Induction of apoptosis and cleavage of poly(ADP-ribose) polymerase by cytopathic bovine viral diarrhea virus infection.

The Pestivirus bovine viral diarrhea virus (BVDV) causes the fatal diarrheal syndrome, mucosal disease, because of mutations in the viral genome which convert the common noncytopathic (ncp) BVDV into a cytopathic (cp) biotype. We examined the nature of the cytopathic effect of cp-BVDV in cultured bovine cells in order to accurately describe the process and to gain insight into the mechanism of cp-BVDV-induced cell death. The findings demonstrate that cells infected with cp-BVDV in vitro die by apoptosis, but cells infected with ncp-BVDV do not. Analysis of nuclear morphology by staining with fluorescent DNA dye and cpi-fluorescence microscopy showed chromatin condensation and margination in cells infected with cp-BVDV. Transmission electron microscopy (TEM) confirmed the condensation of chromatin, as well as cell shrinkage and generation of apoptotic bodies. The chromosomal DNA of cells infected with cp-BVDV undergoes fragmentation, generating the typical oligonucleosomal fragments commonly noted during apoptosis. The fragmented DNA was released from the nucleus to the cytoplasm, and eventually to the culture supernatant. Infection with cp-BVDV activates cellular proteases of the ICE family leading to cleavage of poly(ADP-ribose) polymerase (PARP), a nuclear enzyme implicated in genome maintenance. This demonstration that cp-BVDV kills cells by triggering apoptosis suggests the possibility that cp-BVDV is associated with a fatal disease by the acquisition of a new apoptosis-inducing activity. We consider BVDV to be an excellent model system for studies of the biological and medical relevance of apoptosis in viral infections.

Factor VIII:C assay: influence of buffer components used in immunoaffinity chromatography for purification of factor VIII/von Willebrand factor.

A monoclonal antibody purified factor VIII concentrate containing FVIII/vWF complex has been assayed by one-stage clotting (CA) and chromogenic substrate (CSA) methods. The influences of potassium iodide (KI) and albumin in combination with predilution buffers, standards and storage of samples have been examined. These components are compared for their effect on FVIII potencies in final product and in-process controls. FVIII:C purified by immunoaffinity chromatography can not be measured reliably by CA or CSA, because of KI which interfere on the assay. Overall yield of FVIII, efficiency of IAC step and purity of FVIII can be determined by assaying the desalted samples.