Us mo 14 18947

Three strains of enterovirus D68 were obtained: US/MO/14-18947 (purchased from BEI Resources, NR-49129, lot no. 63205984), US/IL/14-18952 (purchased from BEI Resources, NR-49131, lot no. 63205985), and EV-D68 Fermon strain (generous gift from Charles Y. Chiu at the University of California, San Francisco, CA). Enterovirus D68 strains were grown in human muscle rhabdomyosarcoma (RD) cells (purchased from ATCC CCL-136) for all experiments except those shown in Fig. 5, for which US/MO/14-18947 was grown in SH-SY5Y neuroblastoma cells (purchased from ATCC CRL-2266). Virus was added at a multiplicity of infection (MOI) of 100 to RD cells in a T182 flask for 60 min, followed by a phosphate-buffered saline (PBS) rinse and incubation in 10 mL medium (95% high-glucose Dulbecco’s modified eagle medium [DMEM] + 5% fetal bovine serum [FBS]) for 3 days at 33°C. All strains killed 99% of RD cells in the flask after 3 days.

Enterovirus D68 isolates (EV-D68) US/MO/14-18947 (947), US/MO/14-18949 (949), US/IL-14-18952 (952), US/KY/14-18953 (953), and US/IL/14-18956 (956) were obtained from BEI Resources. The numbers in parentheses are used to refer to each isolate in the text. Rhyne and New York (NY) EV-D68 isolates were kindly provided by Shigeo Yagi (California Department of Public Health, Richmond, CA) and W. Ian Lipkin (Mailman School of Public Health, Columbia University), respectively. The Fermon isolate was purchased from ATCC (American Tissue Culture Collection; Manassas, VA). All viruses were propagated and assayed in RD cells. Viral titers were determined by plaque assay (see below).