Prostaglandin A1 inhibits the replication of bovine viral diarrhea virus

ABSTRACT Bovine viral diarrhea virus can cause acute disease in livestock, leading to economic losses. We show that Prostaglandin A1 inhibits bovine viral diarrhea virus replication in Madin-Darby bovine kidney cells (94% inhibition using 5 µg/mL). Light and electron microscopy of infected cells shows that Prostaglandin A1 also prevents virus-induced vacuolization, but at higher concentrations (10 µg/mL).

Detection and genetic identification of pestiviruses in Brazilian lots of fetal bovine serum collected from 2006 to 2014 / Detecção e identificação genética de pestivírus em lotes brasileiros de soro fetal bovino coletado de 2006 a 2014

The present study performed a genetic identification of pestiviruses contaminating batches of fetal bovine serum (FBS) produced in Brazil from 2006 to 2014. Seventy-three FBS lots were screened by a RT-PCR targeting the 5'untranslated region (UTR) of the pestivirus genome. Thirty-nine lots (53.4%) were positive for pestivirus RNA and one contained infectious virus. Nucleotide sequencing and phylogenetic analysis of the 5'UTR revealed 34 lots (46.6%) containing RNA of bovine viral diarrhea virus type 1 (BVDV-1), being 23 BVDV-1a (5' UTR identity 90.8-98.7%), eight BVDV-1b (93.9-96.7%) and three BVDV-1d (96.2- 97.6%). Six lots (8.2%) contained BVDV-2 (90.3-100% UTR identity) being two BVDV-2a; three BVDV-2b and one undetermined. Four FBS batches (5.5%) were found contaminated with HoBi-like virus (98.3 to 100%). Five batches (6.8%) contained more than one pestivirus. The high frequency of contamination of FBS with pestivirus RNA reinforce the need for systematic and updated guidelines for monitoring this product to reduce the risk of contamination of biologicals and introduction of contaminating agents into free areas.(AU)

No presente estudo foi realizada a identificação genética de pestivírus contaminantes de lotes de soro fetal bovino (SFB) produzidos no Brasil de 2006 a 2014. Setenta e três lotes de SFB foram testados por RT-PCR para a região 5' não traduzida do genoma dos pestivírus. Trinta e nove lotes (53,4%) foram positivos para RNA de pestivírus e um continha vírus infeccioso. O sequenciamento de nucleotídeos e análise filogenética da região 5'UTR revelou que 34 lotes (46,6%) continham RNA do vírus da diarreia viral bovina tipo 1 (BVDV-1), sendo 23 BVDV-1a (identidade na 5' UTR de 90,8-98,7%), oito BVDV-1b (93,9 a 96,7%) e três BVDV-1d (96,2%-97,6%). Seis lotes (8,2%) continham BVDV-2 (90,3 a 100% de identidade), sendo dois BVDV-2a, três BVDV-2b e um de subgenótipo indeterminado. Quatro lotes de SFB (5,5%) estavam contaminados com o vírus HoBi-like (98,3 a 100%). Cinco lotes (6,8%) continham mais do que um pestivírus. A alta frequência de contaminação de SFB com RNA de pestivírus reforça a necessidade para diretrizes sistemáticas atualizadas para a monitoração deste produto com a finalidade de reduzir a contaminação de produtos biológicos e a introdução de agentes contaminantes em áreas livres.(AU)

Use of homologous recombination in yeast to create chimeric bovine viral diarrhea virus cDNA clones

Abstract The open reading frame of a Brazilian bovine viral diarrhea virus (BVDV) strain, IBSP4ncp, was recombined with the untranslated regions of the reference NADL strain by homologous recombination in Saccharomyces cerevisiae, resulting in chimeric full-length cDNA clones of BVDV (chi-NADL/IBSP4ncp#2 and chi-NADL/IBSP4ncp#3). The recombinant clones were successfully recovered, resulting in viable viruses, having the kinetics of replication, focus size, and morphology similar to those of the parental virus, IBSP4ncp. In addition, the chimeric viruses remained stable for at least 10 passages in cell culture, maintaining their replication efficiency unaltered. Nucleotide sequencing revealed a few point mutations; nevertheless, the phenotype of the rescued viruses was nearly identical to that of the parental virus in all experiments. Thus, genetic stability of the chimeric clones and their phenotypic similarity to the parental virus confirm the ability of the yeast-based homologous recombination to maintain characteristics of the parental virus from which the recombinant viruses were derived. The data also support possible use of the yeast system for the manipulation of the BVDV genome.

Computational prediction of MHC class I epitopes for most common viral diseases in cattle (Bos taurus).

Viral diseases like foot-and-mouth disease (FMD), calf scour (CS), bovine viral diarrhea (BVD), infectious bovine rhinotracheitis (IBR) etc. affect the growth and milk production of cattle (Bos taurus) causing severe economic loss. Epitope-based vaccine designing have been evolved to provide a new strategy for therapeutic application of pathogen-specific immunity in animals. Therefore, identification of major histocompatibility complex (MHC) binding peptides as potential T-cell epitopes is widely applied in peptide vaccine designing and immunotherapy. In this study, MetaMHCI tool was used with seven different algorithms to predict the potential T-cell epitopes for FMD, BVD, IBR and CS in cattle. A total of 54 protein sequences were filtered out from a total set of 6351 sequences of the pathogens causing the said diseases using bioinformatics approaches. These selected protein sequences were used as the key inputs for MetaMHCI tool to predict the epitopes for the BoLA-A11 MHC class I allele of B. taurus. Further, the epitopes were ranked based on a proposed principal component analysis based epitope score (PbES). The best epitope for each disease based on its predictability through maximum number of predictors and low PbES was modeled in PEP-FOLD server and docked with the BoLA-A11 protein for understanding the MHC-epitope interaction. Finally, a total of 78 epitopes were predicted, out of which 27 were for FMD, 25 for BVD, 12 for CS and 14 for IBR. These epitopes could be artificially synthesized and recommended to vaccinate the cattle for the considered diseases. Besides, the methodology adapted here could also be used to predict and analyze the epitopes for other microbial diseases of important animal species.

Evaluation of experimental vaccines for bovine viral diarrhea in bovines, ovines and guinea pigs / Evaluación de vacunas experimentales para la diarrea viral bovina en bovinos, ovinos y cobayos

The bovine viral diarrhea virus (BVDV) infection control should be based on elimination of persistently infected animals and on immunization through vaccination, to prevent fetal infection. However, the efficacy of inactivated BVDV vaccines is variable due to its low immunogenicity. This study evaluated the humoral immune response against homologous and heterologous strains of 7 inactivated BVDV vaccines, in bovines and two experimental models (ovine and guinea pig) which might be used to test candidate vaccines. Vaccines formulated with BVDV Singer, Oregon, NADL, and multivalent, induced seroconversion in the three animal models studied, reaching antibody titres higher than 2. Vaccine containing 125C -genotype 2- only induced a low antibody response in ovine, while VS-115 NCP vaccine was not immunogenic. Furthermore, bovine sera at 60 dpv presented homologous as well as heterologous antibody response, indicating a high degree of cross-reactivity among the strains studied. However, when bovine sera were tested against the Argentine field strain 00-693, they showed the lowest levels of cross-reactivity, suggesting the need of continued surveillance to identify and characterize emerging field BVDV strains. Finally, optimal correlations between bovine-ovine and bovine-guinea pig models were observed, indicating that two alternative species could replace bovines when testing the immunogenicity of BVDV candidate vaccines.

El control del virus de la diarrea viral bovina (VDVB) se basa en la eliminación de animales persistentemente infectados, y la inmunización de hembras para prevenir infecciones fetales. La eficiencia de estas vacunas es variable por su baja inmunogenicidad. Se evaluó la respuesta inmune humoral contra virus homólogos y heterólogos de 7 vacunas experimentales inactivadas del VDVB en bovinos y en dos modelos experimentales (ovinos y cobayos). Las vacunas conteniendo VDVB Singer, Oregon, NADL y polivalentes indujeron seroconversión en los tres modelos y se alcanzaron títulos de anticuerpos mayores de 2. La vacuna con VDVB genotipo 2 VS-115, NCP, no resultó inmunogénica. La vacuna genotipo 2 125C sólo indujo baja respuesta humoral en ovinos, mientras que la VS-115, NCP, no resultó inmunogénica. En bovinos se determinó la respuesta a virus homólogos y heterólogos a 60 dpv, lo que indica un alto grado de inmunidad cruzada entre la mayoría de las cepas estudiadas. Cuando los sueros bovinos fueron ensayados con la cepa de campo de Argentina 00-693, los niveles de reacción cruzada fueron más bajos; esto sugiere la necesidad de una vigilancia epidemiológica sostenida a fin de identificar y caracterizar las cepas emergentes del VDVB. La óptima correlación en el modelo bovino-ovino y bovino-cobayo indica su utilidad para evaluar la inmunogenicidad de vacunas inactivadas de VDVB.

Genetic characterization of Brazilian bovine viral diarrhea virus isolates by partial nucleotide sequencing of the 5'-UTR region

Nineteen isolates of bovine viral diarrhea virus (BVDV) from Brazil were genetically characterized through partial nucleotide sequencing and analysis of the 5'UTR region. The isolates were grouped as BVDV-1 (11/19), BVDV-2 (6/19) or "atypical" pestivirus (2/19). Among the BVDV-1, eight isolates were classified as subgenotype BVDV-1a, whereas most (4 out of 6) BVDV-2 belonged to subgenotype 2b. Two isolates from aborted fetuses were not classified into any genetic group, being considered atypical BVDVs. Genetic diversity among Brazilian BVDV isolates may be responsible for vaccination and diag-nostic failure and therefore may influence the control strategies for BVDV infection in the country.

Dezenove amostras do vírus da diarréia viral bovina (BVDV) foram caracterizadas geneticamente através do seqüenciamento parcial de nucleotídeos da Região 5'UTR. As amostras foram agrupadas em BVDV-1 (11/19), BVDV-2 (6/19) e num terceiro grupo de amostras denominadas "atípicas" (2/19). Das onze amostras genotipadas como BVDV-1, oito amostras foram sub-genotipadas como BVDV-1a, enquanto que a maioria (4/6) das amostras de BVDV-2 foi agrupada como BVDV-2b. Duas amostras provenientes de fetos bovinos abortados foram classificadas como atípicas, não BVDV-1 e 2. A presença da diversidade genética de BVDV detectada nas amostras estudadas pode ser responsável por falhas vacinais e de diagnóstico e deve influenciar nas estratégias de controle do BVDV aplicadas nas diferentes regiões brasileiras.

A search for RNA insertions and NS3 gene duplication in the genome of cytopathic isolates of bovine viral diarrhea virus

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 percent) 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.

Expresión y caracterización de dos proteínas estructurales del virus de la diarrea viral bovina (VDVB) / Expression and characterization of 2 bovine viral diarrhea virus structural proteins (]BVDV)

The BVDV glycoproteins gp48 and gp53 were expressed in the baculovirus eukaryotic system. Both recombinant proteins were recognized in western blot analysis by monoclonal antibodies and polyclonal serum. Immunofluorescent test demonstrated that gp53 was localized on the cell surface whereas gp48 was in the cytoplasm. The expressed proteins were extracted by non-denaturing detergent treatment. Rabbit antiserum raised against gp53 recombinant protein efficiently neutralized the virus. These results demonstrate that the recombinant proteins have immunological properties similar to those of the native viral protein and that they can be useful as diagnostic reagents.

Production and characterization of monoclonal antibodies to Brazilian isolates of bovine viral diarrhea virus

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

Characterization of mixed infections with different strains of bovine rotavirus in an outbreak of diarrhea in dairy herds in Goiás, Brazil

Ten faecal samples of bovine rotavirus from calves less than 30 days old from an outbreak of diarrhea in Hidrolândia, Goiás, Brazil were submitted to serological and molecular characterization, using enzyme immonuassay for subgrouping and serotyping, PAGE for determination of electropherotypes and PCR for genome typing. Nine samples belonged to group A/subgroup I rotavirus one samples was group A / soubgroup non-I/non-II. Four samples were characterized as G10P[11] (B223-like), four samples showed a mixture of two rotavirus strains (G6G10 and P[5]P[11]), one sample was characterized as G6P[11] and one sample was characterized only by G serotyping/genotyping, and did not react with any primer used. Two electropherotypes were detected and both were present in the same animal. This study demonstrates that two different electropherotypes and/or serotypes of bovine rotavirus can circulate in the same outbreak.