fragments were amplified, excised, ligated, and purified. In vitro transcription reactions were then preformed to synthesize full-length genomic RNA, which was transfected into Vero E6 cells as previously described28. The media from transfected cells were harvested and served as seed stocks for subsequent experiments. Chimeric and full length viruses were confirmed by sequence analysis prior to use in these studies. Synthetic construction of chimeric mutant and full length SHC014-CoV were approved by the University of North Carolina Institutional Biosafety Committee and the Dual Use Research of Concern committee. Ethics Statement This study was carried out in accordance with the recommendations for care and use of animals by the Office of Laboratory Animal Welfare (OLAW), National Institutes of Health. The Institutional Animal Care and Use Committee (IACUC) of The University of North Carolina at Chapel Hill (UNC, Permit Number A-3410-01) approved the animal study protocol (IACUC #13-033) followed in this manuscript. Mice & In Vivo Infection Female 10 week and 12 month old Balb/cAnNHsD mice were ordered from the Harlan Labs. Mouse infections occurred as previously described29. Briefly, animals were brought into a biosafety lab level 3 and allowed to acclimate for 1 week prior to infection. For infection and live-attenuated virus vaccination, mice were anesthetized with a mixture of ketamine and xylazine and infected intranasally when challenged with 50 μl of phosphatebuffered saline (PBS) or diluted virus with three to four mice per time point, per infection group per dose as described in the figure legends. For individual mice, notations for infection including failure to inhale entire dose, bubbling of inoculum from nose, or infection through the mouth may lead to exclusion of mouse data at discretion of the researcher; post-infection, no other pre-established exclusion/inclusion criteria are defined. No blinding was used in any animal experiments and animals were not randomized. For vaccination, young and aged mice were vaccinated by footpad injection with a 20 μl volume of either 0.2 μg of double-inactivated SARS-CoV vaccine with alum or mock PBS; mice were then boosted with the same regimen 22 days later, and challenged 21 days thereafter. For all groups, as per protocol, animals were monitored daily for clinical signs of disease (hunching, ruffled fur, reduced activity) for the duration of the experiment. Weight loss was monitored daily for the first 7 days after which, weight monitoring continued until the animals recovered to their initial starting weight or displayed three continuous days of weight gain. All mice losing greater than 20% of their starting body weight were ground fed and further monitored multiple times per day as long as they were under the 20% cutoff. Menachery et al. Page 7 Nat Med. Author manuscript; available in PMC 2016 June 01. Author Manuscript Author Manuscript Author Manuscript Author Manuscript Mice losing greater than 30% of their starting body weight were immediately sacrificed as per protocol. Any mouse deemed to be moribund or unlikely to recover were also humanly sacrificed at the discretion of the researcher. Euthanasia was preformed via isoflurane overdose and confirmation of death by cervical dislocation. All mouse studies were performed at the University of North Carolina (Animal Welfare Assurance #A3410-01) using protocols approved by the UNC Institutional Animal Care and Use Committee (IACUC). Histological Analysis The left lung was removed and submerged in 10% buffered formalin (Fisher) without inflation for 1 week. Tissues were embedded in paraffin, and 5 μm sections were prepared by the UNC Lineberger Comprehensive Cancer Center histopathology core facility. To determine the extent of antigen staining, sections were stained for viral antigen using a commercially available polyclonal SARS-CoV anti-nucleocapsid antibody (Imgenex) and scored in a blinded manner by for staining of the airway and parenchyma as previously described29. Images were captured using an Olympus BX41 microscope with an Olympus DP71 camera. Virus Neutralization Assays Plaque reduction neutralization titer assays were performed with previously characterized antibodies against SARS-CoV as previously described30–32. Briefly, nAbs or serum were serially diluted 2-fold and incubated with 100 PFU of the different icSARS-CoV strains for 1 h at 37°C. The virus and antibodies were then added to a 6-well plate with 5 ×105 Vero E6 cells/well with N ≥ 2. After a 1-h incubation at 37°C, cells were overlaid with 3 ml of 0.8% agarose in media. Plates were incubated for two days at 37° C and then stained with neutral red for 3 hours, and plaques were counted. The percentage of plaque reduction was calculated as [1 − (no. of plaques with antibody/no. of plaques without antibody)] × 100. Statistical Analysis All experiments were conducted contrasting two experimental groups (either two viruses, or vaccinated and unvaccinated cohorts). Therefore, significant differences in viral titer and histology scoring were determined by a two-tailed student’s t test at individual time points. Data was normally distributed in each group being compared and had similar variance. Biosafety and biosecurity Reported studies were