Inactivation of Pepper Mild Mottle Virus in Water by Cold Atmospheric Plasma

Abstract

Water scarcity is one of the greatest threats for human survival and quality of life, and this is increasingly contributing to the risk of human, animal and plant infections due to waterborne viruses. Viruses are transmitted through polluted water, where they can survive and cause infections even at low concentrations. Plant viruses from the genus Tobamovirus are highly mechanically transmissible, and cause considerable damage to important crops, such as tomato. The release of infective tobamoviruses into environmental waters has been reported, with the consequent risk for arid regions, where these waters are used for irrigation. Virus inactivation in water is thus very important and cold atmospheric plasma (CAP) is emerging in this field as an efficient, safe, and sustainable alternative to classic waterborne virus inactivation methods. In the present study we evaluated CAP-mediated inactivation of pepper mild mottle virus (PMMoV) in water samples. PMMoV is a very resilient water-transmissible tobamovirus that can survive transit through the human digestive tract. The efficiency of PMMoV inactivation was characterized for infectivity and virion integrity, and at the genome level, using test plant infectivity assays, transmission electron microscopy, and molecular methods, respectively. Additionally, the safety of CAP treatment was determined by testing the cytotoxic and genotoxic properties of CAP-treated water on the HepG2 cell line. 5-min treatment with CAP was sufficient to inactivate PMMoV without introducing any cytotoxic or genotoxic effects in the in-vitro cell model system. These data on inactivation of such stable waterborne virus, PMMoV, will encourage further examination of CAP as an alternative for treatment of potable and irrigation waters, and even for other water sources, with emphasis on inactivation of various viruses including enteric viruses.

Keywords: cold atmospheric plasma; enteric viruses; pepper mild mottle virus; virus inactivation; water decontamination.

FIGURE 1

Schematic representation of a single-electrode cold atmospheric plasma (CAP) jet. During CAP treatment of the infected water samples, the electrode and glass tube are submerged in 10 mL samples and CAP streamers can be seen. Streamers enter the samples in the form of bubbles through the four openings, two on each side of the glass tube.

FIGURE 2

Representative transmission electron microscopy micrographs of pepper mild mottle virus (PMMoV) in water samples. (A) Whole, undamaged PMMoV in untreated PMMoV sample (PMMoV-PC). (B) Damaged (i.e., broken) PMMoV after 1 min cold atmospheric plasma (CAP) treatment. Structures similar to aggregates are associated with the virus particle, which were not seen for the PMMoV-PC or 5-min and 3-min CAP-treated PMMoV samples; these appear to indicate early viral degradation. (C) Damaged PMMoV after 5 min CAP treatment.

FIGURE 3

Representative gel showing pepper mild mottle virus (PMMoV) RNA degradation after cold atmospheric plasma (CAP) treatments. Notable degradation compared to PMMoV-PC (positive control; i.e., untreated PMMoV sample) is seen for all three amplified RNA fragments after two independent 5-min and 3-min CAP treatments. NTC, non-template control (i.e., sterilized water); CAP 3 min^∗, sample with partial PMMoV inactivation; NCI, negative control of isolation (i.e., sterilized water).

FIGURE 4

Quantification of viability of HepG2 cells following exposure to cold atmospheric plasma (CAP)-treated pepper mild mottle virus (PMMoV)-free samples. Influence of 5-min and 3-min CAP-treated PMMoV-free samples on viability of HepG2 cells after 2 h (A) and 24 h (B), expressed as proportions (%) of PMMoV-free-NC (i.e., untreated PMMoV-free sample) in growth medium (1:2). NC, negative control (phosphate buffered saline: growth medium, 1:2); PC, positive control: H₂O₂ (100 μg mL^–1) for the 2 h time point, and etoposide (50 μg mL^–1) for the 24 h time point; ****p < 0.0001 (ANOVA and Dunnett’s multiple comparison tests).

FIGURE 5

Quantification of genotoxic activity of cold atmospheric plasma (CAP)-treated pepper mild mottle virus (PMMoV)-free samples. Influence of 5-min and 3-min CAP-treated PMMoV-free samples on induction of DNA damage in HepG2 cells after 2 h (A) and 24 h (B). Data are expressed as quantile box plots. Edges of the plots represent the 25th and 75th percentiles, with 95% confidence intervals. Solid line through the box is the median. NC, negative control (phosphate buffered saline: growth medium, 1:2); PC, positive control: etoposide (10 μg mL^–1) for the 2 h time point, and etoposide (5 μg mL^–1) for 24 h time point. DNA damage induction by CAP-treated PMMoV-free samples was compared to the induction by the PMMoV-free NC (untreated PMMoV-free sample in growth medium [1:2]); ****p < 0.0001 (Kruskal–Wallis nonparametric tests and Dunn’s multiple comparison tests).