Influenza virus infection presents an ongoing challenge.
Influenza viruses are of major public health importance due to their ongoing transmission in the population and their ability to cause severe illness and mortality. Large and diverse natural reservoirs of influenza A subtypes exist in wild birds with pandemic-causing potential representing an ongoing threat for zoonotic outbreaks. Influenza viruses can also mutate quite rapidly via antigenic shift or antigenic drift, reducing vaccine effectiveness. This means that antiviral drugs, such as M2 blockers, neuraminidase inhibitors, and polymerase acidic inhibitors, continue to play a very important role in the control of influenza infection.
Determining pimodivir efficacy against influenza variants
In the present study, researchers at the Centers for Disease Control in Atlanta, GA, sought to establish a methodology for monitoring the development of resistance to pimodivir, a polymerase basic protein (PB2) inhibitor, among diverse circulating influenza A viruses. MDCK-SIAT1 cells were infected with the virus and treated with serially diluted pimodivir (0.05–1000 nM) for either a focus-reduction assay (FRA) or a high-content imaging neutralization (HINT) assay. In both cases, infected cells were visualized with an antibody against the viral nucleoprotein (NP) followed by incubation with a fluorophore-conjugated secondary antibody. Hoechst was used to visualize the nuclei and give accurate total cell counts and high-throughput imagers, such as the Celigo, were used to quantify the infected cell population. Pimodivir 50% inhibitory concentration (IC50) values were automatically calculated by curve-fitting analysis.
Accurate and reliable quantification of viral infection
When screening for novel drugs and accessing drug effectiveness against viruses, automated high-throughput imaging with the Celigo Image Cytometer can have significant advantages in speed, accuracy, and cost over other methods. The instrument scans the entire well of each plate in under 15 minutes and directly enumerates the infected cells, labeled with a fluorescent antibody against a viral protein, or directly expressing a fluorescent protein, giving an absolute measure of the infectious viral titer. Total cell numbers can be determined with DAPI or Hoechst nuclear staining, or by direct cell counting in brightfield (Figure 1).
Figure 1: Representative viral titer experiment. Total cells are counted in brightfield, virus-infected cells (GFP+) are quantified in the green channel. The Celigo software can calculate the percentage of infected cells by comparing total cells in brightfield vs. cells counted in the green channel.
The capabilities of the Celigo extend beyond the quantification of total uninfected vs. infected cells. Other routine assays in virology, such as plaque/foci, cytopathic effect, and microneutralization assays can be conducted on the instrument with the software providing rapid and consistent data output (Figure 2).
Figure 2. Other assays in virology. The Celigo can rapidly quantify colorimetric plaques (top panel) or morphological changes in infected cells (cytopathic effect, bottom panel).
The Celigo can also be linked with our Nexcelom stacker or other third-party automation platforms to greatly expand throughput and provide true walk-away automation (Figure 3).
Figure 3: Example of an automation workflow for a colorimetric foci assay on Celigo.
The Celigo is a robust platform that completely automates and modernizes traditional virology assays. The F-Theta lens technology provides high-throughput, quality images of the entire well surface proving very suitable for virology assays, among other cell-based assays. Easy to use software with advanced image analysis algorithms provide quantitative data during image acquisition, which can be performed in one click. Finally, connecting with our Nexcelom stacker provides ultra-high-throughput for projects requiring the scanning of many plates. Contact us today for more information or a demonstration!