Fluorescence-based Neuraminidase Inhibition Assay to Assess the Susceptibility of Influenza Viruses to The Neuraminidase Inhibitor Class of Antivirals

1. Determining the Linear Range of Fluorescent Product 4-MU on a Fluorometer

1. Prepare 10 mL of 6.4 mM 4-MU stock solution by dissolving 11.3 mg of 4-MU in 5 mL of absolute ethanol. Add 5 mL of 0.9% NaCl (w/v) to the stock solution to make 10 mL. Prepare a 640 µM working solution by diluting 1 mL of 6.4 mM 4-MU in 9 mL of 1x assay buffer (33.3 mM 2-(N-morpholino)ethanesulfonic acid (MES) and 4 mM CaCl₂, pH 6.5).
   NOTE: Prepare the 2x assay buffer by adding 13 g of MES and 8 mL of 1 M CaCl₂ to 992 mL of distilled water. Adjust the pH to 6.5 using 10 M of NaOH. Filter the buffer using a sterile cellulose acetate filter of pore size 0.2 µm. Caution! Sodium hydroxide is caustic and may cause chemical burns to the skin and eyes. Ensure that full personal protective equipment is worn.
2. Prepare 5 mL of a range of 4-MU concentrations (i.e., 5 µM, 10 µM, 20 µM, 40 µM, 80 µM, 160 µM, and 320 µM) through a two-fold serial dilution of 640 µM 4-MU using 1x assay buffer.
3. Dispense 50 µL of each serial dilution of 4-MU (i.e., 5 µM, 10 µM, 20 µM, 40 µM, 80 µM, 160 µM, 320 µM and 640 µM) (two wells per dilution) into a clear, 96-well flat-bottom plate and 50 µL of 1x assay buffer into the remaining wells (which serve as blanks to measure background signals).
   NOTE: The final concentrations of 4-MU in the reaction volume (50 µL 4-MU + 50 µL 300 µM MUNANA) are 2.5 µM, 5 µM, 10 µM, 20 µM, 40 µM, 80 µM, 160 µM and 320 µM.
4. Prepare a 2.5 mM MUNANA stock solution by reconstituting 25 mg of MUNANA in 20 mL of distilled water. Mix 0.72 mL of 2.5 mM MUNANA with 5.28 mL of 1x assay buffer to obtain a 300 µM MUNANA working solution (sufficient volume for one plate). Cover the tube containing the 300 µM MUNANA working solution with aluminum foil and keep it on ice unless used immediately. Discard any leftover materials.
   NOTE: The 2.5 mM MUNANA stock solution can be stored at -20 °C for 1 month and must be used within one freeze/thaw cycle. The 4-MU and MUNANA solutions are light sensitive and must be protected from prolonged light exposure.
5. Add 50 µL of 300 µM MUNANA to each well, gently tap to mix, and incubate at 37 °C for 30 min. Cover the plate with a plate sealer to prevent evaporation.
   NOTE: This step is to account for the background fluorescence in the NA inhibition assay.
6. Prepare the stop solution by mixing 11 mL of absolute ethanol with 2.225 mL of 0.824 M NaOH (sufficient volume for one plate).
7. Add 100 µL of stop solution to each well to stop the reaction and gently tap to mix.
8. Read the plate using a fluorometer with an excitation wavelength setting of 355 nm and an emission wavelength setting of 460 nm, as per the manufacturer's instructions.
9. Calculate the average background signal using fluorescence signals from all the wells containing no 4-MU. Subtract this average background signal from each of the wells containing 4-MU and calculate the average signals (RFU) for each 4-MU concentration. Plot a standard curve of RFU against the 4-MU concentration (µM), as shown in Figure 1a; a close-up linear section of the curve is shown in Figure 1b.
10. Visualize the plot of RFU against 4-MU concentration (µM) to determine the linear range and the optimal target signal; the linear range is where the fluorescence signal increases proportionately to the increasing concentrations of the 4-MU on the plot, while the optimal target signal is an arbitrary 4-MU concentration within the linear range.
    NOTE: The linear range and the optimal target signal are fluorometer-specific. For example, the fluorometer at the Melbourne WHOCCRRI has a linear range of 2.5-40 µM 4-MU and an optimal target signal of ~30 µM 4-MU, which corresponds to ~1,500 RFU.

2. Determining the NA Activity of the Viruses

NOTE: Influenza viruses are cultured to sufficient titers in Madin-Darby Canine Kidney (MDCK) cells or embryonated chicken eggs⁸.

1. Dispense 120 µL per well of undiluted cultured influenza viruses into column 1 and 60 µL of 1x assay buffer containing 0.1% NP-40 into the remaining 11 columns of a 96-well, U-bottom plate.
2. Serially perform two-fold dilution of the viruses across the plate (i.e., transfer 60 µL from column 1 to column 2 and so forth, up to column 11) using a multichannel pipette, leaving column 12 as a blank containing only 1x assay buffer.
3. Transfer 50 µL from each of the wells (diluted viruses and blanks) into a clear, 96-well, flat-bottom plate.
   NOTE: It is not necessary to change pipette tips if materials are transferred from column 12 through to column 1.
4. Add 50 µL of 300 µM MUNANA (prepared as per step 1.4) per well and gently tap the plate to mix. Incubate the plate at 37 °C for 1 h. Cover the plate with a plate sealer to prevent evaporation.
5. Add 100 µL of stop solution (prepared as per step 1.6) per well to terminate the reaction and gently tap the plate to mix.
6. Read the plate using a fluorometer.
   1. Use an excitation wavelength setting of 355 nm and an emission wavelength setting of 460 nm.
7. Determine the average background signal based on the fluorescence readings in column 12 and subtract the average background signal from each well. Plot a graph of RFU against virus dilutions.
   NOTE: The background values for 100 µM MUNANA at the WHOCCRRI Melbourne are typically between 50 and 120 RFU, but these will differ depending on the fluorometer being used.
8. View the plot of RFU against virus dilutions to determine the mid-point of the linear section of the curve for each virus (Figure 2). Use the optimal target signal (determined in step 1) as the reference point.
   NOTE: This should correspond with the 4-MU linear range of the fluorometer determined in section 1 and will provide the appropriate concentration of viruses to be used in section 3.

3. Assessing Virus Susceptibility to NA Inhibitors Using the NA Inhibition Assay

1. Prepare master stocks of NA inhibitors at concentrations of 300 µM.
   1. Prepare 300 µM zanamivir (molecular weight, MW = 332.32 g/mol) by dissolving 5.0 mg of zanamivir in 50 mL of 2x assay buffer (66.6 mM MES and 8 mM CaCl₂, pH 6.5).
   2. Prepare 300 µM oseltamivir carboxylate (D-tartrate; MW = 386.44 g/mol) by dissolving 5.8 mg in 50 mL of 2x assay buffer.
   3. Prepare 300 µM peramivir trihydrate (MW = 382.45 g/mol) by dissolving 5.7 mg in 50 mL of 2x assay buffer.
   4. Prepare 300 µM laninamivir (MW = 346.34 g/mol) by dissolving 5.2 mg in 50 mL of 2x assay buffer.
      NOTE: The NA inhibitor master stocks can be stored at -20 °C for 12 months. Check the MW of the NA inhibitors to ensure the correct weights and volumes are used in reconstitution. The oseltamivir carboxylate is the active compound of the prodrug oseltamivir phosphate. Therefore, only the oseltamivir carboxylate should be used in the NA inhibition assay.
2. From the master stocks, prepare working stocks of ten-fold serial dilutions of the NA inhibitors in 50 mL centrifuge tubes at concentrations of 0.03 nM, 0.3 nM, 3 nM, 30 nM, 300 nM, 3,000 nM, and 30,000 nM in 2x assay buffer (66.6 mM MES and 8 mM CaCl₂, pH 6.5); this is for use across multiple assays.
   NOTE: The final concentrations of NA inhibitors in the reaction volume (50 µL of virus dilution + 50 µL of NA inhibitor + 50 µL of 300 µM MUNANA) are 0.01 nM, 0.1 nM, 1 nM, 10 nM, 100 nM, 1,000 nM, and 10,000 nM, respectively. The final concentration does not include the 100 µL of stop solution. Store all NA inhibitors dilutions at 2-8 °C. The expiry date is the same as that of the master stocks.
3. Prepare the virus dilutions in 1x assay buffer containing 0.1% NP-40 surfactant in a 96-deep-well block. Use virus dilutions based on the NA activity assay results derived from section 2. Use a total volume of 2 mL per virus to test four NA inhibitors.
   NOTE: Prepare two wells (1 mL per well) per virus in a 96-deep-well block. Examples of virus dilutions for VIRUS 1, 2, and 3 are shown in Table 1.
4. Dispense the required volume of NA inhibitors (prepared as per step 3.2) into an 8 deep-well reservoir. From there, dispense 50 µL of NA inhibitors at dilutions ranging from 0 nM (2x assay buffer only) to 30,000 nM in rows A to H in a clear, 96-well, flat-bottom plate.
   NOTE: The plate layout is illustrated in Figure 3.
5. Add 50 µL of diluted test viruses per well to columns 1-11 and 50 µL per well of 1x assay buffer only to column 12. Gently tap the plate to mix and incubate at room temperature for 45 min. Cover the plate with a plate sealer to prevent evaporation.
6. Add 50 µL of 300 µM MUNANA (prepared as per step 1.4) per well and gently tap the plate to mix. Incubate the plate at 37 °C for 1 h. Cover the plate with a plate sealer to prevent evaporation.
7. Add 100 µL of stop solution (prepared as per step 1.6) to each well and gently tap the plate to mix.
8. Read the plate using a fluorometer.
   1. Use an excitation wavelength of 355 nm and an emission wavelength of 460 nm, as described previously.

4. Calculation of IC₅₀ Values

NOTE: The JASPR v1.2 is curve-fitting software that enables the calculation of IC₅₀ values. The software was developed by the Influenza Division at the CDC, Atlanta, USA. The software utilizes the equation: V=V_max x(1-([I]/(K_i +[I]))), where V_max is the maximum rate of metabolism, [I] is the inhibitor concentration, V is the response being inhibited, and K_i is the IC₅₀ for the inhibition curve.

1. Copy and paste the raw data outputted by the fluorometer into a spreadsheet in a 12-column plate format (96-well), starting with cell A1.
   NOTE: Raw data from each subsequent plate must be staggered by one empty row. If each virus has been tested against four NA inhibitors, paste the raw data in a set of four (with an empty row between each plate).
2. Open the fitting software and click on the "Experiment" tab. Choose "Alternative 2-drug Fluoro 11 Samples."
3. Click on the "Options" tab and tick "Generate Graphs."
4. Click on the "Options" again and click on the "New Key" tab. Save the "inhibition_key" .csv file in the same folder as the raw data spreadsheet file.
5. In the inhibition_key file, list all sample names below the ID. Each sample name must be unique.
6. 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.
7. Return to the "jaspr v 1.2-Inhibition Curve fitting" window and select the raw data file as the "Experiment File" and inhibition_key as the "Key File." Run the analysis. Save the results in .csv and .pdf formats.
   NOTE: The software will automatically plot an inhibition curve (RFU against NA inhibitor concentrations), as shown in Figure 4. The program also calculates IC₅₀ values for each virus against an individual NA inhibitor (Figure 4). Additionally, JASPR presents the IC₅₀ values and the signal-to-background (S/B) ratio in spreadsheet format. The background values can differ from lab to lab depending on the fluorometer used. At the Melbourne WHOCCRRI, the background values for 100 µM MUNANA range from 50 to 120 RFU. For a reliable IC₅₀ value, an S/B ratio of ≥10 is preferred, although a ratio of less than 10 is still acceptable, particularly for mutant viruses that have very low NA activity.
8. Inspect the IC₅₀ 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.
   NOTE: For viruses that show unusually high IC₅₀ values, the assay should be repeated to confirm the result.