A Mouse Model for Betacoronavirus Subgroup 2c Using a Bat Coronavirus Strain HKU5 Variant

Agnihothram, Sudhakar; Yount, Boyd L.; Donaldson, Eric F.; Huynh, Jeremy; Menachery, Vineet D.; Gralinski, Lisa E.; Graham, Rachel L.; Becker, Michelle M.; Tomar, Sakshi; Scobey, Trevor D.; Osswald, Heather L.; Whitmore, Alan; Gopal, Robin; Ghosh, Arun K.; Mesecar, Andrew; Zambon, Maria; Heise, Mark; Denison, Mark R.; Baric, Ralph S.

Generation of polyclonal mouse antisera, neutralization assays, and Western and Northern blot analyses. Genes encoding the CoV S and N proteins indicated above were synthesized by Bio Basic, Inc. (Ontario, Canada) and were packaged into Venezuelan equine encephalitis (VEE) virus replicon particles (VRPs) under BSL2 conditions using attenuated VEE virus 3526 structural protein helpers. Following vaccination, mouse polyclonal sera were generated in BALB/c mice, and neutralization assays involving MERS-CoV isolates and SARS-CoV and using mouse antisera were performed as described previously (49) . For Western blotting, lysates from cells infected with BtCoV HKU5-SE or MERS-CoV isolates were prepared at 12 h p.i. as described previously (36) . The blots were then probed using the mouse polyclonal sera indicated above. Vero cells inoculated with BtCoV HKU5-SE were harvested at 12 h p.i. with TRizol reagent (Invitrogen). Northern blot analysis to describe the genome-and subgenome-length RNAs was performed using a probe for the N gene as described previously (37) .