Purpose: The effect of bromelain, papain, and ficin on cell-culture infectivity of Tulane virus (TV), murine norovirus (MNV), and hepatitis A virus (HAV) were evaluated.
Methods: TV, MNV, and HAV were propagated in LLCMK2, RAW 267.4, and FRhK4 cells, respectively, in the absence of serum, filtered (0.2 µm pore) from cell debris, and treated in duplicate trials (104 to 105 PFU/ml initial concentration) by individual and combined proteases (2500 ppm with 2 mM cysteine-HCl) in HBSS (pH 7) at 45°C for 60 min, 50°C for 10 min, 55°C for 10 min and in citrate buffer (pH 5.5) at 50°C for 10 min. MNV was treated with bromelain at 50°C for various times up to 15 min. Virus infectivity was enumerated by plaque assay or TCID50. Controls included untreated virus, virus heated without proteases, and uninoculated cell culture media and protease solutions.
Results: MNV infectivity was reduced by one and 2.5 logs by papain and bromelain, respectively, at 45°C for 60 min and 3 logs by bromelain at 50°C for 10 min compared to heated and untreated controls (P<0.05). The reduction in MNV at 50°C could be attained within 6 min; no greater reduction was realized with 15 min treatment. HAV infectivity was reduced (one-log) with the combined proteases at 55°C for 10 min (P>0.05). No reduction in TV infectivity was observed. Detection sensitivity was 10 PFU/ml.
Significance: Plant proteases would make desirable antiviral agents for enhanced safety of raw, ready-to-eat produce. Susceptibility of the viruses to the plant proteases suggests variable enzyme access to receptor-binding moieties or sufficient peptide bonds to affect capsid integrity and warrants investigation of human norovirus.