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.

Next, we characterized the expression of structural proteins in cells infected with BtCoV HKU5-SE. Using antisera from mice vaccinated with VRPs expressing BtCoV HKU5 N protein, we demonstrated that BtCoV HKU5 infection of Vero cells produced a discrete N protein band at the predicted molecular mass of 50 kDa in cell lysates (Fig. 2B) . Polyclonal serum specific for SARS-CoV S identified a 180-kDa S glycoprotein, along with a smaller S1 subunit (90 kDa, faster migrating band), in lysates from infected Vero cells (Fig. 2C) , consistent with the expression of the SARS-CoV S ectodomain by the recombinant BtCoV HKU5-SE. In contrast, the polyclonal serum raised against BtCoV HKU5 S did not recognize the S protein (data not shown), supporting our earlier observations that the major antibody epitopes for S proteins are likely concentrated in the N terminus (21) . The N protein of BtCoV HKU5 shares~68% sequence identity with MERS-CoV and BtCoV HKU4 N proteins (7) . To identify any possible crossreactivity with BtCoV HKU5-SE, we generated mouse polyclonal sera using VRPs expressing the N proteins from BtCoV HKU4.2 and the MERS-CoV SA1 isolate and then demonstrated that these sera recognized the BtCoV HKU5-SE N protein ( Fig. S2D and E) . Taken together, these results demonstrate the presence of conserved polyclonal antibody epitopes in subgroup 2c CoV N proteins.