Our investigation verifies the safety margins of plasma derivatives against a potential transmission of WNV and that the model virus concept is valid for predicting the behavior of closely related viruses.
Biopharmaceuticals are of increasing importance in the treatment of a variety of diseases. A remaining concern associated with their production is the potential introduction of adventitious agents into their manufacturing process, which may compromise the pathogen safety of a product and potentially cause stock-out situations for important medical supplies. To ensure the safety of biological therapeutics, regulatory guidance requires adventitious agent testing (AAT) of the bulk harvest. AAT is a deliberately promiscuous assay procedure which has been developed to indicate, ideally, the presence of any viral contaminant. One of the most important cell lines used in the production of biopharmaceuticals is Chinese hamster ovary (CHO) cells and while viral infections of CHO cells have occurred, a systematic screen of their virus susceptibility has never been published. We investigated the susceptibility of CHO cells to infection by 14 different viruses, including members of 12 families and representatives or the very species that were implicated in previously reported production cell infections. Based on our results, four different infection outcomes were distinguished, based on the possible combinations of the two factors (i) the induction, or not, of a cytopathic effect and (ii) the ability, or not, to replicate in CHO cells. Our results demonstrate that the current AAT is effective for the detection of viruses which are able to replicate in CHO cells. Due to the restricted virus susceptibility of CHO cells and the routine AAT of bulk harvests, our results provide re-assurance for the very high safety margins of CHO cell-derived biopharmaceuticals.
The genome of hepatitis B virus (HBV) encodes two transcriptional activators: the HBx protein and the PreS2-activator large surface protein (LHBs). Both proteins trigger activation of c-Raf-1/MEK kinase cascade. In case of HBx this can be mediated by a PKC-independent and Ras-dependent mechanism, in case of LHBs activation is PKC-dependent and does not require Ras. Selective destruction of either LHBs-or of HBx-specific activation does not result in significant decrease of viral production from transfected HepG2 cells. Simultaneous inhibition of LHBs-and HBx-dependent activation by blocking signaling steps common to both activators, using trans dominant negative c-Raf-1-or MEK-specific inhibitors, abolished HBV gene expression. In accordance with this no HBV propagation was observed after transfection of a mutated HBV genome defective for HBx-and PreS2-activator function. A detailed analysis revealed that the observed inhibition of HBV-propagation is because of a significant reduction of HBV-specific RNA resulting in an inhibition of the de novo synthesis of viral compounds (viral proteins and nucleic acid) and not by blocking secretion or assembly of the virus. Based on these results we conclude that transcriptional-activator function, mediated by the cRaf-1/MEK signaling cascade, is essential for HBV gene expression.
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