in cerebellum (5/8), anterior cerebral cortex and pons-medulla (3/8) and occasionally in dorsolateral (2/8), ventrolateral and posterior cerebral cortices, midbrain and thalamus (1/8). Viral DNA of group B rabbits showed a broader distribution, being detected at higher frequency in ventrolateral (6/7) and posterior cerebral cortices (5/7), pons-medulla (6/7), thalamus (4/7), and midbrain (3/7). In contrast, rabbits inoculated with BHV-1 harbored viral DNA almost completely restricted to trigeminal ganglia and the distribution did not change post-reactivation. These results demonstrate that latency by BHV-5 is established in several areas of the rabbit's brain and that virus reactivation leads to a broader distribution of latent viral DNA. Spread of virus from trigeminal ganglia and other areas of the brain likely contributes to this dissemination and may contribute to the recrudescence of neurological disease frequently observed upon BHV-5 reactivation.

O presente estudo teve como objetivo a caracterização das propriedades de crescimento in vitro de uma amostra brasileira de herpesvírus bovino tipo 1.2a que apresenta uma deleção no gene que codifica a glicoproteína E (BHV-1.2a gE-). Os tamanhos de placa, cinética de penetração e cinética de multiplicação do vírus BHV-1.2a gE- foram estudados e comparados com o vírus parental, bem como com um vírus BHV-1.1 gE- recombinante, o qual é derivado de uma amostra européia de BHV-1.1. Em termos de cinética de penetração, não foram observadas diferenças significativas quando comparados os vírus gE- com os parentais. A determinação da cinética de multiplicação não demonstrou diferenças significativas entre os quatro vírus estudados. Foi entretanto observado que 11 horas pós infecção os dois vírus gE- foram excretados das células em títulos significativamente maiores do que os vírus parentais. Não foram observadas diferenças significativas quando comparados os diâmetros de placas formadas pelos dois vírus parentais. Da mesma forma, os diâmetros de placas dos vírus gE- foram semelhantes nos três tipos celulares estudados. Entretanto, a comparação dos diâmetros de placas entre os vírus gE- e os parentais mostrou uma redução significativa das placas dos vírus gE- em todos os tipos celulares. Esta característica indica que a falta da gE teve o mesmo efeito em ambos os subtipos de BHV-1, representado por uma disseminação viral célula-célula reduzida.

This study aimed the in vitro growth characterization of a previously constructed Brazilian bovine herpesvirus 1.2a with a deletion in the glycoprotein E gene (BHV-1.2a gE-). The plaque sizes, penetration and growth kinetics of the Brazilian BHV-1.2a gE- were studied and compared with the parental virus, as well as with a BHV-1.1 gE- recombinant derived from an European BHV-1.1 strain. No statistical differences were observed between the gE- recombinants and the respective parental viruses penetration assays were performed. When single step growth curves were studied, no statistical differences were observed between gE- and parental viruses. However, it was observed that both gE- viruses were excreted from cells in significantly higher titres at 11 hours post infection in comparison with parental viruses. No statistical differences were observed when plaque sizes of parental viruses or gE- viruses we analyzed separately in each cell type. However, both gE- recombinants displayed a significantly reduced plaque areas on three different cell cultures, in comparison with parental viruses, indicating that the lack of gE had the same effect on both BHV-1 subtypes, manifested by a restricted cell-to-cell spread in infected cells.

Antigens of a bovine herpesvirus type 5 (BHV-5), isolated from a cow with a neurological infection in Rio Grande do Sul State, Brazil, were used to immunize BALB/c mice to produce monoclonal antibodies (mAbs). Eleven hybridomas secreting mAbs directed at BHV-5 antigens were obtained after two fusions and screening of 356 hypoxanthine-aminopterin-thymidine-resistant clones. The mAbs reacted at dilutions up to 1:500 (hybridoma culture supernatant) and up to >1:10,000 (ascitic fluid) in an indirect fluorescent antibody assay (IFA) and in immunoperoxidase staining of BHV-5-infected cells. Four mAbs (1D12, 2E2, 2G10 and 4E4) showed virus-neutralizing activity against the parental BHV-5 isolate. Five mAbs (1F3, 2A6, 2F9, 2G10 and HB24L) reacted in Western immunoblotting with a protein of approximately 90 kDa. Three other mAbs (2E2, 3D6 and 4E4) reacted in IFA with antigens of a BHV-1 mutant glycoprotein C- negative strain, demonstrating that they are directed at a viral antigen other than glycoprotein C. The eleven mAbs tested reacted with 20 BHV-5 field isolates and nine mAbs reacted with 10 BHV-1 isolates. Two mAbs (1F3 and 2F9) failed to react with BHV-1 field isolates, although they displayed a weak and nonreproducible reaction with the BHV-1 reference strain Los Angeles. These mAbs may be very useful in distinguishing between BHV-1 and BHV-5 infections since most of the traditional reagents and techniques are unable to do so. One mAb (2F9) was shown to bind to viral antigens by immunohistochemistry of histological sections of the brain of a BHV-5-infected calf. These results demonstrate that the mAbs produced here are suitable for use in a variety of immunological techniques and therefore may be useful for diagnostic and research purposes.

Duas amostras do vírus da Diarréia Viral Bovina (BVDV) submetidas a múltiplas passagens em cultivo celular e exposição à radiação ultravioleta (UV) a cada passagem foram avaliadas como candidatos a vírus vacinais. As amostras foram testadas quanto à sua atenuação para bezerros e fetos ovinos, reatividade antigênica contra isolados de campo, e capacidade de induzir proteção fetal em ovelhas prenhes. Inoculação intramuscular (IM) dos vírus modificados em quatro bezerros produziu apenas uma elevação discreta e passageira da temperatura corporal, seguida de produção de altos títulos de anticorpos neutralizantes. O vírus não foi detectado em secreções nasais ou sangue nos dias seguintes à inoculação. Porém, a inoculação IM desses vírus em quatro ovelhas prenhes foi seguida de transmissão transplacentária e infecção em todos os fetos. Para os testes de proteção fetal, ovelhas prenhes previamente imunizadas com duas doses vacinais, foram inoculadas por via intranasal com amostras de BVDV-1 (SV-126.8, n=6) ou BVDV-2 (SV-260, n=5). No dia do desafio (134 dias após a segunda dose), todos os animais apresentavam altos títulos de anticorpos neutralizantes (256 a >4096) contra os vírus vacinais; além de títulos variados (8 a >4096) contra várias isolados brasileiros de BVDV-1 e BVDV-2. Quinze dias após o desafio, as ovelhas foram sacrificadas e os tecidos fetais foram examinados para a presença de vírus. Todos os fetos das ovelhas controle não-vacinadas apresentaram-se (n=4) positivos para os vírus utilizados no desafio. Em contraste, nenhum feto das ovelhas imunizadas (n=11) foi positivo para vírus, indicando que a resposta imunológica induzida pela vacinação com os vírus modificados foi capaz de prevenir a infecção fetal. Estes resultados indicam que é possível obter-se forte resposta imunológica e proteção fetal contra o BVDV com o uso de vacinas vivas modificadas.

Two isolates of bovine viral diarrhea virus (BVDV) submitted to multiple passages in tissue culture associated with ultraviolet irradiation were evaluated as vaccine virus candidates. The attenuation of the modified viruses was assessed in calves and in pregnant ewes. Intramuscular inoculation of the viruses in four seronegative calves produced only a mild and transient rise in body temperature, followed by the production of high titers of neutralizing antibodies. The viruses were not detected in nasal secretions or in the blood following inoculation. However, intramuscular inoculation of these viruses in four pregnant ewes resulted in transplacental transmission and infection of all fetuses. To assess fetal protection conferred by immunization, pregnant ewes immunized twice with the modified viruses were subsequently challenged by intranasal inoculation of BVDV-1 (SV-126.8, n=6) or BVDV-2 (SV-260, n=5). At the day of challenge (134 days after the second immunization), all ewes had high titers of neutralizing antibodies (256 to >4096) to the vaccine viruses and variable titers (8 to >4096) to Brazilian BVDV-1 and BVDV-2 field isolates. Fifteen days after challenge, the ewes were euthanized and fetal tissues were examined for infectivity. All fetuses from non-vaccinated, challenged ewes (n=4) were infected. In contrast, none of the fetuses from the immunized dams (n=11) were positive for virus, indicating that the immunological response induced by immunization with the vaccine candidate viruses was capable of preventing fetal infection. These results indicate that it is possible to achieve fetal protection to BVDV by induction of a strong immunological response using modified live vaccines.

Com o objetivo de avaliar a capacidade dos herpesvírus bovinos tipos 1 e 5 (BHV-1 e BHV-5) de invadir e replicar no sistema nervoso central (SNC) (neuroinvasividade), bem como sua capacidade de induzir doença neurológica (neurovirulência), coelhos com 30 a 35 dias de idade foram inoculados com uma amostra do Herpesvírus da Encefalite Bovina (BHV-5; amostra EVI 88/95) ou com amostras de BHV-1 (Los Angeles ou Cooper), pelas vias intratecal (IT) e intranasal (IN). A inoculação da amostra de BHV-5, tanto pela via IT como IN, induziu sinais clínicos neurológicos em 100% (12/12) dos coelhos inoculados. Os exames histopatológicos revelaram um quadro de meningoencefalite não-purulenta multifocal, caracterizada por gliose multifocal e infiltrados perivasculares. O vírus foi isolado de várias áreas do SNC desses animais. As amostras de BHV-1, quando inoculadas pela via IT, não foram neurovirulentas. A amostra Los Angeles de BHV-1, quando administrada pela via IN, induziu sinais respiratórios severos, além de sinais neurológicos em 57% (4/7) dos animais inoculados. Entretanto, o exame histopatológico destes quatro animais revelou vasculite e trombose no pulmão e cérebro, este último apresentando focos de necrose neuronal, porém sem lesões indicativas de encefalite. Isso sugere que os sinais neurológicos foram, provavelmente, conseqüentes a prejuízos no fluxo sangüíneo encefálico, e não a danos neuronais provocados pela inoculação desse vírus. A amostra Cooper de BHV-1, quando inoculada pela via IN, induziu apenas sinais leves de infecção respiratória. Estes resultados indicam que apenas a amostra de BHV-5 foi capaz de invadir e replicar no encéfalo dos coelhos quando inoculada tanto por via IN como IT, apresentando neuroinvasividade e neurovirulência. É possível que estas observações tenham relação com o fato de amostras de BHV-5 freqüentemente causarem encefalites, em contraposição a infecções pelo BHV-1, onde encefalites são raramente observadas.

In order to determine the capacity of bovine herpesvirus type 1 and 5 (BHV-1 and BHV-5) to invade, multiply and spread along the central nervous system (CNS) (neuroinvasiveness), as well as their potential to induce neurological illness (neurovirulence), 30 to 35 days old rabbits were inoculated with the BHV-5 strain EVI 88 / 95 and Los Angeles and Cooper BHV-1 strains, by the intrathecal (IT) and intranasal (IN) routes. The BHV-5 strain induced severe neurological clinical signs in 100% (12/12) of the rabbits inoculated by both routes. Histopathological examination revealed multifocal non-suppurative meningoencephalitis, characterized by multifocal gliosis and perivascular cuffing. Virus was recovered from many parts of the brain. Both BHV-1 strains, when inoculated via IT route, were not neurovirulent. The strain Los Angeles, after IN inoculation, induced signs of severe respiratory disease (7/7), as well as signs of neurological impairment, indistinguishable from those induced by BHV-5, in 57% (4/7) of the infected rabbits. However, the rabbits with nervous signs revealed at histopathology vasculitis and thrombosis in lungs and brain, the latter with foci of neuronal necrosis, but no lesions indicative of encephalitis, suggesting that neural damage was probably consequent to tissue anoxia. The BHV-1 strain Cooper, after IN inoculation, induced only mild signs of respiratory disease. These findings indicate that the BHV-5 strain was both neuroinvasive and neurovirulent, since it was capable of invading, spreading and multiplying in the rabbits' brains by both routes of inoculation, yet causing neurological disease, apparently consequent to virus induced neural damage. The BHV-1 Los Angeles strain was not neuroinvasive, whereas its neurovirulence was probably consequent to tissue anoxia, which histologically seemed not to be related to direct viral pathogenic effect. The BHV-1 strain Cooper was neither neurovirulent nor neuroinvasive for rabbits. It is possible that these observations bear relationship with the frequent association of BHV-5 with encephalitis in cattle, as opposed to BHV-1 encephalitis, which is a rare event in nature.

O perfil antigênico de 45 herpesvírus (44 de bovinos, sendo seis amostras de referência de BHV-1 e 15 prováveis BHV-1; três amostras de referência de BHV-5 e 20 prováveis BHV-5) e uma amostra de herpesvírus bubalino (BuHV) foi examinado com um painel de anticorpos monoclonais (Acms) produzidos contra antígenos de herpesvírus bovinos. Para os exames, foi utilizada a prova de imunoperoxidase (IPX) sobre cultivos de células infectadas, tendo os Acms como anticorpos primários. A determinação dos padrões de reatividade das amostras de vírus frente aos Acms permitiu a diferenciação entre os tipos 1 e 5. Todas as amostras isoladas de casos de encefalite apresentaram perfil de BHV-5. Quatro amostras de BHV-5 isoladas de áreas geograficamente distintas apresentaram perfís de reatividade diferenciados em relação às demais amostras do tipo 5. Duas amostras de vírus com perfil antigênico de BHV-5 foram isoladas de sêmen de animais infectados. Estes resultados comprovam a utilidade da caracterização antigênica com este painel de Acms na tipagem de amostras de BHV-1 e BHV-5.

The antigenic profile of 45 herpesviruses (44 viruses from cattle, including six reference BHV-1 strains and 15 putative BHV-1; three reference BHV-5 strains and 20 putative BHV-5) and one buffalo isolate (BuHV) were examined with a panel of monoclonal antibodies (Mabs) prepared against bovine herpesvirus antigens. Tests were performed by immunoperoxidase (IPX) on infected cell cultures, with the Mabs as primary antibodies. Immunostaining allowed the differentiation between types 1 and 5 viruses. All isolates from cases of encephalitis displayed BHV-5 profiles. Four BHV-5 isolates obtained from geographically distinct areas displayed different and highly variable IPX patterns of reactivity. Two viruses with BHV-5 antigenic profile were isolated from semen of asymptomatic bulls. The results showed that the antigenic characterization with the Mab panel employed here is useful for typing BHV-1 and BHV-5 isolates.

Three Brazilian isolates of bovine viral diarrhea virus (BVDV), antigenically distinct from the standard North American isolates, were selected to immunize BALB/c mice in order to obtain hybridoma cells secreting anti-BVDV monoclonal antibodies (mAbs). Two hybridoma clones secreting mAbs, reacting specifically with BVDV-infected cells (mAbs 3.1C4 and 6.F11), were selected after five fusions and screening of 1001 hypoxanthine-aminopterin-thymidine-resistant clones. These mAbs reacted in an indirect fluorescent antibody (IFA) assay with all 39 South and North American BVDV field isolates and reference strains available in our laboratory, yet failed to recognize other pestiviruses, namely the hog cholera virus. The mAbs reacted at dilutions up to 1:25,600 (ascitic fluid) and 1:100 (hybridoma culture supernatant) in IFA and immunoperoxidase (IPX) staining of BVDV-infected cells but only mAb 3.1C4 neutralized virus infectivity. Furthermore, both mAbs failed to recognize BVDV proteins by IPX in formalin-fixed paraffin-embedded tissues and following SDS-PAGE and immunoblot analysis of virus-infected cells, suggesting they are probably directed to conformational-type epitopes. The protein specificity of these mAbs was then determined by IFA staining of CV-1 cells transiently expressing each of the BVDV proteins: mAb 3.1C4 reacted with the structural protein E2/gp53 and mAb 6.F11 reacted with the structural protein E1/gp25. Both mAbs were shown to be of the IgG2a isotype. To our knowledge, these are the first mAbs produced against South American BVDV isolates and will certainly be useful for research and diagnostic purposes.