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.

Highly potent and selective inhibition of bovine viral diarrhea virus replication by γ-carboline derivatives.

Several novel γ-carboline derivatives were identified as selective inhibitors of bovine viral diarrhea virus (BVDV) replication in cell cultures. Among them, 3,4,5-trimethyl-γ-carboline (SK3M4M5M) was the most active against BVDV (Nose strain) in MDBK cells, with a 50% effective concentration of 0.017±0.005µM and a selectivity index of 435. The compound inhibited viral RNA synthesis in a dose-dependent fashion. In a time of drug-addition experiment during a single viral replication cycle, SK3M4M5M lost its antiviral activity when first added at 8h or later after infection, which coincides with the onset of viral RNA synthesis. When selected γ-carboline derivatives, including SK3M4M5M, were examined for their inhibitory effect on the mutant strains resistant to some classes of nonnucleoside BVDV RNA-dependent RNA polymerase inhibitors, all of which target the top of the finger domain of the polymerase, the strains displayed cross-resistance to the γ-carboline derivatives. These results indicate that the γ-carboline derivatives may possibly target a hot spot of the RNA-dependent RNA polymerase. Although SK3M4M5M was highly active against BVDV, the compound proved inactive against hepatitis C virus (HCV) in HCV RNA replicon cells.

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.

Bovine viral diarrhea viruses differentially alter the expression of the protein kinases and related proteins affecting the development of infection and anti-viral mechanisms in bovine monocytes.

Using a proteomics approach, we evaluated the effect of cytopathic (cp), and non-cytopathic (ncp) bovine viral diarrhea viruses (BVDV) on the expression of protein kinases and related proteins in bovine monocytes. Proteins were isolated from membrane and cytosolic fractions with the differential detergent fractionation (DDF) method and identified with 2D-LC ESI MS2. Of approximately 10,000 proteins identified, 378 proteins had homology with known protein kinases or related proteins. Eighteen proteins involved in cell differentiation and activation, migration, anti-viral mechanisms (interferon/apoptosis), biosynthesis, sugar metabolism and oncogenic transformation were significantly altered in BVDV-infected monocytes compared to the uninfected controls. Six proteins, mostly related to cell migration, anti-viral mechanisms, sugar metabolism and possibly tumor resistance were differentially expressed between the ncp and cp BVDV-infected monocytes. Particularly, the expression of the receptor of activated C kinase (RACK), of pyridoxal kinase (PK), diacyglycerol kinase (DGK) and Brutons tyrosine kinase (BTK) was decreased in monocytes infected with cp BVDV compared to ncp BVDV, possibly contributing to the cytopathic effect of the virus. This and other findings are discussed in view of the possible role the identified proteins play in the development of viral infection and oncogenic transformation of cells.

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.