Chinese Academy of Sciences, Wuhan, China Abstract The emergence of Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV) and Middle East Respiratory Syndrome (MERS)-CoV underscores the threat of cross-species transmission events leading to outbreaks in humans. In this study, we examine the disease potential for SARSlike CoVs currently circulating in Chinese horseshoe bat populations. Utilizing the SARS-CoV infectious clone, we generated and characterized a chimeric virus expressing the spike of bat coronavirus SHC014 in a mouse adapted SARS-CoV backbone. The results indicate that group 2b viruses encoding the SHC014 spike in a wild type backbone can efficiently utilize multiple ACE2 receptor orthologs, replicate efficiently in primary human airway cells, and achieve in vitro titers equivalent to epidemic strains of SARS-CoV. Additionally, in vivo experiments demonstrate replication of the chimeric virus in mouse lung with notable pathogenesis. Evaluation of available SARS-based immune-therapeutic and prophylactic modalities revealed poor efficacy; both monoclonal antibody and vaccine approaches failed to neutralize and protect from CoVs utilizing the novel spike protein. Importantly, based on these findings, we synthetically rederived an Corresponding Authors: Ralph S. Baric (rbaric@email.unc.edu); Vineet D. Menachery (vineet@email.unc.edu). Author Contributions VDM designed, coordinated, performed experiment, completed analysis, and wrote the manuscript. BLY designed infectious clone and recovered chimeric viruses. SA completed neutralization assays. LEG helped perform mouse experiments, TS and JAP completed mouse experiments and plaque assays. XG performed pseudotyping experiments. KD generated structural figures and predictions. ED generated phylogenetic analysis. RLG completed RNA analysis. SHR provided primary human airway epithelial cultures. AL and WM provided critical monoclonal antibody reagents. ZLS provided SHC014 spike sequences and plasmids. RSB designed experiments and wrote manuscript. The authors declare no competing financial interest. HHS Public Access Author manuscript Nat Med. Author manuscript; available in PMC 2016 June 01. Published in final edited form as: Nat Med. 2015 December ; 21(12): 1508–1513. doi:10.1038/nm.3985. Author Manuscript Author Manuscript Author Manuscript Author Manuscript infectious full length SHC014 recombinant virus and demonstrate robust viral replication both in vitro and in vivo. Together, the work highlights a continued risk of SARS-CoV reemergence from viruses currently circulating in bat populations. Introduction Emergence of Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV) heralded a new era in the cross-species transmission of severe respiratory illness1,2 . Since then, several strains, including influenza A H5N1, H1N1, H7N9, and Middle East Respiratory Syndrome (MERS) CoV have emerged from animal populations causing considerable disease and mortality3 . While public health measures silenced the SARS-CoV outbreak2 , recent metagenomics studies have identified sequences of closely related SARS-like viruses circulating in Chinese bat populations that may pose a future threat4,5 . However, sequence data alone provides minimal insights to identify and prepare for future pre-pandemic viruses. Therefore, to examine emergence potential of circulating CoVs, we built a chimeric virus that encodes a novel, zoonotic spike protein in the context of a viable CoV backbone. This approach characterized the threat posed by SHC014-CoV spike in primary human airway cells, in vivo, as well as the efficacy of available immune therapeutics. Together, the strategy translates metagenomics data to help predict and prepare for future emergent viruses. Results SHC014 and WIV1 sequences represent the closest relatives to the epidemic SARS-CoV strains (Fig. 1 a,b), but maintain important differences in the 14 residues that bind human ACE2, including the five critical for host range: Y442, L472, N479, T487, and Y4916 . In WIV1, three of these residues vary from SARS-CoV Urbani, but were not expected to alter binding (Supplementary Fig. 1a, b, Supplementary Table 1). This fact is confirmed by both pseudotyping experiments (Supplementary Fig. 1d) and in vitro replication of WIV1-CoV5 . In contrast, seven of the 14 ACE2 interaction residues in SHC014 are different than SARSCoV, including all five critical residues (Supplementary Fig. 1c, Supplementary Table 1). These changes, coupled with failure of pseudotyping (Supplementary Fig. 1d), suggested that SHC014 spike is unable to bind human ACE2. However, similar changes had been reported to convey ACE2 binding in related SARS-CoV strains6,7 and thus suggested functional testing was required for verification. Therefore, we synthesized the SHC014 spike in the context of the replication competent, mouse-adapted SARS-CoV backbone (SHC014- MA15) (Supplementary Fig. 2a). Despite predictions from both structure-based modeling and pseudotyping experiments, SHC014-MA15 was viable and replicated to high titers in Vero cells (Supplementary Fig. 2b). Similar to SARS, SHC014-MA15 also required a functional ACE2 molecule for entry, but uses human, civet, and bat orthologs (Supplementary Fig. 2c, d). To test the ability of