Detection of negative-strand RNA viruses
AssigneesWashington University, National Institute of Health, Rush-Presbyterian-St. Luke's Medical Center
InventorsPaul D. Olivo, Sondra Schlesinger, Mark E. Peeples, Peter Collins
Life of this patent
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Abstract
A diagnostic assay for detecting a negative-strand RNA virus in a sample and a genetically engineered cell for use in the assay are disclosed. The cell expresses a heterologous DNA-dependent RNA polymerase that synthesizes a minigenome or miniantigenome of the RNA virus from a cDNA template present in the cell. The cell also expresses the nucleocapsid proteins of the negative-strand virus that are necessary for replication of the minigenome or miniantigenome. Infection of the cell by the negative-strand virus results in expression of a reporter gene product encoded by the miniantigenome.
Claims21
A genetically engineered cell for detecting a negative-strand RNA virus, the cell comprising:
- a polynucleotide encoding a DNA-dependent RNA polymerase;
- a cDNA comprising a minigenome and a miniantigenome of the negative-strand RNA virus operably linked to a promoter for the DNA-dependent RNA polymerase, wherein the miniantigenome comprises a nucleotide sequence encoding a reporter gene product, and wherein expression of the reporter gene product is dependent upon the presence of the negative-strand RNA virus;
- and
- one or more nucleotide sequences encoding each of the nucleocapsid proteins of the negative-strand RNA virus which are necessary and sufficient for replication of minigenome RNA or miniantigenome RNA synthesized by the DNA-dependent RNA polymerase.
A method for detecting a negative-strand RNA virus in a sample, the method comprising:
- (a) providing a genetically engineered cell which comprises
- a polynucleotide encoding a DNA-dependent RNA polymerase;
- a cDNA comprising a minigenome and a miniantigenome of the negative-strand RNA virus operably linked to a promoter for the DNA-dependent RNA polymerase, wherein the miniantigenome comprises a nucleotide sequence encoding a reporter gene product, and wherein expression of the reporter gene product is dependent upon the presence of the negative-strand RNA virus;
- and
- one or more nucleotide sequences encoding each of the nucleocapsid proteins of the negative-strand RNA virus which are necessary and sufficient for replication of minigenome RNA or miniantigenome RNA synthesized by the DNA-dependent RNA polymerase;
- (b) culturing the cell for a time sufficient to synthesize the minigenome RNA or the miniantigenome RNA and to express the nucleocapsid proteins;
- (c) incubating the cell with the sample;
- and
- (d) detecting expression of the reporter gene product.
A kit for detecting a negative-strand virus in a sample, the kit comprising:
- a container;
- said container containing a supply of genetically engineered cells which comprise:
- a polynucleotide encoding a DNA-dependent RNA polymerase;
- a cDNA comprising a minigenome and a miniantigenome of the negative-strand RNA virus operably linked to a promoter for the DNA-dependent RNA polymerase, wherein the miniantigenome comprises a nucleotide sequence encoding a reporter gene product, and wherein expression of he reporter gene product is dependent upon the presence of the negative-strand RNA virus;
- and,
- one or more nucleotide sequences encoding each of the nucleocapsid proteins of the negative-strand RNA virus which are necessary and sufficient for replication of minigenome RNA or miniantigenome RNA synthesized by the DNA-dependent RNA polymerase.
Dependencies are inferred from the claim text.