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.

Systematic assembly and recovery of full-length infectious clones of BtCoV HKU5 variants. The BtCoV HKU5 molecular clone was designed based on a consensus sequence of BtCoV HKU5 isolates available in Gen-Bank (nucleotide sequence accession number EF065512.1). The gene encoding the BtCoV HKU5 S protein (ORF2) was also replaced with the full-length ectodomain of the SARS-CoV S protein (residues 1 to 1190) using previously described strategies (16, 19) . The mouse-adapted substitution Y436H in the SARS-CoV spike protein (20, 25) was also included in the chimeric S construct to promote replication in mice. This clone was designated BtCoV HKU5-SE. The wild-type virus and chimeric BtCoV HKU5 E fragments were synthesized by Bio Basic, Inc. (Ontario, Canada) as six contiguous cDNAs. After assembly and in vitro transcription, recombinant virus was recovered on Vero cells through transfection of the full-length transcripts, as previously described in detail (16, 18) .