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.

Mouse adaptation of BtCoV HKU5-SE. Although the S glycoprotein is a critical determinant for cross-species transmission, additional mutations across the genome have been shown to contribute to host switching and increased disease severity (20, 25) . To identify potential virulence alleles in BtCoV HKU5-SE, we serially passaged BtCoV HKU5-SE in the lungs of 10-week-old BALB/c mice over 2-day intervals. Virus from passage 9 (BtCoV HKU5-SE MA [mouse adapted]) was plaque purified and used to inoculate young (10-week-old) and aged (1-year-old) animals. BtCoV HKU5-SE MA did not cause any significant weight loss in young mice, but it caused Ͼ20% weight loss in aged mice (Fig. 6A) . The virus titers in lungs were significantly increased in young and aged animals at days 2 and 4 p.i. (Ͼ10 7 PFU/g and 10 6 PFU/g, respectively) (Fig. 6B ). Although young animals did not exhibit any significant increases in pathology compared to that in mice inoculated with nonadapted strains (Fig. 3D ), all aged animals developed acute interstitial pneumonia with fibrin deposition and hyaline membrane formation (Fig. 6C) . Genome sequencing in comparison to the sequence of the parent virus revealed changes in the nsp13, nsp14, ORF5, and ORF7 (M) genes as a result of mouse adaptation ( Fig. 7A and B, top) . It was noteworthy that a frameshift mutation resulted in a truncated ORF5 polyprotein (insertion of nucleotide G between 26,897 and 26,898 bp) (Fig. 7B, top) . Furthermore, virus was also detected in blood at 2 days p.i. (4 ϫ 10 5 PFU/ml), but not in any other organs tested (brain, liver, and spleen) (Fig. 7B, bottom) .