Genetic diversity of bovine viral diarrhoea virus (BVDV) from Peru and Chile

Twenty-five BVDV strains, detected in serum from persistently infected cattle from Peru (n=15) and Chile (n=10) were genetically characterized. The phylogenetic analysis based on the 5' UTR showed that all 25 strains belonged to genotype 1. Twenty-three of the strains could further be subdivided into subtype 1b, and two out of ten Chilean strains into subtype 1a. In conclusion, in total 23 out of 25 strains analyzed were of genotype 1, subtype 1b. This is the predominant BVDV subtype in many countries all over the world, including USA. The close homology with previously described strains reflects the influence of livestock trade on the diversity of BVDV circulating within and between countries and continents. Peru and Chile have imported large numbers of cattle from USA and Europe, mostly with insufficient or lacking health documentation.

The glycosylation pattern of baculovirus expressed envelope protein E2 affects its ability to prevent infection with bovine viral diarrhoea virus

We have investigated the role of glycosylation of the envelope glycoprotein E2 of bovine viral diarrhoea virus (BVDV), produced in insect cells, in BVDV infection. When amino acids predicated to code for the C-terminal N-linked glycosylation site were mutated the resulting protein was less efficient than wild type protein at preventing infection of susceptible cells with BVDV. In addition, mutational analysis showed that a further two predicted N-terminal N-linked glycosylation sites of E2 are required for efficient production of recombinant protein. (c) 2005 Elsevier B.V. All rights reserved.

Expression, purification and crystallization of the ectodomain of the envelope glycoprotein E2 from Bovine viral diarrhoea virus

Bovine viral diarrhoea virus (BVDV) is an economically important animal pathogen which is closely related to Hepatitis C virus. Of the structural proteins, the envelope glycoprotein E2 of BVDV is the major antigen which induces neutralizing antibodies; thus, BVDV E2 is considered as an ideal target for use in subunit vaccines. Here, the expression, purification of wild-type and mutant forms of the ectodomain of BVDV E2 and subsequent crystallization and data collection of two crystal forms grown at low and neutral pH are reported. Native and multiple-wavelength anomalous dispersion (MAD) data sets have been collected and structure determination is in progress.

Persistent infections after natural transmission of bovine viral diarrhoea virus from cattle to goats and among goats

Bovine viral diarrhoea virus (BVDV) is an economically important pathogen of cattle worldwide. Infection of a pregnant animal may lead to persistent infection of the foetus and birth of a persistently infected (PI) calf that sheds the virus throughout its life. However, BVD viruses are not strictly species specific. BVDV has been isolated from many domesticated and wild ruminants. This is of practical importance as virus reservoirs in non-bovine hosts may hamper BVDV control in cattle. A goat given as a social companion to a BVDV PI calf gave birth to a PI goat kid. In order to test if goat to goat infections were possible, seronegative pregnant goats were exposed to the PI goat. In parallel, seronegative pregnant goats were kept together with the PI calf. Only the goat to goat transmission resulted in the birth of a next generation of BVDV PI kids whereas all goats kept together with the PI calf aborted. To our knowledge, this is the first report which shows that a PI goat cannot only transmit BVD virus to other goats but that such transmission may indeed lead to the birth of a second generation of PI goats. Genetic analyses indicated that establishment in the new host species may be associated with step-wise adaptations in the viral genome. Thus, goats have the potential to be a reservoir for BVDV. However, the PI goats showed growth retardation and anaemia and their survival under natural conditions remains questionable.

Effects of exposure to Bovine viral diarrhoea virus 1 on risk of bovine respiratory disease in Australian feedlot cattle

Viruses play a key role in the complex aetiology of bovine respiratory disease (BRD). Bovine viral diarrhoea virus 1 (BVDV-1) is widespread in Australia and has been shown to contribute to BRD occurrence. As part of a prospective longitudinal study on BRD, effects of exposure to BVDV-1 on risk of BRD in Australian feedlot cattle were investigated. A total of 35,160 animals were enrolled at induction (when animals were identified and characteristics recorded), held in feedlot pens with other cattle (cohorts) and monitored for occurrence of BRD over the first 50 days following induction. Biological samples collected from all animals were tested to determine which animals were persistently infected (PI) with BVDV-1. Data obtained from the Australian National Livestock Identification System database were used to determine which groups of animals that were together at the farm of origin and at 28 days prior to induction (and were enrolled in the study) contained a PI animal and hence to identify animals that had probably been exposed to a PI animal prior to induction. Multi-level Bayesian logistic regression models were fitted to estimate the effects of exposure to BVDV-1 on the risk of occurrence of BRD.Although only a total of 85 study animals (0.24%) were identified as being PI with BVDV-1, BVDV-1 was detected on quantitative polymerase chain reaction in 59% of cohorts. The PI animals were at moderately increased risk of BRD (OR 1.9; 95% credible interval 1.0-3.2). Exposure to BVDV-1 in the cohort was also associated with a moderately increased risk of BRD (OR 1.7; 95% credible interval 1.1-2.5) regardless of whether or not a PI animal was identified within the cohort. Additional analyses indicated that a single quantitative real-time PCR test is useful for distinguishing PI animals from transiently infected animals.The results of the study suggest that removal of PI animals and/or vaccination, both before feedlot entry, would reduce the impact of BVDV-1 on BRD risk in cattle in Australian feedlots. Economic assessment of these strategies under Australian conditions is required. © 2016 Elsevier B.V.

Monitoring bovine viral diarrhea virus (BVDV) infection status in dairy herds

This study was designed to assess the relationship between antibodies against bovine viral diarrhea virus (BVDV) determined in the bulk tank milk (BTM) and the within-herd seroprevalence. We also assessed the efficiency of measuring antibody levels in BTM samples to monitor BVDV infection status in a herd. In the 81 farms included in the study, BTM samples were obtained and blood samples withdrawn from all cattle older than one year. The infection status was then determined in serum and milk using a commercial blocking ELISA based on the detection of anti-p80 antibodies. Apart from these baseline serum and milk samples, another BTM sample was collected from each herd 9 months later, and a third BTM sample obtained 9 months after this. In these second and third milk samples, anti-BVDV antibodies were determined using the same ELISA kit. Statistical tests revealed good agreement between herd seroprevalences (% seropositive animals in the herd) and the antibody levels detected in the BTM samples. During the 18 months of follow-up, the farms with persistently infected cattle at the study outset (14.8% of the herds) showed a significant decrease in BTM antibody titers after virus clearance. Conversely, a significant increase in BTM antibody levels was observed in the herds infected with BVDV during the follow-up period. Our findings indicate that monitoring antibody levels in the BTM is a useful method of identifying changes in the BVDV infection status of a herd.

Serological response of guinea pigs to oily and aqueous inactivated vaccines containing a Brazilian isolate of the Bovine Viral Diarrhea Virus (BVDV)

Bovine Viral Diarrhea Virus (BVDV) is widespread in cattle in Brazil and research shows its large antigenic variability. Available vaccines are produced with virus strains isolated in other countries and may not be effective. In this study, inactivated vaccines containing the Brazilian BVDV-Ib IBSP11 isolate were developed and tested on 6 groups of 10 guinea pigs (Cavia porcellus). Animals in groups A and C received an aqueous vaccine (aluminum hydroxide); B and D groups received an oily vaccine (Montanide ISA50); Group E positive-control animals were given an imported commercial vaccine with BVDV-la Singer; Group F animals were sham vaccinated (negative control). Groups A, B and E received two doses, and Groups C and D, three, every 21 days. Twelve blood samples were taken, at 21-day intervals over 231 days, and evaluated for antibody titer through virus-neutralization (VN), using a homologous strain (IBSP11), and a heterologous strain (BVDV-la NADL). Most animals, 42 days following the first dose, seroconverted to both strains and, after the second dose, there was a significant increase of titers in all groups. The oily formulation induced greater response after the third administration. This increase was not observed with the aqueous vaccines, regardless of the virus used in the VN. Antibody decline was more rapid in animals that received aqueous vaccines. The results showed the importance of studying the influence of endemic strains of commercial vaccines, to improve the efficacy of BVD vaccination. Use of the endemic strain in vaccine formulation presented promising results, as well as the use of guinea pigs as a laboratory model. (C) 2011 Elsevier Ltd. All rights reserved.

Immunohistochemical diagnosis of persistent infection with Bovine Viral Diarrhea Virus (BVDV) on skin biopsies

Detection of persistent infection with BovineViral Diarrhea Virus (BVDV) is essential for both epidemiological and clinical reasons. In addition to the classical virological methods such as virus isolation in tissue culture, ELISA and RT-PCR, immunohistochemistry of skin biopsies has become a useful and reliable tool. Assuming that the presence of BVDV antigen in skin structures is restricted to persistent infection, this method could differentiate from transient infection. In order to answer this question, 6 calves were experimentally infected orally with a non-cytopathic genotype 1 BVDV strain belonging to the subtype k.The calves developed fever, mucopurulent nasal discharge, coughing and leucopenia with relative lymphopenia. Immunohistochemistry of skin biopsies taken daily up to day 13-post infection did not reveal any evidence of BVDV infection. BVDV was, however, isolated from blood samples on cell cultures. Anti-NS3-antibody-ELISA and serum neutralization tests showed that all six calves seroconverted. We conclude that in acute BVDV infections, with genotype 1 and the subtypes found in Switzerland (b, e, h and k) viral antigen is not found in epidermal structures of the skin. In contrast, persistently infected animals test positive for BVD viral antigen by immunohistochemistry of the skin.

Apoptosis in Bovine viral diarrhea virus (BVDV)-induced mucosal disease lesions: a histological, immunohistological, and virological investigation

Cattle persistently infected with a noncytopathic Bovine viral diarrhea virus (BVDV) are at risk of developing fatal "mucosal disease" (MD). The authors investigated the role of various apoptosis pathways in the pathogenesis of lesions in animals suffering from MD. Therefore, they compared the expression of caspase-3, caspase-8, caspase-9, and Bcl-2L1 (Bcl-x) in tissues of 6 BVDV-free control animals, 7 persistently infected (PI) animals that showed no signs of MD (non-MD PI animals), and 11 animals with MD and correlated the staining with the localization of mucosal lesions. Caspase-3 and -9 staining were markedly stronger in MD cases and were associated with mucosal lesions, even though non-MD PI animals and negative controls also expressed caspase-9. Conversely, caspase-8 was not elevated in any of the animals analyzed. Interestingly, Bcl-x also colocalized with mucosal lesions in the MD cases. However, Bcl-x was similarly expressed in tissues from all 3 groups, and thus, its role in apoptosis needs to be clarified. This study clearly illustrates ex vivo that the activation of the intrinsic, but not the extrinsic, apoptosis pathway is a key element in the pathogenesis of MD lesions observed in cattle persistently infected with BVDV. However, whether direct induction of apoptosis in infected cells or indirect effects induced by the virus are responsible for the lesions observed remains to be established.

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.

Evidence of mixed persistent infections in calves born to cows challenged with a pool of bovine viral diarrhea virus isolates

Pregnant cows infected with noncytopathic (NCP) isolates of bovine viral diarrhea virus (BVDV) between days 40 and 120 days of gestation frequently deliver immunotolerant, persistently infected (PI) calves. We herein report the characterization of PI calves produced experimentally through inoculation of pregnant cows with a pool of Brazilian BVDV-1 (n=2) and BVDV-2 isolates (n=2) between days 60 and 90 of gestation. Two calves were born virus positive, lacked BVDV antibodies, but died 7 and 15 days after birth, respectively. Six other calves were born healthy, seronegative to BVDV, harbored and shed virus in secretions for up to 210 days. Analysis of the antigenic profile of viruses infecting these calves at birth and 30 days later with a panel of monoclonal antibodies indicated two patterns of infection. Whereas three calves apparently harbored only one isolate (either a BVDV-1 or BVDV-2), co-infection by two antigenically distinct challenge viruses was demonstrated in three PI calves. Moreover, testing the viruses obtained from the blood of PI calves by an RT-PCR able to differentiate between BVDV-1 and BVDV-2 confirmed the presence/persistence of two co-infecting viruses of different genotypes (BVDV-1 and BVDV-2) in these animals. These findings indicate that persistent infection of fetuses/calves - a well characterized consequence of fetal infection by BVDV - may be established concomitantly by more than one isolate, upon experimental inoculation. In this sense, mixed persistent infections with antigenically distinct isolates may help in understanding the immunological and molecular basis of BVDV immunotolerance and persistence.

Antigenic characterization of Brazilian bovine viral diarrhea virus isolates by monoclonal antibodies and cross-neutralization

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.

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.

Recombinant E2 glycoprotein of bovine viral diarrhea virus induces a solid humoral neutralizing immune response but fails to confer total protection in cattle

Two recombinant baculoviruses were produced in order to obtain a bovine viral diarrhea virus (BVDV) immunogen: AcNPV/E2 expressing E2 glycoprotein, and AcNPV/E0E1E2 expressing the polyprotein region coding for the three structural proteins of BVDV (E0, E1, and E2). Mice were immunized with Sf9 cells infected with the recombinant baculoviruses in a water in oil formulation and the production of neutralizing antibodies was evaluated. Since E2 elicited higher neutralizing antibody titers than E0-E1-E2 polyprotein, it was selected to immunize cattle. Calves received two doses of recombinant E2 vaccine and were challenged with homologous BVDV 37 days later. The recombinant immunogen induced neutralizing titers which showed a mean value of 1.5 ± 0.27 on the day of challenge and reached a top value of 3.36 ± 0.36, 47 days later (84 days post-vaccination). On the other hand, sera from animals which received mock-infected Sf9 cells did not show neutralizing activity until 25 days post-challenge (62 days post-vaccination), suggesting that these antibodies were produced as a consequence of BVDV challenge. Even when no total protection was observed in cattle, in vitro viral neutralization assays revealed that the recombinant immunogen was able to induce neutralizing antibody synthesis against the homologous strain as well as against heterologous strains in a very efficient way.

INFLUENCE OF TYPE 2 BOVINE VIRAL DIARRHEA VIRUS NPRO ON ENHANCEMENT OF BOVINE RESPIRATORY SYNCYTIAL VIRUS REPLICATION MEDIATED BY ANTAGONISM OF HOST CELL INTERFERON TYPE I RESPONSES

Bovine viral diarrhea virus (BVDV) is a member of the genus Pestivirus, Family Flaviviridae. The virus can infect many species of animals of the order Artiodactyla. The BVDV genome encodes an auto protease, Npro, that degrades interferon regulatory factor-3 (IRF-3) reducing type I interferon (IFN-I) production from host cells. Bovine respiratory syncytial virus (BRSV) is a member of the genus Pneumovirus, Family Paramyxoviridae. Concurrent infection with BVDV and BRSV causes more severe respiratory and enteric disease than infection with either virus alone. Our hypothesis was that Npro modulates the innate immune responses to BVDV infection and enhances replication of BVDV or BRSV co-infection. The noncytopathic BVDV2 viruses NY93/c N- Npro 18 EGFP (a mutant with modified Npro fused with enhanced green fluorescent protein), NY93 infectious clone (NY93/c), wild-type NY93-BVDV2 (NY93-wt), and BRSV were evaluated in this study. The objectives of this study were: (1) to characterize the replication kinetics and IFN-I induction in Madin-Darby bovine kidney (MDBK) cells following infection with each of the BVDV isolates, and (2) to characterize the influence of BVDV-mediated IFN-I antagonism on enhancement of BRSV replication in bovine turbinate (BT) cells. NY93/c N- Npro 18 EGFP replicated 0.4 – 1.6 TCID50 logs lower than NY93-wt in MDBK cells. NY93/c N- Npro 18 EGFP-infected MDBK cells synthesized IFN-I significantly higher than NY93/c- and NY93-wt-infected MDBK cells. BT cells co-infected with NY93/c N- Npro 18 EGFP/BRSV or NY93-wt/BRSV were evaluated to determine the effects of co-infection on BRSV replication and IFN-I induction in BT cells. BRSV RNA levels in NY93-wt/BRSV co-infected BT cells were 2.49, 2.79, and 2.89 copy number logs significantly greater than in NY93/c N- Npro 18 EGFP/BRSV co-infected BT cells on days 5, 7, and 9 post-infection, respectively. BVDV RNA levels in NY93/c N- Npro 18 EGFP-infected BT cells were 1.64 – 4.38 copy number logs lower than in NY93-wt-infected BT cells. NY93/c N- Npro 18 EGFP single and co-infected BT cells synthesized IFN-I significantly higher than NY93-wt single and co-infected BT cells. In summary, these findings suggest: (1) NY93/c N- Npro 18 EGFP BVDV2 induced higher levels of IFN-I than BVDV2-wt and may be useful as a safer, replicating BVDV vaccine, and (2) Enhancement of BRSV infection by BVDV co-infection is mediated by antagonism of IFN-I.