# US 6,750,009 B2 · Multiple viral replicon culture systems > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 6,750,009 B2 - Document ID: US6750009B2 - Status: Granted - Kind: B2 - Application number: 10/060,941 - Filed: 2002-01-29 - Published: 2003-08-14 - Granted: 2004-06-15 - Earliest claimed: 2002-01-29 - Assignees: Apath, LLC - Inventors: Julie Dyall; Charles P. Romano; Paul D. Olivo; Robert M. Roth - Web page: https://patentgrep.com/patent/US6750009B2 ## CPC - G01N 33/502: {for testing non-proliferative effects} - G01N 33/5008: {for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics} - G01N 2500/10: involving cells - G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q) ## 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. ## Claims ### Claim 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, and wherein the first and second cell cultures are combined before step (b). ### Claim 2 (depends on 1) The method of claim 1, further comprising, in step (a), preparing a third cell culture comprising cells not containing a subgenomic viral replication system. ### Claim 3 (depends on 1) The method of claim 1, wherein at least one of the subgenomic viral replication systems is a replicon. ### Claim 4 (depends on 1) The method of claim 1, wherein at least one of the subgenomic viral replication systems is a defective genome. ### Claim 5 (depends on 1) The method of claim 1, wherein the cells in the first and second cell cultures are mammalian cells and the first and second subgenomic viral replication systems are from mammalian viruses. ### Claim 6 (depends on 5) The method of claim 5, wherein the mammalian cells are human cells and the mammalian viruses are human viruses. ### Claim 7 (depends on 6) The method of claim 6, wherein the human viruses are selected from the group consisting of hepatitis C virus, yellow fever virus, respiratory syncytial virus, Sindbis virus, poliovirus, Japanese encephalitis virus, hepatitis B virus, human papilloma virus, herpes simplex virus type 1, Epstein-Barr virus, adeno-associated virus, Venezuela encephalitis virus, rubella, coxsackevirus, enterovirus, hepatitis A virus, Dengue fever virus, West Nile virus, tick-borne encephalitis virus, astrovirus, rabies virus, influenza virus A, influenza virus B, measles, mumps, Ebola virus, Marburg virus, La Crosse virus, California encephalitis virus, Hantaan virus, Crimean-Congo virus, Rift Valley fever, Lassa fever, Argentine hemorrhagic fever virus, Bolivian hemorrhagic fever virus, Colorado tick fever, JC virus, BK virus, herpes simplex virus type two, human cytomegalovirus, varicella-zoster virus, human herpes simplex virus type six, human herpes virus type seven, human herpes virus type eight, human adenovirus, HIV-1, HIV-2, HTLV-1, HTLV-2, and human parovirus. ### Claim 8 (depends on 6) The method of claim 6, wherein the human viruses are selected from the group consisting of hepatitis C virus, yellow fever virus, respiratory syncytial virus, Sindbis virus, poliovirus, Japanese encephalitis virus, hepatitis B virus, human papilloma virus, herpes simplex virus type 1, Epstein-Barr virus, and adeno-associated virus. ### Claim 9 (depends on 6) The method of claim 6, wherein the human viruses are selected from the group consisting of hepatitis C virus, respiratory syncytial virus, yellow fever virus and Sindbis virus. ### Claim 10 (depends on 1) 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 each subgenomic viral replication system is genetically distinct from every other subgenomic viral replication system. ### Claim 11 (depends on 10) The method of claim 10, wherein all cell cultures comprising a subgenomic viral replication system are combined before step (b). ### Claim 12 A mixed cell culture comprising 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. ### Claim 13 (depends on 12) The mixed cell culture of claim 12, wherein at least one of the subgenomic viral replication systems is a replicon. ### Claim 14 (depends on 12) The mixed cell culture of claim 12, wherein at least one of the subgenomic viral replication systems is a defective genome. ### Claim 15 (depends on 12) The mixed cell culture of claim 12, wherein all of the cells of the mixed cell culture are the same cell line. ### Claim 16 (depends on 12) The mixed cell culture of claim 12, wherein the cells of the mixed cell culture comprise more than one cell line. ### Claim 17 (depends on 12) The mixed cell culture of claim 12, wherein all of the cells in the mixed cell culture are mammalian cells and all of the subgenomic viral replication systems are from mammalian viruses. ### Claim 18 (depends on 17) The mixed cell culture of claim 17, wherein the mammalian cells are human cells and the mammalian viruses are human viruses. ### Claim 19 (depends on 18) The mixed cell culture of claim 18, wherein the human viruses are selected from the group consisting of hepatitis C virus, yellow fever virus, respiratory syncytial virus, Sindbis virus, poliovirus, Japanese encephalitis virus, hepatitis B virus, human papilloma virus, herpes simplex virus type 1, Epstein-Barr virus, adeno-associated virus, Venezuela encephalitis virus, rubella, coxsackievirus, enterovirus, hepatitis A virus, Dengue fever virus, West Nile virus, tick-borne encephalitis virus, astrovirus, rabies virus, influenza virus A, influenza virus B, measles, mumps, Ebola virus, Marburg virus, La Crosse virus, California encephalitis virus, Hantaan virus, Crimean-Congo virus, Rift Valley fever, Lassa fever, Argentine hemorrhagic fever virus, Bolivian hemorrhagic fever virus, Colorado tick fever, JC virus, BK virus, herpes simplex virus type two, human cytomegalovirus, varicella-zoster virus, human herpes simplex virus type six, human herpes virus type seven, human herpes virus type eight, human adenovirus, HIV-1, HIV-2, HTLV-1, HTLV-2, and human parvovirus. ### Claim 20 (depends on 18) The mixed cell culture of claim 18, wherein the human viruses are selected from the group consisting of hepatitis C virus, yellow fever virus, respiratory syncytial virus, Sindbis virus, poliovirus, Japanese encephalitis virus, hepatitis B virus, human papilloma virus, herpes simplex virus type 1, Epstein-Barr virus, and adeno-associated virus. ### Claim 21 (depends on 18) The mixed cell culture of claim 18, wherein the human viruses are selected from the group consisting of hepatitis C virus, respiratory syncytial virus, yellow fever virus and Sindbis virus. ### Claim 22 (depends on 12) The mixed cell culture of claim 12, further comprising a third cell culture comprising cells containing a third subgenomic viral replication system. ### Claim 23 (depends on 22) The mixed cell culture of claim 22, wherein the mixed cell culture further comprises a fourth cell culture comprising cells containing a fourth subgenomic viral replication system.