Plaque Reduction Neutralisation Tests (PRNTs)

PRNTs were performed essentially as previously described [44], but utilising the SARS-CoV-2-ZsGreen reporter virus. In brief, VeroE6 cells were seeded in 48-well plates to obtain a confluent monolayer at the time of infection. SARS-CoV-2-ZsGreen viral stock (MOI = 0.001) was pre-incubated with a 3-fold dilution series of the heat-inactivated serum for 2 h at 37°C, before addition to the cells. A semi-solid overlay, made of 0.2% agarose in complete DMEM, was added after 2 h. 48 h post-infection, plates were fixed for 30 min in 4% PFA. Fixative was replaced with PBS containing a 1:5,000 dilution of DAPI, and ZsGreen fluorescence analysed by automated microscopy (Cellomics). 36 images were acquired for each well of a 48-well plate at a magnification of 5x, allowing full area coverage (1,728 images per 48-well plate). The 36 images for each well were stitched together using a Fiji (ImageJ) [43] macro, and the resulting 48 composite images stacked together in a single file from which the background was subtracted. Then, montages (6 rows, 8 columns) were generated for each stack and inverted. Illustrative PRNT data is shown in S6 Fig.