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.

We report the in vitro reconstruction and biological characterization, using reverse genetics and synthetic-genome design, of an infectious clone of BtCoV HKU5 (icBtCoV HKU5) containing the ectodomain from the SARS spike (S) protein (BtCoV HKU5-SE). We show that BtCoV HKU5-SE replicated efficiently and demonstrate that a small molecule inhibitor targeting 3CLpro effectively inhibited BtCoV HKU5-SE and MERS-CoV replication in cell culture. In addition, we report that BtCoV HKU5-SE replicated to high titers in both young and aged mice but did not cause lifethreatening disease. Virus replication and disease were dependent on the presence of the mouse angiotensin-converting enzyme 2 (ACE2) receptor, and immunohistochemistry staining demonstrated the presence of viral antigen in epithelial cells lining the small airways and alveoli. BtCoV HKU5-SE replication was inhibited by SARS-CoV S protein-based vaccines but was not sensitive to vaccines based on S proteins from subgroup 2c CoVs. Unlike mice vaccinated with SARS-CoV N-expressing Venezuelan equine encephalitis (VEE) virus replicon particles (VRP) (15), a severe eosinophilic immune pathology was not observed following BtCoV HKU5-SE challenge of animals vaccinated with VRP expressing HKU5 N or MERS-CoV N, supporting the development of killed and recombinant protein vaccines for MERS-CoV. In vivo passage of BtCoV HKU5-SE in young mice resulted in selection for a mouse-adapted strain (BtCoV HKU5-SE MA) that replicated more efficiently and caused significant clinical disease that progressed to interstitial pneumonia and diffuse alveolar damage (DAD) in aged mice. Mutations in nsp13, nsp14, open reading frame 5 (ORF5), and the M protein conferred increased virulence in mice. Taken together, our results illustrate the utility of synthetic-genomics-based platforms for rapidly generating a set of related zoonotic viruses, identifying viral determinants of increased virulence, assisting in the development of broadly active drugs, and previewing potential vaccine-related complications, which could provide invaluable directions for investigations during the early phases of an epidemic.