Inactivation of Human Norovirus Surrogates, Feline Calicivirus and Tulane Virus, by Ozonated Water

Human noroviruses (HuNoVs) are globally established as the leading non-bacterial acute gastroenteritis agents. Numerous control strategies have been evaluated to determine their effectiveness against HuNoVs, that have some disadvantages. Aqueous ozone is an aqueous chlorine alternate for microbial inactivation without known carcinogenic by-product formation.This study evaluated the effect of ~1 ppm aqueous ozone against cultivable HuNoV surrogates, feline calicivirus (FCV-F9) and Tulane virus (TV). The objectives were to 1) determine the inactivation kinetics of FCV-F9 and TV using ~1 ppm aqueous ozone with and without organicmatter in water at room temperature using linear and Weibull distribution models; and 2) investigate the mode of action of ozonated water to inactivate FCV-F9 and TV using Transmission Electron Microscopy (TEM), SDS-PAGE, Western blot analysis and RTPCR. FCV at 8.10 ± 0.13 log PFU/ml was reduced by 6.07±0.34 log PFU/mL after 5 min with ~1 ppm aqueous ozone, while TV at 6.27±0.05 log PFU/ml was non-detectable (ND; <100 >particles) after 4.5 min in clean water. The linear model D-values were 0.82±0.01 and 0.96±0.08 min, while Weibull model td=1 values were 1.59±0.25 and 1.79±0.10 min for FCV and TV, respectively. Organic matter increased the treatment time to 36 and 22.5 min to achieve nondetectable levels, while the D-values increased to 6.26±1.27 and 5.08±0.33 min and td=1 values were 10.05±3.11 and 8.28±0.25 min for FCV and TV, respectively. Overall, Weibull distribution resulted in a shoulder (β >1), indicating an initial lag resistant phase during inactivation. TEM displayed subtle distortions in FCV and TV structures, and SDS-PAGE displayed possible degradation of major capsid proteins (VP1; 58 kDa) for both viruses. Short-template RT-PCR (~100 bp) indicated damage to the non-structural ORF1 sequence. Thus, results indicate that longer treatment times are needed for inactivation under soiled conditions. However, removal/reduction of organic load prior to ozonation may be warranted. The results show that aqueous ozone causes damage to viral structure, potentially destroying viral capsid protein, followed by viral nucleic acid degradation. Overall, this system shows promise for inactivation of the tested viruses in water using longer treatment times with ~1 ppm aqueous ozone.