Materials

2.1. Critical Equipment 1. 96-Well plate reader for luminescence with injectors [such as the Victor™ (PerkinElmer, Waltham, MA)].

2. Class II biological safety cabinet.

3. Freezer (-80°C) for long-term storage of virus isolates.

4. Refrigerator (4°C) for short-term storage of thawed virus isolates.

5. 37°C Incubator (cabinet) with thermometer.

6. Microcentrifuge (with rotor for 1.5-2 ml centrifuge tubes).

7. Vortex mixer.

2.2. Software Software such as Robosage (in-house, GlaxoSmithKline) (25),

JASPR (in-house, CDC) (34), or others such as Graphpad Prism and Sigmaplot (35) for curve fitting and calculation of the IC50 (drug concentration required to inhibit NA enzyme activity by 50%).

2.3. Viruses 1. Test virus isolates, propagated in cell culture (e.g., MDCK

cells, ATCC, Manassas, VA) or in embryonated chicken eggs. Viruses are stored at -80°C.

2. Reference (control) virus strains, propagated in MDCK cells (ATCC, Manassas, VA) and stored at -80°C. Preferably, a pair of references including NAI-sensitive and NAI-resistant viruses.

Influenza viruses pose a biosafety hazard, therefore protocols described in this chapter must be performed in a containment laboratory according to institutional requirements.

• Zanamivir (GlaxoSmithKline, Uxbridge, UK).

• Oseltamivir carboxylate, active metabolite of the prodrug oseltamivir (Hoffman-La Roche, Basel, Switzerland).

NAIs are acquired from their respective manufacturers via Material Transfer Agreements.

2. NA-Star® Influenza Neuraminidase Inhibitor Resistance Detection Kit (Applied Biosystems, Foster City, CA) which includes:

• NA-Star® Buffer (Part No. 4374345). Stored at 4°C.

• NA-Star® Substrate (Part No. 4374347). Stored at 4°C.

• NA-Star® Accelerator (Part No. 4374346). Stored at 4°C.

• 96-Well NA-Star® Detection Microplates (Part No. 4374349).

2.5. Other Materials 1. 1.5-ml Eppendorf tubes (Fisher Scientific, Pittsburgh, PA).

2. Assorted pipettes and pipette tips (Rainin, Oakland, CA).

3. Reagent reservoirs (ISC BioExpress, Raleigh, NC).

4. 15- and 50-ml Falcon conical tubes (Fisher Scientific, Pittsburgh, PA).

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