Mechanisms of Zoonotic SARS-CoV Host Range Expansion in Human Airway Epithelium

Department of Microbiology and Immunology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina; Department of Epidemiology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina; Cystic Fibrosis/Pulmonary Research and Treatment Center, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina; Institute for Research in Biomedicine, Bellinzona, Switzerland

^* To whom correspondence should be addressed. Email: rbaric{at}email.unc.edu.

	Abstract

In 2003, SARS Coronavirus (SARS-CoV) emerged and caused over 8000 human cases and greater than 700 deaths worldwide. Zoonotic SARS-CoV likely evolved to infect humans by a series of transmission events between humans and animals for sale in China. Using synthetic biology, we engineered the spike protein (S) from a civet strain, SZ16, into our epidemic strain infectious clone creating the chimeric virus, icSZ16-S, which was infectious but yielded progeny viruses incapable of propagating in vitro. After introducing a K479N mutation within the S receptor binding domain (RBD) of SZ16, the recombinant virus (icSZ16-S K479N) replicated in Vero cells but was severely debilitated in growth. The in vitro evolution of icSZ16-S K479N on human airway epithelial cells (HAE) produced two viruses (icSZ16-S K479N D8 and D22) with enhanced growth on HAE and on DBT cells expressing the SARS-CoV receptor (hACE2). icSZ16-S K479N D8 (D8) and D22 (D22) virus RBDs contained mutations in ACE2 contact residues Y442F and L472F that remodeled S interactions with hACE2. Further, these viruses were neutralized by a human monoclonal antibody (mAb), S230.15, while the parent icSZ16-S K479N strain was eight times more resistant. These data suggest that human adaptation of zoonotic SARS-CoV strains may select for some variants that are highly susceptible to select mABs that bind to the RBD. Epidemic, icSZ16-S K479N and D22 viruses replicate similarly in the BALB/c mouse lung highlighting the potential use of these zoonotic spike SARS-CoV to assess vaccine or sero therapy efficacy in vivo.