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

Hamada A Aboubakr; Fernando Sampedro; James Collins; Peter J Bruggeman; Sagar M Goyal

doi:10.1016/j.fm.2019.103307

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

Hamada A Aboubakr, Fernando Sampedro, James Collins, Peter J Bruggeman, Sagar M Goyal

Research output: Contribution to journal › Article › peer-review

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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.

Original language	English (US)
Article number	103307
Journal	Food Microbiology
Volume	85
Issue number	103307
DOIs	https://doi.org/10.1016/j.fm.2019.103307
State	Published - Feb 2020

Bibliographical note

Funding Information:
This project was partially funded by the Agriculture and Food Research Initiative of the USDA's National Institute of Food and Agriculture , grant number # 2017-67017-26172 . The authors acknowledge Kyocera Inc., Japan for providing the electrode array used in this research and Dr. Jan Vinjé (National Calicivirus Laboratory, Centers for Disease Control and Prevention, Atlanta) for providing HuNoV GII.4-positive stool sample. We also thank Dr. Sunil Mor from Veterinary Diagnostic Lab, College of Veterinary Medicine, University of Minnesota for technical advice during the development of RT-qPCR quantification method of HuNoV. Thanks are also due to Dr. Yishan Yang, USDA, for technical help.

Funding Information:
This project was partially funded by the Agriculture and Food Research Initiative of the USDA's National Institute of Food and Agriculture, grant number # 2017-67017-26172. The authors acknowledge Kyocera Inc. Japan for providing the electrode array used in this research and Dr. Jan Vinj? (National Calicivirus Laboratory, Centers for Disease Control and Prevention, Atlanta) for providing HuNoV GII.4-positive stool sample. We also thank Dr. Sunil Mor from Veterinary Diagnostic Lab, College of Veterinary Medicine, University of Minnesota for technical advice during the development of RT-qPCR quantification method of HuNoV. Thanks are also due to Dr. Yishan Yang, USDA, for technical help.

Publisher Copyright:
© 2019

Keywords

Cold plasma
EMA-coupled RT-qPCR
FCV versus HuNoV
HuNoV inactivation
Lettuce
RNase-coupled RT-qPCR
Steel surface

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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. / Aboubakr, Hamada A; Sampedro, Fernando; Collins, James et al.
In: Food Microbiology, Vol. 85, No. 103307, 103307, 02.2020.

Research output: Contribution to journal › Article › peer-review

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title = "{\`I}n 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",

author = "Aboubakr, {Hamada A} and Fernando Sampedro and James Collins and Bruggeman, {Peter J} and Goyal, {Sagar M}",

note = "Funding Information: This project was partially funded by the Agriculture and Food Research Initiative of the USDA's National Institute of Food and Agriculture , grant number # 2017-67017-26172 . The authors acknowledge Kyocera Inc., Japan for providing the electrode array used in this research and Dr. Jan Vinj{\'e} (National Calicivirus Laboratory, Centers for Disease Control and Prevention, Atlanta) for providing HuNoV GII.4-positive stool sample. We also thank Dr. Sunil Mor from Veterinary Diagnostic Lab, College of Veterinary Medicine, University of Minnesota for technical advice during the development of RT-qPCR quantification method of HuNoV. Thanks are also due to Dr. Yishan Yang, USDA, for technical help. Funding Information: This project was partially funded by the Agriculture and Food Research Initiative of the USDA's National Institute of Food and Agriculture, grant number # 2017-67017-26172. The authors acknowledge Kyocera Inc. Japan for providing the electrode array used in this research and Dr. Jan Vinj? (National Calicivirus Laboratory, Centers for Disease Control and Prevention, Atlanta) for providing HuNoV GII.4-positive stool sample. We also thank Dr. Sunil Mor from Veterinary Diagnostic Lab, College of Veterinary Medicine, University of Minnesota for technical advice during the development of RT-qPCR quantification method of HuNoV. Thanks are also due to Dr. Yishan Yang, USDA, for technical help. Publisher Copyright: {\textcopyright} 2019",

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AU - Sampedro, Fernando

AU - Collins, James

AU - Bruggeman, Peter J

AU - Goyal, Sagar M

N1 - Funding Information: This project was partially funded by the Agriculture and Food Research Initiative of the USDA's National Institute of Food and Agriculture , grant number # 2017-67017-26172 . The authors acknowledge Kyocera Inc., Japan for providing the electrode array used in this research and Dr. Jan Vinjé (National Calicivirus Laboratory, Centers for Disease Control and Prevention, Atlanta) for providing HuNoV GII.4-positive stool sample. We also thank Dr. Sunil Mor from Veterinary Diagnostic Lab, College of Veterinary Medicine, University of Minnesota for technical advice during the development of RT-qPCR quantification method of HuNoV. Thanks are also due to Dr. Yishan Yang, USDA, for technical help. Funding Information: This project was partially funded by the Agriculture and Food Research Initiative of the USDA's National Institute of Food and Agriculture, grant number # 2017-67017-26172. The authors acknowledge Kyocera Inc. Japan for providing the electrode array used in this research and Dr. Jan Vinj? (National Calicivirus Laboratory, Centers for Disease Control and Prevention, Atlanta) for providing HuNoV GII.4-positive stool sample. We also thank Dr. Sunil Mor from Veterinary Diagnostic Lab, College of Veterinary Medicine, University of Minnesota for technical advice during the development of RT-qPCR quantification method of HuNoV. Thanks are also due to Dr. Yishan Yang, USDA, for technical help. Publisher Copyright: © 2019

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N2 - 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.

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KW - HuNoV inactivation

KW - Lettuce

KW - RNase-coupled RT-qPCR

KW - Steel surface

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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

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