Decontamination of food and food-processing surfaces from norovirus by cold atmospheric-pressure gaseous plasma

University of Minnesota Ph.D. dissertation. December 2017. Major: Veterinary Medicine. Advisor: Sagar Goyal. 1 computer file (PDF); x, 327 pages + 1 supplemental data table.Human Norovirus (HuNoV) is a leading cause of acute foodborne gastroenteritis outbreaks and sporadic cases. The estimated burden of HuNoV on public health is 685 million and 20 million illnesses each year in the world and the US, respectively. This results in approximately $4.3 billion and $60.3 billion in direct and indirect annual costs worldwide. In the USA, the annual economic burden of foodborne gastroenteritis illness has been estimated to be $77 billion. Most of the foodborne outbreaks are due to fresh produce and contaminated food contact surfaces. Obviously, heat cannot be used to decontaminate fresh fruits and vegetables and hence there is a need for novel non-thermal technologies that can inactivate noroviruses on food and food-processing surfaces. Cold atmospheric pressure gaseous plasma (CAP) is one such technology that has showed strong antibacterial and antifungal effects. However, studies on virucidal activity of CAP are lacking. In this thesis, we studied the in vitro virucidal efficacy of CAP against feline calicivirus (FCV), as a cultivable surrogate of HuNoV. Our results showed that >6 log10 TCID50 of FCV could be inactivated in liquid by a radio-frequency driven plasma jet after 15s exposure to Ar-1%O2 plasma. CAP generated from other feed gas mixtures (i.e. pure Ar, Ar+1% air, Ar+0.27% water) showed lower efficacies than Ar+1% O2. The virucidal efficacy is dependent on the exposure power, exposure distance, and virus-suspending medium. A combination of detailed scavenger measurements combined with positive controls of CAP reactive species allowed us to identify two distinctive pathways that lead to virus inactivation. The first mechanism depends on singlet oxygen in case of Ar+1% O2 plasma. In the presence of air, however, there is a significant reduction in pH and peroxynitrous acid is the key virus inactivating species. Smaller effects of H2O2, O3 and NO2− were also found. By employing different cultural, morphological, molecular and proteomic techniques, we found that reactive species of CAP generated from Ar+O2 are targeting mainly the virus capsid. The CAP was found to inactivate FCV by damaging and disintegrating the virus capsid and oxidizing specific functional peptide residues on major capsid proteins that are important for the attachment and entry of virus into the host cell. Using another CAP generation setup (two-dimensional air-based plasma microdischarge array or 2D-APMA), we were able to inactivate >5 logs of FCV on the surface of stainless steel discs after 3 min of wet exposure at 12.9 W discharge power, and 16.4 slm air flow rate without any significant effect of exposure distances (from 0.5 to 40 cm). Unlike wet exposure, the 2D-APMA showed no efficacy against FCV on dry surface. By using ethidium monoazido-coupled RT-qPCR (EMA-RT-qPCR) method for titrating HuNoV inactivation, CAP showed only 2.6 log reduction in HuNoV GII-4 on the surface of stainless steel and Romaine lettuce leaves under the same operational conditions as mentioned above. The lower inactivation of HuNoV GII-4 by CAP as compared to FCV was due to underestimation by the EMA-RT-qPCR method. The presence of fecal impurities in the HuNoV sample was also found to partially suppress the effect of CAP against HuNoV. This may be due to the competition of organic impurities with the virus particles on reaction with the reactive species of CAP. In a comparison study between the virucidal efficacy of CAP and UVC, as another non-thermal technology, we found that UVC could inactivate FCV on both dry and wet surfaces while CAP was effective only under wet conditions. However, CAP was able to inactivate FCV at both direct and indirect exposures while UVC could inactivate the virus under direct exposure only. This indicates that CAP may be useful for decontamination of rough surfaces with irregularities, which UV may not be able to penetrate. In terms of the power consumption, UVC inactivated FCV at much lower power than needed by CAP to inactivate the same titers of FCV. However, this limitation can be overcome by additional modifications in the design of the CAP setup to be less power consuming. In general, CAP has many advantages and holds promise as a viral decontamination technique