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

Eichelberger MC, Hassantoufighi A, Wu M, Li M - Virol. J. (2008)

Bottom Line: 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.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.This assay is suitable for high throughput screening to identify potential antivirals or can be used to identify drug-resistant influenza strains.

View Article: PubMed Central - HTML - PubMed

Affiliation: Center for Biologics Evaluation and Research, Food and Drug Administration, Bethesda, MD, USA. Maryna.Eichelberger@fda.hhs.gov

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.

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Related in: MedlinePlus

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.
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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.

Mentions: Final assay conditions were used in a blind screen of an ion channel inhibitor panel. The assay includes negative (quadruplicate wells that contained no inhibitor), and positive control wells (duplicate wells with 330 nM zanamivir or 33 μM amantadine), as shown in Figure 6. The results identified amantadine (Figure 6, well A5) and an amantadine derivative (Figure 6, well D6) as inhibitors of A/Memphis/14/98.


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

Eichelberger MC, Hassantoufighi A, Wu M, Li M - Virol. J. (2008)

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.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2562994&req=5

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.
Mentions: Final assay conditions were used in a blind screen of an ion channel inhibitor panel. The assay includes negative (quadruplicate wells that contained no inhibitor), and positive control wells (duplicate wells with 330 nM zanamivir or 33 μM amantadine), as shown in Figure 6. The results identified amantadine (Figure 6, well A5) and an amantadine derivative (Figure 6, well D6) as inhibitors of A/Memphis/14/98.

Bottom Line: 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.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.This assay is suitable for high throughput screening to identify potential antivirals or can be used to identify drug-resistant influenza strains.

View Article: PubMed Central - HTML - PubMed

Affiliation: Center for Biologics Evaluation and Research, Food and Drug Administration, Bethesda, MD, USA. Maryna.Eichelberger@fda.hhs.gov

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.

Show MeSH
Related in: MedlinePlus