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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

 Antiviral susceptibility testing using ELVIRA Flu A‐luc and ELVIRA Flu B‐Rluc cells Bafilomycin (0·78–30 nm) and amantadine (4 –62 μm) were added to ELVIRA® Flu A‐luc cells and incubated at 37°C for 30 minutes and then the cells were infected with influenza A or B virus. Luciferase activity was measured after 24 hours. Data expressed as per cent no‐drug control. (A) Bafilomycin inhibition curve of influenza A virus (A/MHIA0009) ELVIRA® Flu A‐luc cells. (B) Amantadine inhibition curve of influenza A virus (A/MHIA0009) on ELVIRA® Flu A‐luc cells. (C) Bafilomycin inhibition of influenza B virus (B/Malaysia/2506/04) on ELVIRA® Flu B‐Rluc cells. (D) Amantadine inhibition of influenza B virus (B/Malaysia/2506/04) on ELVIRA® Flu B‐Rluc cells.
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f5:  Antiviral susceptibility testing using ELVIRA Flu A‐luc and ELVIRA Flu B‐Rluc cells Bafilomycin (0·78–30 nm) and amantadine (4 –62 μm) were added to ELVIRA® Flu A‐luc cells and incubated at 37°C for 30 minutes and then the cells were infected with influenza A or B virus. Luciferase activity was measured after 24 hours. Data expressed as per cent no‐drug control. (A) Bafilomycin inhibition curve of influenza A virus (A/MHIA0009) ELVIRA® Flu A‐luc cells. (B) Amantadine inhibition curve of influenza A virus (A/MHIA0009) on ELVIRA® Flu A‐luc cells. (C) Bafilomycin inhibition of influenza B virus (B/Malaysia/2506/04) on ELVIRA® Flu B‐Rluc cells. (D) Amantadine inhibition of influenza B virus (B/Malaysia/2506/04) on ELVIRA® Flu B‐Rluc cells.

Mentions: The applicability of this methodology for antiviral susceptibility testing was also evaluated. Figure 5 shows the effect of various concentrations of two known anti‐influenza compounds on virus‐induced luciferase expression. The graphic display of the results shows classic sigmoidal curves and the IC50 determinations calculated using this method were similar to results reported in the literature for amantadine‐sensitive influenza strains.31 Influenza B virus is resistant to amantadine and this is reflected in the lack of reduction of luciferase expression following amantadine treatment of infected ELVIRA® Flu B‐Rluc cells (Figure 5D).


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)

 Antiviral susceptibility testing using ELVIRA Flu A‐luc and ELVIRA Flu B‐Rluc cells Bafilomycin (0·78–30 nm) and amantadine (4 –62 μm) were added to ELVIRA® Flu A‐luc cells and incubated at 37°C for 30 minutes and then the cells were infected with influenza A or B virus. Luciferase activity was measured after 24 hours. Data expressed as per cent no‐drug control. (A) Bafilomycin inhibition curve of influenza A virus (A/MHIA0009) ELVIRA® Flu A‐luc cells. (B) Amantadine inhibition curve of influenza A virus (A/MHIA0009) on ELVIRA® Flu A‐luc cells. (C) Bafilomycin inhibition of influenza B virus (B/Malaysia/2506/04) on ELVIRA® Flu B‐Rluc cells. (D) Amantadine inhibition of influenza B virus (B/Malaysia/2506/04) on ELVIRA® Flu B‐Rluc cells.
© Copyright Policy
Related In: Results  -  Collection

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

f5:  Antiviral susceptibility testing using ELVIRA Flu A‐luc and ELVIRA Flu B‐Rluc cells Bafilomycin (0·78–30 nm) and amantadine (4 –62 μm) were added to ELVIRA® Flu A‐luc cells and incubated at 37°C for 30 minutes and then the cells were infected with influenza A or B virus. Luciferase activity was measured after 24 hours. Data expressed as per cent no‐drug control. (A) Bafilomycin inhibition curve of influenza A virus (A/MHIA0009) ELVIRA® Flu A‐luc cells. (B) Amantadine inhibition curve of influenza A virus (A/MHIA0009) on ELVIRA® Flu A‐luc cells. (C) Bafilomycin inhibition of influenza B virus (B/Malaysia/2506/04) on ELVIRA® Flu B‐Rluc cells. (D) Amantadine inhibition of influenza B virus (B/Malaysia/2506/04) on ELVIRA® Flu B‐Rluc cells.
Mentions: The applicability of this methodology for antiviral susceptibility testing was also evaluated. Figure 5 shows the effect of various concentrations of two known anti‐influenza compounds on virus‐induced luciferase expression. The graphic display of the results shows classic sigmoidal curves and the IC50 determinations calculated using this method were similar to results reported in the literature for amantadine‐sensitive influenza strains.31 Influenza B virus is resistant to amantadine and this is reflected in the lack of reduction of luciferase expression following amantadine treatment of infected ELVIRA® Flu B‐Rluc cells (Figure 5D).

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