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Panel of Influenza A Viruses for Assessment
of Resistance to Neuraminidase Inhibitors

Panel Description:
This panel of influenza A viruses is for the evaluation of resistance to neuraminidase(NA) inhibitors and for the standardization of IC50 values. The package includes fourhuman influenza A viruses isolated, plaque purified and cultured in Madin Darby caninekidney (MDCK) cells. There are two vials of each virus (each vial containing 250 µl ofvirus-containing phenol red-free culture medium).
1. A/MISSISSIPPI/3/01 (H1N1) wild-type virus (A/New Caledonia/20/99-like) -
carrying histidine at position 274 (274H) of neuraminidase glycoprotein (zanamivir and
oseltamivir carboxylate susceptible).
2. A/MISSISSIPPI/3/01 (H1N1) resistant virus (A/New Caledonia/20/99-like)
carrying tyrosine at position 274 (274Y) of neuraminidase glycoprotein (reduced
susceptibility to oseltamivir carboxylate).
3. A/FUKUI/20/04 (H3N2) wild-type virus (A/Fujian/411/2002-like) - carrying glutamic
acid at position 119 (119E) of neuraminidase glycoprotein (zanamivir and oseltamivir
carboxylate susceptible).
4. A/FUKUI/45/04 (H3N2) resistant virus (A/Fujian/411/2002-like) - carrying valine at
position 119 (119V) of neuraminidase glycoprotein (reduced susceptibility to oseltamivir
Storage Conditions:
The influenza A viruses included in the panel were dispensed aseptically. Viruses are
provided on dry ice and should be stored at -70°C immediately upon arrival.
Growing the Viruses:
Important note before proceeding: It is strongly recommended to limit the number of
passages of the influenza viruses included in the panel because some NA inhibitor-
resistant viruses can be unstable following cell culture passage, resulting in a mixture of
drug-resistant and drug-sensitive variants. Existence of a mixture of variants may have a
direct affect on the drug susceptibility of the reference virus (IC50 values). Therefore,
before use in a NA inhibition assay the viruses included in the panel should be cultured
only once in an influenza virus permissive cell line such as MDCK, aliquoted and stored
at -700C until used.
All of the following procedures should be conducted under normal aseptic cell cultureconditions: Note: Infection of MDCK cells with each of the four influenza A viruses must be
performed separately and all appropriate safety procedures and virological
requirements should be followed.

A. For each virus, prepare two 75cm2 cell culture flasks of MDCK cells to near 100%confluence.
B. Prepare 1:100 dilution of virus in sterile phosphate-buffered saline (PBS), pH 7.2-7.4.
Label sterile tubes with appropriate dilutions, and add 1800 µl PBS to the first tube (1:10dilution) and 9000 µl PBS to the second tube (1:100 dilution). Take 200 µl of the virusand add it to the first tube to give 1:10 dilution, mix thoroughly. Take 1000 µl of the 1:10dilution and add it to the second tube to give 1:100 dilution, mix thoroughly. Theremaining virus stock should be kept and stored at -700C.
C. Wash the MDCK cell monolayer twice with sterile PBS, and then inoculate the 5 mlsof diluted virus (1:100 dilution) onto each of the two 75cm2 cell culture flasks of MDCKcells.
D. Incubate the 75cm2 flasks for 30 minutes at 350C for virus adsorption. Remove thevirus inoculum and add 20 mls per flask of an appropriate maintenance medium withTPCK-trypsin and then incubate at 350C for 3 days (up to 5 days of incubation ispossible). Observe the cell monolayer daily for cytopathic effect. Virus growth should beconfirmed by haemagglutination assay or another assay.
Storage Conditions of Influenza A Viruses for Use in NA
Inhibition Assays:

To avoid repeated freeze-thaw steps it is recommended that influenza viruses for use inNA inhibition assays should be stored in cryogenic tubes at -70 From the 40 ml of cell culture supernatant harvested, the recommendedvolumes for the storage of each virus is: (100ul of virus should be sufficient for a single assay) As needed, one of the medium sized tubes (1000ul or 2000ul) can be thawed andrefrozen into volumes enough for use in only a single assay (for example 100 µl).
Determination of IC50 Values:
The Panel of Influenza A Viruses for Assessment of Resistance to Neuraminidase
Inhibitors have been grown and tested by four different laboratories.The protocols for NA
enzyme inhibition assays can be provided from NISN upon request. The drug
concentration that inhibited 50% of the NA enzymatic activity (IC50) was determined from
the dose-response curve. IC50 values can be determined using various software
programs such as GraphPad Prism® 4, GraphPad, San Diego, CA. IC50 values
determined by the four laboratories using both a chemiluminescence- and a
fluorescence-based NA enzyme inhibition assays are listed in Table 1.
Table 1. Evaluation of the panel of influenza A viruses in NA enzyme inhibition

a IC50 values presented as the range of values determined by three different laboratories.
b IC50 values presented as the mean  1 standard deviation determined by multiple assays from onelaboratory Use Restrictions:
The Panel of Influenza A Viruses for Assessment of Resistance to Neuraminidase
Inhibitors are being provided by the Neuraminidase Inhibitor Susceptibility Network
(NISN) and are distributed for internal research, non-commercial purposes only. The
viruses are provided without material transfer agreement, however, NISN require that
the viruses, their products or its derivatives may not be distributed to third parties. All
requests for these viruses must be addressed directly to NISN.
acknowledged as the source of these viruses


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