Prism version

The proportion of viruses isolated in eggs as compared to cells was assessed using Chi-squared analysis. The difference in variation in isolation rates over the years studied between egg and cells was assessed using the F-test, calculated in Excel (Microsoft, Australia). The difference in HA titres or Cts between viruses isolated only in cells as compared to viruses isolated in both cells and eggs was assessed using an unpaired t-test, using GraphPad Prism Version 7. The difference in HI titres between cell and egg-grown isolates was assessed using a multiple t-test, using GraphPad Prism Version 7.

WST-1 cell proliferation assays were carried out as previously described (Scott et al. 2022 ; Scott, Hodgson, et al. 2023 (link)). For the colony formation assays cells were drugged at the indicated concentrations for 72 h with their respective DMSO controls, then 100 cells/well were seeded in 6-well dishes with the treatment and maintained for 14 days until colonies of more than 50 cells/colony had formed. The culture medium and treatment were changed on day 7. Colonies were fixed with ice-cold absolute methanol for 10 min then stained with 0.5% w/v crystal violet. For CellTiter-Glo® assays, cells were seeded in Nunc™ MicroWell™ 96-well plates (Thermo Scientific, 10072151) at 10,000 cells per well in 100 μL complete media. After 24 h, cells were drugged at the range of concentrations indicated. Cell viability was assessed at 72 h with the CellTiter-Glo® Luminescent Cell Viability Assay (Promega, G7571). Luminescence was recorded with the Varioskan™ LUX microplate reader. Dose response curves, unpaired t-tests and bar plots were generated using GraphPad PRISM version 9.5.0. Data are presented as the mean of three biological repeats ± standard error of the mean (SEM). At least 3 technical and 3 biological repeats were performed for each experiment, with representative data shown in the figures.

Statistical analyses were performed using Prism Version software (GraphPad, San Diego, USA). ns = not significant; P > 0.05; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001 in the figures.

Detection of plasma antibodies that can neutralize a pseudotyped VSV virus expressing the SARS-CoV-2 S protein was performed as previously described (8 (link)). Briefly, various dilutions of heat-inactivated plasma were incubated with 10⁴ PFU of VSV-SARS-CoV-2-S_△21 virus for 1 hour at 37°C. Antibody-virus complexes were added to Vero E6 cells and incubated for 7.5 h at 37°C. Cells were then fixed and stained with Hoechst 33342 nuclear stain (Invitrogen). Images were acquired with the InCell 2000 Analyzer (GE Healthcare) automated microscope to visualize nuclei and infected cells (eGFP-positive cells). Images were analyzed in InCell Analyzer 1000 Workstation Software (GE Healthcare) and data were processed using Prism version 8 (GraphPad Software, Inc).

Statistical significance was assessed by two-tailed Student’s t-test using Prism version 5.04 (GraphPad, La Jolla, CA, USA). A p ≤ 0.05 was considered statistically significant. For multiple comparisons, we utilized ANOVA. The CompuSyn software (Version 3.0.1, ComboSyn, Inc., Paramus, NJ, USA, www.combosyn.com) was used for the drug combination analysis including the calculation of the combination index (CI). A CI < 1 was considered as synergistic, a CI = 1 as additive, and a CI > 1 as antagonistic.

Statistical analysis of in vitro data was performed using PRISM version 5.0 (GraphPad Software). Data are expressed as means ± SD (Student's t-test). Statistical analysis of in vivo data was performed using the Mann–Whitney U test. A P-value < 0.05 was considered significant for all comparisons.