- Frequently Asked Questions on virological assay methodology
Q. How do I test for Adamantane resistance? 
Resistance to adamantanes (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. This genotyping can be done in two ways: Sanger sequencing of the M gene or pyrosequencing of the ion channel region 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 the amantadine and rimantadine IC50s 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?
The neuraminidase inhibitors 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 at varying rates (<1% in adults versus 4-18% in children) and resistance to zanamivir has been seen although only in immunocompromised individuals. In the untreated population, resistance has been found at a level of less than 1%. However, this situation changed in 2008 when H1N1 viruses carrying the H275Y (274 in N2 numbering) mutation were shown to be circulating globally by surveillance programs.
As NI 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 sensitivity of circulating strains.
Q. Do I need to monitor both Oseltamivir and Zanamivir?
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?
The simplest and clearest method to measure NI susceptibility is by enzyme inhibition assay. There are two commercially available substrates suitable for this type of test, MUNANA and NA-Star.
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 Sigma-Aldrich (catalogue number M8639). All other reagents needed (assay buffer, stop solution) need to be made in house.
NA Star is a chemiluminescent substrate and is sold as part of a dedicated kit (Applied Biosystems, USA). The kit is specifically designed for NI susceptibility testing and contains ten 96 well plates, assay buffer, substrate, and accelerator.
NA-Star® Influenza Neuraminidase Inhibitor Resistance Detection Reagent Set (Part number: 4374348)
Protocol (Part number: 4375714)
NA-Star™ Detection Microplates (Part number: 4374349)
Q. Can I determine NI susceptibility by sequencing the NA gene?
Not entirely. NI resistance is drug and influenza subtype specific. Whilst we do know some common mutations in the NA which cause resistance (H274Y in H1N1; E119V I222V orR292K in H3N2; R152K, D198E or I222T in Flu B all given in N2 numbering) the mechanism is not fully understood and there may be more not yet characterised. Sequencing can and should be used alongside phenotypic testing (enzyme inhibition tests) to characterise isolates with high IC50 values i.e. reduced NI susceptibility.
Q. Are there cell based assays to measure NI sensitivity?
The use of cell based assays to carry out widespread surveillance for NI 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 useful to characterise NI resistant viruses or those with reduced sensitivity.
Q. How can I perform plaque reduction assays for NI susceptibility?
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 point at which the number of plaques formed is reduced to 50% of that of the virus control is the IC50 for that 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, or the inclusion of replicate titrations to give more robust data.
Q. Do I need both fluorescence and luminescence enzyme inhibition tests?
Not necessarily. Surveillance can be carried out using one method. IC50 values generated from fluorescence and luminescence tests cannot be directly compared as the chemistry is slightly different, leading to different values. It is important that one method is used consistently in a lab to allow evaluation of trends. The alternate assay can be useful for research purposes, and to investigate outliers or resistance, but not interchangeably for surveillance.
Q. What is the cost per sample of the enzyme inhibition tests?
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 viral titration and IC50 measurements.
Luminescence Assay: £10-20 per sample
Fluorescence Assays: £2-4 per sample
Q. What equipment do I need for enzyme inhibition tests?
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 (+4ooC)
Freezer (- 20ooC and - 80ooC)
Desirable: Multi-well reservoirs
(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 (+4ooC)
Freezer (- 20ooC and - 80ooC)
Black 96 well flat bottom plates (Corning 3915 or similar)
Adhesive plate sealers (or plastic lids are also suitable)
Desirable: Multi-well reservoirs
(Thermo Electron Cat. No. RTP/08200-10)
Q. What machine should I buy?
The majority of commercially available fluorimeters and luminometers are suitable for application to these tests. Dual reading machines are available and again most of these are suitable for use, you should ensure that sensitivity is not lost in either function. There are several machines in use in laboratories worldwide that have been shown to be suitable for both tests, 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?
The reagents vary depending on the type of test you choose to perform (chemiluminescence or fluorescence):
Luminescence Testing Applied Biosystems (ABI) supply a kit which contains all of the reagents needed for this test. The kit can be purchased directly from ABI (4374348: NA-Star® Influenza Neuraminidase Inhibitor Resistance Detection Reagent Set). In addition to the kit the following are required:
- Influenza virus isolates derived from passage in tissue culture or egg fluids with an HA titre of 20 Units or higher
- Subtype matched reference viruses
- Oseltamivir Carboxylate (Roche. Product no. GS4071 or Ro64-0802)
- Zanamivir (Glaxo-Smithkline Product no. GR121167X or GG167)
Fluorescence Testing The exact reagents are dependent on the protocol in use, however, the following are critical to all protocols:
- Influenza virus isolates derived from passage in tissue culture or egg fluids with an HA titre of 20 Units or higher
- Subtype matched reference viruses
- 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 NI drugs?
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 out.
Neuraminidase Inhibitor Susceptibility Monitoring: Which Samples?
Q. Which viruses are suitable to test?
NIs 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.
Q. Can I test original clinical material?
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 cultured material?
Yes, egg allantoic and amniotic fluids and cell culture supernatants are suitable for use in the fluorescence and chemiluminescence tests.
Q. Do I need to use phenol red free media to culture viruses for use in the chemiluminescence tests?
No. Viruses are diluted in assay buffer before performing the IC50 test, meaning that the phenol red is diluted enough so as not to interfere with the chemiluminescence signal.
Q. How do I know how much virus to use in the test?
There are several protocols for the MUNANA assay which follow the same principles with minor variations in the details. 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.
Neuraminidase Inhibitor Susceptibility Monitoring: Validation and Reference Strains
Q. Where can I get reference strains?
There is a panel of virus strains available on request from NISN. Included in this panel are four human influenza A 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?
Instructions how to grow stocks of the virus reference strains are included with the panel in an information leaflet.
Q. How should I validate my test?
Tests should be validated by inclusion of wild type and resistant paired reference strains. The IC50 values for each reference should be measured several times to identify limits in your assay conditions. In each test thereafter the reference strain IC50 values should lie within the predefined limits. Additional factors to validate a test should be an examination of each enzyme curve to ensure an S shaped curve is seen. Samples yielding curves with have spurious points or shapes should be repeated. Examples of valid and invalid curves are shown in the ‘Interpretation of raw data’ guidance.
Information on which cells to use to grow viruses is available here.
