Patent US5591579A successfully refreshed from Google Patents.
US5591579A
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Indicator cell line for detecting RNA viruses and method therefor
Assignee: Washington University in St. Louis
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Filing: 1993-12-21
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Granted: 1997-01-07
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Expires: 2013-12-21
Abstract
Abstract Cell lines and methods are disclosed for detecting the presence of RNA viruses in a specimen. The cell lines are stably transformed with a DNA molecule that includes a promoter capable of being recognized by the DNA dependent RNA polymerase of the cell capable of directing the transcription of a cDNA of a structurally defective RNA virus genome operably coupled to the promoter. The cDNA contains a structural coding sequence encoding a selected reporter gene product. The RNA molecules transcribed by the DNA dependent RNA polymerase are not capable of causing the translation of the reporter gene in the cell except when an active related virus that provides the necessary trans-acting enzymes to cause the increased replication of the RNA containing the reporter gene which is then translated into the reporter gene product is provided. Methods utilizing the cell lines of this invention to detect RNA viruses in a specimen by incubating the specimen with the cell line and assaying for expression of the reporter gene and a kit containing a supply of the cells and a supply of the reagents necessary for the detection of the reporter gene product are also provided.
Inventors
- 👤 Paul D. Olivo
- 👤 Sondra Schlesinger
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-11-07 13:06
CPC Classifications
C; C07; C07K; C07K14/00; C07K14/005; C12; C12Q; C12Q1/00; C12Q1/68; C12Q1/6897; G; G01; G01N; G01N33/00; G01N33/48; G01N33/50; G01N33/53; G01N33/569; G01N33/56983; C12N; C12N2760/00; C12N2760/00011; C12N2760/18011; C12N2760/18811; C12N2760/18822; G01N2333/00; G01N2333/005; G01N2333/08; G01N2333/085; G01N2333/18; Y; Y10; Y10S; Y10S435/00; Y10S435/975
Claims Analysis
Total Claims: 24
Claim 1
Independent Claim
205 words
A method for the detection of a positive strand RNA virus in a specimen, the method comprising: incubating a mammalian cell stably transformed with a DNA molecule, said DNA molecule containing a cDNA of a structurally-defective positive strand RNA virus genome including a junction region wherein the cDNA is operably linked to a promoter capable of causing transcription of said cDNA into an RNA molecule in a mammalian cell wherein said cDNA contains a structural coding sequence encoding a reporter gene product which is downstream of and under the regulatory control of said junction region and which is sufficiently downstream from said promoter that the reporter gene product is not translated in the absence of an active virus which replicates through an RNA intermediate wherein the transcribed RNA molecule containing the reporter gone is replicated and the reporter gene product is translated only in the presence of said active virus, with a specimen suspected of containing a positive strand RNA virus for a period of time sufficient to permit said RNA virus suspected of being in said specimen to synthesize its transacting enzymes; and detecting the expression of said reporter gene product which indicates the presence of a positive strand RNA virus in said specimen.
Claim 2
Independent Claim
205 words
A method for the detection of a positive strand RNA virus in a specimen, the method comprising: incubating a mammalian cell stably transformed with a DNA molecule, said DNA molecule containing a cDNA of a structurally-defective positive strand RNA virus genome including a junction region wherein the cDNA is operably linked to a promoter capable of causing transcription of said cDNA into an RNA molecule in a mammalian cell wherein said cDNA contains a structural coding sequence encoding a reporter gene product which is downstream of and under the regulatory control of said junction region and which is sufficiently downstream from said promoter that the reporter gene product is not translated in the absence of an active virus which replicates through an RNA intermediate wherein the transcribed RNA molecule containing the reporter gone is replicated and the reporter gene product is translated only in the presence of said active virus, with a specimen suspected of containing a positive strand RNA virus for a period of time sufficient to permit said RNA virus suspected of being in said specimen to synthesize its transacting enzymes; and detecting the expression of said reporter gene product which indicates the presence of a positive strand RNA virus in said specimen.
Claim 3
Dependent Claim
23 words
The method of claim 1 wherein said structural coding sequence encoding a reporter gent product is located immediately 3' of said junction region.
Claim 4
Dependent Claim
24 words
The method of claim 2 wherein said RNA virus is selected from the groups consisting of the alphaviruses, rubella viruses, coronaviruses, astroviruses and caliciviruses.
Claim 5
Dependent Claim
12 words
The method of claim 3 wherein said RNA virus is an alphavirus.
Claim 6
Dependent Claim
29 words
The method of claim 3 wherein said structural coding sequence encoding a reporter gene product encodes an enzyme capable of being detected by a colorimetric, fluorimetric, or luminometric assay.
Claim 7
Dependent Claim
19 words
The method of claim 5 wherein said structural coding sequence encoding a reporter gene product encodes luciferase or β-galactosidase.
Claim 8
Dependent Claim
18 words
The method of claim 6 wherein the detecting step comprises assaying for the presence of luciferase or β-galactosidase.
Claim 9
Dependent Claim
23 words
The method of claim 1 wherein the incubating step comprises incubation of the mammalian cell with a biological fluid obtained from an individual.
Claim 10
Dependent Claim
11 words
The method of claim 1 wherein said mammalian cell is BHKSINLuc2.
Claim 11
Dependent Claim
149 words
A mammalian cell line stably transformed with a DNA molecule which permits the detection of the presence of a positive strand RNA virus in a specimen, wherein said cells comprise: a cDNA of a structurally-defective positive strand RNA virus genome including a junction region wherein the cDNA is operably linked to a promoter capable of causing transcription of said cDNA into an RNA molecule in a mammalian cell wherein said cDNA contains a structural coding sequence encoding a reporter gene product which is downstream of and under the regulatory control of said junction region and which is sufficiently downstream from said promoter that the reporter gene product is not translated in the absence of an active virus which replicates through an RNA intermediate wherein the transcribed RNA molecule containing the reporter gene is replicated and the reporter gene product is translated only in the presence of said active virus.
Claim 12
Dependent Claim
149 words
A mammalian cell line stably transformed with a DNA molecule which permits the detection of the presence of a positive strand RNA virus in a specimen, wherein said cells comprise: a cDNA of a structurally-defective positive strand RNA virus genome including a junction region wherein the cDNA is operably linked to a promoter capable of causing transcription of said cDNA into an RNA molecule in a mammalian cell wherein said cDNA contains a structural coding sequence encoding a reporter gene product which is downstream of and under the regulatory control of said junction region and which is sufficiently downstream from said promoter that the reporter gene product is not translated in the absence of an active virus which replicates through an RNA intermediate wherein the transcribed RNA molecule containing the reporter gene is replicated and the reporter gene product is translated only in the presence of said active virus.
Claim 13
Dependent Claim
24 words
The stably transformed mammalian cell of claim 10 wherein said junction region is upstream of said structural coding sequence encoding the reporter gene product.
Claim 14
Dependent Claim
30 words
The stably transformed mammalian cell of claim 11 wherein said structurally defective RNA virus genome is selected from the group consisting of the alphaviruses, coronaviruses, astroviruses, caliciviruses, and rubella viruses.
Claim 15
Dependent Claim
27 words
The stably transformed mammalian cell of claim 10 wherein said regulatory region is selected from the group consisting of Rous sarcoma virus promoter or SV40 virus promoter.
Claim 16
Dependent Claim
22 words
The stably transformed mammalian cell of claim 13 wherein said structural coding sequence encoding a reporter gene product encodes β-galactosidase or luciferase.
Claim 17
Dependent Claim
14 words
The stably transformed mammalian cell of claim 14 wherein said mammalian cell is BHKSINLuc2.
Claim 18
Dependent Claim
165 words
A kit for conducting assays for the presence of a positive strand RNA virus in a specimen, said kit comprising: a supply of mammalian cells stably transformed with a DNA molecule having a cDNA of a structurally-defective positive strand RNA virus genome including a junction region wherein the cDNA is operably linked to a promoter capable of causing transcription of said cDNA into an RNA molecule in a mammalian cell wherein said cDNA contains a structural coding sequence encoding a reporter gene product which is downstream of and under the regulatory control of said junction region and which is sufficiently downstream from said promoter that the reporter gene product is not translated in the absence of active virus which replicates through an RNA intermediate wherein the transcribed RNA molecule containing the reporter gene is replicated and the reporter gene product is translated only in the presence of said active virus; and a supply of reagents necessary to detect the expression of said reporter gene product.
Claim 19
Dependent Claim
165 words
A kit for conducting assays for the presence of a positive strand RNA virus in a specimen, said kit comprising: a supply of mammalian cells stably transformed with a DNA molecule having a cDNA of a structurally-defective positive strand RNA virus genome including a junction region wherein the cDNA is operably linked to a promoter capable of causing transcription of said cDNA into an RNA molecule in a mammalian cell wherein said cDNA contains a structural coding sequence encoding a reporter gene product which is downstream of and under the regulatory control of said junction region and which is sufficiently downstream from said promoter that the reporter gene product is not translated in the absence of active virus which replicates through an RNA intermediate wherein the transcribed RNA molecule containing the reporter gene is replicated and the reporter gene product is translated only in the presence of said active virus; and a supply of reagents necessary to detect the expression of said reporter gene product.
Claim 20
Dependent Claim
21 words
The kit of claim 16 wherein said junction region is upstream of said structural coding sequence encoding the reporter gene product.
Claim 21
Dependent Claim
27 words
The kit of claim 17 wherein said structurally defective RNA virus genome is selected from the group consisting of the alphaviruses, coronaviruses, astroviruses, caliciviruses and rubella viruses.
Claim 22
Dependent Claim
24 words
The kit of claim 16 wherein said regulatory region is selected from the group consisting of Rous sarcoma virus promoter or SV40 virus promoter.
Claim 23
Dependent Claim
19 words
The kit of claim 19 wherein said structural coding sequence encoding a reporter gene product encodes β-galactosidase or luciferase.
Claim 24
Dependent Claim
11 words
The kit of claim 20 wherein said mammalian cell is BHKSINLuc2.
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