Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Reexamination Certificate
1999-01-19
2001-12-04
Park, Hankyel T. (Department: 1648)
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
Reexamination Certificate
active
06326480
ABSTRACT:
TECHNICAL FIELD
The present invention relates generally to the detection of RNA viruses. The invention is more particularly related to plasmid systems comprising an antisense reporter construct, which may be used to assay positive sense RNA virus replication.
BACKGROUND OF THE INVENTION
Many pathological conditions affecting humans and other animals are caused by infection with an RNA virus. Such viruses may be positive sense viruses (in which the viral genome has the same polarity as viral mRNA and may be directly translated), negative sense viruses (in which the viral genome is the complement of viral mRNA, and must be transcribed prior to translation) or double-stranded RNA viruses. Positive sense viruses include flaviviruses (e.g., hepatitis C virus); togaviruses (e.g., rubella virus, Sindbis virus, eastern and western encephalitis viruses) and picornaviruses (e.g., the enterovirus, polio virus, coxsackievirus, echovirus and rhinovirus).
In order to accurately diagnose and treat conditions caused by positive sense RNA virus infection, assays that are capable of sensitive detection of such viruses are needed. Most such assays focus on detecting the presence of viral nucleic acid and/or antigens. Such systems have the disadvantage that they cannot distinguish between active virus (capable of replication) and inactive virus, and they often lack the sensitivity that is necessary for a clinically reliable assay.
More recently, detection methods that take advantage of unique pathways of viral gene expression have been proposed. Such methods rely on the use of transgenic cell lines in which a virus-specific event triggers the production of a reporter protein. See Olivo,
Clinical Microbiology Reviews
9:321-334, 1996; Park et al.,
Proc. Natl. Acad. Sci. USA
88:5537-5543, 1991; U.S. Pat. Nos. 5,591,579 and 5,418,132. However, it is unclear whether such systems can detect positive sense RNA viruses with the necessary sensitivity.
Accordingly, there is a need in the art for a system that permits the sensitive detection of active positive sense RNA viruses. The present invention fulfills this need and further provides other related advantages.
SUMMARY OF THE INVENTION
Briefly stated, the present invention provides antisense reporter systems for use in detecting active positive sense RNA viruses. Within certain aspects, the present invention provides antisense reporter plasmids, comprising a promoter operably linked to a DNA sequence encoding: (a) a sequence complementary to the 3′ end of a viral genome; (b) a reporter gene in antisense orientation; and (c) a sequence complementary to the 5′ end of the viral genome.
Within related aspects, the present invention provides antisense reporter mRNAs encoding: (a) a sequence complementary to a 3′ end of a viral genome; (b) a reporter gene in antisense orientation; and (c) a sequence complementary to a 5′ end of the viral genome.
Within certain embodiments of the above antisense reporter plasmids and antisense reporter mRNAs, the viral genome is a Hepatitis C virus genome. Reporter genes for use within such systems include, but are not limited to, chloramphenicol acetyl transferase, beta-galactosidase, alkaline phosphatase, green fluorescent protein, human growth factor and luciferase.
The present invention further provides, within other aspects, host cells transformed or transfected with an antisense reporter plasmid or antisense reporter mRNA as described above.
Within further aspects, the present invention provides methods for monitoring the level of RNA virus replication in an in vitro system, comprising the steps of: (a) contacting a cell as described above with a viral culture; and (b) determining a level of reporter gene expression in the cell, relative to a predetermined level in the absence of viral culture; and therefrom determining the level of RNA virus replication in an in vitro system.
Methods are also provided, within further aspects, for determining the effect of an agent on RNA virus replication in an in vitro system, comprising the steps of: (a) contacting a cell as described above with a viral culture and an agent; and (b) determining a level of reporter gene expression in the cell, relative to a predetermined level in the absence of agent; and therefrom determining the effect of an agent on RNA virus replication in an in vitro system.
Within further aspects, methods are provided for determining the presence or absence of an RNA virus in a sample, comprising the steps of: (a) contacting a cell as described above with a sample; and (b) determining a level of reporter gene expression in the cell, relative to a predetermined level in the absence of sample, and therefrom determining the presence or absence of an RNA virus in the sample. Suitable samples include biological samples isolated from a patient.
The present invention further provides, within other aspects, kits for determining the presence or absence of an RNA virus in a sample, comprising: (a) an antisense reporter plasmid as described above; and (b) a supply of reagents for detecting expression of the reporter gene.
Within further aspects, the present invention provides methods for monitoring the effectiveness of a therapy for RNA virus infection, comprising: (a) exposing a patient infected with an RNA virus to a candidate therapy; (b) contacting a sample obtained from the patient with a cell transformed or transfected as described above; and (c) determining a level of expression of the reporter gene, relative to a predetermined level for cells contacted with a second sample obtained from the patient, wherein the second sample was obtained prior to the candidate therapy, and therefrom monitoring the effectiveness of the candidate therapy.
Within further aspects, methods are provided for detecting a drug resistant RNA virus, comprising: (a) exposing a sample obtained from a patient infected with an RNA virus to a drug; (b) contacting the sample with a cell as described above; and (c) determining a level of expression of the reporter gene, relative to a predetermined level for cells contacted with a second sample obtained from the patient, wherein the second sample is not exposed to the drug, and therefrom identifying a drug resistant RNA virus.
These and other aspects of the present invention will become apparent upon reference to the following detailed description and attached drawings. All references disclosed herein are hereby incorporated by reference in their entirety as if each was incorporated individually.
REFERENCES:
patent: 5418132 (1995-05-01), Olivo
patent: 5591579 (1997-01-01), Olivo et al.
Olivo, “Transgenic Cell Lines for Detection of Animal Viruses,”Clinical Microbiology Reviews 9(3): 321-334, 1996.
Olivo et al., “A Cell Line That Expresses a Reporter Gene in Response to Infection by Sindbis Virus: A Prototype for Detection of Positive Strand RNA Viruses,”Virology 198: 381-384, 1994.
Park et al., “Rescue of a foreign gene by Sendai virus,”Proc. Natl. Acad. Sci. USA 88: 5537-5541, 1991.
Barbosa Miguel
Kovelman Robert
Park Hankyel T.
Signal Pharmaceuticals Inc.
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