Methods
Deaths and confirmed cases data from the European CDC (https://www.ecdc.europa.eu/en/covid-19-pandemic)1, which is updated daily, was accessed on April 6, 2020. SARS-CoV-2 viral spike sequences were accessed from the GISAID database (https://www.gisaid.org) on April 6, 2020. Assuming that a patient was tested and accounted as a confirmed case 11 days before death, the case fatality rate (CFR) was calculated from total cases and total deaths per day in each country, considering an average of 11 days from hospitalization to death (medRxiv 2020.01.29.20019547). The analysis was restricted to a time window consisting of the last 8 days from the data accession date (3/30/2020-4/6/2020). Both the average and median CFR were calculated for analysis. Standard linear regression was performed for CFR vs D614 percentage from this data using GraphPad Prism, including and excluding the data from the United Kingdom, which exhibited an unusually low number of cases due to an unusually low level of testing/diagnosis and an unusually high level of death reporting.
For structural analysis of the impact of 614 D/G identity, we used published cryo-electron microscopy structures of the SARS-CoV-2 trimeric viral spike in its “down”, or unliganded, state, wherein the host angiotensin converting enzyme 2 (ACE-2) binding site is buried and inaccessible (PDB ID 6VXX), and its “up” state, wherein this host receptor binding site that is necessary for viral infection is exposed (PDB ID 6VYB, 6VSB)2,3. A single monomer (chain A) in the trimer was mutated in silico to G614. For both 614 identities, the neighboring sidechains (10.0 Å) were minimized using the Biased-Probability Monte Carlo algorithm for 106steps4. Van der Waals energy, vacuum Coulomb electrostatics, solvation electrostatics, hydrogen bonding, torsional energy and entropy were calculated for each conformation searched and the lowest energy conformation was identified. The free energy change caused by the mutation of D614 to G was then calculated as: ΔΔG = ΔGG-Mutant – ΔGD-WildType. Thus, a positive value indicates destabilization of the mutant G relative to the wild-type D, since ΔG was a negative value for both forms. All biophysical calculations, molecular modeling and molecular graphics were performed using ICM-Pro (Molsoft LLC, La Jolla, CA).