Quantitative determination of the electron beam radiation dose for SARS-CoV-2 inactivation to decontaminate frozen food packaging

Abstract

The spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) from cold-chain foods to frontline workers poses a serious public health threat during the current global pandemic. There is an urgent need to design concise approaches for effective virus inactivation under different physicochemical conditions to reduce the risk of contagion through viral contaminated surfaces of cold-chain foods. By employing a time course of electron beam exposure to a high titer of SARS-CoV-2 at cold-chain temperatures, a radiation dose of 2 ​kGy was demonstrated to reduce the viral titer from 10^4.5 to 0 median tissue culture infectious dose (TCID₅₀)/mL. Next, using human coronavirus OC43 (HCoV-OC43) as a suitable SARS-CoV-2 surrogate, 3 ​kGy of high-energy electron radiation was defined as the inactivation dose for a titer reduction of more than 4 log units on tested packaging materials. Furthermore, quantitative reverse transcription PCR (RT-qPCR) was used to test three viral genes, namely, E, N, and ORF1ab. There was a strong correlation between TCID₅₀ and RT-qPCR for SARS-CoV-2 detection. However, RT-qPCR could not differentiate between the infectivity of the radiation-inactivated and nonirradiated control viruses. As the defined radiation dose for effective viral inactivation fell far below the upper safe dose limit for food processing, our results provide a basis for designing radiation-based approaches for the decontamination of SARS-CoV-2 in frozen food products. We further demonstrate that cell-based virus assays are essential to evaluate the SARS-CoV-2 inactivation efficiency for the decontaminating strategies.

Keywords: Cold-chain; Electron beam radiation; Food decontamination; Inactivation; Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

Fig. 1

Workflow of inactivation of viruses using electron beam radiation. A. The radiation dose required for the inactivation of SARS-CoV-2 was determined using low-energy electron irradiation (LEEI). B. HCoV-OC43 was used as a surrogate for SARS-CoV-2 and treated with both LEEI and high-energy electron irradiation (HEEI) to determine the radiation dose required for the inactivation of the virus in different materials.

Fig. 2

Effect of LEEI on the infectivity of SARS-CoV-2 and HCoV-OC43. Up to 10 ​μL of clarified viral supernatant of 10^4.6 TCID₅₀/mL of SARS-CoV-2 (A) or 10^4.75 TCID₅₀/mL of HCoV-OC43 (B) was placed on stainless steel sheet and exposed to increasing doses of LEEI at 4 ​°C or −20 ​°C, respectively. The stainless steel sheet was washed thoroughly with serum-free DMEM, and the solution containing eluted viruses was tested in duplicates using the TCID₅₀ assay. Representative microphotographs (magnification ​× ​100) of Vero E6 (C) and BHK-21 (D) monolayers 72 ​h post-infection with SARS-CoV-2 or HCoV-OC43 under treatment with the indicated doses of radiation at −20 ​°C are shown. Complete viral inactivation was determined using three consecutive passages of cell culture supernatants on corresponding cells with no cytopathic effect. The data are the mean of triplicate measurements. The experiments were repeated three times with similar results.

Fig. 3

Effect of the tested materials on HCoV-OC43 inactivation using HEEI. Ten microliters of viral stock containing 10⁴−10⁵ TCID₅₀/mL were placed on different materials at 4 ​°C (A) or −20 ​°C (B). Virus particles were carefully recovered after the indicated doses of radiation and processed for TCID₅₀ assay. Complete viral inactivation was verified using three consecutive passages of cell culture supernatants on corresponding cells with no cytopathic effect. The mean ​± ​SD of triplicate measurements is shown. ∗P ​< ​0.05, ∗∗P ​< ​0.01.

Fig. 4

Influence of LEEI on the sensitivity of RT-qPCR assay for SARS-CoV-2 detection. Ten microliters of serially diluted (10-fold) viral samples on stainless steel surfaces were exposed to 2 ​kGy of radiation or not irradiated, and then the viral particles were recovered. SARS-CoV-2 RNA was extracted and subjected to RT-qPCR assays. At each dilution, the Cq detection threshold and linear relationship between TCID₅₀ and Cq values for viral E (A and D), N (B and E), and ORF1ab (C and F) genes are shown. The mean ​± ​SD of triplicate measurements is shown.

Fig. 5

Combination of virus culture and RT-qPCR methods. The SARS-CoV-2 with indicated titers placed on stainless steel surfaces were recovered, and the viral suspensions were incubated with Vero E6 monolayers for 72 ​h. Viral RNA extracted from the cell supernatants was subjected to RT-qPCR assays of the E, N, and ORF1ab genes. SARS-CoV-2 inactivated using 2 ​kGy of radiation could not be recovered (A). Correlation analysis was performed between viral TCID₅₀ and Cq values of the E (B), N (C), and ORF1ab (D) genes. The dashed grey line represents the RT-qPCR limit of detection. The data are the mean of triplicate measurements.