C - Chemistry – Metallurgy – 07 – K
Patent
C - Chemistry, Metallurgy
07
K
C07K 14/14 (2006.01) A61K 39/15 (2006.01) C07K 14/18 (2006.01) C07K 16/10 (2006.01) C12N 9/50 (2006.01) A61K 38/00 (2006.01) A61K 48/00 (2006.01)
Patent
CA 2383607
Bovine viral diarrhea virus (BVDV), which belongs to the Pestivirus genus of the Flaviviridae family, is one of the most important pathogen in cattle. BVDV strains exist as two biotypes, cytopathogenic (cp) and noncytopathogenic (ncp), according to their effects on tissue culture cells. It has been previously reported that cell death associated to cp BVDV in vitro is mediated by apoptosis. In contrast to the situations described for other members of the Flaviviridae family (for instance, classical swine fever virus), only cp strains of BVDV express the p80 (also designated NS3 according to internationnaly recognized nomenclature) protein in virus-infected cells in vitro, suggesting its role in the cytophatogenicity of the virus. In order to determine whether the BVDV p80 is a key-factor invoved in the induction of cell apoptosis, the p80- and p80.DELTA.50 (which is the p80 deleted from the NH2-terminal 50 amico acids)-cDNA encoding sequences of BVDV NADL, cp strain were cloned into AdTR5-DC-GFPq transfer vector for the generation of recombinant adenoviruses (rec-adenovirus) from which the BVDV gene of interest could be expressed from a tetracycline-responsive promoter in a dicistronic means coexpressing the green fluorescent protein (GFP). A549 (human lung carcinoma cells) tTA (A549tTA) infected in vitro with p80 or p80.DELTA.50-expressing rec- adenoviruses showed cytopathogenic changes (e.g cell rounding and detachment, and nucleus chromatin condensation) beginning as early as 18 to 24 h post infection (pi) with the p80 or p80.DELTA.50-expressing rec- Adenovirus. DNA fragmentation assays (oligonucleosomal DNA ladder formation on agarose gel and TUNEL assay) performed on these infected cells clearly correlated the observed cytopathogenic changes with apoptosis. Moreover, we were able to correlate the BVDV p80 or 80.DELTA.50-induced apoptosis process with the activation of cellular proteases of the ICE family (caspases), as determined by cleavage of the death substrate poly(ADP-ribose) polymerase (PARP). This result suggests that the BVDV p80 or p80.DELTA.50 induced apoptosis through the caspase activation pathway. Together with the flow cytometry results on the DNA content of infected cells to quantitate apoptotic cells, the results of our study have shown, for the first time ever, that the p80 of BVDV is an inducer of cell apoptosis in vitro, and should be a determinant factor in the cytophatogenicity of cp BVDV in vivo. Moreover, the results have also shown that the 50 amino acids located at the NH2-terminal of the protein are not involved in BVDV p80- induced apoptosis, thereby identifying the BVDV p80.DELTA.50 as also a potent inducer of apoptosis.
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Universite Du Quebec A. Montreal
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