SARS-CoV-2 Virus Neutralization Assay

VNT on pig sera was done as described previously^{36 (link)}. Briefly, Vero E6 cells were seeded in 96-well flat-bottom plates (1 × 10⁵ cells/mL) and incubated at 37 °C overnight prior to the assays. Two-fold serial dilutions of sera (starting dilution 1 in 5) in quadruplicate were prepared in 96-well round-bottom plates using Dulbecco’s modified Eagle medium (DMEM) with 1% (v/v) FBS and 1% antibiotic–antimycotic (Gibco). 75 µL of the diluted sera was mixed with an equal volume of media containing 64 PFU of SARS-CoV-2 virus (hCoV-19/England/02/2020, EPI_ISL407073) and incubated for 1 h at 37 °C. Media in the wells seeded with Vero E6 was replaced with 100 µL DMEM with 10% (v/v) FBS and 1% antibiotic–antimycotic (Gibco) and 100 µL of the sera–virus mixture was added into the wells. The plates were incubated for 6 days at 37 °C. Cytopathic effect (CPE) was monitored on a brightfield microscopy, and by fixation using formaldehyde (VWR) and staining using 0.1% (w/v) Toluidine Blue (Sigma-Aldrich). CPE was scored by researchers who were blinded to the identity of the samples. No sera or no virus controls were run in parallel on each plate. Neutralisation titres (ND5₀) were expressed as the reciprocal of the serum dilution that prevented CPE in 50% of the wells.