Materials and Reagents

1. Centrifuge tube, 14 ml (Corning, Falcon^®, catalog number: 352059 )
   
2. 1.5 ml Eppendorf Snap-Cap microcentrifuge tubes (VWR, catalog number: 21008-959 )
   
3. 4 ml Screw neck vials, amber glass (VWR, catalog number: 548-0052 )
   
4. Transparent 96-well (flat-bottom) tissue culture plates (Corning, Falcon^®, catalog number: 353072 )
   
5. Tissue culture flasks, 150 cm² (TPP, catalog number: 90856 )
   
6. Pipette tips (10 µl, 100 µl, 1,000 µl)
   
7. Disposable serological pipets (5 ml, 10 ml and 25 ml)
8. Reagent reservoir, 25 ml (Biotix, catalog number: SR-0025-5SWM )
   
9. Nalgene rapid-flow sterile disposable filter units with SFCA membrane (Thermo Fisher Scientific, Thermo Scientific^TM, catalog number: 156-4020 )
   
10. Hela Rh cell line (a Hela subclone, highly susceptible and permissible to rhinovirus-induced CPE, a gift from Janssen Pharmaceutica, Belgium) (Lacroix et al., 2014)
    
11. All EV-68 strains were cultivated on HeLa Rh cells and stored at -80 °C
    
    1. EV-D68 strains (742, 947, 2042, 1348, 2284 and 670) were isolated from the Netherlands (a kind of gift from Adam Meijer, Diagnostics and Screening National Institute for Public Health and the Environment [RIVM], Bilthoven)
       
    2. EV-D68 strains (US/KY/14-18953, US/IL/14-18952 and US/MO14-18947) – these strains were originally obtained from BEI Resources
    Note: The lysate of all virus strains were used for antiviral assay, and virus titer was measured by endpoint titration assay.
    
12. Dulbecco’s phosphate-buffered saline (DPBS), no calcium, no magnesium (Thermo Fisher Scientific, Gibco^TM, catalog number: 14190144 )
    
13. 0.05% trypsin-EDTA (Thermo Fisher Scientific, Gibco^TM, catalog number: 25300054 )
    
14. Minimum essential media (MEM) Rega-3 (Thermo Fisher Scientific, Gibco^TM, catalog number: 19993013 )
    
15. Potential antiviral compounds
    
    1. SG85*
       
    2. Pleconaril*
       
    3. Vapendavir*
       
    4. Pirodavir*
       
    5. Rupintrivir*
       
    6. Enviroxime*
       
    7. Favipiravir*
    Note: All *compounds were dissolved in DMSO at a concentration of 10 mg/ml and stored in screw neck vials at 4 or -20 °C.
    
16. Fetal bovine serum (GE Healthcare, Hyclone^TM, catalog number: SH30084.03 )
    
17. L-glutamine 200 mM (Thermo Fisher Scientific, Gibco^TM, catalog number: 25030081 )
    
18. Sodium bicarbonate 7.5% solution (Thermo Fisher Scientific, Gibco^TM, catalog number: 25080060 )
    
19. Minimum essential media (MEM), no glutamine, no phenol red (Thermo Fisher Scientific, Gibco^TM, catalog number: 51200038 )
    
20. Magnesium chloride (MgCl₂) (Sigma-Aldrich, catalog number: M8266 )
    
21. CellTiter 96^® AQueous MTS reagent powder (Promega, catalog number: G1111 )
    
22. HCl
    
23. Dimethyl sulfoxide (VWR, catalog number: 67-68-5 )
    
24. Growth culture medium (see Recipes)
    
25. Assay medium (see Recipes)
    
26. MTS/PMS solution (see Recipes)
    

Equipment

1. Pipette controller, PIPETBOY acu2 (VWR, catalog number: 612-0927 )
   
2. Multichannel pipette, 10-100 µl (Eppendorf)
   
3. Incubator at 37 °C (and 35 °C ) and 5% CO₂ (Binder)
   
4. Mid bench centrifuge (Sigma Laborzentrifugen, mode: 4K15C )
   
5. Moxi^TM Z Mini Automated Cell Counter (ORFLO Technologies, catalog number: MXZ001 )
   
6. SpectraMax 190 microplate reader (Molecular Devices, model: SpectraMax 190 )
   
7. pH bench meter (Xylem Analytics, WTW, model: inoLab^® 9310 IDS )
   
8. Inverted microscope (Motic, model: AE21 )
   
9. Safety cabinet (Telstar)
   

Software

1. GraphPad Prism

Procedure