2.2 Plaque reduction neutralisation assay
SARS-CoV-2 Victoria/01/2020 (Caly, Druce et al., 2020) was generously provided by The Doherty Institute, Melbourne, Australia at P1 and passaged twice in Vero/hSLAM cells [ECACC 04091501]. Whole genome sequencing was performed, on the working stock at Passage 3, using both Nanopore and Illumina as described previously (Lewandowski, Xu et al., 2019). Virus titre was determined by plaque assay on Vero E6 cells [ECACC 85020206]. Cell lines were obtained from the European Collection of Authenticated Cell Cultures (ECACC) PHE, Porton Down, UK. Cell cultures were maintained at 37oC in minimal essential media (MEM) (Life Technologies, California, USA) supplemented with 10% foetal bovine serum (FCS) (Sigma, Dorset, UK) and 25 mM HEPES (Life Technologies).
All heparin compounds were diluted, 5-fold in MEM (Life Technologies) containing 1% (v/v) FCS (Life Technologies), 1x antibiotic/antimycotic (Life Technologies) and 25 mM HEPES buffer (Sigma) (PRNT media) by diluting down in a 96 well plate. Dilutions were made fresh on day of assay. SARS-CoV-2 was diluted to a concentration of 933 pfu/mL (70 pfu/75µl) in PRNT media and mixed 50:50 with heparin dilutions, in a 96-well V-bottomed plate. The plate was incubated at 37°C in a humidified box for 1 hour to allow the virus to be exposed to heparin. The neutralised virus was transferred onto the wells of a washed 24-well plate that had been seeded with Vero E6 cells the previous day at 1.5 x 105 cells/well. The virus/heparin mixture was left to adsorb for an hour at 37°C, then plaque assay overlay media was applied (MEM containing 1.5% carboxymethylcellulose (Sigma), 4% (v/v) FCS and 25mM HEPES buffer). After incubation at 37ºC in a humidified box, for 5 days, plates were fixed overnight with 20% (v/v) formalin/PBS, washed with tap water and then stained with methyl crystal violet solution (0.2% v/v) (Sigma) and plaques were counted. Heparin dilutions were performed in either triplicate or quadruplicate. Heparin dilutions with cells only were also run in duplicate, to determine if there was any cell cytotoxicity.
An internal positive control for the PRNT assay was run in duplicate using a sample of heat-inactivated human MERS convalescent serum known to neutralise SARS-CoV-2 (National Institute for Biological Standards and Control [NIBSC], UK).