Noncytopathogenic Bovine Viral Diarrhea Virus Research Articles

Infection of polarized bovine respiratory epithelial cells by bovine viral diarrhea virus (BVDV)

ABSTRACT Bovine viral diarrhea virus (BVDV) is affecting cattle populations all over the world causing acute disease, immunosuppressive effects, respiratory diseases, gastrointestinal, and reproductive failure in cattle. The virus is taken up via the oronasal route and infection of epithelial and immune cells contributes to the dissemination of the virus throughout the body. However, it is not known how the virus gets across the barrier of epithelial cells encountered in the airways. Here, we analyzed the infection of polarized primary bovine airway epithelial cells (BAEC). Infection of BAEC by a non-cytopathogenic BVDV was possible via both the apical and the basolateral plasma membrane, but the infection was most efficient when the virus was applied to the basolateral plasma membrane. Irrespective of the site of infection, BVDV was efficiently released to the apical site, while only minor amounts of virus were detected in the basal medium. This indicates that the respiratory epithelium can release large amounts of BVDV to the environment and susceptible animals via respiratory fluids and aerosols, but BVDV cannot cross the airway epithelial cells to infect subepithelial cells and establish systemic infection. Further experiments showed that the receptor, bovine CD46, for BVDV is expressed predominantly on the apical membrane domain of the polarized epithelial cells. In a CD46 blocking experiment, the addition of an antibody directed against CD46 almost completely inhibited apical infection, whereas basolateral infection was not affected. While CD46 serves as a receptor for apical infection of BAEC by BVDV, the receptor for basolateral infection remains to be elucidated.

Interaction of bovine viral diarrhea virus with bovine cumulus–oocyte complex during IVM: Detection in permissive cells

Structural changes in the zona pellucida (ZP) of bovine oocytes seem to modulate their interaction with various viral agents, facilitating the viral infection in in vitro production systems. To evaluate the susceptibility of bovine oocytes to noncytopathogenic bovine viral diarrhea virus (ncp-BVDV), cumulus–oocyte complexes were exposed to 107 ​tissue culture-infective doses (TCID50)/mL of an ncp-BVDV strain during IVM (in vitro maturation). After that, cumulus cells and the ZP were removed by hyaluronidase and pronase treatment, respectively, and the percentages of oocytes with polar body were analyzed as a sign of nuclear maturation. After passage through cell culture, the virus was isolated from granulosa cells, ZP-free mature oocytes, and ZP-intact mature oocytes. These results were confirmed by reverse transcription–polymerase chain reaction. After consecutive washes, the virus remained associated with ZP-free oocytes, maintaining its replication and infectivity in permissive cells. Based on these findings, it is concluded that the classical viral isolation procedure has a predictive value to detect BVDV associated with ZP-free oocytes and that it was novelty demonstrated that both washing and trypsin treatment of oocytes were ineffective to remove BVDV infection.

Experimental infection of pregnant cows with noncytopathogenic bovine viral diarrhoea virus between days 26 and 50 postbreeding

The effect of bovine viral diarrhoea virus (BVDV) infection on early pregnant cows between 10 and 24days after virus inoculation at day 26 of pregnancy was determined. Four cows were inoculated intravenously with either BVDV (treated, n=3) or growth medium (control, n=1). The treated cows were euthanized on either day 10, 17 or 24 post-infection and the control cow was euthanized on day 24 post-infection. The level of serum 2-5A synthetase increased in all of the three treated cows. Progesterone levels decreased to below 1.0ng/ml between 10 and 22days after inoculation in two of the three treated cows and the embryos/foetuses of two cows died. Therefore, BVDV may be a cause of early embryonic or feotal loss in early pregnant cows and serum 2-5A synthetase may be useful as an indicator of viral infection in cows.

Identification and characterization of a novel subgenotype of bovine viral diarrhea virus isolated from dairy cattle in Northwestern China

Bovine viral diarrhea virus (BVDV) was detected by RT-PCR in 105 out of 391 samples which were collected from five dairy farms in Ningxia, China during 2010-2011. Non-cytopathogenic BVDV was isolated from 13 samples and a 230-bp fragment of the 5'-untranslated region was amplified and sequenced. While the predominant subgenotypes were BVDV-1b and BVDV-1d, a potentially novel subgenotype was identified by phylogenetic analysis, which may have implications for vaccine development.

Biological properties of bovine viral diarrhea virus quasispecies detected in the RK13 cell line

The rabbit kidney cell line RK13 has been reported to be contaminated with noncytopathogenic (ncp) bovine viral diarrhea virus (BVDV). Persistent infection was confirmed by demonstrating the stability of virus titers (10(4.6±0.5) TCID50/ml) and BVDV positive cells (71.9±3.12%), over six successive passages. Based on the "exaltation of Newcastle disease virus" (END) and reverse plaque formation methods, two types of ncp viruses were isolated, END-phenomenon-positive and negative. Isolates, RK13/E(+) and RK13/E(-), demonstrated (1) differing levels of reproducibility in cell cultures, (2) similar antigenicity against BVDV antisera, (3) identical 5'-UTR region nucleotide sequences, (4) four amino acid differences throughout the genomic open reading frame, and (5) better growth ability in primary rabbit cells than other laboratory strains when inoculated in parallel at an MOI of 0.01. Overall, the BVDV population in RK13 cells consists of at least two different END characteristic quasispecies that are adapted to cultures of rabbit origin, giving rise to naturally attenuated BVDV strains that can be used in vaccine development.

Virus Distribution and Role of Thymic Macrophages During Experimental Infection With Noncytopathogenic Bovine Viral Diarrhea Virus Type 1

Thymic depletion, presence of viral antigen, and changes in distribution and cytokine production of thymic macrophages were investigated in calves experimentally infected with a noncytopathogenic bovine viral diarrhea virus type (BVDV) 1 strain. Ten clinically healthy colostrum-deprived calves were used. Eight calves were inoculated with the virus and two were used as uninfected controls. Calves were sedated and euthanized in batches between 3 and 14 days postinoculation. At necropsy, thymus samples were collected for structural, immunohistochemical, and ultrastructural study and TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labeling). From 6 days postinoculation, the thymic cortex was multifocally depleted with increased frequency of pyknosis and karyorrhexis, suggestive of apoptosis and confirmed by the TUNEL technique. Although the onset of lymphoid depletion was coincident with the detection of viral antigen by immunohistochemistry, the number of infected lymphocytes was very low through the experiment. There was an increase in number of macrophages in cortex and medulla, accompanied by ultrastructural changes indicative of phagocyte activation, and a decrease in cells expressing tumor necrosis factor-alpha (TNF-α) and IL-1α. These results suggest that the increase in number of these cells could be related to phagocytosis of cell debris and apoptotic lymphocytes. Furthermore, the results imply that, in contrast to the situation with classical swine fever virus, the lymphocyte apoptosis resulting from bovine viral diarrhea virus infection is not mediated by TNF-α or interleukin-1 alpha (IL-1α) production by virus-infected macrophages. This is the first study that describes this decrease in the number of thymic cells expressing TNF-α and IL-1α in cattle experimentally infected with bovine viral diarrhea virus type 1.

Bovine Viral Diarrhea Virus Strain- and Cell Type-Specific Inhibition of Type I Interferon Pathways

The interaction of noncytopathogenic bovine viral diarrhea virus type 1 (BVDV-1ncp) with antigen-presenting cell (APC) subsets is of great interest due to the recent increase in severe acute BVDV outbreaks attributed to this genotype (1, 12, 13). We report the effect of a BVDV-1ncp strain (Ho916ncp [13]) causing severe acute infection on in vitro type I interferon (alpha/beta interferon [IFN-α/β]) production compared to that of a mild-acute strain (Ky1203ncp) (15). Ho916ncp but not Ky1203ncp induced IFN-α/β in only lineage-negative (LIN−) cells, a recently described enriched IFN-α/β-producing cell population in cattle (A. Gibson, S. Miah, P. Griebel, J. Brownlie, and D. Werling, submitted for publication). In agreement with published data, monocytes and monocyte-derived dendritic cells (moDC) did not produce IFN-α/β in response to either strain (4, 11, 17). Experimental infection of calves with BVDVncp results in the detection of IFN-α/β and the IFN-α/β-inducible gene Mx (5, 20) as well as serum IFN-α/β activity in pregnant dams and the fetus (22). The identification of IFN-α/β-producing cells within the lymph nodes of BVDVncp-infected calves (4) and of enriched circulating IFN-α/β-producing cells (Gibson et al., submitted) points to an APC subset as a cellular source of IFN-α/β during in vivo BVDVncp infections. In the present study, monocytes, moDC, and LIN− cells, generated from the same animal (11, 24-26, 28; Gibson et al., submitted), were exposed to either strain at a multiplicity of infection (MOI) of 0.1 and IFN-α/β was assessed after 48 h, using a previously described Mx-CAT (chloramphenicol acetyltransferase) reporter assay (4-6, 9). LIN− cells, but not monocytes or moDC, produced significant amounts of IFN-α/β in response to Ho916ncp but not to Ky1203ncp or mock-infected control (Fig. ​(Fig.1).1). To assess whether these observed differences in IFN-α/β production were due to differences in viral replication, the presence of BVDV was determined by immunoperoxidase staining using a bovine hyperimmune serum, similar to what was described previously (11). Astonishingly, no clear differences in abilities to infect the different cell types were seen for the two BVDVncp strains (Fig. ​(Fig.2).2). As differences in viral replication did not explain differences in IFN-α/β production, we next assessed the potential effects of both BVDVncp strains on interferon response factors (IRFs). BVDVncp is known to inhibit IFN-α/β production through viral proteins Npro (2, 3, 10, 14) and Erns (16, 18, 19), which block IFN-α/β production by targeting IRF-3 for degradation by polyubiquitination (7, 14) or by degrading viral RNA (19). Translocation of IRF-3 occurs during infection but not binding to DNA (2). In contrast, IRF-7 is neither activated nor translocated to the nucleus (2). FIG. 1. Type I interferon response to BVDVncp. Monocytes (MON), moDC, and LIN− cells (LIN) were isolated from the same animal and stimulated with 50 μg ml−1 of poly(I:C), BVDVncp strains Ho916ncp and Ky1203ncp at an MOI of 0.1, and corresponding ... FIG. 2. Antigen-presenting cell susceptibility to BVDVncp. Monocytes (A), moDC (B), and LIN− cells (C) were infected with BVDVncp strains Ho916ncp and Ky1203ncp for 48 h (MOI, 0.1). Cells were fixed and stained for the presence of BVDV antigen using a ... We assessed the impact of both strains on IFN-α/β signaling components IRF-3 and IRF-7 by Western blotting, which showed that IRF-3 and IRF-7 were increased in all three cell types infected with Ho916ncp compared to mock-infected controls. In contrast, Ky1203ncp induced a reduction of IRF-7 expression in monocytes and moDC and a reduction in IRF-3 expression in all three cell types. IRF-7 remained unchanged in LIN− cells, potentially indicating cell type-specific degradation in monocytes and moDC for Ky1203ncp (Fig. ​(Fig.3).3). Ho916ncp-induced IFN-α/β production by LIN− cells appears to be independent of IRF-3 or IRF-7 modulation; however, LIN− cells, unlike monocytes or moDC, express TLR7 (27; Gibson et al., submitted), which has been recently implicated in the recognition of other pestiviruses, such as West Nile virus and dengue virus (8, 21, 23). Ho916ncp, as a severe acute BVDV-1ncp strain could be more readily accessible to the endosomal compartment within LIN− cells, thus producing cell type-restricted IFN-α/β through TLR7. Our data show for the first time differences in BVDV-1ncp strains with regard to a cell-specific IFN-α/β response and offer some suggestions for this modulation. FIG. 3. Relative expression of IRF-3 and IRF-7 in response to BVDVncp. Monocytes (MON), moDC, and LIN− (LIN) cells were isolated from the same animal and stimulated with Ho916ncp and Ky1203ncp alongside mock-infected controls for 48 h. Protein lysates ...

Hypomyelination Associated With Bovine Viral Diarrhea Virus Type 2 Infection in a Longhorn Calf

A newborn Longhorn heifer calf presented with generalized tremors, muscle fasciculations, ataxia, and nystagmus. At necropsy, no gross central nervous system lesions were observed. Histologically, the brain and spinal cord had mild to moderate diffuse microgliosis and astrocytosis, minimal nonsuppurative encephalitis, and decreased myelin staining. Ultrastructural examination revealed thinning and absence of myelin sheaths. Various cell types were immunohistochemically positive for bovine viral diarrhea virus (BVDV). Noncytopathogenic BVDV was isolated from the brain and identified as BVDV type 2 by phylogenetic analysis. BVDV-induced hypomyelination is rare and analogous to lesions in neonates infected with border disease and classical swine fever viruses. This is the first documented case of hypomyelination in a calf specifically attributed to BVDV type 2 and the first description of the ultrastructural appearance of BVDV-induced hypomyelination.

Activation of extracellular signal-regulated kinase in MDBK cells infected with bovine viral diarrhea virus

Our efforts to identify the cellular signaling cascades triggered by bovine viral diarrhea virus (BVDV) infection in MDBK cells revealed marked activation of extracellular signal-regulated kinase 1/2 (ERK). Enhanced phosphorylation of ERK was detected following infection with cytopathogenic (cp) BVDV, but not with noncytopathogenic BVDV. It appears that cp BVDV-induced ERK phosphorylation is caused by oxidative stress, because ERK phosphorylation was inducible by treatment with hydrogen peroxide or serum deprivation and was attenuated by addition of antioxidants. These results indicate that BVDV infection influences the ERK signaling pathway via oxidative stress, depending on the biotype.

Cleavage of the NS2-3 protein in the cells of cattle persistently infected with non-cytopathogenic bovine viral diarrhea virus

The NS2-3 of BVDV is cleaved in cultured cells infected with cp BVDV but not in those infected with ncp BVDV when tested more than 10 hours post infection. However, it is not known whether cleavage of NS2-3 occurs in vivo. In the present study, cleavage of NS2-3 in cattle persistently infected with BVDV was investigated. All BVDV isolated from PI animals were of the ncp biotype, and NS2-3 proteins were detected in bovine fetal muscular cells infected with these viruses. On the other hand, in the leukocytes of those PI animals, NS3 proteins, products of the cleavage of NS2-3 proteins, were detected. In addition, the NS3 proteins were also detected in leukocytes artificially infected with ncp BVDV. These results reveal that the NS2-3 protein of BVDV is cleaved in leukocytes. Furthermore, NS3 proteins were detected in many tissues of PI cattle, such as lymphoid tissue, brain, thyroid, lung, and kidney. These results suggest that the NS2-3 protein of ncp BVDV cleaves in vivo.

Experimental Superinfection of a Lesser Malayan Mousedeer (Tragulus javanicus) Persistently Infected with Bovine Viral Diarrhea Virus

A Lesser Malayan mousedeer (Tragulus javanicus), persistently infected with noncytopathogenic bovine viral diarrhea virus (BVDV) type 1f, was experimentally superinfected with a cytopathogenic isolate of BVDV type 1c, which antigenically partially matched the endogenous strain. Within the observational period of 125 days after superinfection, the animal did not demonstrate any clinical signs of the disease and/or significant changes in blood values. Neutralizing antibodies were detected at 35 and 42 days postinfection. The isolate causing the superinfection was found in feces, nasal swabs, and saliva starting from day 29 and at various times postchallenge. Macroscopic or histologic examination did not reveal mucosal disease-like lesions, despite the detection of the cytopathogenic isolate in the salivary gland, rumen, abomasum, kidney, and superficial prescapular lymph node. Results indicate that the cytopathogenic BVDV strain, which was used in the superinfection, persisted in the viremic animal without causing disease within the observation period.

The relationship between the viral RNA level and upregulation of innate immunity in spleen of cattle persistently infected with bovine viral diarrhea virus

Persistent infection in cattle with bovine viral diarrhea virus (BVDV), which is classified in the genus Pestivirus, family Flaviviridae, may result in growth retardation and immunosuppression. In vitro infection with noncytopathogenic (ncp) BVDV has been shown to suppress interferon (IFN) responses, whereas ncpBVDV induces transient strong IFN responses in vivo following acute infection of naïve cattle. In this study, the innate immune response of the spleen, a crucial organ for immune system homeostasis, from PI cattle was analyzed. The transcription of five IFN- and apoptosis-related mRNAs (Mx1, iNOS, OAS-1, PKR, and TNF-α), which was upregulated in response to BVDV replication in our previous in vitro studies, and the level of viral RNA were quantified using real-time RT-PCR. Upregulation of Mx1, OAS-1, PKR, and TNF-α mRNA expression was detected in the spleens of PI cattle regardless of their age, and induction of apoptosis was also upregulated in the spleens of PI cattle compared with those of nonPI cattle. Although it cannot be excluded that the innate immune responses may be activated in response to any secondary infections in immunosuppressed cattle, the absence of any pathogenic microorganisms in the PI cattle and the statistically significant correlation between innate immune responses and the viral RNA level indicates that there may be a positive relationship between the increased level of viral RNA replication and upregulation of innate immunity in vivo.

Bovine Viral Diarrhea Virus: Prevention of Persistent Fetal Infection by a Combination of Two Mutations Affecting ErnsRNase and NproProtease

Different genetically engineered mutants of bovine viral diarrhea virus (BVDV) were analyzed for the ability to establish infection in the fetuses of pregnant heifers. The virus mutants exhibited either a deletion of the overwhelming part of the genomic region coding for the N-terminal protease N(pro), a deletion of codon 349, which abrogates the RNase activity of the structural glycoprotein E(rns), or a combination of both mutations. Two months after infection of pregnant cattle with wild-type virus or either of the single mutants, the majority of the fetuses contained virus or were aborted or found dead in the uterus. In contrast, the double mutant was not recovered from fetal tissues after a similar challenge, and no dead fetuses were found. This result was verified with a nonrelated BVDV containing similar mutations. After intrauterine challenge with wild-type virus, mutated viruses, and cytopathogenic BVDV, all viruses could be detected in fetal tissue after 5, 7, and 14 days. Type 1 interferon (IFN) could be detected in fetal serum after challenge, except with wild-type noncytopathogenic BVDV. On days 7 and 14 after challenge, the largest quantities of IFN in fetal serum were induced by the N(pro) and RNase-negative double mutant virus. The longer duration of fetal infection with the double mutant resulted in abortion. Therefore, for the first time, we have demonstrated the essential role of both N(pro) and E(rns) RNase in blocking interferon induction and establishing persistent infection by a pestivirus in the natural host.

Genetic and Pathobiological Characterization of Bovine Viral Diarrhea Viruses Recently Isolated from Cattle in Japan

The 475 strains of bovine viral diarrhea virus (BVDV) isolated from cattle in 12 prefectures of Japan in the last 7 years were phylogenetically classified as BVDV-1 or BVDV-2 on the basis of the nucleotide sequence of the 5'-untranslated region. BVDV-1 strains were further subtyped as 1a (101 strains), 1b (163), 1c (128), 1j (3), and So CP/75-like (1), and all of the 79 BVDV-2 strains belonged to subtype 2a. These 2a BVDVs contain two isolates that had high nucleotide identities with those of highly pathogenic BVDV-2 strains reported in North America (Pellerin et al., 1994). However, acute infection with severe mortality like North American outbreak was not observed and most of the present BVDV-2 strains were isolated from persistently infected (PI) cattle showing mild or no clinical sign. Moreover, it was revealed that 61.5% of the 39 PI cattle with cytopathogenic BVDVs did not show typical mucosal disease and 54.6% of the 405 PI animals only with non-cytopathogenic BVDVs were apparently healthy. The present results indicate that the prevention of the infection with an appropriate vaccine and active surveillance covering healthy cattle are required for the control of BVD.

The double-stranded RNA-induced apoptosis pathway is involved in the cytopathogenicity of cytopathogenic Bovine viral diarrhea virus

Bovine viral diarrhea virus (BVDV), which is classified in the genus Pestivirus, family Flaviviridae, can be divided into two biotypes according to its ability to induce a cytopathic effect in tissue culture cells. The mechanisms through which cytopathogenic (cp) BVDV induces cell death and non-cytopathogenic (ncp) BVDV causes persistent infection without producing cell death remain unclear. Here, it was found that the overexpression of four apoptosis-related cellular mRNAs in cells infected with cpBVDV could also be caused by synthetic dsRNA. In fact, it was found that the amount of dsRNA produced by cpBVDV considerably exceeded the amount yielded by ncpBVDV. To evaluate the possible involvement of dsRNA in the induction of apoptosis, this study examined whether RNAi-mediated depletion of two dsRNA-reactive cellular factors, dsRNA-dependent protein kinase and 2',5'-oligoadenylate synthetase 1, resulted in the prevention of cpBVDV-induced apoptosis. Although the induction of apoptosis was reduced after the suppression of either factor alone, the simultaneous silencing of both factors resulted in an almost complete inhibition of apoptosis without affecting viral titre. These results showed that dsRNA is the main trigger of apoptosis in cpBVDV-infected cells and that the cytopathogenicity of BVDV depends on the yield potential of dsRNA. In contrast, ncpBVDV yielded minimal levels of dsRNA, thereby establishing a persistent infection without inducing apoptosis. This report supports the significance of viral dsRNA as a trigger of innate immune responses.

Genetic recombination at different points in the N pro-coding region of bovine viral diarrhea viruses and the potentials to change their antigenicities and pathogenicities

Cytopathogenic (cp) bovine viral diarrhea virus (BVDV) strain KS86-1cp was isolated from a cow persistently infected with non-cytopathogenic (ncp) BVDV strain KS86-1ncp after development of mucosal disease by superinfection with cp BVDV strain Nose. cp BVDV strains 799cp and 839cp were also isolated from independent cattle that developed mucosal disease by superinfection with cp BVDV KS86-1cp. In the present study, genetic analysis revealed that the genes of cp BVDV strains 799cp and 839cp were chimeras between the genes of the persisting ncp BVDVs and that of superinfecting KS86-1cp. The genetic recombination that generates 799cp occurred between the identical points in the N pro gene region, whereas genetic recombination that generates 839cp occurred between different points in the N pro gene region. Both 799cp and 839cp were inherited Jiv gene of KS86-1cp strain and envelope protein genes of the persisting viruses. In addition, neutralization test disclosed that antigenicities of 799cp, 839cp, and KS86-1cp were also similar to each persisting virus. These findings indicate that exogenous cp BVDV containing insertion of Jiv gene in the 5 terminal region can induce genetic recombination with the original ncp BVDV at different points in the N pro gene region, and those viruses have high potential to change those antigenicities and pathogenicities by RNA recombination.

Ability to differentiate between cp and ncp BVDV by microarrays: Towards an application in clinical veterinary medicine?

Microarray expression profiling provides a comprehensive portrait of the transcriptional world enabling us to view the organism as a ‘system’ that is more than the sum of its parts. The vigilance of cells to environmental change, the alacrity of the transcriptional response, the short half-life of cellular mRNA and the genome-scale nature of the investigation collectively explain the power of this method. These same features pose the most significant experimental design and execution issues which, unless surmounted, predictably generate a distorted image of the transcriptome. Conversely, the expression profile of a properly conceived and conducted microarray experiment can be used for hypothesis testing: disclosure of the metabolic and biosynthetic pathways that underlie adaptation of the organism to infectious processes; the identification of co-ordinately regulated genes; the regulatory circuits and signal transduction systems that mediate the adaptive response; and temporal features of developmental programmes. The study of viral pathogenesis by microarray expression profiling poses special challenges and opportunities. Although the technical hurdles are many, obtaining expression profiles of an organism growing in tissue will probably reveal strategies for growth and survival of the virus in the host's cells. Here, we show data obtained using a tailored microarray system based on synthetic polynucleotides derived from human sequences (SIRS-Lab GmbH, Jena, Germany) to study the effect of cytopathogenic (cpe) and non-cytopathogenic (ncp) bovine viral diarrhoea virus (BVDV) infection of bovine macrophages, focusing on intracellular signalling molecules. Of the 575 genes present on the array, more than 70% showed a reaction with the oligonuleotides spotted on the array, and 26 genes were differentially expressed comparing cDNA derived from cpe and ncp infected cells. These data will help to further understand our knowledge regarding BVDV infection, and will especially help to understand differences in cellular responses to cpe and ncp biotypes.

Differential activation of interferon regulatory factors-3 and -7 by non-cytopathogenic and cytopathogenic bovine viral diarrhoea virus.

Non-cytopathogenic bovine viral diarrhoea virus (ncpBVDV) has previously been shown to inhibit the function of interferon regulatory factor-3 in cultured cells [J. Virol. 76 (2002) 8979]. In this study, we show that, like ncpBVDV, when cells were previously exposed to cytopathogenic BVDV (cpBVDV) the appearance of an IRF-3-DNA complex from nuclear extracts that can be induced by heterologous virus infection was not observed. Infection of cells with ncpBVDV or cpBVDV resulted in neither the translocation of IRF-7 from the cytoplasm to the nucleus of infected cells, nor an inhibition of its nuclear translocation in cells super-infected by Semliki Forest Virus. We conclude that cpBVDV and ncpBVDV both share the ability to inhibit the full function of IRF-3 but neither stimulate or block the nuclear uptake of IRF-7.

Isolation and confirmation of bovine viral diarrhoea virus in Serbia and comparative typing with recent Slovenian isolates

The results of an investigation on bovine viral diarrhoea virus (BVDV) in fetal calf serum (FCS), whole blood and pathological material obtained from sick or dead cattle in Serbia are presented. Whole blood and FCS from sick animals were screened for BVDV antigen (Erns) by ELISA. ELISA positive samples and pathological material from dead animals were inoculated into primary cell cultures of fetal calf testis (FTTe). After threefold passage in FTTe cells, BVDV was detected by direct immunofluorescence and indirect immunoperoxidase tests and by reverse transcriptase-polymerase chain reaction (RT-PCR). Among 64 individual samples of FCS, two were positive for noncytopathogenic BVDV. One cytopathogenic BVDV was isolated from a whole blood sample from a heifer with clinical signs of mucosal disease. The 5'-untranslated region (5'-UTR) of three Serbian BVDV isolates was amplified by RT-PCR, sequenced and, together with 15 recent Slovenian BVDV isolates characterized by phylogenetic analysis. All isolates were classified as BVDV genotype 1 viruses. The majority of the BVDV isolates were of the 1f (Serbia - 2 isolates, Slovenia - 7 isolates) and 1d subtypes (Slovenia - 7 isolates) whilst one Serbian and one Slovenian isolate were genotyped as BVDV 1b.

Inhibition of beta interferon transcription by noncytopathogenic bovine viral diarrhea virus is through an interferon regulatory factor 3-dependent mechanism.

The induction and inhibition of the interferon (IFN) response and apoptosis by bovine viral diarrhea virus (BVDV) has been examined. Here we show that prior infection of cells by noncytopathogenic BVDV (ncp BVDV) fails to block transcriptional responses to alpha/beta IFN. In contrast, ncp BVDV-infected cells fail to produce IFN-alpha/beta or MxA in response to double-stranded RNA (dsRNA) or infection with a heterologous virus (Semliki Forest virus [SFV]). ncp BVDV preinfection is unable to block cp BVDV- or SFV-induced apoptosis. The effects of ncp BVDV infection on the transcription factors controlling the IFN-beta induction pathway have been analyzed. The transcription factor NF-kappa B was not activated following ncp BVDV infection, but ncp BVDV infection was not able to block the activation of NF-kappa B by either SFV or tumor necrosis factor alpha. Furthermore, ncp BVDV infection did not result in the activation of stress kinases (JNK1 and JNK2) or the phosphorylation of transcription factors ATF-2 and c-Jun; again, ncp BVDV infection was not able to block their activation by SFV. Interferon regulatory factor 3 (IRF-3) was shown to be translocated to the nuclei of infected cells in response to ncp BVDV, although DNA-binding of IRF-3 was not seen in nuclear extracts. In contrast, an IRF-3-DNA complex was observed in nuclear extracts from cells infected with SFV, but the appearance of this complex was blocked when cells were previously exposed to ncp BVDV. We conclude that the inhibition of IFN induction by this pestivirus involves a block to IRF-3 function, and we speculate that this may be a key characteristic for the survival of pestiviruses in nature.