Limits...
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.

Show MeSH

Related in: MedlinePlus

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

License
getmorefigures.php?uid=PMC2562994&req=5

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.

Mentions: The primary inhibitors used to determine assay conditions suitable to identify antivirals were amantadine, an inhibitor of M2 ion channel activity [22] and zanamivir, an inhibitor of NA activity [23]. We showed inhibition by zanamivir and amantadine when cells were infected with influenza at a MOI between 0.01 and 0.1 for 16–24 hr. After incubation with virus, consistent results were obtained when the substrate was added to either the original MDCK-containing wells or the supernatants from infected cells. Figure. 2 shows data from an experiment that titrated zanamivir against A/Memphis/14/98. As expected, NA activity in cells with residual supernatant was greater than a small volume of supernatant alone (in the absence of inhibitor, relative fluorescence units (RFU) was 300,000 and 250,000 respectively). This difference reflects the additional activity of NA that is expressed on the surface of infected cells.


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)

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

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

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.
Mentions: The primary inhibitors used to determine assay conditions suitable to identify antivirals were amantadine, an inhibitor of M2 ion channel activity [22] and zanamivir, an inhibitor of NA activity [23]. We showed inhibition by zanamivir and amantadine when cells were infected with influenza at a MOI between 0.01 and 0.1 for 16–24 hr. After incubation with virus, consistent results were obtained when the substrate was added to either the original MDCK-containing wells or the supernatants from infected cells. Figure. 2 shows data from an experiment that titrated zanamivir against A/Memphis/14/98. As expected, NA activity in cells with residual supernatant was greater than a small volume of supernatant alone (in the absence of inhibitor, relative fluorescence units (RFU) was 300,000 and 250,000 respectively). This difference reflects the additional activity of NA that is expressed on the surface of infected cells.

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