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.

Synthetic reconstruction of BtCoV HKU5. We have previously described the synthetic reconstruction of a noncultivatable virus, BtCoV HKU3 (16) . As phylogenetic studies indicate that BtCoV HKU5 is most closely related to MERS-CoV, we used published sequence information to synthesize an infectious molecular clone for BtCoV HKU5 (icBtCoV HKU5) (16, 17) . The genome of the virus was synthesized as six contiguous cDNA fragments, as shown in Fig. 1A , and each subclone was flanked by class II restriction endonucleases that allow for directed assembly of a fulllength cDNA genome, which encodes a T7 start at the 5= end and a poly(T) tract at the 3= end. Full-length RNAs were generated using in vitro transcription of ligated cDNAs (16, 18) . Upon electroporation of Vero cells with the full-length RNA, no infectious virus was recoverable, despite robust expression of BtCoV HKU5 N subgenomic mRNAs through 4 days postelectroporation (see Fig. S1B in the supplemental material). Passage of cell culture supernatants resulted in diminished viral subgenomic N mRNA expression over time, and eventually, the recombinant virus went extinct in culture (Fig. S1C) .