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Influenza virus assays based on virus-inducible reporter cell lines.

Li Y, Larrimer A, Curtiss T, Kim J, Jones A, Baird-Tomlinson H, Pekosz A, Olivo PD - Influenza Other Respir Viruses (2009)

Bottom Line: A strategy for influenza A virus-induction of a reporter gene was recently described.Upon infection of the cells with influenza virus the virus-inducible reporter gene segment (VIRGS) is replicated and transcribed by the viral polymerase complex resulting in reporter gene expression.Reporter gene induction occurs after infection with a number of laboratory strains and clinical isolates of influenza virus including several H5N1 strains.

View Article: PubMed Central - PubMed

Affiliation: Diagnostic Hybrids Inc., Athens, OH, USA.

ABSTRACT

Background: Virus-inducible reporter genes have been used as the basis of virus detection and quantitation assays for a number of viruses. A strategy for influenza A virus-induction of a reporter gene was recently described. In this report, we describe the extension of this strategy to influenza B virus, the generation of stable cell lines with influenza A and B virus-inducible reporter genes, and the use of these cells in various clinically relevant viral assays. Each of the cell lines described herein constitutively express an RNA transcript that contains a reporter gene coding region flanked by viral 5¢- and 3¢-untranslated regions (UTR) and therefore mimics an influenza virus genomic segment. Upon infection of the cells with influenza virus the virus-inducible reporter gene segment (VIRGS) is replicated and transcribed by the viral polymerase complex resulting in reporter gene expression.

Findings: Reporter gene induction occurs after infection with a number of laboratory strains and clinical isolates of influenza virus including several H5N1 strains. The induction is dose-dependent and highly specific for influenza A or influenza B viruses.

Conclusions: These cell lines provide the basis of simple, rapid, and objective assays that involve virus quantitation such as determination of viral titer, assessment of antiviral susceptibility, and determination of antibody neutralization titer. These cell lines could be very useful for influenza virus researchers and vaccine manufacturers.

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Related in: MedlinePlus

 Dose–response of luciferase expression of ELVIRA® Flu A‐luc or ELVIRA® Flu B‐Rluc detecting cell line following infection with either Influenza A or B virus. Cell lines were infected with their corresponding virus and analyzed for luciferase activity 24 hours after infection (RLU/well). Results are the average of triplicates ± SD. (A) Luciferase activity of ELVIRA® Flu A‐luc cell line after infection with 12 different amounts of Influenza A/Wisconsin67/05 from 100–75 000 infectious units. (B) Luciferase activity of ELVIRA® Flu B‐Rluc cell line infected with seven different concentrations of influenza B/Malaysia/2506/04 ranging from 100 to 7500 infectious units.
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f2:  Dose–response of luciferase expression of ELVIRA® Flu A‐luc or ELVIRA® Flu B‐Rluc detecting cell line following infection with either Influenza A or B virus. Cell lines were infected with their corresponding virus and analyzed for luciferase activity 24 hours after infection (RLU/well). Results are the average of triplicates ± SD. (A) Luciferase activity of ELVIRA® Flu A‐luc cell line after infection with 12 different amounts of Influenza A/Wisconsin67/05 from 100–75 000 infectious units. (B) Luciferase activity of ELVIRA® Flu B‐Rluc cell line infected with seven different concentrations of influenza B/Malaysia/2506/04 ranging from 100 to 7500 infectious units.

Mentions: A virus dose–response was performed by infecting the ELVIRA® Flu A‐luc and ELVIRA® Flu B‐Rluc cell lines with various amounts of influenza A and B viruses and measuring luciferase activity after 24 hours (Figure 2). For both cell lines there was a linear dose–response curve with a wide dynamic range. This result suggested that this methodology can be used to accurately titer virus stocks. We next determined the titer of stocks of several influenza A and B virus strains using the ELVIRA® Flu A‐luc and ELVIRA® Flu B‐Rluc cells and determined the titers at the same time with MDCK cells using a TCID50 method. We included in this analysis four strains of highly pathogenic avian (H5N1) influenza A virus which all were able to induce luciferase in the ELVIRA® Flu A‐luc cells (data not shown). As shown in Table 1 the titers generated with both cell lines were very similar.


Influenza virus assays based on virus-inducible reporter cell lines.

Li Y, Larrimer A, Curtiss T, Kim J, Jones A, Baird-Tomlinson H, Pekosz A, Olivo PD - Influenza Other Respir Viruses (2009)

 Dose–response of luciferase expression of ELVIRA® Flu A‐luc or ELVIRA® Flu B‐Rluc detecting cell line following infection with either Influenza A or B virus. Cell lines were infected with their corresponding virus and analyzed for luciferase activity 24 hours after infection (RLU/well). Results are the average of triplicates ± SD. (A) Luciferase activity of ELVIRA® Flu A‐luc cell line after infection with 12 different amounts of Influenza A/Wisconsin67/05 from 100–75 000 infectious units. (B) Luciferase activity of ELVIRA® Flu B‐Rluc cell line infected with seven different concentrations of influenza B/Malaysia/2506/04 ranging from 100 to 7500 infectious units.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4940803&req=5

f2:  Dose–response of luciferase expression of ELVIRA® Flu A‐luc or ELVIRA® Flu B‐Rluc detecting cell line following infection with either Influenza A or B virus. Cell lines were infected with their corresponding virus and analyzed for luciferase activity 24 hours after infection (RLU/well). Results are the average of triplicates ± SD. (A) Luciferase activity of ELVIRA® Flu A‐luc cell line after infection with 12 different amounts of Influenza A/Wisconsin67/05 from 100–75 000 infectious units. (B) Luciferase activity of ELVIRA® Flu B‐Rluc cell line infected with seven different concentrations of influenza B/Malaysia/2506/04 ranging from 100 to 7500 infectious units.
Mentions: A virus dose–response was performed by infecting the ELVIRA® Flu A‐luc and ELVIRA® Flu B‐Rluc cell lines with various amounts of influenza A and B viruses and measuring luciferase activity after 24 hours (Figure 2). For both cell lines there was a linear dose–response curve with a wide dynamic range. This result suggested that this methodology can be used to accurately titer virus stocks. We next determined the titer of stocks of several influenza A and B virus strains using the ELVIRA® Flu A‐luc and ELVIRA® Flu B‐Rluc cells and determined the titers at the same time with MDCK cells using a TCID50 method. We included in this analysis four strains of highly pathogenic avian (H5N1) influenza A virus which all were able to induce luciferase in the ELVIRA® Flu A‐luc cells (data not shown). As shown in Table 1 the titers generated with both cell lines were very similar.

Bottom Line: A strategy for influenza A virus-induction of a reporter gene was recently described.Upon infection of the cells with influenza virus the virus-inducible reporter gene segment (VIRGS) is replicated and transcribed by the viral polymerase complex resulting in reporter gene expression.Reporter gene induction occurs after infection with a number of laboratory strains and clinical isolates of influenza virus including several H5N1 strains.

View Article: PubMed Central - PubMed

Affiliation: Diagnostic Hybrids Inc., Athens, OH, USA.

ABSTRACT

Background: Virus-inducible reporter genes have been used as the basis of virus detection and quantitation assays for a number of viruses. A strategy for influenza A virus-induction of a reporter gene was recently described. In this report, we describe the extension of this strategy to influenza B virus, the generation of stable cell lines with influenza A and B virus-inducible reporter genes, and the use of these cells in various clinically relevant viral assays. Each of the cell lines described herein constitutively express an RNA transcript that contains a reporter gene coding region flanked by viral 5¢- and 3¢-untranslated regions (UTR) and therefore mimics an influenza virus genomic segment. Upon infection of the cells with influenza virus the virus-inducible reporter gene segment (VIRGS) is replicated and transcribed by the viral polymerase complex resulting in reporter gene expression.

Findings: Reporter gene induction occurs after infection with a number of laboratory strains and clinical isolates of influenza virus including several H5N1 strains. The induction is dose-dependent and highly specific for influenza A or influenza B viruses.

Conclusions: These cell lines provide the basis of simple, rapid, and objective assays that involve virus quantitation such as determination of viral titer, assessment of antiviral susceptibility, and determination of antibody neutralization titer. These cell lines could be very useful for influenza virus researchers and vaccine manufacturers.

Show MeSH
Related in: MedlinePlus