2.5 Virus Neutralization Assay
To determine the presence of neutralizing SARS-CoV-2 antibodies in the sera of chickens and turkeys, a plaque reduction neutralization test was used. Serum samples collected at 0, 7, 14 and 21 days after infection were heat inactivated at 56°C for half an hour. Serial five-fold dilutions of the inactivate sera were incubated with virus for 1 hour at 37°C. Each virus-serum mixture was then added to duplicate wells of Vero E6 cells in a 48-well format, incubated for 1 hour at 37°C, and overlaid with 500 μl of 2.00% carboxymethyl cellulose (Sigma) in Dulbecco’s Modified Eagle Medium (Thermo-Fisher) per well. Plates were then incubated at 37°C for 72 hours, fixed with 10% buffered formalin and stained with 0.5% crystal violet. Serum dilutions resulting in >70% reduction of plaque counts compared to virus controls were considered positive for virus neutralization.