Patent ID: 6699657
Filing Date: 2004-03-02
Classification: C12N,C12Q,Y02A

Abstract:
An in vitro method for measuring the replication of a genomic sequence of a virus that is dependent for replication upon RNA-dependent RNA polymerase (an RDRP virus) comprising the steps of:a) culturing virally-compatible eukaryotic cells which have been transfected with the cDNA of the genomic sequence of said RDRP virus; b) transfecting said cultured cells with a construct comprising a cDNA, in antisense orientation, of a reporter gene sequence wherein said reporter gene cDNA sequence is operably linked on its 5â€² end with cDNA of the untranslated region (UTR) comprising an HCV IRES element and a HCV replication element, in antisense orientation, of the native 3â€² end of said RDRP virus and is operably linked on its 3â€² end with the cDNA of the UTR, in antisense orientation, of the native 5â€² end of said RDRP virus wherein the construct further comprises the cDNA, in the sense orientation, of a delta ribozyme operably linked to the 3â€² end of the 5â€²UTR sequence; c) culturing said cells for a sufficient period of time under conditions which are permissive for replication of said RDRP virus; and d) analyzing the cells for the presence of the protein encoded by the reporter gene sequence, whereby detection of said protein provides a means to measure the genomic replication of said RDRP virus.