GENETICALLY ENGINEERED CELL LINES EXPRESSING INFLUENZA VIRUS-INDUCIBLE REPORTER GENES AS TOOLS FOR DETECTING AND QUANTIFYING INFLUENZA A OR B VIRUS

Objectives: To develop genetically engineered human cell lines for detecting various strains of influenza A (Flu A) or B (Flu B) viruses. To use these cell lines as a tool for screening anti-influenza drugs, for antibody neutralization studies, and for virus titration.

Methods: 293T cells were engineered to express (i) the firefly luciferase gene under the control of the untranslated region (UTR) of the influenza A virus nucleoprotein (NP) RNA segment, as described in Lutz et al. 2005 JVM 126:13 or (ii) the Renilla luciferase gene under the control of the UTR of the influenza B virus NP RNA segment. The RNA transcript is expressed via a human RNA polymerase I promoter/terminator cassette.  As a result, the expression of each reporter gene is inducible by, respectively, Flu A or Flu B.

Results: The Flu A or B cell lines were able to detect many of the laboratory adapted strains or clinical isolates of Flu A or Flu B viruses tested, respectively. Interestingly, Flu B viruses did not induce the expression of luciferase in the Flu A cell line; however, certain strains of Flu A virus did minimally trigger the expression of the reporter gene in the Flu B cell line.  More importantly, the Flu A and B cell lines did not show cross-reactivity with other respiratory viruses, such as Parainfluenza or RSV, as shown by their inability to activate luciferase expression. Virus dose-dependent activity of the reporter genes was detected as early as 8 hrs post-infection with the Flu A virus and 12 hours with the Flu B virus.  Both cell lines showed high Flu A or Flu B detection sensitivity and were able to detect an infection with an MOI as low as 0.005.  Finally, these cells were successfully used to quantify Flu A and B viruses for establishing TCID₅₀ of antibody neutralization titers of human donors and IC₅₀ for the antiviral drugs.

Conclusions: We have developed novel and sensitive Flu A and B detecting cell lines and have demonstrated their utility in drug screening and antibody neutralization assays. Further studies are being conducted to demonstrate the utility of these cell lines as a faster and more efficient (more sensitive and reliable) method for titering the influenza A and B viruses, including H5N1, when compared with current methods (e.g., CPE, MTT, EIA, or plaque assays).