Activation of cell signaling pathways is dependant on the biotype of bovine viral diarrhea viruses type 2.

Bovine viral diarrhea virus (BVDV), a pestivirus of the Flaviviridae family, is an economically important cattle pathogen with a worldwide distribution. Besides the segregation into two distinct species (BVDV1/BVDV2) two different biotypes, a cytopathic (cp) and a noncytopathic (ncp) biotype, are distinguished based on their behavior in epithelial cell cultures. One of the most serious forms of BVDV infection affecting immunocompetent animals of all ages is severe acute BVD (sa BVD) which is caused by highly virulent ncp BVDV2 strains. Previous studies revealed that these highly virulent ncp viruses cause cell death in a lymphoid cell line (BL3) which is not clearly associated with typical apoptotic changes (e.g. PARP cleavage) observed after infection with cp BVDV. To further characterize the underlying molecular mechanisms, we first analyzed the role of the mitochondria and caspases as key mediators of apoptosis. Compared to infection with cp BVDV2, infection with highly virulent ncp BVDV2 resulted in a delayed and less pronounced disruption of the mitochondrial transmembrane potential (DeltaPsi(m)) and a weaker activation of the caspase cascade. In contrast, infection with low virulence ncp BVDV2 showed no significant differences from the uninfected control cells. Since different pro- and anti-apoptotic cellular signaling pathways may become activated upon virus infection, we compared the effect of different BVDV2 strains on cellular signaling pathways in BL3 cells. Stress-mediated p38 MAPK phosphorylation was detected only in cells infected with cp BVDV2. Interestingly, infection with highly virulent ncp BVDV2 was found to influence the phosphoinositide 3-kinase (PI3K)-Akt pathway. This indicates that BL3 cells respond differently to infection with BVDV depending on virulence and biotype.

[Analysis of bulk milk samples using polymerase chain reaction: an additional tool for bovine viral diarrhea monitoring]. / Untersuchung von Sammelmilchproben mittels Polymerasekettenreaktion: Eine ergänzende Methode für die BVD-Uberwachung.

Programmes for the eradication and control of infections with bovine viral diarrhea virus (BVDV) concentrate on the identification and elimination of persistently infected (PI) animals. The identification of these animals is mainly based on the detection of viral antigen using ELISA techniques. Protocols detecting viral nucleic acid using RT-PCR have been described recently. Due to high costs the German model recommends screening of animals of 9 up to 36 months of age. Screening of bulk milk samples using RT-PCR technology would allow a system independent of age. The aim of the present study was to test whether bulk milk samples (1433 including max. 50 animals each) collected in four counties of Lower Saxony are suitable for a complementary identification of PI animals via RT-PCR. Thirty-one bulk milk samples derived from 27 dairy herds were BVDV positive, corresponding to 2.3 % of the herds analysed in this study. Two samples first scored doubtful. Follow up tests revealed lactating PI animals in most cases (18). In other cases the epidemiological status of the herd, i.e. high sero-prevalence and/or presence of PI animals among non-lactating cattle, suggested a transient infection detected in the first bulk milk sample. These results demonstrate that monitoring of lactating cattle of any age using RT-PCR is a very sensitive, economically effective additional method for the identification of PI animals.

No caspase activation but overexpression of Bcl-2 in bovine cells infected with noncytopathic bovine virus diarrhoea virus.

Cytopathic bovine viral diarrhoea viruses (cp BVDV) induce apoptosis in permissible cell cultures via the intrinsic pathway, which involves the mitochondria as key organelles. An important event is the irreversible opening of the permeability transition pore (PTP) and the breakdown of the transmembrane potential DeltaPsi(m). The resulting release of cytochrome C from the mitochondria serves as a trigger to form the apoptosome which then leads to caspase activation and cell death. In contrast, noncytopathic (ncp) BVDV do not seem to affect cells in vivo or in vitro, suggesting that they inhibit apoptosis. Interestingly, inhibition of caspases in cells infected with cp BVDV delayed the apoptotic cascade but did not prevent the cytopathic effect (CPE). This suggests that the induction of apoptosis and the processes finally leading to the CPE may proceed separately, implying that the inhibition of apoptosis by ncp BVDV has to start earlier in the cascade. In this study we show that in fact apoptosis inhibition in cells infected with ncp BVDV must occur at the mitochondrial level, before the activation of the caspase cascade occurs. To elucidate the role of mitochondria after infection of cells with ncp BVDV, expression of Bcl-2 and Bax were analysed. It was shown that while Bax expression was not affected, the anti-apoptotic Bcl-2 protein was upregulated, presumably suppressing initiation of cell death and enabling persistent infection in vitro.

Induction of the intrinsic apoptotic pathway in cells infected with cytopathic bovine virus diarrhoea virus.

Cytopathic bovine viral diarrhoea virus (cp BVDV) induces apoptosis in bovine cell cultures. This also seems to be a prominent feature in the pathogenesis of mucosal disease. To gain an insight into the molecular pathways of the cell alterations, the involvement of different members of the apoptotic cascade was analyzed. It was shown that inhibition of the mitochondrial permeability transition pore significantly delayed the cytopathic effect without affecting virus replication. Moreover, the membrane potential (deltapsi(m)) was affected, and translocation of cytochrome c to the cytosol, overexpression of apoptotic protease-activating factor 1 and a significant increase of caspase-9 activity were demonstrated, indicating that the apoptosome is formed. We conclude that at least in vitro, infection of cells with cp BVDV leads to the activation of the intrinsic pathway of apoptosis.

Apoptosis inhibitors delay the cytopathic effect of bovine viral diarrhoea virus (BVDV).

Based on their action in cell culture, two biotypes of bovine viral diarrhoea virus (BVDV) can be distinguished. The noncytopathic (ncp) BVDV isolated from persistently infected animals cause no visible damage to cultured bovine cells. In contrast, cytopathic (cp) BVDV induces severe damage and apoptosis in cell cultures. Cp BVDV can be isolated from cattle suffering from mucosal disease (MD) and is associated with the severe lesions that primarily affect the gastrointestinal tract. To get an insight into the molecular events during BVDV induced cytopathic effect (CPE), the effect of three chemical reagents (3-aminobenzamide, ascorbic acid and N-acetyl-leucyl-leucyl-methional) with completely different mode of actions in infected cells was analysed. All three substances were able to delay the cytopathic effect induced in permissive bovine cells.