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.

Similar to our previous results with the subgroup 2b BtCoV strain HKU3 (16), the recombinant BtCoV HKU5 replicated but did not spread efficiently between cells, suggesting an entry block. This observation could reflect a low level or lack of appropriate receptor expression and/or other critical entry cofactors, such as cathepsins or TMPSSR2 proteases (35) . Using a chimeric BtCoV HKU3 virus, we have previously demonstrated that the RBD of SARS-CoV S protein alone is sufficient for binding to mouse ACE2, enabling virus replication in mice (36) . Inclusion of the mouse-adapted Y436H substitution in S (37) enhanced BtCoV HKU3 virus replication but not disease severity in aged-animal models (16) . In SARS-CoV recombinant viruses, S amino acid substitutions are sufficient to produce severe disease in aged BALB/c mouse models (20) . In BtCoV HKU5, our attempts to replace the putative HKU5 RBD with the SARS-CoV RBD were unsuccessful, most likely reflecting the high antigenic distance and structural incompatibilities that hinder genetic exchange of some domains between subgroup 2b and 2c S glycoproteins. Supporting earlier work with mouse hepatitis virus and feline coronavirus (19) , we introduced the SARS S glycoprotein ectodomain with the Y436H substitution into the BtCoV HKU5 genome (BtCoV HKU5-SE), and a recombinant chimeric virus was isolated that was capable of productive infection in culture and in young and aged BALB/c mice.