Multiple viral replicon culture systems

Abstract

Methods and compositions are provided for screening candidate antiviral agents using cells containing subgenomic viral replication systems such as replicons and minigenomes. The methods involve the simultaneous assay of more than one subgenomic viral replication system. Compositions useful for these methods are also provided.

1 . A method of screening a candidate antiviral agent for antiviral activity comprising 
 (a) preparing a first cell culture comprising cells containing a first subgenomic viral replication system, and a second cell culture comprising cells containing a second subgenomic viral replication system; then    (b) adding the candidate antiviral agent to each cell culture; then    (c) incubating the cell cultures under conditions and for a time sufficient to detect an antiviral effect by the candidate antiviral agent on the subgenomic viral replication systems; and    (d) determining the effect of the candidate antiviral agent on each viral replication system,    wherein the first subgenomic viral replication system is genetically distinct from the second subgenomic viral replication system.    
     
     
         2 - 10 . (canceled)  
     
     
         11 . The method of  claim 1 , wherein the candidate antiviral agent is a chemical that does not comprise an oligopeptide or an oligonucleotide.  
     
     
         12 . The method of  claim 1 , wherein the candidate antiviral agent comprises an oligopeptide or an oligonucleotide.  
     
     
         13 . The method of  claim 1 , wherein the candidate antiviral agent comprises an oligonucleotide or a polynucleotide.  
     
     
         14 . The method of  claim 1 , wherein the candidate antiviral agent comprises a protein.  
     
     
         15 . The method of  claim 14 , wherein the candidate antiviral agent comprises an antibody binding domain.  
     
     
         16 . The method of  claim 1 , wherein the effect of the candidate antiviral agent on at least one of the subgenomic viral replication systems is determined by quantitation of a portion of the nucleic acid of the at least one subgenomic viral replication system.  
     
     
         17 . The method of  claim 16 , wherein the quantitation is performed by nucleic acid amplification.  
     
     
         18 . The method of  claim 17 , wherein the nucleic acid amplification is by RT-PCR.  
     
     
         19 . The method of  claim 1 , wherein the effect of the antiviral agent on at least one of the subgenomic viral replication systems is determined by quantitation of a reporter gene or assayable portion of a fusion protein that is transcribed along with other viral proteins.  
     
     
         20 . The method of  claim 1 , wherein the effect of the antiviral agent on at least one of the subgenomic viral replication systems is determined by quantitation of a viral protein.  
     
     
         21 . The method of  claim 20 , wherein the viral protein is an enzyme and the quantitation is by assay of the activity of the enzyme.  
     
     
         22 . The method of  claim 1 , wherein at least one of the cell cultures comprises cells wherein the subgenomic viral replication system is stably maintained.  
     
     
         23 . The method of  claim 1 , wherein at least one of the cell cultures comprises cells wherein the subgenomic viral replication system is not stably maintained.  
     
     
         24 . The method of  claim 1 , wherein at least one of the cell cultures comprises primary cells.  
     
     
         25 . The method of  claim 1 , wherein the cell cultures are incubated at least 20 h.  
     
     
         26 . The method of  claim 1 , further comprising at least a third cell culture comprising cells containing a third subgenomic viral replication system, wherein the third cell culture is also subjected to steps (a), (b), (c) and (d), 
 wherein the each subgenomic viral replication system is genetically distinct from every other subgenomic viral replication system.    
     
     
         27 - 69 . (canceled)