restrictions on mouse adapted strains and monoclonal antibodies generated against escape mutants, research into CoV emergence and therapeutic efficacy may be severely limited moving forward. Together, these data and restrictions represent a crossroads of GOF research concerns; the potential to prepare and mitigate future outbreaks must be weighed against the risk of creating more dangerous pathogens. In developing policies moving forward, it is important to consider the value of the data generated by these studies and if they warrant further study or the inherent risks involved. Overall, our approach has used metagenomics data to identify a threat posed by circulating bat SARS-like CoV SHC014. With the ability to replicate in human airway cultures, produce in vivo pathogenesis, and escape current therapeutics, SHC014 chimeric viruses illustrate the need for both surveillance and improved therapeutics against circulating SARSMenachery et al. Page 5 Nat Med. Author manuscript; available in PMC 2016 June 01. Author Manuscript Author Manuscript Author Manuscript Author Manuscript like viruses. The approach also unlocks metagenomics data to predict viral emergence with possible applications for preparing to treat future emerging virus infections. Online Methods Viruses, Cells, In Vitro Infection, and Plaque Assays. Wild-type SARS-CoV (Urbani), mouse adapted SARS-CoV (MA15) and chimeric SARS-like CoVs were cultured on Vero E6 cells, grown in DMEM (Gibco, CA) and 5% Fetal Clone Serum (Hyclone, South Logan, UT) along with anti/anti (Gibco, Carlsbad, CA). DBT cells expressing ACE2 orthologs have been previously described for both human and civet; bat ACE2 sequence based on Rhinolophus leschenaulti and established as described previously22. Pseudotyping experiments were based on HIV-based pseudovirus prepared as previously described23 and examined on HeLa cells expressing ACE2 orthologs grown in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal calf serum (Gibco) as previously described24. Growth curves in Vero, DBT, Calu-3 2B4, and primary human airway epithelial cells were performed as previously described22, 25. Vero E6 cells were originally obtained from USAMRIID; Calu3 cells were originally provided by Dr. CT Tseng, Universtiy of Texas Medical Branch; none of the cell line working stocks have not been recently authenticated or tested for mycoplasma, although the original seed stocks used to create the working stocks are free from contamination. Human lungs for HAE cultures were procured under University of North Carolina at Chapel Hill Institutional Review Board-approved protocols and represent highly differentiated human airway epithelium containing ciliated and non-ciliated epithelial cells as well as goblet cells. The cultures are also grown on an air-liquid interface for several weeks prior to use as previously described26. Briefly, cells were washed with PBS, and inoculated with virus or mock diluted in PBS for 40 minutes at 37 °C. Following inoculation, cells were washed 3 times, and fresh media added to signify time 0. Three or more biological replicates were harvested at each described time point. No blinding was used in any sample collections nor were samples randomized. All virus cultivation was performed in a BSL3 laboratory with redundant fans in Biosafety Cabinets as described previously by our group. All personnel wore Powdered Air Purifying Respirator (3M breathe easy) with Tyvek suits, aprons, booties and were double-gloved. Sequence Clustering and Structural Modeling The full-length genome sequences and S1 domains of spike amino acid sequences of representative CoVs were downloaded from Genbank or PATRIC, aligned with ClustalX, and phylogenetically compared by Maximum Likelihood using 100 bootstraps or with the PhyML package respectively. The tree was generated using Maximum Likelihood with the PhyML package. The scale bar represents nucleotide substitutions. Only nodes with bootstrap support above 70% are labeled. The tree shows that CoVs are divided into three distinct phylogenetic groups defined as α, β, and γ. Classical subgroup clusters are marked as 2a–2d for β CoVs and 1a and 1b for the α CoVs. Structural models were generated using Modeller (Max Planck Institute Bioinformatics Toolkit) to generate homology models for SHC014 and Rs3367 of the SARS RBD in complex with ACE2 based on crystal structure 2AJF (RCSB PBD identifier). Homology models were visualized and manipulated in MacPyMol (version 1.3). Menachery et al. Page 6 Nat Med. Author manuscript; available in PMC 2016 June 01. Author Manuscript Author Manuscript Author Manuscript Author Manuscript Construction of chimeric SL-Viruses Both wild-type and chimeric viruses were derived from either SARS-CoV Urbani or corresponding mouse adapted (MA15) infectious clone as previously described27. Plasmids containing spike sequences for SHC014 were extracted by restriction digest and ligated into the E and F plasmid of the MA15 infectious clone. The clone was designed and purchased from Bio Basic as six contiguous cDNAs using published sequences flanked by unique class II restriction endonuclease sites (BglI). Thereafter, plasmids containing wild-type, chimeric SARS-CoV and SHC014-CoV genome