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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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 The effect of neutralizing antibody on influenza A virus‐induced luciferase activity of ELVIRA® Flu A‐luc cells. Naive and immunized mouse sera were heat‐treated at 56°C for 30 minutes and then serial diluted from 1:10 to 1:640 (non‐immune serum) and 1:10 to 1:2560 (immune serum). Influenza A virus (WS/33; H1N1) (5000 infectious units) was incubated 1:1 (vol/vol) with each dilution of the sera for 60 minutes at room temperature. The virus was then used to infect ELVIRA® Flu A‐luc cells and luciferase activity was measured after 24 hours. (A) Data expressed as total RLU per well. (B) Data expressed as a percentage of luciferase activity in virus‐only wells.
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f4:  The effect of neutralizing antibody on influenza A virus‐induced luciferase activity of ELVIRA® Flu A‐luc cells. Naive and immunized mouse sera were heat‐treated at 56°C for 30 minutes and then serial diluted from 1:10 to 1:640 (non‐immune serum) and 1:10 to 1:2560 (immune serum). Influenza A virus (WS/33; H1N1) (5000 infectious units) was incubated 1:1 (vol/vol) with each dilution of the sera for 60 minutes at room temperature. The virus was then used to infect ELVIRA® Flu A‐luc cells and luciferase activity was measured after 24 hours. (A) Data expressed as total RLU per well. (B) Data expressed as a percentage of luciferase activity in virus‐only wells.

Mentions: The results of Figure 2 show that luciferase activity is proportional to the virus inoculum and suggest that this system could be applied to any influenza assay that depends on a quantitative assessment of virus infectivity. We therefore tested the usefulness of this system to titer neutralizing serum. Dilutions of immune or non‐immune mouse sera were added to a fixed amount of influenza virus. As shown in Figure 4 immune serum reduced luciferase activity in a dose‐dependent manner to nearly background levels. We recorded the titer as the reciprocal of the lowest dilution of serum that resulted in at least a 99% reduction in luciferase compared with the virus‐only control. We then compared the use of this assay to a hemagglutination inhibition assay to assess neutralization titer of nine human sera against influenza A virus (A/Wisconsin/56/2005) and five sera against influenza B virus (B/Malaysia2506/04). As shown in Table 3, the titers were comparable, although not identical which is not uncommon when comparing microneutralization to HI.30


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)

 The effect of neutralizing antibody on influenza A virus‐induced luciferase activity of ELVIRA® Flu A‐luc cells. Naive and immunized mouse sera were heat‐treated at 56°C for 30 minutes and then serial diluted from 1:10 to 1:640 (non‐immune serum) and 1:10 to 1:2560 (immune serum). Influenza A virus (WS/33; H1N1) (5000 infectious units) was incubated 1:1 (vol/vol) with each dilution of the sera for 60 minutes at room temperature. The virus was then used to infect ELVIRA® Flu A‐luc cells and luciferase activity was measured after 24 hours. (A) Data expressed as total RLU per well. (B) Data expressed as a percentage of luciferase activity in virus‐only wells.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4940803&req=5

f4:  The effect of neutralizing antibody on influenza A virus‐induced luciferase activity of ELVIRA® Flu A‐luc cells. Naive and immunized mouse sera were heat‐treated at 56°C for 30 minutes and then serial diluted from 1:10 to 1:640 (non‐immune serum) and 1:10 to 1:2560 (immune serum). Influenza A virus (WS/33; H1N1) (5000 infectious units) was incubated 1:1 (vol/vol) with each dilution of the sera for 60 minutes at room temperature. The virus was then used to infect ELVIRA® Flu A‐luc cells and luciferase activity was measured after 24 hours. (A) Data expressed as total RLU per well. (B) Data expressed as a percentage of luciferase activity in virus‐only wells.
Mentions: The results of Figure 2 show that luciferase activity is proportional to the virus inoculum and suggest that this system could be applied to any influenza assay that depends on a quantitative assessment of virus infectivity. We therefore tested the usefulness of this system to titer neutralizing serum. Dilutions of immune or non‐immune mouse sera were added to a fixed amount of influenza virus. As shown in Figure 4 immune serum reduced luciferase activity in a dose‐dependent manner to nearly background levels. We recorded the titer as the reciprocal of the lowest dilution of serum that resulted in at least a 99% reduction in luciferase compared with the virus‐only control. We then compared the use of this assay to a hemagglutination inhibition assay to assess neutralization titer of nine human sera against influenza A virus (A/Wisconsin/56/2005) and five sera against influenza B virus (B/Malaysia2506/04). As shown in Table 3, the titers were comparable, although not identical which is not uncommon when comparing microneutralization to HI.30

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