Application of an In-Cell Western assay for measurement of influenza A virus replication.

Influenza A pandemics present enormous challenges to modern medicine. To control such pandemics, quantitative assays characterised by rapidity, high sensitivity, and high-throughput are critical in determining the susceptibility of the influenza A virus to antiviral drugs and for screening chemicals that can inhibit viral replication effectively. In the present study, a rapid and quantitative method to determine influenza A virus replication was developed by an In-Cell Western (ICW) assay. This assay was found to be useful for monitoring the kinetics of influenza A virus replication, as viral nucleoprotein production could be correlated to both increasing doses of viral infection and to the lapse of time during viral infection. Compared to other conventional assays, such as TCID(50), quantitative real-time RT-PCR, and the indirect immunofluorescence assay, the ICW assay exhibits high accuracy, reproducibility, and ease of use. The antiviral effect of amantadine and ribavirin can be determined readily by the ICW assay in 96-well formats, providing a means of rapid antiviral drug screening. Thus, the ICW assay can be used for detecting viral replication, quantifying virus production, and assessing drug-susceptibility in high-throughput applications.