Ìn situ inactivation of human norovirus GII.4 by cold plasma: Ethidium monoazide (EMA)-coupled RT-qPCR underestimates virus reduction and fecal material suppresses inactivation

Abstract

Cold atmospheric-gaseous plasma (CAP) is an emerging non-thermal technology for decontamination of foodborne bacterial and viral pathogens. We obtained a >5 log₁₀ reduction in the titer (TCID₅₀) of feline calicivirus (FCV) on stainless steel discs and Romaine lettuce leaves after 3 min wet exposure to air plasma generated by a two-dimensional array of integrated coaxial-microhollow dielectric barrier discharge (2D-AICM-DBD). However, when human norovirus (HuNoV GII.4) was treated for 5 min under the same conditions, ~2.6 log₁₀ (>99.5%) reduction in genome copy number was observed as measured by ethidium monoazide-coupled RT-qPCR (EMA-RT-qPCR). To assess this discrepancy, we studied CAP's effect on FCV by the cell culture method and by the EMA-coupled RT-qPCR method. It was found that the molecular titration method (EMA-RT-qPCR) underestimates the level of virus reduction by CAP. Additionally, the fecal matter present in HuNoV samples partially suppressed virucidal activity of CAP. Assuming that the lower virus reduction measured by EMA-RT-qPCR method compared to cell culture method for FCV is the same as for HuNoV, we can conclude that FCV may be used as a surrogate for HuNoV to assess the virucidal effect of CAP. CAP is able to inactivate 3.5 Log₁₀ units of HuNoV at low titers after 2 min of exposure.

Keywords: Cold plasma; EMA-coupled RT-qPCR; FCV versus HuNoV; HuNoV inactivation; Lettuce; RNase-coupled RT-qPCR; Steel surface.