Fig. 1. SARS-CoV-2 D614G variant shows enhanced infectivity in immortalized cell lines and replication fitness in upper human respiratory epithelia compared with the ancestral WT virus.

(A) Genomes of recombinant SARS-CoV-2 D614G variants based on the backbone of a D614-form strain WA1. E, envelope; M, membrane; N, nucleocapsid; nLuc, nanoluciferase; S, spike. (B) Entry efficiency of WT-nLuc and D614G-nLuc in multiple susceptible cell lines at a MOI of 0.5. After 1-hour of infection, cells were treated with neutralization Abs to minimize the secondary round of infection. The relative light unit (RLU) representing the nLuc expression level was measured at 8 hours after infection. RLU values were normalized with background (Bkgd) residual luciferase signals in both viral inocula. (C) Growth curves of the two viruses in Vero-E6 (i), Vero-81 (ii), A549-ACE2 (iii), and Huh7 (iv) cell lines at a MOI = 0.5. (D to F) Comparison of 24-, 48-, and 72-hour titers between the two variants in infected primary nasal (D), large airway (E), and small airway (F) cells at a MOI of 0.1. Triplicated titers of the two viruses in the cultures from the same donor were analyzed by paired t test. (G) Schematic of competition assays on LAE cells. Cultures were infected with a 1:1 or 10:1 ratio of WT and D614G mixture at a MOI of 0.5, and the supernatants were serially passaged three times in naïve cultures. PBS, phosphate-buffered saline. (H and I) BtsCI digestion (H) and Sanger sequencing chromatogram (I) of S gene fragments amplified from viral samples in the LAE competition assay. The 1.5-kb fragments containing the residue 614 were amplified from the total RNA of individual samples collected in each passage. P, passage. Data in (B) and (C) are indicated as mean ± SD and were analyzed by unpaired t test between both viruses; data in (D) to (F) were analyzed by paired t test. N.S., not significantly different; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.