Influenza virus assays based on virus-inducible reporter cell lines

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.

Figure 1

Time course of luciferase expression of ELVIRA^® Flu A‐luc or Flu B‐Rluc cells following infection with influenza viruses. ELVIRA^® Flu A‐luc or Flu B‐Rluc cells were infected with influenza A/Wisconsin67/05 or influenza B/Malaysia2506/04 at the indicated MOI. Luciferase activity was measured at various time points after virus infection and expressed as RLU/well. Results shown are mean of 3 replicates. (A) Luciferase activity of ELVIRA^® Flu A‐luc cells infected with influenza A/Wisconsin67/05. The insert shows data from the early time points. (B) Luciferase activity of ELVIRA^® Flu B‐Rluc cells infected with influenza B Malaysia2506/04.

Figure 2

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.

Figure 3

Photomicrographs (100×) showing GFP expression from ELVIRA^® Flu‐GFP cell lines infected with influenza A or B viruses. GFP‐expressing cells were detected by fluorescent microscopy at 24 or 48 hours after infection. (A) The ELVIRA^® Flu A‐GFP cell line was infected with influenza A virus strains A/Wisconsin/67/05 and A/MHIA0009, and influenza B virus strains B/Maryland/1/59 (ATCC VR‐296) and B/Massachusetts (ATCC VR‐523) at 1 MOI. Photomicrographs were taken at 24 hours after infection. (B) The ELVIRA^® Flu B‐GFP cell line was infected with influenza B virus B/ODH0149 and B/GL/1739/54 at 0·5 MOI and influenza A virus influenza A virus A/MIHA0009 and A/PortChalmers/1/73(H3N2) at 1 MOI. Photomicrographs were taken at 48 hours after infection.

Figure 4

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.

Figure 5

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.