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.

Therefore, we analyzed whether the small molecule inhibitor GRL-001 could inhibit the replication of BtCoV HKU5-SE and MERS-CoV in cell culture (23, 24) . The enzyme was purified using published methods (detailed in Methods in Text S1 in the supplemental material), resulting in high yield (28 mg/ liter) and purity, and inhibition assays were conducted using a fluorimetric substrate as described previously (22, 23) . GRL-001 was very potent in inhibiting BtCoV HKU5 3CLpro activity, with an inhibition constant (K i ) of 5.9 Ϯ 0. BtCoV HKU5-SE replication in vivo. To examine whether BtCoV HKU5-SE replicates and causes disease in mice, we intranasally infected young (~10-weekold) and old (1-year-old) BALB/c mice with 1 ϫ 10 5 PFU of virus. BtCoV HKU5-SE did not cause weight loss in young animals, but aged BALB/c mice ex-perienced~10% weight loss by 4 days p.i. (Fig. 3A ). In the aged-mouse model, clinical signs of disease included lethargy, ruffling of fur, and mildly labored breathing patterns. More importantly, in both young and aged mice, the virus replicated to 10 6 and Ͼ10 4 PFU/g in the lungs at 2 and 4 days p.i. (Fig. 3B ). Weight loss and pathology changes were not noted in mock-infected young and aged animals (data not shown). Because the Y436H substitution in the SARS-CoV S ectodomain enhances binding to the mouse ACE2 receptor (20), we infected 1-yearold ACE2 Ϫ/Ϫ mice with BtCoV HKU5-SE to assess whether ACE2 was required for virus entry and disease. No weight loss or virus replication was observed in ACE2 Ϫ/Ϫ mice ( Fig. 3A and B) , clearly identifying the requirement of mouse ACE2 in BtCoV HKU5-SE entry, replication, and disease.