The overall goal of this methodology is to assess the susceptibility of influenza A and B viruses to the neuraminidase inhibitors by using a fluorescence-based assay. This method can help determine whether an influenza virus is resistant or sensitive to the neuraminidase inhibitor class of antivirals. The main principle of the assay is to see whether the neuraminidase inhibitor antiviral blocks the action of the neuraminidase enzyme activity.
Prior to antiviral susceptibility testing, the virus must be grown to high viral titer by either passaging in cell cultures such as the MDCK cells or in embryonate chicken eggs. In this demonstration, I will be using live influenza A viruses of the H1N1 and H3N2 subtypes. Beginning with a 96 well U bottom plate, dispense 120 microliters per well of undiluted cultured influenza viruses into column one.
And 60 microliters of one times assay buffer containing 0.1%NP40 into the remaining 11 columns. Using a multi-channel pipette, serially perform two full dilutions across the plate, leaving column 12 as a blank containing only one times assay buffer. Transfer 50 microliters from each of the wells into a clear 96 well flat bottom plate.
Add 50 microliters of 300 micromolar MUNANA per well. And gently tap the plate to mix. Then use a plate sealer to cover the plate to prevent evaporation.
And incubate the plate at 37 degrees celsius for one hour. Add 100 microliters of stop solution per well to terminate the reaction. And gently tap the plate to mix.
Use a fluorometer to read the plate. Then after plotting a graph of RFU against virus dilutions according to the text protocol, determine the appropriate concentration of viruses to assess viral susceptibility to NA inhibitors by using the optimal target signal as a reference point, to select the midpoint of the linear section of the virus NA activity curves. To prepare 300 micromolars in amivir, dissolve five milligrams of zanamivir in 50 milliliters of two-times assay buffer.
Prepare 300 micromolar oseltamivir carboxylate by dissolving 5.8 milligrams of oseltamivir carboxylate in 50 milliliters of two-times assay buffer. Dissolve 5.7 milligrams of peramivir trihydrate in 50 milliliters two times assay buffer for a stock concentration of 300 micromolar. Then prepare 300 micromolar laninamivir by dissolving 5.2 milligrams in 50 milliliters two times assay buffer.
From the master stocks, prepare working stocks of ten-fold dilutions of the NA inhibitors in 50 milliliter centrifuge tubes with the following concentrations. Attention must be given when preparing the series of NA inhibitors'concentrations. Any irregularity in the NA inhibitor concentrations will be evident in the inhibition curves.
Using virus dilutions based on the NA activity as determined earlier in this video, prepare two milliliters of virus dilutions in one-times assay buffer containing 0.1%NP40 surfactant in a 96-deep well block. When diluting the virus, ensure it is added directly into the one-times assay buffer. This is crucial particularly when the virus volume is very low.
Dispense the required volume of NA inhibitors into an eight-deep well reservoir. Then dispense 50 microliters of NA inhibitors at dilutions ranging from zero to 30, 000 nanomolar. Add 50 microliters of diluted test viruses per well to columns one through 11.
And 50 microliters per well of one-times assay buffer to column 12. Gently tap the plate to mix and cover it to prevent evaporation. Then incubate the plate at room temperature for 45 minutes.
Following the incubation, add 50 microliters of 300 micromolar MUNANA to each well. Then gently tap the plate to mix and cover it. Incubate the plate at 37 degrees celsius for one hour.
Add 100 microliters of stop solution to each well. And gently tap the plate to mix it. Then use a fluorometer to read the plate.
To calculate IC50 values, copy and paste the raw data outputted by the fluorometer into a spreadsheet in a 12 column plate format starting with cell A1.Open the fitting software and click on the experiment tab. Choose Alternative 2-drug Fluoro 11 Samples. Click on the options tab and tick Generate Graphs.
Click on options again and select the New Key tab. Then save the inhibition key SEV file in the same folder as the raw data spreadsheet file. In the inhibition_key file, list all sample names below the ID.If four NA inhibitors were tested, insert spaces for two additional rows below oseltamivir and type in peramivir and laninamivir.
Save the changes made to the inhibition_key file. Return to the jasprv1.2 Inhibition Curve Fitting window and select the raw data file as the experiment file. And the inhibition_key as the key file.
Run the analysis. Then, save the results in CSV and PDF formats. Inspect the IS50 values and the curve shapes generated by the software.
All the data points should fall on or close to the curve. If they do not, repeat the NA inhibition assay. As shown in this inhibition curve, the fluorescence against the increasing concentration of NA inhibitor is plotted for an A(H1N1)pdn09 virus, A/Perth/82/2015.
The IC50 is indicated here. A result of normal inhibition, reduced inhibition or highly reduced inhibition is assigned to the test viruses by comparing the IC50 with the median. Median IC50 values should be derived in each laboratory using data generated from testing viruses with normal inhibition.
And example of median IC50 values determined at the WHO Collaborating Centre for Influenza in Melbourne for respective types and subtypes. For influenza A viruses, normal inhibition viruses are those with IC50 values less than 10 fold compared to the reference median IC50 for their respective subtype. Reduced inhibition viruses are those with IC50 values between 10 and 100 fold above the reference median IC50 for their respective subtypes.
Whilst highly reduced inhibition viruses have IC50 values of 100 fold above the reference median IC50. For influenza Bs, normal inhibition viruses have IC50 values less than five fold compared to the reference median IC50. Reduced inhibition viruses fall between five and 50 fold, and HRI viruses have values 50 fold above the reference median IC50.
This table presents a list of NA amino acid substitutions that have been detected over the last five years that confer reduced inhibition to the neuraminidase inhibitors. The fluorescent based NA inhibition assay shown in this video is a highly robust and reproducible assay. After watching this video and reading the protocol, you should have a good understanding of how to determine the sensitivity of influenza viruses to the neuraminidase inhibitor class of antivirals.
