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Frequently Asked Questions - Methodology

Q. How do I test for resistance to M2 inhibitors?

A. Resistance to amantadine and rimantadine is very well characterised and is known to be caused by mutation at one or more of five residues in the M2 ion channel. 

Genotyping of viruses is a widely accepted surveillance method for amantadine susceptibility, and can be done in two ways: Sanger sequencing of the M gene or pyrosequencing of the region encoding the transmembrane domain, specifically to monitor these five mutations (Bright et al, 2006).  Restriction fragment length polymorphism (RFLP) assay can also identify resistance mutations.  There are phenotypic assays to determine IC50s for amantadine and rimantadine such as plaque reduction assays (using agarose or avicell), or AVINA. 

Neuraminidase Inhibitor Susceptibility Monitoring: Setting up 

Q. Why do I need to perform Neuraminidase Inhibitor Susceptibility Monitoring? 

A. The neuraminidase inhibitors (NAIs) were introduced into clinical practice in 1999.  No naturally occurring resistance was found in isolates prior to this introduction.  Resistance to oseltamivir has occurred following treatment with varying frequency (<1% in adults versus 4-18% in children) and resistance to zanamivir has been seen infrequently, and only in immunocompromised individuals.  In untreated populations, resistance has generally been detected at levels of less than 1%.  However, an unusual situation occurred in 2008 when oseltamivir-resistant A(H1N1) viruses, carrying the H275Y mutation, emerged and circulated globally, in the absence of drug pressure, until replaced by the drug sensitive 2009 pandemic A(H1N1) virus.  There have been sporadic detections of oseltamivir resistant A(H1N1)pdm09, due to the H275Y mutation, as well as several incidents of limited  person-to-person transmission, and more recently in Australia, a cluster of unrelated cases, indicating probable community transmission of the resistant virus.

As NAI drugs are the treatment of choice for seasonal influenza and for first line defence against avian influenza infection of humans and stockpiling in the event of a pandemic, it is imperative that we are aware of the antiviral sensitivity of circulating strains.

Q. Do I need to monitor both Oseltamivir and Zanamivir?       

A. Monitor susceptibility to both drugs if you are able to do so.  Development of resistance is drug specific and therefore may arise to either oseltamivir or zanamivir independently. Samples tested against one drug cannot be assumed to be sensitive to the other.  Resistance has developed more readily to oseltamivir than to zanamivir, albeit infrequently and therefore if you cannot monitor both drugs, oseltamivir testing is more relevant.


Q. What tests can I use to determine neuraminidase inhibitor susceptibility?       

A. The simplest and clearest method to measure NAI susceptibility is by enzyme inhibition assay.  Several in-house protocols are available here, as well as three commercial kits (Life Technologies), using one of two available substrates, one fluorescent (MUNANA) and two chemiluniescent (NA-Star and NA-XTD). 

MUNANA is the neuraminidase substrate linked to the fluorophore, methylumbelliferone. Cleavage of MUNANA by neuraminidase releases the methylumbelliferone which then fluoresces.  The amount of fluorescence therefore directly relates to the amount of enzyme activity.

MUNANA is sold by several companies; the most widely used source is Sigma-Aldrich (catalogue number M8639).  All buffers and solutions required for this test can be made in-house and there are several protocols available for use.

The MUNANA substrate is now also available in a kit format, with all reagents provided with the exception of the neuraminidase inhibitors.

NA Star and NA-XTD are chemiluminescent substrates, and both are sold as part of dedicated kits (Life technologies USA).  These kits are specifically designed for NAI susceptibility testing. 

QCan I determine NAI susceptibility by sequencing the NA gene?

A. Not entirely.  NAI resistance is drug and influenza type and subtype specific.  Whilst we do know a number of common mutations in the NA which cause resistance, or reduced susceptibility, (H275Y in A(H1N1); E119V, I222V or R292K in A(H3N2); R152K, D198E or I222T in influenza B) the mechanisms of resistance are not fully understood and other mutations, not yet characterised, may emerge.  Sequencing can and should be used alongside phenotypic testing (enzyme inhibition tests) to characterise isolates with high IC50 values i.e. reduced NAI susceptibility. 

Q. Are there cell based assays to measure NAI sensitivity?

A. The use of cell based assays to carry out widespread surveillance for NAI susceptibility is not recommended as these tend to generate variable data and can be difficult to interpret.  Cell based assays such as plaque reduction assays and AVINA are, however, useful to characterise NAI resistant viruses or those with reduced sensitivity. 

Q. How can I perform plaque reduction assays for NAI susceptibility?

A. Plaque reduction assays involve plaquing a standard amount of virus (typically 50-100pfu) in the presence of a titration of the desired drug and a virus control (without drug). The concentration which reduces the number of plaques by 50% of that of the virus control is the IC50 for the drug. This assay can be performed on 6 well plates, using agarose as the overlay. Alternatively, the semi solid overlay Avicel (a microcrystalline cellulose) can be used to perform this assay on 96 well plates to allow greater throughput and the inclusion of replicate titrations to give more robust data. 

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Q. Do I need both fluorescence and luminescence enzyme inhibition tests?       

A. No. Surveillance can be carried out using one method. IC50 values generated from fluorescence and luminescence assays cannot be directly compared as differences in the chemistry cause differences in results. It is important that one method is used consistently in a lab to allow evaluation of trends. The alternative assay can be useful for research purposes, and to investigate outliers or resistance, but not interchangeably for surveillance. 

Q:  Why does the IC50 value determined in the chemiluminescent assay differ from that in the fluorescent assay?

A. NAI assay choice does affect the IC50 value.  This is due to several factors (concentration of substrate, structure of substrate, amongst others). Typically, the difference is more pronounced when testing drug-resistant viruses.
Q. What is the cost per sample of the enzyme inhibition tests?   

A. Prices below are a guide based on testing each virus in duplicate and given on a cost per sample basis, and are the combined cost of the virus titration and IC50 measurement.

NA-Fluor™ assay: $0.50/well, $24 per sample

NA-XTD™ assay: $1.10/well, $26-$52 per sample

NA-Star® assay: $1.00/well, $24-$48 per sample

MUNANA in-house assay: $3-$6 per sample

Q:  Should testing be done in duplicate?
A. Testing in duplicate is preferable, and gives greater confidence in the results.  However, kit based methods are high cost and this may necessitate single testing of viruses for surveillance purposes.  However, for purposes other than screening, for example in-depth analysis or characterization of a new mutant, viruses should be tested in duplicate.

The inhibitory curve (graph) produced for each virus is inspected carefully to determine if there are any deviations from the expected shape.  This allows for the detection of problems with a particular well and, in such instances, testing is repeated.

Q. What equipment do I need for enzyme inhibition tests?

A. The equipment needed for enzyme inhibition tests depends on your choice of assay (chemiluminescence or fluorescence):

Luminescence Testing

Specifically required:      Luminescence plate reader (Injectors are desirable but not essential if multi-channel pipettes are available)

Required:                           96 well plate shaker
                                           Single channel pipettes (10μl to 900μl volumes)
                                           Multi-channel pipettes (10μl to 150μl volumes)
                                           Filtered tips (10μl-900μl volumes)
                                           10 and 25 ml disposable pipettes
                                           Warm Room (+37oC)
                                           Fridge (+4oC)
                                           Freezer (- 20oC and - 80oC)

Desirable:                           Multi-well reservoirs (e.g. Thermo Electron Cat. No. RTP/08200-10)

Fluorescence Testing

Specifically required:            Fluorescence plate reader with 355nm and 460nm filters

Required:                            96 well plate shaker 

                                          Single channel pipettes(10μl to 900μl volumes)
                                          Multi-channel pipettes (10μl to 150μl volumes)
                                          Filtered tips (10μl-900μl volumes)
                                          10 and 25 ml disposable pipettes
                                          Warm Room (+37oC)
                                          Fridge (+4oC)
                                          Freezer (- 20oC and - 80oC)
                                          Black 96 well flat bottom plates (Corning 3915 or similar)
                                          Adhesive plate sealers (or plastic lids are also suitable)

Desirable:                           Multi-well reservoirs (e.g. Thermo Electron Cat. No. RTP/08200-10)

Q. What plate reader instruments are suitable for fluorescence or chemiluminscence detection?

A. The majority of commercially available fluorimeters and luminometers are suitable for application to these tests.  Dual reading instruments are available and again most of these are suitable for use, but you should ensure that sensitivity is not lost in either function.  There are several instruments in use in laboratories worldwide that have been shown to be suitable for both tests, including e.g. those listed below for reference.

BMG LabTech               FluroStar Optima (Dual reader, fluorescence and luminescence)

Thermo electron           Fluoroskan ascent FL (Dual reader, fluorescence and luminescence)


Q. What reagents do I need?       

A. The reagents differ depending on the type of test you choose to perform (chemiluminescence or fluorescence):

Chemiluminescence Testing

Life Technologies supply two kits which contain all of the reagents needed for this test:

NA-Star® Influenza NAI Resistance Detection Kit (Part number: 4374348)

NA-XTD® Influenza NAI Resistance Detection Kit (Part number: 4457534)

Detection Microplates (Part number: 4374349)

In addition to the kits the following are required:

  • Influenza virus isolates derived from passage in cell culture or eggs; fluids with sufficient HA titre
  • Reference viruses (subtype matched, if possible)
  • Oseltamivir Carboxylate (Roche. Product no. GS4071 or Ro64-0802)
  • Zanamivir (Glaxo-Smithkline Product no. GR121167X or GG167)


Fluorescence Testing

Life Technologies supply a kit which contains all of the reagents needed for this test.

NA-FLUOR® Influenza NAI Resistance Detection Kit (Part number: 4457091)

In addition to the kit, the four items detailed above are required.

Alternatively, the test can be done using an in-house method.  The exact reagents are dependent on the protocol used; however, the following are essential for all protocols:

  • Influenza virus isolates derived from passage in cell culture or eggs; fluids with sufficient HA titre
  • Reference viruses (subtype matched, if possible)
  • 2-Morpholinoethanesulfonic acid (MES) (Sigma-Aldrich M3671 or similar)
  • Calcium chloride (VWR 5701 or similar)
  • Oseltamivir Carboxylate (Roche. Product no. GS4071 or Ro64-0802)
  • Zanamivir (Glaxo-Smithkline Product no. GR121167X or GG167)
  • MUNANA (2’ 2′-(4-Methylumbelliferyl)-α-D-N-acetylneuraminic acid sodium salt hydrate) (Sigma-Aldrich M8639 or similar)
  • 4-Methylumbelliferone sodium salt (Sigma-Aldrich M1508)
  • Absolute Ethanol (VWR 101077Y or similar)
  • Sodium Hydroxide (VWR 101182 or similar)
  • Distilled water

 Used in some protocols: 

  • Glycine (VWR 1517 or similar)

Q. Where can I get the NAI drugs?

A. Oseltamivir carboxylate (the biologically active form of the drug) is needed for in vitro tests. This is available on request from Roche.  Zanamivir is available on request from Glaxo SmithKline.  For each drug, a basic material transfer agreement must be signed before the drug is shipped.  Further information about obtaining NAI drugs for testing can be found here.

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Neuraminidase Inhibitor Susceptibility Monitoring: Which Samples? 
Q. Which viruses are suitable to test?       

A. NAIs are active against all influenza A subtypes and Influenza B viruses and the fluorescent and chemiluminescent tests can be performed on all influenza viruses.  Both human and avian viruses can be tested, however, suitable containment facilities are required for avian virus testing as testing protocols use live virus.  There are some methods of virus inactivation which do not affect the neuraminidase activity and are thus suitable for use with NAI tests for IC50 determination.  

A validated procedure is available hereadobe pdf icon2  The published reference for the protocol is available hereadobe pdf icon2 

Q. Can I test original clinical material in NAI (IC50) assays?       

A. No. Due to the amount of virus needed for both the fluorescence and chemiluminescence tests, it is unlikely that clinical material would have sufficient virus present to yield good signals.

Q. Can I test egg-grown and cell-culture material in NAI (IC50) assays

A. Yes, egg allantoic and amniotic fluids and cell culture supernatants are suitable for use in the fluorescence and chemiluminescence tests.  Note: Inactivation procedures have not been validated for use on egg fluids, and may not be suitable due to the high protein content.

Q. Do I need to use phenol red free media to culture viruses for use in the chemiluminescence tests?       

A. No. As long as viruses are diluted at least 1 in 5 in assay buffer before performing the test, the phenol red is diluted sufficiently so as not to interfere with the chemiluminescence signal. 

Q:  Do I need to pre-titrate the virus, and add a standard amount of virus to the inhibition assay?

A. Most protocols, particularly those using MUNANA as the substrate, require titration and addition of a standard amount of virus, based on NA activity, to the inhibition assay.  Greater robustness and accuracy of IC50 determination is achieved by using a standard NA activity for each virus.  Additionally, cross-talk between neighboring wells containing viruses with high and low NA activity could interfere with signals if the high activity virus is undiluted.

Q. How do I determine the standard virus dose to use in the test? 

A. There are several protocols for the MUNANA assay which follow the same principles, with minor variations in detail.  Further information can be found within the IC50 methodology section of the website.  All of the protocols provided are used extensively and validated for use by those who submitted them. 

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Neuraminidase Inhibitor Susceptibility Monitoring: Validation and Reference Strains

Q. Where can I get reference strains?     

A. There is a panel of virus strains available on request from the isirv-AVG.   Included in this panel are four pairs of sensitive and resistant human influenza A (two H1N1 and one H3N2) and B viruses, isolated, plaque purified and cultured in MDCK cells, which can be used for the evaluation of resistance to NA inhibitors and for the standardisation of IC50 values.  

Q. How should I grow stocks of reference viruses? 
A. Instructions on how to grow stocks of the reference viruses are included in an information leaflet provided with the panel.


Q. How should I validate my IC50 test?       

A. Tests should be validated by inclusion of a sensitive and resistant pair of reference strains. The IC50 values for each reference virus should be measured several times to identify the extent of variation in your assay conditions. In each test thereafter the reference strain IC50 values should lie within the predefined limits.  Additional criteria to validate a test should include examination of each enzyme inhibition curve to ensure the data fit an S shaped curve.  Samples yielding curves which have spurious points or shapes should be repeated. Examples of valid and invalid curves are shown in the ‘Interpretation of raw data guidance.

Q:  Can the same reference viruses be used in chemiluminescent and fluorescent assays?

A:  Yes, but their IC50 values will differ between the two assay types. Validation is required independently for both assay types.

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