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 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.

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.

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.

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.

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.

Seqüenciamento e análise filogenética de 17 amostras do vírus da diarréia viral bovina (BVDV) isoladas no Brasil identificaram quatro amostras (23,5%) do genótipo 1a (BVDV-1a), nove amostras (52,9%) do genótipo 1b (BVDV tipo 1b) e quatro amostras (23,5%) do genótipo 2 (BVDV tipo 2). As amostras brasileiras de BVDV tipo 2 apresentaram-se genotipicamente distintas dos BVDV tipo 2 até então identificados na América do Norte e Europa, sugerindo pertencerem a um novo subgenótipo. A caracterização antigênica dessas amostras por neutralização cruzada revelou reatividade sorológica muito reduzida com cepas vacinais do BVDV. O anti-soro produzido contra três cepas vacinais do BVDV apresentou atividade neutralizante muito reduzida contra várias amostras brasileiras de BVDV tipos 1 e 2. Diferenças de até 128 vezes nos títulos de anticorpos neutralizantes foram observadas entre cepas vacinais e amostras brasileiras do BVDV. Nos testes de soroneutralização (SN) contra o vírus dos tipos 1 e 2, de 1134 amostras testadas, 280 (24,7%) possuiam anticorpos neutralizantes anti-BVDV e dessas, 215 (76,8%) apresentaram atividade neutralizante contra ambos os vírus, 37 (13,2%) reagiram apenas contra o BVDV tipo 2 e 28 amostras (10%) foram positivas apenas contra o BVDV tipo 1. Esses resultados demonstram que testes de SN utilizando vírus de apenas um genótipo podem resultar em um número significativo de falsos-negativos e indica a necessidade da formulação de vacinas com amostras locais de BVDV e/ou contendo vírus dos dois genótipos.

Nucleotide sequencing and phylogenetic analysis of 17 Brazilian isolates of bovine viral diarrhea virus (BVDV) identified four isolates (23.5%) belonging to genotype 1a (BVDV-1a), nine isolates (52.9%) of the genotype 1b (BVDV-1b) and four isolates (23.5%) belonging to genotype 2 (BVDV-2). The Brazilian BVDV type 2 isolates were shown to be genotypically different from the BVDV type 2 identified so far in North America and Europe, suggesting they might belong to a new subgenotype. Antigenic characterization of these isolates by cross-neutralization revealed a very low serologic cross-reactivity with vaccine BVDV strains. The immune serum raised in lambs to three vaccine strains presented a very low and even negligible neutralizing activity against some Brazilian BVDV type 1 and 2 isolates. Up to 128-fold differences in serum neutralizing antibody titers were observed between the vaccine strains and some Brazilian BVDV isolates. The potential impact of the antigenic variability of BVDV on serologic diagnosis was investigated by testing field serum samples by serum-neutralization (SN) against viruses of both genotypes. Two hundred and eighty samples (24.7%) had neutralizing antibodies against BVDV. Out of these, 215 (76.8%) had neutralizing activity against viruses of both genotypes, 37 samples (13.2%) reacted only against BVDV type 2 and 28 (10%) were positive only to BVDV type 1. These results demonstrate that SN tests using only one virus may result in a significant number of false-negative results. The reduced neutralizing activity of the antisera to vaccine strains against some Brazilian isolates raises the question about the degree of protection conferred by these vaccines and indicates the need of producing vaccines based on local viruses and/or including viruses of both genotypes.

Nineteen Brazilian isolates of bovine viral diarrhea virus (BVDV) were characterized antigenically with a panel of 19 monoclonal antibodies (mAbs) (Corapi WV, Donis RO and Dubovi EJ (1990) American Journal of Veterinary Research, 55: 1388-1394). Eight isolates were further characterized by cross-neutralization using sheep monospecific antisera. Analysis of mAb binding to viral antigens by indirect immunofluorescence revealed distinct patterns of reactivity among the native viruses. Local isolates differed from the prototype Singer strain in recognition by up to 14 mAbs. Only two mAbs - one to the non-structural protein NS23/p125 and another to the envelope glycoprotein E0/gp48 - recognized 100% of the isolates. No isolate was recognized by more than 14 mAbs and twelve viruses reacted with 10 or less mAbs. mAbs to the major envelope glycoprotein E2/gp53 revealed a particularly high degree of antigenic variability in this glycoprotein. Nine isolates (47.3%) reacted with three or less of 10 E2/gp53 mAbs, and one isolate was not recognized by any of these mAbs. Virus-specific antisera to eight isolates plus three standard BVDV strains raised in lambs had virus-neutralizing titers ranging from 400 to 3200 against the homologous virus. Nonetheless, many antisera showed significantly reduced neutralizing activity when tested against heterologous viruses. Up to 128-fold differences in cross-neutralization titers were observed for some pairs of viruses. When the coefficient of antigenic similarity (R) was calculated, 49 of 66 comparisons (74.24%) between viruses resulted in R values that antigenically distinguish strains. Moreover, one isolate had R values suggesting that it belongs to a distinct serologic group. The marked antigenic diversity observed among Brazilian BVDV isolates should be considered when planning diagnostic and immunization strategies.