Neuraminidase activity provides a practical read-out for a high throughput influenza antiviral screening assay

Abstract

Background: The emergence of influenza strains that are resistant to commonly used antivirals has highlighted the need to develop new compounds that target viral gene products or host mechanisms that are essential for effective virus replication. Existing assays to identify potential antiviral compounds often use high throughput screening assays that target specific viral replication steps. To broaden the search for antivirals, cell-based replication assays can be performed, but these are often labor intensive and have limited throughput.

Results: We have adapted a traditional virus neutralization assay to develop a practical, cell-based, high throughput screening assay. This assay uses viral neuraminidase (NA) as a read-out to quantify influenza replication, thereby offering an assay that is both rapid and sensitive. In addition to identification of inhibitors that target either viral or host factors, the assay allows simultaneous evaluation of drug toxicity. Antiviral activity was demonstrated for a number of known influenza inhibitors including amantadine that targets the M2 ion channel, zanamivir that targets NA, ribavirin that targets IMP dehydrogenase, and bis-indolyl maleimide that targets protein kinase A/C. Amantadine-resistant strains were identified by comparing IC50 with that of the wild-type virus.

Conclusion: Antivirals with specificity for a broad range of targets are easily identified in an accelerated viral inhibition assay that uses NA as a read-out of replication. This assay is suitable for high throughput screening to identify potential antivirals or can be used to identify drug-resistant influenza strains.

Figure 1

NA activity can be used to quantify virus. A/Wisconsin/67/2005 was serially diluted in PBS and 50 μl incubated with an equal volume of 20 μM MU-NANA for 1 hour at 37°C. Stop solution was added before reading fluorescence. The signal to background ratio at each concentration is shown. Under these conditions enzyme activity reaches a plateau with > 5 × 10⁵ TCID₅₀ due to limiting substrate.

Figure 2

NA activity in cell culture wells and in the supernatants of cells infected 20 hr earlier in the presence of different amounts of zanamivir. In this experiment, MDCK cells were infected with 400 TCID₅₀ A/Memphis/14/98 (MOI = 0.01). The inoculated amount of virus is not sufficient to measure NA activity at the selected conditions.

Figure 3

Titration of amantadine against influenza A and B viruses in the AVINA assay. Cells were infected with A/PR/8/34, A/Memphis/14/98 and B/Jiangsu/10/2003 at 0.01 MOI in the presence of serial dilutions of amantadine. The next day, NA activity was measured in the supernatant and IC₅₀ calculated by GraphPad Prism software.

Figure 4

Titration of ribavirin against A/Memphis/14/98 in the AVINA assay. Cells were infected with 0.01 MOI A/Memphis/14/98 in the presence of serial dilutions of ribavirin. The next day, NA activity was measured in the supernatant and IC₅₀ calculated by GraphPad Prism software.

Figure 5

Titration of bis-indolyl maleimide (BIM) against A/Memphis/14/98 in the AVINA assay. Cells were infected with 0.01 MOI A/Memphis/14/98 in the presence of serial dilutions of BIM. The next day, NA activity was measured in the supernatant (shown as relative fluorescence units) and IC₅₀ calculated by GraphPad Prism software. Cell viability was determined by ATPlite assay (shown as relative light units).

Figure 6

Example of an HTS assay. The plate map is shown in the upper panel, with wells set aside for background and virus controls. Each plate also includes known inhibitors, zanamivir and amantadine, at concentrations known to inhibit virus replication. The lower panel shows the results of NA activity for an assay that evaluated the antiviral activity of a panel of ion channel inhibitors. Activity is represented by a range of color; blue representing low relative fluorescence units (RFU), that is, no or little NA activity, and red representing high RFU, that is, a high amount of NA activity.

Figure 7

Identification of amantadine-resistant virus preparations. A/Memphis/14/98 was cultured in the presence of amantadine for one passage (A/Mem/98 p1), two passages (A/Mem/98 p2), or 4 passages (A/Mem/98 p4) in tissue culture. The TCID₅₀ of each virus passage was determined, and 0.02 MOI added to serial dilutions of amantadine in the AVINA assay. The percent inhibition of virus replication is shown at each concentration of amantadine was calculated by: 100 × (average RFU at each amantadine concentration/average RFU in the absence of amantadine).