The Melbourne WHO Collaborating Centre for Reference and Research on Influenza is supported by the Australian Government Department of Health.

1. Determining the Linear Range of Fluorescent Product 4-MU on a Fluorometer
<ol>
	<li>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 &#181;M 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 &#181;m. Caution! Sodium hydroxide is caustic and may cause chemical burns to the skin and eyes. Ensure that full personal protective equipment is worn.</li>
	<li>Prepare 5 mL of a range of 4-MU concentrations (i.e., 5 &#181;M, 10 &#181;M, 20 &#181;M, 40 &#181;M, 80 &#181;M, 160 &#181;M, and 320 &#181;M) through a two-fold serial dilution of 640 &#181;M 4-MU using 1x assay buffer.</li>
	<li>Dispense 50 &#181;L of each serial dilution of 4-MU&#160;(i.e., 5 &#181;M, 10 &#181;M, 20 &#181;M, 40 &#181;M, 80 &#181;M, 160 &#181;M, 320 &#181;M and 640 &#181;M) (two wells per dilution) into a clear, 96-well flat-bottom plate and 50 &#181;L 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 &#181;L 4-MU + 50 &#181;L 300 &#181;M MUNANA) are 2.5 &#181;M, 5 &#181;M, 10 &#181;M, 20 &#181;M, 40 &#181;M, 80 &#181;M, 160 &#181;M and 320 &#181;M.</li>
	<li>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 &#181;M MUNANA working solution (sufficient volume for one plate). Cover the tube containing the 300 &#181;M 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 &#176;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.</li>
	<li>Add 50 &#181;L of 300 &#181;M MUNANA to each well, gently tap to mix, and incubate at 37 &#176;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.</li>
	<li>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).</li>
	<li>Add 100 &#181;L of stop solution to each well to stop the reaction and gently tap to mix.</li>
	<li>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&#39;s instructions.</li>
	<li>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 (&#181;M), as shown in Figure 1a; a close-up linear section of the curve is shown in Figure 1b.</li>
	<li>Visualize the plot of RFU against 4-MU concentration (&#181;M) 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 &#181;M 4-MU and an optimal target signal of ~30 &#181;M 4-MU, which corresponds to ~1,500 RFU.</li>
</ol>
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.
<ol>
	<li>Dispense 120 &#181;L per well of undiluted cultured influenza viruses into column 1 and 60 &#181;L of 1x assay buffer containing 0.1% NP-40 into the remaining 11 columns of a 96-well, U-bottom plate.</li>
	<li>Serially perform two-fold dilution of the viruses across the plate (i.e., transfer 60 &#181;L 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.</li>
	<li>Transfer 50 &#181;L 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.</li>
	<li>Add 50 &#181;L of 300 &#181;M MUNANA (prepared as per step 1.4) per well and gently tap the plate to mix. Incubate the plate at 37 &#176;C for 1 h. Cover the plate with a plate sealer to prevent evaporation.</li>
	<li>Add 100 &#181;L of stop solution (prepared as per step 1.6) per well to terminate the reaction and gently tap the plate to mix.</li>
	<li>Read the plate using a fluorometer.
	<ol>
		<li>Use an excitation wavelength setting of 355 nm and an emission wavelength setting of 460 nm.</li>
	</ol>
	</li>
	<li>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 &#181;M MUNANA at the WHOCCRRI Melbourne are typically between 50 and 120 RFU, but these will differ depending on the fluorometer being used.</li>
	<li>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.</li>
</ol>
3. Assessing Virus Susceptibility to NA Inhibitors Using the NA Inhibition Assay
<ol>
	<li>Prepare master stocks of NA inhibitors at concentrations of 300 &#181;M.
	<ol>
		<li>Prepare 300 &#181;M 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).</li>
		<li>Prepare 300 &#181;M oseltamivir carboxylate (D-tartrate; MW = 386.44 g/mol) by dissolving 5.8 mg in 50 mL of 2x assay buffer.</li>
		<li>Prepare 300 &#181;M peramivir trihydrate (MW = 382.45 g/mol) by dissolving 5.7 mg in 50 mL of 2x assay buffer.</li>
		<li>Prepare 300 &#181;M 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 &#176;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.</li>
	</ol>
	</li>
	<li>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 &#181;L of virus dilution + 50 &#181;L of NA inhibitor + 50 &#181;L of 300 &#181;M 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 &#181;L of stop solution. Store all NA inhibitors dilutions at 2-8 &#176;C. The expiry date is the same as that of the master stocks.</li>
	<li>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.</li>
	<li>Dispense the required volume of NA inhibitors (prepared as per step 3.2) into an 8 deep-well reservoir. From there, dispense 50 &#181;L 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.</li>
	<li>Add 50 &#181;L of diluted test viruses per well to columns 1-11 and 50 &#181;L 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.</li>
	<li>Add 50 &#181;L of 300 &#181;M MUNANA (prepared as per step 1.4) per well and gently tap the plate to mix. Incubate the plate at 37 &#176;C for 1 h. Cover the plate with a plate sealer to prevent evaporation.</li>
	<li>Add 100 &#181;L of stop solution (prepared as per step 1.6) to each well and gently tap the plate to mix.</li>
	<li>Read the plate using a fluorometer.
	<ol>
		<li>Use an excitation wavelength of 355 nm and an emission wavelength of 460 nm, as described previously.</li>
	</ol>
	</li>
</ol>
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=Vmax x(1-([I]/(Ki +[I]))), where Vmax is the maximum rate of metabolism, [I] is the inhibitor concentration, V is the response being inhibited, and Ki is the IC50 for the inhibition curve.
<ol>
	<li>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).</li>
	<li>Open the fitting software and click on the &#34;Experiment&#34; tab. Choose &#34;Alternative 2-drug Fluoro 11 Samples.&#34;</li>
	<li>Click on the &#34;Options&#34; tab and tick &#34;Generate Graphs.&#34;</li>
	<li>Click on the &#34;Options&#34; again and click on the &#34;New Key&#34; tab. Save the &#34;inhibition_key&#34; .csv file in the same folder as the raw data spreadsheet file.</li>
	<li>In the inhibition_key file, list all sample names below the ID. Each sample name must be unique.</li>
	<li>If four NA inhibitors were tested, insert spaces for two additional rows below Oseltamivir and type in &#34;Peramivir&#34; and &#34;Laninamivir.&#34; Save the changes made to the inhibition_key file.</li>
	<li>Return to the &#34;jaspr v 1.2-Inhibition Curve fitting&#34; window and select the raw data file as the &#34;Experiment File&#34; and inhibition_key as the &#34;Key File.&#34; 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 &#181;M MUNANA range from 50 to 120 RFU. For a reliable IC50 value, an S/B ratio of &#8805;10 is preferred, although a ratio of less than 10 is still acceptable, particularly for mutant viruses that have very low NA activity.</li>
	<li>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.</li>
</ol>

<ol>
	<li>Moscona, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuraminidase+inhibitors+for+influenza.">Neuraminidase inhibitors for influenza.</a> N.Engl.J.Med. 353 (13), 1363-1373 (2005).</li><li>Hurt, A. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Global+update+on+the+susceptibility+of+human+influenza+viruses+to+neuraminidase+inhibitors.">Global update on the susceptibility of human influenza viruses to neuraminidase inhibitors.</a> Antiviral Res. 132, 178-185 (2016).</li><li>Meijer, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Global+update+on+the+susceptibility+of+human+influenza+viruses+to+neuraminidase+inhibitors.">Global update on the susceptibility of human influenza viruses to neuraminidase inhibitors.</a> Antiviral Res. 110, 31-41 (2014).</li><li>Takashita, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Global+update+on+the+susceptibility+of+human+influenza+viruses+to+neuraminidase+inhibitors.">Global update on the susceptibility of human influenza viruses to neuraminidase inhibitors.</a> Antiviral Res. 117, 27-38 (2015).</li><li>Lackenby, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Emergence+of+resistance+to+oseltamivir+among+influenza+A(H1N1)+viruses+in+Europe.">Emergence of resistance to oseltamivir among influenza A(H1N1) viruses in Europe.</a> Euro Surveill. 13 (5), (2008).</li><li>Hurt, A. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Community+transmission+of+oseltamivir-resistant+A(H1N1)pdm09+influenza.">Community transmission of oseltamivir-resistant A(H1N1)pdm09 influenza.</a> N Engl J Med. 365 (26), 2541-2542 (2011).</li><li>Takashita, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+a+large+cluster+of+influenza+A(H1N1)pdm09+viruses+cross-resistant+to+oseltamivir+and+peramivir+during+the+2013-2014+influenza+season+in+Japan.">Characterization of a large cluster of influenza A(H1N1)pdm09 viruses cross-resistant to oseltamivir and peramivir during the 2013-2014 influenza season in Japan.</a> Antimicrob Agents Chemother. 59 (5), 2607-2617 (2015).</li><li>Eisfeld, A. J., Neumann, G., Kawaoka, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Influenza+A+virus+isolation,+culture+and+identification.">Influenza A virus isolation, culture and identification.</a> Nat Protoc. 9 (11), 2663-2681 (2014).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Meetings+of+the+WHO+working+group+on+surveillance+of+influenza+antiviral+susceptibility+-+Geneva,+November+2011+and+June+2012.">Meetings of the WHO working group on surveillance of influenza antiviral susceptibility - Geneva, November 2011 and June 2012.</a> Wkly Epidemiol Rec. 87 (39), 369-374 (2012).</li><li>Okomo-Adhiambo, M., Hurt, A. C., Gubareva, L. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+chemiluminescent+neuraminidase+inhibition+assay:+a+functional+method+for+detection+of+influenza+virus+resistance+to+the+neuraminidase+inhibitors.">The chemiluminescent neuraminidase inhibition assay: a functional method for detection of influenza virus resistance to the neuraminidase inhibitors.</a> Methods Mol Biol. 865, 95-113 (2012).</li><li>Hurt, A. C., Okomo-Adhiambo, M., Gubareva, L. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+fluorescence+neuraminidase+inhibition+assay:+a+functional+method+for+detection+of+influenza+virus+resistance+to+the+neuraminidase+inhibitors.">The fluorescence neuraminidase inhibition assay: a functional method for detection of influenza virus resistance to the neuraminidase inhibitors.</a> Methods Mol Biol. 865, 115-125 (2012).</li><li>Analysis of IC50 data. isirv Antiviral Group (isirv-AVG) Available from: <a target="_blank" href="https://isirv.org/site/index.php/methodology/analysis-of-ic50-data">https://isirv.org/site/index.php/methodology/analysis-of-ic50-data</a> (2016)</li><li>Sleeman, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=R292K+substitution+and+drug+susceptibility+of+influenza+A(H7N9)+viruses.">R292K substitution and drug susceptibility of influenza A(H7N9) viruses.</a> Emerg Infect Dis. 19 (9), 1521-1524 (2013).</li><li>Gubareva, L. V., Robinson, M. J., Bethell, R. C., Webster, R. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Catalytic+and+framework+mutations+in+the+neuraminidase+active+site+of+influenza+viruses+that+are+resistant+to+4-guanidino-Neu5Ac2en.">Catalytic and framework mutations in the neuraminidase active site of influenza viruses that are resistant to 4-guanidino-Neu5Ac2en.</a> J Virol. 71 (5), 3385-3390 (1997).</li></ol>

The authors have nothing to disclose.

The global monitoring of influenza virus susceptibility to NA inhibitors is currently being conducted by a number of laboratories using either fluorescent or chemiluminescent NA inhibition assays11,12. The fluorescent assay is more commonly used than the chemiluminescent assay. Although both assays are robust and reproducible, the IC50 values obtained from the fluorescence-based assay are often higher than the chemiluminescence-based assay, making a direct comparison of the data from the two assays difficult13. Even with the use of the same protocol, data generated from one laboratory may vary from another. Because of these variations between laboratories, the WHO Working Group on Surveillance of Influenza Antiviral Susceptibility produced a guideline to assist in inter-laboratory comparisons. Rather than comparing the absolute IC50 values, this guideline uses a comparison based on the IC50 fold difference to the median IC50 of the NI influenza viruses tested in each particular laboratory. The ability to compare data from the five collaborating centers has resulted in the annual publication of global influenza antiviral susceptibility data2,3,4. The availability of the large amount of influenza susceptibility data in the public domain allows researchers to compare IC50 data from those studies with that generated in their own laboratories.
Other NA inhibition assays that adopt a similar concept are also commercially available. These commercial kits that contain ready-to-use reagents (NA inhibitors not included) are equally reproducible. However, the in-house NA inhibition assay is substantially cheaper than the commercial kits, because the majority of the reagents can be made in-house in larger quantities and the MUNANA substrate, which previously made up the major cost of the assay, can now be purchased from various sources at competitive prices. The cost of testing one influenza isolate per drug is approximately $1 (USD). At the Melbourne WHOCCRRI, improvements have been made to the in-house NA inhibition assay after the incorporation of a robotic platform for the liquid handling components of the assay. Apart from the manual preparation of virus dilutions, the majority of the procedures are performed using the liquid-handling robot. Not only does this minimize manual handling, but it also increases the numbers of assays that can be run in a day.
Although the NA inhibition assay is highly robust, there are a number of critical steps that need to be completed with additional care. First, any irregularity in the NA inhibitor concentrations can shift the inhibition curves and the IC50 values; therefore, careful attention should be paid when preparing the NA inhibitor concentrations. Second, accurate pipetting and precise incubation periods are crucial to maintaining consistent results across assays; this can be achieved by using calibrated pipettes and timers. The inclusion of control viruses in every assay also enables the monitoring of assay performance from assay to assay and over long periods of time. Third, because the NA enzyme activity of seasonal influenza viruses is optimal at pH 6.5, the correct pH of the assay buffer is important. Some reports have found that the use of lower pH conditions may improves the identification of influenza variants, such as the A(H7N9) variant containing the R292K mutation14,15. However, the modification to the pH of the assay buffer will shift the IC50 values, and this may complicate the comparison of data within laboratories and between laboratories. Other modifications and troubleshooting that can be performed are listed in Table 5.
The NA inhibitors are the only class of approved antivirals that are currently effective against circulating influenza viruses. Until other antiviral classes become available for clinical use, the antiviral susceptibility surveillance of circulating influenza viruses will be focused on NA inhibitors alone. Because of the simplicity and reproducibility of results, the use of the NA inhibition assay to assess influenza virus susceptibility to NA inhibitors will continue.

Haemagglutinin (HA) and neuraminidase (NA) are the two major surface glycoproteins of influenza A and B viruses. HA binds to the sialic acid-galactose of cell surface glycoproteins or glycolipids, while the NA releases virus by cleaving the sialic acid from the galactose on the cell surface1. The NA inhibitors are a class of influenza antivirals that were rationally designed to bind tightly to the NA enzymatic active site, thereby preventing the release and spread of virus progeny. Oseltamivir and zanamivir are two NA inhibitors that have been approved in many countries worldwide for the treatment and prophylaxis of influenza. In recent years, two additional NA inhibitors, peramivir and laninamivir, have been approved for use in a limited number of countries. Screening of influenza viruses for susceptibility to NA inhibitors and the identification of mutations that confer resistance are important in determining and monitoring the effectiveness of this class of antivirals.
In the last 16 years, the fluorescence-based NA inhibition assay has been performed routinely at the WHO Collaborating Centre for Reference and Research on Influenza, Melbourne (Melbourne WHOCCRRI) to monitor the changing trend of antiviral susceptibility among circulating influenza viruses. Annually, more than 2,000 influenza viruses are tested for antiviral susceptibility. In most influenza seasons, &#62;98% of the viruses are susceptible to all four NA inhibitors2,3,4, although during the 2007-2008 northern hemisphere influenza season, there was a surge in the number of former seasonal A(H1N1) viruses that had reduced susceptibility to oseltamivir5. This group of viruses, which contained the NA amino acid substitution H275Y, spread to the rest of the world by the end of 2008, making oseltamivir inappropriate globally for the treatment of this virus. The vast majority of currently circulating influenza B, influenza A(H3N2), and influenza A(H1N1)pdm09 strains are susceptible to oseltamivir, although community clusters of A(H1N1)pdm09 variants containing the NA amino acid substitution H275Y that confers reduced oseltamivir and peramivir susceptibility, have been reported in various parts of the world6,7.
Because of the need for a sufficiently high virus titer, clinical specimens (including animal nasal washes) must be passaged in either cell culture or embryonated chicken eggs prior to antiviral susceptibility testing. The NA inhibition assay described in this article can be divided into three sections:
Determining the linear range for the fluorescent product 4-methylumbelliferone (4-MU) on a particular fluorometer 
Due to the inherent differences between fluorometers, the linear range for the fluorescent end-product, 4-MU, and the relative fluorescence unit (RFU), need to be established. Once the linear range for 4-MU is established, the optimal target signal is selected, to which the concentration of the influenza viruses is adjusted in the NA activity assay. Once completed for a particular fluorometer, this should not need to be repeated.
Determining the NA activity of the viruses 
The NA activity assay is a simple assay that involves the addition of the 2&#8242;-(4-Methylumbelliferyl)-&#945;-D-N-acetylneuraminic acid (MUNANA) substrate to serially diluted viruses. The amount of fluorescent end-product 4-MU generated from the cleavage of MUNANA by the NA is measured using a fluorometer. The appropriate virus dilution to use in the NA inhibition assay is selected by plotting fluorescence units against virus dilution. From the sigmoidal curve produced, the mid-point of the linear section should correspond to the 4-MU linear range of the fluorometer determined in section 1 and will inform the appropriate concentration of viruses to be used in section 3.
Assessing virus susceptibility to NA inhibitors using the NA inhibition assay 
To assess the susceptibility of viruses to a particular NA inhibitor, viruses at the dilution determined in section 2 are incubated with a range of NA inhibitor concentrations. Following a subsequent incubation with MUNANA, the 4-MU generated by uninhibited viruses is measured in RFU by the fluorometer. The inhibitory effect of the NA inhibitor on the NA enzyme activity of a virus is calculated according to the NA inhibitor concentration required to reduce 50% of the NA activity, given as an IC50 value.

<table><tbody><tr><td>Influenza A and B viruses</td><td></td><td>Cultured in MDCK cells or 9 day old embryonated specific pathogen free (SPF) eggs</td><td></td></tr><tr><td>Madin-Darby Canine Kidney (MDCK) cells</td><td>ATCC</td><td>PTA-6500</td><td></td></tr><tr><td>2-(4-methylumbelliferyl)-a-D-N-acetylneuraminic acid (MUNANA)&amp;nbsp;</td><td>Biosynth AG</td><td>M-5507</td><td></td></tr><tr><td>2-(4-methylumbelliferyl)-a-D-N-acetylneuraminic acid (MUNANA)&amp;nbsp;</td><td>Sigma</td><td>M8639</td><td></td></tr><tr><td>4-Methylumbelliferone (4-MU)</td><td>Sigma</td><td>M1381-25G</td><td></td></tr><tr><td>2-[N-morpholino]ethanesulphonic acid (MES hydrate) (free acid)&amp;nbsp;</td><td>Sigma</td><td>M8250-250G</td><td></td></tr><tr><td>Calcium Chloride (Ca Cl&lt;sub&gt;2&lt;/sub&gt;)&amp;nbsp;</td><td>APS AJAX Finechem&amp;nbsp;</td><td>127-500G</td><td></td></tr><tr><td>Surfactant-Amps-NP-40 (10% solution)&amp;nbsp;</td><td>Thermo Fisher Scientific&amp;nbsp;</td><td>PIE28324</td><td></td></tr><tr><td>Sodium Hydroxide (NaOH)&amp;nbsp;</td><td>APS AJAX Finechem</td><td>482-2.5KG</td><td></td></tr><tr><td>Absolute Ethanol&amp;nbsp;</td><td>APS AJAX Finechem</td><td>214-2.5L GL</td><td></td></tr><tr><td>96-well clear flat-bottom plates</td><td>NUNC</td><td>456537</td><td></td></tr><tr><td>96-well U-bottom plates</td><td>Greiner Bio-one&amp;nbsp;</td><td>4650101</td><td></td></tr><tr><td>8 channel deep well block</td><td>Pacific Laboratory Products</td><td>RES-MW8-HP</td><td></td></tr><tr><td>96-well deep plates, 2.0 mL square wells</td><td>Pacific Laboratory Products</td><td>P-2ML-SQ-C</td><td></td></tr><tr><td>Plate sealers</td><td>Thermo Fisher Scientific&amp;nbsp;</td><td>236366</td><td></td></tr><tr><td>Bottle-top vacuum filter system (cellulose membrane (nitrate), pore size 0.2 &amp;mu;m, membrane area 33.2 cm&lt;sup&gt;2&lt;/sup&gt;, filter capacity 500 mL)</td><td>Sigma-Aldrich</td><td>CLS430758-12EA</td><td></td></tr><tr><td>Single-channel pipettes (1 &amp;micro;L - 1,000 &amp;micro;L)</td><td>Variety of suppliers (eg. Eppendorf, Sartorius)</td><td></td><td></td></tr><tr><td>Multi-channel pipettes</td><td>Variety of suppliers (eg. Eppendorf, Sartorius)</td><td>8 or 12 channel electronic and manual pipette (5 - 1250 &amp;micro;L volume)</td><td></td></tr><tr><td>Pipette tips (1 &amp;micro;L - 1,250 &amp;micro;L)</td><td>Variety of suppliers (eg. Eppendorf, Sartorius)</td><td></td><td></td></tr><tr><td>Disposable pipettes (10 mL and 25 mL)</td><td>Greiner Bio-one&amp;nbsp;</td><td>P7740-200EA and P7865-200EA</td><td></td></tr><tr><td>Pipette controller</td><td>Eppendorf</td><td>4430000018</td><td></td></tr><tr><td>Centrifuge tubes 50 mL</td><td>BD Bioscience</td><td>352070</td><td></td></tr><tr><td>Racked tubes</td><td>Scientific Specialties, Inc.</td><td>1750-00</td><td></td></tr><tr><td>Fluorometer with excitation wavelength setting of 355 nm and an emission wavelength setting of 460 nm</td><td>TermoFisher Scientific</td><td>ASCENT FL 374</td><td></td></tr><tr><td>Ascent software</td><td>TermoFisher Scientific</td><td>5185410CD</td><td></td></tr><tr><td>Incubator set at 37 &amp;deg;C</td><td>Lab Supply</td><td>Biocell 1000</td><td></td></tr><tr><td>Zanamivir</td><td>GlaxoSmithKline</td><td>Request directly from the company</td><td></td></tr><tr><td>Oseltamivir carboxylate</td><td>Roche</td><td></td><td></td></tr><tr><td>Peramivir (BCX-1812)</td><td>BioCryst</td><td></td><td></td></tr><tr><td>Laninamivir (R-125489)</td><td>Daiichi-Sankyo</td><td></td><td></td></tr><tr><td>&amp;nbsp;JASPR v1.2</td><td>Influenza Division at the CDC Atlanta, USA&amp;nbsp;</td><td>freely available upon request (fluantiviral@cdc.gov)</td><td></td></tr></tbody></table>

fluorescence-based neuraminidase inhibition assay to assess the susceptibility of influenza viruses to the neuraminidase inhibitor class of antivirals

The neuraminidase (NA) inhibitors are the only class of antivirals approved for the treatment and prophylaxis of influenza that are effective against currently circulating strains. In addition to their use in treating seasonal influenza, the NA inhibitors have been stockpiled by a number of countries for use in the event of a pandemic. It is therefore important to monitor the susceptibility of circulating influenza viruses to this class of antivirals. There are different types of assays that can be used to assess the susceptibility of influenza viruses to the NA inhibitors, but the enzyme inhibition assays using either a fluorescent substrate or a chemiluminescent substrate are the most widely used and recommended. This protocol describes the use of a fluorescence-based assay to assess influenza virus susceptibility to NA inhibitors. The assay is based on the NA enzyme cleaving the 2&#8242;-(4-Methylumbelliferyl)-&#945;-D-N-acetylneuraminic acid (MUNANA) substrate to release the fluorescent product 4-methylumbelliferone (4-MU). Therefore, the inhibitory effect of an NA inhibitor on the influenza virus NA is determined based on the concentration of the NA inhibitor that is required to reduce 50% of the NA activity, given as an IC50 value.

Using standardized reporting guidelines from the WHO Working Group on Surveillance of Influenza Antiviral Susceptibility9, the susceptibility of influenza viruses to the NA inhibitors are reported using the terms normal inhibition (NI), reduced inhibition (RI), and highly reduced inhibition (HRI). NI viruses are those with IC50 values less than 10-fold compared to the reference median IC50 for influenza A viruses (or less than 5-fold for influenza B viruses). RI viruses are those with IC50 values between 10- and 100-fold above the reference median IC50 for influenza A viruses (or 5- and 50-fold for influenza B). HRI viruses are those with IC50 values of 100-fold above the reference median IC50 for influenza A viruses (or above 50-fold for influenza B viruses); see Table 2.
The reference median IC50 values for A(H1N1)pdm09, A(H3N2), and B Yamagata/B Victoria viruses are calculated and updated annually at the Melbourne WHOCCRRI to reflect minor changes in the IC50 values of circulating influenza strains to the NA inhibitors (Table 3). The median IC50 values in influenza A(H1N1)pdm09 viruses are almost the same across the four NA inhibitors, but the median zanamivir and laninamivir IC50 values for A(H3N2) viruses are 2- to 4-fold higher compared to oseltamivir and peramivir IC50 values (Table 3). The median oseltamivir IC50 value for influenza B viruses is generally 5- to 10-fold higher than the zanamivir, peramivir, and laninamivir IC50 values (Table 3).
The NA inhibition assay is a phenotypic assay that does not provide information on the genetic changes associated with RI or HRI. Therefore, it is important that genetic analysis is performed following the identification of viruses with RI or HRI. At the Melbourne WHOCCRRI, the NA gene of variants is analyzed using Sanger sequencing and pyro-sequencing. A representative list of amino acid substitutions that can be found in the NA gene of viruses with RI and HRI variants is presented in Table 4. A more extensive list of amino acid substitutions that can alter NAI susceptibility is also available on the WHO website10.
<img alt="Figure 1" src="/files/ftp_upload/55570/55570fig1.jpg" /> 
Figure 1: RFU against 4-MU concentration. (a) Standard curve of RFU against 4-MU concentration (&#181;M). The dotted box shows the linear range of 4-MU for the fluorometer. The fluorescence signals above the linear range may be saturated, and therefore, any small changes in fluorescence may not be detected by the fluorometer. (b) Close-up linear section of the standard curve of Figure 1a for the identification of the &#34;optimal target signal.&#34; The fluorometer at the Melbourne WHOCCRRI has a linear range of 2.5-40 &#181;M 4-MU and an optimal target signal of ~30 &#181;M 4-MU, which corresponds to ~1,500 RFU. <a href="http://cloudflare.jove.com/files/ftp_upload/55570/55570fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/55570/55570fig2.jpg" /> 
Figure 2: Example of the NA activity curves of influenza viruses. The average background value of 50.61 RFU has been subtracted from every dilution point on the NA activity curves. The arrows indicate the appropriate virus dilution to use in the NA inhibition assay for each virus. For easier preparation of virus dilutions, one may choose to perform a 1/100 dilution for VIRUS 3 instead of a 1/96 dilution. <a href="http://cloudflare.jove.com/files/ftp_upload/55570/55570fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/55570/55570fig3.jpg" /> 
Figure 3: Plate layout for the setup of the NA inhibition assay. Each plate includes the last column, which acts as a negative control that contains no virus but only 1x assay buffer (AB), NA inhibitor, MUNANA, and stop solution. NOTE: JASPR uses readings from column 12 of each plate to determine the average blank signal used in the calculation of the IC50 values. <a href="http://cloudflare.jove.com/files/ftp_upload/55570/55570fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" src="/files/ftp_upload/55570/55570fig4.jpg" /> 
Figure 4: Example of an inhibition curve and IC50 value of an A(H1N1)pdm09 virus, A/Perth/82/2015. The JASPR software presents the inhibition curve as fluorescence (RFU) against the increasing concentration (nM) of NA inhibitor, with every point fit within the curve. Based on the inhibition curve, the IC50 value is determined as the concentration of NA inhibitor to reduce 50% of the virus NA activity. <a href="http://cloudflare.jove.com/files/ftp_upload/55570/55570fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Virus</td>
			<td>Virus dilution required&#160;</td>
			<td>1x assay buffer volume (&#181;L)</td>
			<td>Surfactant-Amps-NP-40 (10%) (mL)</td>
			<td>Virus volume (mL)</td>
		</tr>
		<tr>
			<td>1</td>
			<td>1/20</td>
			<td>940</td>
			<td>10</td>
			<td>50</td>
		</tr>
		<tr>
			<td>2</td>
			<td>1/40</td>
			<td>965</td>
			<td>10</td>
			<td>25</td>
		</tr>
		<tr>
			<td>3</td>
			<td>1/100</td>
			<td>890</td>
			<td>10</td>
			<td>10</td>
		</tr>
	</tbody>
</table>
Table 1: Preparation of virus dilutions for VIRUS 1, 2, and 3 in the NA inhibition assay.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td rowspan="2">Virus type/subtype/lineage</td>
			<td>Normal inhibition&#160;</td>
			<td>Reduced inhibition&#160;</td>
			<td>Highly reduced inhibition&#160;</td>
		</tr>
		<tr>
			<td>(NI)</td>
			<td>(RI)</td>
			<td>(HRI)</td>
		</tr>
		<tr>
			<td>A(H1N1)pdm09</td>
			<td>&#60;10 fold</td>
			<td>10-100 fold</td>
			<td>&#62;100 fold</td>
		</tr>
		<tr>
			<td>A(H3N2)</td>
			<td>&#60;10 fold</td>
			<td>10-100 fold</td>
			<td>&#62;100 fold</td>
		</tr>
		<tr>
			<td>B Yamagata and B Victoria</td>
			<td>&#60;5 fold</td>
			<td>5-50 fold</td>
			<td>&#62;50 fold</td>
		</tr>
	</tbody>
</table>
Table 2: The WHO Antiviral Working Group recommended guidelines for the classification of influenza virus susceptibility to NA inhibitors.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td rowspan="2">Virus type/subtype/lineage</td>
			<td rowspan="2">N</td>
			<td>Zanamivir</td>
			<td>Oseltamivir</td>
			<td>Peramivir</td>
			<td>Laninamivir</td>
		</tr>
		<tr>
			<td>Median (range) IC50 nM</td>
			<td>Median (range) IC50 nM</td>
			<td>Median (range) IC50 nM</td>
			<td>Median (range) IC50 nM</td>
		</tr>
		<tr>
			<td>A(H1N1)pdm09</td>
			<td>1,326</td>
			<td>0.42 (0.1-3.43)</td>
			<td>0.36 (0.01-3.48)</td>
			<td>0.19 (0.07-1.60)</td>
			<td>0.55 (0.05-2.29)</td>
		</tr>
		<tr>
			<td>A(H3N2)</td>
			<td>1,654</td>
			<td>0.9 (0.11-4.0)</td>
			<td>0.38 (0.01-3.65)</td>
			<td>0.33 (0.12-3.06)</td>
			<td>1.38 (0.01-9.38)</td>
		</tr>
		<tr>
			<td>B Yamagata and B Victoria</td>
			<td>1,115</td>
			<td>2.2 (1.24-10.72)</td>
			<td>15.12 (2.39-70.75)</td>
			<td>1.36 (0.57-6.67)</td>
			<td>2.89 (1.62-9.15)</td>
		</tr>
	</tbody>
</table>
Table 3: Median IC50 and IC50 range of normal inhibition (NI) viruses from 2015 derived in the WHO CCRRI, Melbourne.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td rowspan="2">Amino acid substitution</td>
			<td rowspan="2">Type/subtype/lineage</td>
			<td colspan="4">IC50 fold-change compared to reference median IC50 values.</td>
		</tr>
		<tr>
			<td>Zanamivir&#160;</td>
			<td>Oseltamivir&#160;</td>
			<td>Peramivir&#160;</td>
			<td>Laninamivir&#160;</td>
		</tr>
		<tr>
			<td>H275Y</td>
			<td>A(H1N1)pdm09</td>
			<td>1</td>
			<td>557 (HRI)</td>
			<td>123 (HRI)</td>
			<td>2</td>
		</tr>
		<tr>
			<td>E119V</td>
			<td>A(H3N2)</td>
			<td>1</td>
			<td>63 (RI)</td>
			<td>1</td>
			<td>1</td>
		</tr>
		<tr>
			<td>H134Y</td>
			<td>B Victoria</td>
			<td>1</td>
			<td>4</td>
			<td>76 (HRI)</td>
			<td>2</td>
		</tr>
		<tr>
			<td>N151T</td>
			<td>B Victoria</td>
			<td>4</td>
			<td>4</td>
			<td>42 (HRI)</td>
			<td>1</td>
		</tr>
		<tr>
			<td>G104E</td>
			<td>B Victoria</td>
			<td>1,220 (HRI)</td>
			<td>87 (HRI)</td>
			<td>17,724 (HRI)</td>
			<td>701 (HRI)</td>
		</tr>
		<tr>
			<td>E105K</td>
			<td>B Victoria</td>
			<td>3</td>
			<td>5 (RI)</td>
			<td>59 (HRI)</td>
			<td>2</td>
		</tr>
		<tr>
			<td>I222T</td>
			<td>B Victoria</td>
			<td>2</td>
			<td>7 (RI)</td>
			<td>8 (RI)</td>
			<td>3</td>
		</tr>
		<tr>
			<td>H273Y</td>
			<td>B Yamagata</td>
			<td>1</td>
			<td>230 (HRI)</td>
			<td>377 (HRI)</td>
			<td>2</td>
		</tr>
		<tr>
			<td>D197N</td>
			<td>B Yamagata</td>
			<td>4</td>
			<td>7 (RI)</td>
			<td>32 (RI)</td>
			<td>3</td>
		</tr>
	</tbody>
</table>
Table 4: Representative list of amino acid substitutions linked to reduced inhibition (RI) or highly reduced inhibition (HRI) to NA inhibitors.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Problem</td>
			<td>Possible reason(s)</td>
			<td>Solution(s)</td>
		</tr>
		<tr>
			<td rowspan="2">No or low NA activity</td>
			<td>No virus was present or low virus yield.</td>
			<td>Clinical specimen must be cultured in cell lines (i.e. Madin-Darby Canine Kidney cells) or in embryonated chicken eggs&#160; to a higher virus load for use in the NA inhibition assay.&#160;</td>
		</tr>
		<tr>
			<td>Some mutant viruses have extremely low NA activity despite at high virus load.</td>
			<td>Use neat virus concentration for testing. Lower pH assay buffer (e.g. pH 5.3) may be used. However, caution must be taken when comparing data.</td>
		</tr>
		<tr>
			<td rowspan="3">No or low NA activity in NA inhibition assay</td>
			<td>No virus was added.</td>
			<td>Re-dilute the virus. Ensure the virus is directly added into the 1x assay buffer.</td>
		</tr>
		<tr>
			<td>Wrong virus dilution was used.</td>
			<td>Repeat the NA activity assay.</td>
		</tr>
		<tr>
			<td>Insufficient incubation time.</td>
			<td>Ensure the incubation time is followed.</td>
		</tr>
		<tr>
			<td rowspan="3">Data points fall outside the IC50 curve</td>
			<td rowspan="2">Cross contamination of NA inhibitor of higher concentration.</td>
			<td>Ensure that the tips are not in contact with the NA inhibitor when dispensing diluted viruses into the 96 well plate.</td>
		</tr>
		<tr>
			<td>If an 8 deep well reservoir was used, discard and re-dispense the NA inhibitor concentrations into a fresh 8 deep well reservoir.&#160;</td>
		</tr>
		<tr>
			<td>The volume of the NA inhibitor or MUNANA or diluted virus was not added equally into each well.</td>
			<td>Repeat the assay with a calibrated multi-channel pipette. Ensure equal volume of each reagent is dispensed into each well.&#160;</td>
		</tr>
		<tr>
			<td rowspan="3">Unusually high IC50 values</td>
			<td>Too high concentration of virus was added.</td>
			<td>Repeat the NA activity assay and NA inhibition assay.</td>
		</tr>
		<tr>
			<td>Test sample contained mixtures of influenza A and influenza B.</td>
			<td>Perform real-time PCR to identify the presence of virus mixtures.</td>
		</tr>
		<tr>
			<td>Bacterial contamination in the sample</td>
			<td>Culture virus in the sterile condition with the presence of antibiotic.&#160;</td>
		</tr>
		<tr>
			<td rowspan="2">High background fluorescence signal</td>
			<td>MUNANA substrate may degrade over time.&#160;</td>
			<td>Use a new batch of MUNANA subtrate.</td>
		</tr>
		<tr>
			<td>Detection of fluorescence from neighboring wells.&#160;</td>
			<td>Use black 96-well flat-bottom plates</td>
		</tr>
	</tbody>
</table>
Table 5: Troubleshooting for potential problems in the NA inhibition assay.

Watch this Scientific Journal Video about Fluorescence-based Neuraminidase Inhibition Assay to Assess the Susceptibility of Influenza Viruses to The Neuraminidase Inhibitor Class of Antivirals at JoVE

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

We describe the use of a phenotypic fluorescence-based neuraminidase inhibition assay to assess the susceptibility of influenza A and B viruses to the neuraminidase inhibitor class of antivirals.

The neuraminidase (NA) inhibitors are the only class of antivirals approved for the treatment and prophylaxis of influenza that are effective against ...

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JoVE Journal

Immunology and Infection

17.7K Views.  Peter Doherty Institute for Infection and Immunity. We describe the use of a phenotypic fluorescence-based neuraminidase inhibition assay to assess the susceptibility of influenza A and B viruses to the neuraminidase inhibitor class of antivirals. 

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

Detection of  Neu1 Sialidase Activity in Regulating TOLL-like Receptor Activation

Mammalian Toll-like receptors (TLRs) are a family of receptors that recognize pathogen-associated molecular patterns. Not only are TLRs crucial sensors of microbial (e.g., viruses, bacteria and parasite) infections, they also play an important role in the pathophysiology of infectious diseases, inflammatory diseases, and possibly in autoimmune diseases. Thus, the intensity and duration of TLR responses against infectious diseases must be tightly controlled. It follows that understanding the structural integrity of sensor receptors, their ligand interactions and signaling components is essential for subsequent immunological protection. It would also provide important opportunities for disease modification through sensor manipulation. Although the signaling pathways of TLR sensors are well characterized, the parameters controlling interactions between the sensors and their ligands still remain poorly defined. We have recently identified a novel mechanism of TLR activation by its natural ligand, which has not been previously observed 1,2. It suggests that ligand-induced TLR activation is tightly controlled by Neu1 sialidase activation. We have also reported that Neu1 tightly regulates neurotrophin receptors like TrkA and TrkB 3, which involve Neu1 and matrix metalloproteinase-9 (MMP-9) cross-talk in complex with the receptors 4. The sialidase assay has been initially use to find a novel ligand, thymoquinone, in the activation of Neu4 sialidase on the cell surface of macrophages, dendritic cells and fibroblast cells via GPCR G&alpha;i proteins and MMP-9 5. For TLR receptors, our data indicate that Neu1 sialidase is already in complex with TLR-2, -3 and -4 receptors, and is induced upon ligand binding to either receptor. Activated Neu1 sialidase hydrolyzes sialyl &alpha;-2,3-linked &beta;-galactosyl residues distant from ligand binding to remove steric hinderance to TLR-4 dimerization, MyD88/TLR4 complex recruitment, NFkB activation and pro-inflammatory cell responses. In a collaborative report, Neu1 sialidase has been shown to regulate phagocytosis in macrophage cells 6. Taken together, the sialidase assay has provided us with powerful insights to the molecular mechanisms of ligand-induced receptor activation. Although the precise relationship between Neu1 sialidase and the activation of TLR, Trk receptors has yet to be fully elucidated, it would represent a new or pioneering approach to cell regulation pathways.

The sialidase assay is a simple technical approach that will elucidate novel molecular mechanism(s) of TLR sensors of microbial infections and involvement in inflammatory diseases at the receptor level on the cell surface of live macrophages.

Mammalian Toll-like receptors (TLRs) are a family of receptors that recognize pathogen-associated molecular patterns. Not only are TLRs crucial sensors of ...

Quantitative Analyses of all Influenza Type A Viral Hemagglutinins and Neuraminidases using Universal Antibodies in Simple Slot Blot Assays

Hemagglutinin (HA) and neuraminidase (NA) are two surface proteins of influenza viruses which are known to play important roles in the viral life cycle and the induction of protective immune responses1,2. As the main target for neutralizing antibodies, HA is currently used as the influenza vaccine potency marker and is measured by single radial immunodiffusion (SRID)3. However, the dependence of SRID on the availability of the corresponding subtype-specific antisera causes a minimum of 2-3 months delay for the release of every new vaccine. Moreover, despite evidence that NA also induces protective immunity4, the amount of NA in influenza vaccines is not yet standardized due to a lack of appropriate reagents or analytical method5. Thus, simple alternative methods capable of quantifying HA and NA antigens are desirable for rapid release and better quality control of influenza vaccines.
Universally conserved regions in all available influenza A HA and NA sequences were identified by bioinformatics analyses6-7. One sequence (designated as Uni-1) was identified in the only universally conserved epitope of HA, the fusion peptide6, while two conserved sequences were identified in neuraminidases, one close to the enzymatic active site (designated as HCA-2) and the other close to the N-terminus (designated as HCA-3)7. Peptides with these amino acid sequences were synthesized and used to immunize rabbits for the production of antibodies. The antibody against the Uni-1 epitope of HA was able to bind to 13 subtypes of influenza A HA (H1-H13) while the antibodies against the HCA-2 and HCA-3 regions of NA were capable of binding all 9 NA subtypes. All antibodies showed remarkable specificity against the viral sequences as evidenced by the observation that no cross-reactivity to allantoic proteins was detected. These universal antibodies were then used to develop slot blot assays to quantify HA and NA in influenza A vaccines without the need for specific antisera7,8. Vaccine samples were applied onto a PVDF membrane using a slot blot apparatus along with reference standards diluted to various concentrations. For the detection of HA, samples and standard were first diluted in Tris-buffered saline (TBS) containing 4M urea while for the measurement of NA they were diluted in TBS containing 0.01% Zwittergent as these conditions significantly improved the detection sensitivity. Following the detection of the HA and NA antigens by immunoblotting with their respective universal antibodies, signal intensities were quantified by densitometry. Amounts of HA and NA in the vaccines were then calculated using a standard curve established with the signal intensities of the various concentrations of the references used. 
Given that these antibodies bind to universal epitopes in HA or NA, interested investigators could use them as research tools in immunoassays other than the slot blot only.

A simple slot blot method was developed for the quantification of influenza viral hemagglutinin and neuraminidase using universal antibodies targeting their most conserved sequences identified through bioinformatics analyses. This innovative approach may provide a useful alternative to quantitative determination of all viral hemagglutinin and neuraminidase.

Hemagglutinin (HA) and neuraminidase (NA) are two surface proteins of influenza viruses which are known to play important roles in the viral life cycle ...

High-throughput Detection Method for Influenza Virus

Influenza virus is a respiratory pathogen that causes a high degree of morbidity and mortality every year in multiple parts of the world. Therefore, precise diagnosis of the infecting strain and rapid high-throughput screening of vast numbers of clinical samples is paramount to control the spread of pandemic infections. Current clinical diagnoses of influenza infections are based on serologic testing, polymerase chain reaction, direct specimen immunofluorescence and cell culture 1,2.
Here, we report the development of a novel diagnostic technique used to detect live influenza viruses. We used the mouse-adapted human A/PR/8/34 (PR8, H1N1) virus 3 to test the efficacy of this technique using MDCK cells 4. MDCK cells (104 or 5 x 103 per well) were cultured in 96- or 384-well plates, infected with PR8 and viral proteins were detected using anti-M2 followed by an IR dye-conjugated secondary antibody. M2 5 and hemagglutinin 1 are two major marker proteins used in many different diagnostic assays. Employing IR-dye-conjugated secondary antibodies minimized the autofluorescence associated with other fluorescent dyes. The use of anti-M2 antibody allowed us to use the antigen-specific fluorescence intensity as a direct metric of viral quantity. To enumerate the fluorescence intensity, we used the LI-COR Odyssey-based IR scanner. This system uses two channel laser-based IR detections to identify fluorophores and differentiate them from background noise. The first channel excites at 680 nm and emits at 700 nm to help quantify the background. The second channel detects fluorophores that excite at 780 nm and emit at 800 nm. Scanning of PR8-infected MDCK cells in the IR scanner indicated a viral titer-dependent bright fluorescence. A positive correlation of fluorescence intensity to virus titer starting from 102-105 PFU could be consistently observed. Minimal but detectable positivity consistently seen with 102-103 PFU PR8 viral titers demonstrated the high sensitivity of the near-IR dyes. The signal-to-noise ratio was determined by comparing the mock-infected or isotype antibody-treated MDCK cells.
Using the fluorescence intensities from 96- or 384-well plate formats, we constructed standard titration curves. In these calculations, the first variable is the viral titer while the second variable is the fluorescence intensity. Therefore, we used the exponential distribution to generate a curve-fit to determine the polynomial relationship between the viral titers and fluorescence intensities. Collectively, we conclude that IR dye-based protein detection system can help diagnose infecting viral strains and precisely enumerate the titer of the infecting pathogens.

This method describes the use of Infrared dye based imaging system for detection of H1N1 in bronchioalveolar lavage (BAL) fluid of infected mice at a high sensitivity. This methodology can be performed in a 96- or 384-well plate, requires &lt;10 &mu;l volume of test material and has the potential for concurrent screening of multiple pathogens.

Influenza virus is a respiratory pathogen that causes a high degree of morbidity and mortality every year in multiple parts of the world. Therefore, ...

A Miniaturized Glycan Microarray Assay for Assessing Avidity and Specificity of Influenza A Virus Hemagglutinins

Influenza A virus (IAV) hemagglutinins recognize sialic acids on the cell surface as functional receptors to gain entry into cells. Wild waterfowl are the natural reservoir for IAV, but IAV can cross the species barrier to poultry, swine, horses and humans. Avian viruses recognize sialic acid attached to a penultimate galactose by a &#945;2-3 linkage (avian-type receptors) whereas human viruses preferentially recognize sialic acid with a &#945;2-6 linkage (human-type receptors). To monitor if avian viruses are adapting to human type receptors, several methods can be used. Glycan microarrays with diverse libraries of synthetic sialosides are increasingly used to evaluate receptor specificity. However, this technique is not used for measuring avidities. Measurement of avidity is typically achieved by evaluating the binding of serially diluted hemagglutinin or virus to glycans adsorbed to conventional polypropylene 96-well plates. In this assay, glycans with &#945;2-3 or &#945;2-6 sialic acids are coupled to biotin and adsorbed to streptavidin plates, or are coupled to polyacrylamide (PAA) which directly adsorb to the plastic. We have significantly miniaturized this assay by directly printing PAA-linked sialosides and their non PAA-linked counterparts on micro-well glass slides. This set-up, with 48 arrays on a single slide, enables simultaneous assays of 6 glycan binding proteins at 8 dilutions, interrogating 6 different glycans, including two non-sialylated controls. This is equivalent to 18x 96-well plates in the traditional plate assay. The glycan array format decreases consumption of compounds and biologicals and thus greatly enhances efficiency.

Using a printed glycan microarray strategy, a conventional 96-well plate assay was miniaturized for analysis of influenza A virus hemagglutinin avidity and specificity for sialic acid containing receptors.

Influenza A virus (IAV) hemagglutinins recognize sialic acids on the cell surface as functional receptors to gain entry into cells. Wild waterfowl are the ...

Rapid Molecular Detection and Differentiation of Influenza Viruses A and B

Influenza is a contagious respiratory illness caused by influenza viruses A and B in humans and causes a significant amount of morbidity and mortality every year. The Influenza A and B assay was the first CLIA-waived molecular rapid flu test available. The Influenza A and B test works by employing isothermal amplification with influenza-specific primers followed by target detection with molecular beacon probes. Here, the performance of the Influenza A and B assay on frozen, archived nasopharyngeal swab (NPS) specimens stored in viral transport medium (VTM) were compared to a respiratory panel assay.
The performance of the Influenza A and B assay was evaluated by comparing the results to the respiratory panel reference method. The sensitivity for total influenza virus A was 67.5% (95% CI (CI), 56.6-78.5) and the specificity was 86.9% (CI, 71.0-100). For influenza virus B testing, the sensitivity and specificity were 90.2% (CI, 68.5-100) and 98.8% (CI, 68.5-100), respectively.
This system has the advantage of a significantly shorter test time than any other currently available molecular assay and the simple, pipette-free procedure runs on a fully integrated, closed, small-footprint system. Overall, the Influenza A and B assay evaluated in this study has the potential to serve as a point-of-care rapid influenza diagnostic test.

We describe a rapid, molecular-based Influenza A and B assay. The Influenza assay detects each target within 15 min by employing isothermal amplification with influenza-specific primers followed by target detection with molecular beacon probes. The Influenza A and B assay is user-friendly and required minimal hands-on time to perform.

Influenza is a contagious respiratory illness caused by influenza viruses A and B in humans and causes a significant amount of morbidity and mortality ...

Measuring Influenza Neuraminidase Inhibition Antibody Titers by Enzyme-linked Lectin Assay

Antibodies to neuraminidase (NA), the second most abundant surface protein on influenza virus, contribute toward protection against influenza. Traditional methods to measure NA inhibiting (NI) antibody titers are not practical for routine serology. This protocol describes the enzyme-linked lectin assay (ELLA), a practical alternative method to measure NI titers that is performed in 96 well plates coated with a large glycoprotein substrate, fetuin. NA cleaves terminal sialic acids from fetuin, exposing the penultimate sugar, galactose. Peanut agglutinin (PNA) is a lectin with specificity for galactose and therefore the extent of desialylation can be quantified using a PNA-horseradish peroxidase conjugate, followed by addition of a chromogenic peroxidase substrate. The optical density that is measured is proportional to NA activity. To measure NI antibody titers, serial dilutions of sera are incubated at 37 &#176;C O/N on fetuin-coated plates with a fixed amount of NA. The reciprocal of the highest serum dilution that results in &#8805;50% inhibition of NA activity is designated as the NI antibody titer. The ELLA provides a practical format for routine evaluation of human antibody responses following influenza infection or vaccination.

We describe the enzyme-linked lectin assay (ELLA) for measuring influenza neuraminidase (NA)-inhibition antibody titers in sera. The assay uses peanut agglutinin to quantify galactose residues that become accessible when NA removes sialic acid from fetuin-coated, 96-well plates.

Antibodies to neuraminidase (NA), the second most abundant surface protein on influenza virus, contribute toward protection against influenza. Traditional ...

Optimization of a Quantitative Micro-neutralization Assay

The micro-neutralization (MN) assay is a standard technique for measuring the infectivity of the influenza virus and the inhibition of virus replication. In this study, we present the protocol of an imaging-based MN assay to quantify the true antigenic relationships between viruses. Unlike typical plaque reduction assays that rely on visible plaques, this assay quantitates the entire infected cell population of each well. The protocol matches the virus type or subtype with the selection of cell lines to achieve maximum infectivity, which enhances sample contrast during imaging and image processing. The introduction of quantitative titration defines the amount of input viruses of neutralization and enables the results from different experiments to be comparable. The imaging setup with a flatbed scanner and free downloadable software makes the approach high throughput, cost effective, user friendly, and easy to deploy in most laboratories. Our study demonstrates that the improved MN assay works well with the current circulating influenza A(H1N1)pdm09, A(H3N2), and B viruses, without being significantly influenced by amino acid substitutions in the neuraminidase (NA) of A(H3N2) viruses. It is particularly useful for the characterization of viruses that either grow to low HA titer and/or undergo an abortive infection resulting in an inability to form plaques in cultured cells.

This study describes an imaging-based micro-neutralization assay to analyze the antigenic relationships between viruses. The protocol employs a flatbed scanner and has four steps, including titration, titration quantitation, neutralization, and neutralization quantitation. The assay works well with current circulating influenza A(H1N1)pdm09, A(H3N2), and B viruses.

The micro-neutralization (MN) assay is a standard technique for measuring the infectivity of the influenza virus and the inhibition of virus replication. ...

Measuring Influenza Neutralizing Antibody Responses to A(H3N2) Viruses in Human Sera by Microneutralization Assays Using MDCK-SIAT1 Cells

Neutralizing antibodies against hemagglutinin (HA) of influenza viruses are considered the main immune mechanism that correlates with protection for influenza infections. Microneutralization (MN) assays are often used to measure neutralizing antibody responses in human sera after influenza vaccination or infection. Madine Darby Canine Kidney (MDCK) cells are the commonly used cell substrate for MN assays. However, currently circulating 3C.2a and 3C.3a A(H3N2) influenza viruses have acquired altered receptor binding specificity. The MDCK-SIAT1 cell line with increased &#945;-2,6 sialic galactose moieties on the surface has proven to provide improved infectivity and more faithful replications than conventional MDCK cells for these contemporary A(H3N2) viruses. Here, we describe a MN assay using MDCK-SIAT1 cells that has been optimized to quantify neutralizing antibody titers to these contemporary A(H3N2) viruses. In this protocol, heat inactivated sera containing neutralizing antibodies are first serially diluted, then incubated with 100 TCID50/well of influenza A(H3N2) viruses to allow antibodies in the sera to bind to the viruses. MDCK-SIAT1 cells are then added to the virus-antibody mixture, and incubated for 18 - 20 h at 37 &#176;C, 5% CO2 to allow A(H3N2) viruses to infect MDCK-SIAT1 cells. After overnight incubation, plates are fixed and the amount of virus in each well is quantified by an enzyme-linked immunosorbent assay (ELISA) using anti-influenza A nucleoprotein (NP) monoclonal antibodies. Neutralizing antibody titer is defined as the reciprocal of the highest serum dilution that provides &#8805;50% inhibition of virus infectivity.

Measuring Influenza Neutralizing Antibody Responses to AH3N2 Viruses in Human Sera by Microneutralization Assays Using MDCK-SIAT1 Cells

Influenza neutralizing antibodies correlate with protection of influenza infections. Microneutralization assays measure neutralizing antibodies in human sera and are often used for influenza human serology. We describe a microneutralization assay using MDCK-SIAT1 cells to measure neutralizing antibody titers to contemporary 3C.2a and 3C.3a A(H3N2) viruses following influenza vaccination or infection.

Neutralizing antibodies against hemagglutinin (HA) of influenza viruses are considered the main immune mechanism that correlates with protection for ...

High-throughput Antiviral Assays to Screen for Inhibitors of Zika Virus Replication

Antiviral drug discovery requires the development of reliable biochemical and cellular assays that can be performed in high-throughput screening (HTS) formats. The flavivirus non-structural (NS) proteins are thought to co-translationally assemble on the endoplasmic reticulum (ER) membranes, forming the replication complex (RC). The NS3 and NS5 are the most studied enzymes of the RC and constitute the main targets for drug development due to their crucial roles in viral genome replication. NS3 protease domain, which requires NS2B as its cofactor, is responsible for the cleavage of the immature viral polyprotein into the mature NS proteins, whereas NS5 RdRp domain is responsible for the RNA replication. Herein, we describe in detail the protocols used in replicon-based screenings and enzymatic assays to test large compound libraries for inhibitors of the Zika virus (ZIKV) replication. Replicons are self-replicating subgenomic systems expressed in mammalian cells, in which the viral structural genes are replaced by a reporter gene. The inhibitory effects of compounds on viral RNA replication can be easily evaluated by measuring the reduction in the reporter protein activity. The replicon-based screenings were performed using a BHK-21 ZIKV replicon cell line expressing Renilla luciferase as a reporter gene. To characterize the specific targets of identified compounds, we established in-vitro fluorescence-based assays for recombinantly expressed NS3 protease and NS5 RdRp. The proteolytic activity of the viral protease was measured by using the fluorogenic peptide substrate Bz-nKRR-AMC, while the NS5 RdRp elongation activity was directly detected by the increase of the fluorescent signal of SYBR Green I during RNA elongation, using the synthetic biotinylated self-priming template 3&#8242;UTR-U30 (5&#39;-biotin-U30-ACUGGAGAUCGAUCUCCAGU-3&#39;).

In this work, we describe the protocols used in replicon-based and viral enzyme-based assays to screen for inhibitors of Zika virus replication in a high-throughput screening format.

Antiviral drug discovery requires the development of reliable biochemical and cellular assays that can be performed in high-throughput screening (HTS) ...

An Assay to Measure Influenza Neuraminidase Inhibition Antibody Titers

Source: Gao, J., et al. <a href="https://app.jove.com/v/54573">Measuring Influenza Neuraminidase Inhibition Antibody Titers by Enzyme-linked Lectin Assay.</a> J. Vis. Exp. (2016).
This video demonstrates an assay measuring influenza neuraminidase (NA) inhibiting antibody titers in a serum sample. A serum sample containing antibodies against NA is added to a fetuin-coated microwell plate. Upon adding the virus, the antibodies prevent viral NA from cleaving fetuin and exposing its galactose moiety. A peroxidase-conjugated lectin is then added, which binds to the exposed galactose. The NA-inhibiting antibody titer is determined using a colorimetric test, where a peroxidase substrate gets cleaved by the immobilized lectin-conjugated peroxidase to produce color.

Test do pomiaru miana przeciwciał hamujących neuraminidazę grypy

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
1. Preparation of Reagents and ...