Virus dilutions for the NI assay

One NAI Two NAIs Three NAIs

Virus dilution Virus (ml) Buffer (ml) Virus (ml) Buffer (ml) Virus (ml) Buffer (ml)

1:5 80 320 160 640 240 960

1:10 40 360 80 720 120 1,080

1:20 20 380 40 760 60 1,140

1:40 10 390 20 780 30 1,170

a When making virus dilutions, it is not advisable to make dilutions greater than 1:600 or pipette volumes less than 2 ml into 1 ml (or greater volume) due to possible pipetting error. If a dilution greater than 1:600 is needed, then a two-step dilution should be performed. For example, if a 1:1,000 dilution is required, first make a 1:100 dilution followed by a 1:10 dilution of the initial 1:100 dilution

It is good laboratory practice to run the NI assay in duplicate, to reduce testing errors. The settings of curve-fitting software such as JASPR require that NI assay testing of different NAIs be conducted on separate 96-well plates.

1. Aliquot the respective diluted NAIs (see Subheading 3.1, steps 2-4) and NA-Star® Buffer into 96-well plates as follows:

• To the wells in column 12, add 50 ml NA-Star® Buffer as a blank (background) control.

• To the wells in column 11, add 25 ml of NA-Star® Buffer. This is a control for uninhibited (100%) NA activity. The activity of this well also will be used to determine the signal to background (S/B) for each virus tested in the NI assay. This helps ensure that the appropriate amount of virus was used and that the NA activity is within linear range.

• To the wells of columns 1-10, add 25 ml of the corresponding drug dilution. Add the drug starting with the lowest concentration in column 10 through to the highest concentration in column 1. The same tips can be used if adding from lowest to highest drug concentrations.

2. Add diluted viruses to the plates containing NAI as follows:

• Using an 8-channel pipette, add 25 ml of the diluted viruses to appropriate rows of the 96-well plates containing NAIs, starting at column 11 toward column 1 (from lowest to highest NAI dilution). Change tips between each addition of virus dilution. Following addition of the virus dilutions, tap the plates gently on each side to mix virus and NAIs.

• If using an 8-channel 200 ml electronic repeater pipette, set it to dispense 25 ml 6 times. Aspirate the diluted virus and visually inspect the volume in the pipette tips. Dispense 25 ml of the diluted viruses to columns 11 to column 6, visually inspect tips to ensure that remaining volume is equal for each tip. Change tips, reset the pipetter and aspirate another 150 ml (6 x25 ml) of virus diluted and visually inspect the volume in the pipette tips. Dispense 25 ml of the diluted viruses from columns 5 to column 1 as described above, then discard tips, including the excess volume of virus dilution. It is not critical that pipette tips touch drug solution in the wells while using a repeater pipette when adding diluted virus starting from low-drug concentration. It is better to use technique with tips touching to the side, bottom, or corner of the well while dispensing diluted virus rather than dispensing into the air.

3. Add substrate to the wells containing virus and NAI as follows:

• Using an 8-channel pipette add 10 ml of NA-Star® Substrate (diluted 1:1,000) to the bottom of each well, starting at column 12 toward column 1 (from lowest to highest NAI dilution). Change tips between each addition of substrate.

• If using an 8-channel 200-ml electronic repeater pipette, set it to dispense 10 ml 12 times, then aspirate 120 ml of the substrate. Visually inspect the volume in the pipette tips. Dispense 10 ml of NA-Star substrate to each column, starting from column 12 toward column 1. Ensure that the pipette tips are in the bottom of each well to guarantee correct dispensation of the substrate.

• After the addition of the NA-Star® Substrate, tap the plate gently on each side to mix the virus and NA-Star® Substrate.

4. Incubate the plates at room temperature for 30 min.

5. Set up the plate reader in accordance with the manufacturer's instructions.

6. Following the 30 min incubation, place the 96-well plate containing substrate, virus, and NAI on the plate reader and measure NA activity.

3.6. Data Analysis Susceptibility of the tested virus isolates to NAIs is assessed based on the IC50 value, the drug concentration required to inhibit NA

enzyme activity by 50% which is determined through curve fitting by plotting raw data (RLUs) against drug concentration (nM) (see Note 9).

1. The curve-fitting software, Robosage (in-house, GlaxoSmithKline) generates IC50 values using the equation: y = ymax(1-(x / (K + x))), where V is the maximum rate of metabolism, x is the inhibi-

tor concentration, y is the response being inhibited, and K is the IC®„ for the inhibition curve (i.e., y = 50% V when x = K).

50 max

2. The JASPR software (in-house, CDC), can also be used for curve fitting and IC5 0 calculation purposes. Curve fitting in JASPR is done using the equation: V= Vmax(1 - ([I] / (K® + [I]))), where V is the maximum rate of metabolism, [I] is the max inhibitor concentration, V is the response being inhibited, and K® is the IC50 for the inhibition curve.

3. The JASPR and Robosage software compute comparative IC50 values (see Fig. 2), with JASPR providing a faster and higher throughput method of IC5 0 determination and a more user-friendly format. Attention must be paid to IC5 0 values and curve shapes generated by both these software; the inhibition curves should always be visually inspected to ensure that all data points are on or close to the curve.

4. Notes

1. When diluting the NA-Star® Substrate (see Subheading 3.1, step 1), take note that each 96-well plate used in the NA activity assay (see Subheading 3.3) and chemiluminescent NI assay (see Subheading 3.5) requires approximately 1.2 ml ofNA-Star® Substrate; therefore, appropriate amounts of substrate should be diluted, depending on the number of viruses to be tested. The final concentration of NA-Star® Substrate used in the assay is 1.67 mM. A fresh working dilution of substrate should be prepared daily.

2. When reconstituting NAIs (see Subheading 3.1, step 2), always check the molecular weight (MW) of NAI compounds to ensure that appropriate drug weights and water volumes are used in reconstitution. A reliable molarity calculator should be used to determine appropriate volumes, such as the Tocris Molarity Calculator (36). Additionally, NAIs are also available as prodrug form. Oseltamivir carboxylate is the active compound of the ethyl ester prodrug oseltamivir phosphate (Tamiflu, MW 410.4), therefore, oseltamivir car-boxylate, and not oseltamivir phosphate, should be used in the NI assay.

Fig. 2. Chemiluminescent NA inhibition assay: NA activity inhibition curves and IC50 values for oseltamivir generated with the use of either Robosage (a-c) or JASPR (d-f) softwares: an oseltamivir sensitive A/H1N1 virus (a and d), an oseltamivir-resistant virus with the H275Y mutation in the NA (b and e) and examples of curves with scattered points may lead to inaccurate IC values (c and f).

Fig. 2. Chemiluminescent NA inhibition assay: NA activity inhibition curves and IC50 values for oseltamivir generated with the use of either Robosage (a-c) or JASPR (d-f) softwares: an oseltamivir sensitive A/H1N1 virus (a and d), an oseltamivir-resistant virus with the H275Y mutation in the NA (b and e) and examples of curves with scattered points may lead to inaccurate IC values (c and f).

3. Repeated freeze/thaw of test and reference viruses or extended time at 4°C may alter NA activity for particular viruses, thus the NA activity (see Subheading 3.3) and chemiluminescent NI assays (see Subheading 3.5) should be conducted on the same day.

4. If testing multiple plates (each with eight viruses) in the NA activity (see Subheading 3.3) and chemiluminescent NI assays (see Subheading 3.5), the addition of NA-Star® Substrate needs to be staggered to account for the time needed to dispense NA-Star® Accelerator and read each plate. For example, with the Victor 3V plate reader, it takes approximately 7 min to dispense the accelerator and to read each plate; therefore, the addition of NA-Star® Substrate to each plate should be staggered by approximately 7 min.

5. Chemiluminescence is the imission of light as a result of a chemical reaction that does not involve a change in temperature. NA activity is measured as light emission, which is triggered by the addition of 60 ml of NA-Star® Accelerator into each reaction well. The light emission from each well is recorded 0.1 s after the addition of the NA-Star® Accelerator.

6. When diluting viruses (see Subheading 3.4), if phenol red is present in virus growth media, the lowest dilution of virus that should be used is 1:5 so that the quenching effect caused by the presence of phenol red is minimized.

7. Some laboratories use a dilution calculator spreadsheet for accurately determining virus dilutions (see Subheading 3.4) following NA activity assessment.

8. When diluting viruses (see Subheading 3.4), ifviruses are to be tested with more than one NAI, multiply 400 ml (volume required for 1 NAI) by the number of NAIs to be tested. For example, if testing two NAIs (zanamivir and oseltamivir) carboxylate, prepare 800 ml of the working dilution for each virus to be tested. If testing three NAIs (zanamivir, oseltamivir carboxylate, and peramivir), prepare 1,200 ml of the working dilution for each virus to be tested.

9. The critical parameters measured in the chemiluminescent NI assay (see Subheading 3.6) for each test virus include an IC50 value (drug concentration that reduces NA enzymatic activity by 50%), a fold difference in IC50 (IC50 test virus/IC50 sensitive reference virus), and a signal-to-background (S/B) ratio. A significantly elevated IC5 0 value in the test virus compared to the reference virus is an important indicator of potential NAI resistance.

The IC50 values provide valuable information for detecting resistant viruses as well as for comparing potencies of different NAIs. However, elevated ICi0 values alone are not sufficient criteria for defining NAI resistance and should be combined with the detection of known (e.g., H275Y) or novel molecular markers of resistance in the NA genes by conventional sequencing (17), pyrosequencing (37, 38), or other genotypic assays.

IC50 values may be affected by assay conditions (substrate concentration, buffer, etc.) and they typically vary by influenza types/subtypes for the different NAIs. Therefore, it is essential to establish baseline IC50 values for a particular NAI, NI assay, as well as NA type and subtype. This allows detection of viruses with slightly reduced susceptibility to NAIs (mild outliers). Statistical analysis is typically used to identify such mild outliers, whereas extreme outliers can be easily detected due to a great increase in IC50 values compared with an NAI-sensitive control (17, 25, 34). It is essential to confirm, when possible, that the NA change(s) responsible for the elevated IC50 values of the virus isolate is also present in the matching clinical specimen.

10. Certain problems may arise when performing the chemilumi-nescent NI inhibition assay, such as a lack of or weak NA activity (signal), which may be caused by a variety offactors including (1) using the wrong virus dilution and thereby adding too little virus to the assay, (2) adding too little NA-Star® Substrate to the assay, (3) using reagents that are too cold for the chemistry to be accurately executed, (4) using insufficient incubation times, or (5) not adding NA-Star® Accelerator to the assay. These problems are mainly technical and can be solved by identifying and correcting the error that the operator may have made while performing the assay. This would include rechecking NA activity data and recalculating virus dilutions, repeating NA activity determination if necessary, repeating the NI assay with correct addition of substrate to the bottom of each well, ensuring that all reagents used are at room temperature, correct incubation times are adhered to and enough volume of accelerator is present in the plate reader. A weak signal may also be the result of too low NA content in the virus preparation used in the NI assay, which can be corrected by regrowing the virus isolate and retesting it.

Conversely, an abnormally high signal may also be encountered as a result of using the wrong virus dilution or adding too much NA-Star® Substrate to the assay. This can be remedied by rechecking NA activity data and recalculating virus dilutions, repeating NA activity if necessary or repeating the NI assay with the addition of correct volume of substrate to the bottom of each well.

An abnormally high IC50 value generated in the chemilu-minescent NI assay may result from factors such as (1) adding too little virus to the assay, (2) incorrect concentrations of NAI within the drug dilutions, (3) testing virus samples that contain a mixed infection with another pathogen that has NA activity (e.g., parainfluenza viruses). An abnormally high IC50

values for influenza A viruses could also be encountered when samples contain a mix of influenza A and influenza B viruses. These problems may be solved by repeating the NI assay using sufficient amounts of virus, checking the possible mix of influenza A and B in virus samples by real-time PCR analysis, repeating the assay using correctly prepared drug dilutions and performing real-time PCR analysis with primers specific for the detection of parainfluenza viruses, respectively.

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