Inactivation of a human norovirus surrogate, human norovirus virus-like particles, and vesicular stomatitis virus by gamma irradiation

Abstract

Gamma irradiation is a nonthermal processing technology that has been used for the preservation of a variety of food products. This technology has been shown to effectively inactivate bacterial pathogens. Currently, the FDA has approved doses of up to 4.0 kGy to control food-borne pathogens in fresh iceberg lettuce and spinach. However, whether this dose range effectively inactivates food-borne viruses is less understood. We have performed a systematic study on the inactivation of a human norovirus surrogate (murine norovirus 1 [MNV-1]), human norovirus virus-like particles (VLPs), and vesicular stomatitis virus (VSV) by gamma irradiation. We demonstrated that MNV-1 and human norovirus VLPs were resistant to gamma irradiation. For MNV-1, only a 1.7- to 2.4-log virus reduction in fresh produce at the dose of 5.6 kGy was observed. However, VSV was more susceptible to gamma irradiation, and a 3.3-log virus reduction at a dose of 5.6 kGy in Dulbecco's modified Eagle medium (DMEM) was achieved. We further demonstrated that gamma irradiation disrupted virion structure and degraded viral proteins and genomic RNA, which resulted in virus inactivation. Using human norovirus VLPs as a model, we provide the first evidence that the capsid of human norovirus has stability similar to that of MNV-1 after exposure to gamma irradiation. Overall, our results suggest that viruses are much more resistant to irradiation than bacterial pathogens. Although gamma irradiation used to eliminate the virus contaminants in fresh produce by the FDA-approved irradiation dose limits seems impractical, this technology may be practical to inactivate viruses for other purposes, such as sterilization of medical equipment.

Fig. 1.

Gamma irradiation of MNV-1 in fresh produce and cell culture medium. MNV-1 stock solutions (10⁸ PFU/ml) were inoculated into spinach, lettuce, strawberries, and DMEM to achieve an inoculation level of 10⁷ PFU/g or 10⁷ PFU/ml. Prepared samples were irradiated with up to 22.4 kGy and were stomached for 2 min. The survival plot was determined by plaque assays. Data points were averages of three replicates. Error bars represent ±1 standard deviations.

Fig. 2.

Comparison of the sensitivities of MNV-1 and VSV to gamma irradiation. MNV-1 stock (10⁸ PFU/ml) and VSV stock (10⁹ PFU/ml) were inoculated in DMEM and exposed to irradiation up to 22.4 kGy. The survival plot was determined by plaque assays. Data points were averages of three replicates. Error bars represent ±1 standard deviations.

Fig. 3.

Stability of MNV-1 and VSV in different buffers treated by gamma irradiation. MNV-1 and VSV stocks were inoculated into four different buffers (water, PBS, DMEM, and DMEM plus 10% FBS) at final concentrations of 10⁷ and 10⁸ PFU/ml, respectively. The samples were exposed to 2.8 and 5.6 kGy of irradiation. Virus survivors were determined by plaque assays. Data points were averages of three replicates. (A) Stability of MNV-1 in different buffer; (B) stability of VSV in different buffer. The inactivation kinetics of VSV in water and PBS were indistinguishable.

Fig. 4.

Gamma irradiation degrades MNV-1 and VSV structural proteins. (A) SDS-PAGE analysis of purified MNV-1 irradiated at 2.8 kGy and 5.6 kGy. Total viral proteins were analyzed by 12% SDS-PAGE, followed by Coomassie staining. VP1 = MNV-1 capsid protein. (B) SDS-PAGE analysis of purified MNV-1 irradiated at 22.4 kGy. No VP1 protein was present after the treatment. (C) SDS-PAGE analysis of purified VSV irradiated at 2.8 kGy and 5.6 kGy. Five structural proteins of VSV, L, G, P, N, and M, were visualized after Coomassie blue staining. (D) SDS-PAGE analysis of purified VSV irradiated at 22.4 kGy. Only the VSV N and M proteins were visualized after the treatment.

Fig. 5.

Western blot analysis of the MNV-1 capsid protein and VSV G protein after gamma irradiation. (A) Western blot analysis of the MNV-1 capsid protein. Purified MNV-1 was irradiated at doses of 2.8 and 5.6 kGy. Total proteins were separated by SDS-PAGE and subjected to Western blotting using rabbit anti-MNV VP1 polyclonal antibody. (B) Western blot analysis of VSV G protein. Purified VSV was irradiated at doses of 2.8 and 5.6 kGy. Total proteins were separated by SDS-PAGE and subjected to Western blotting using monoclonal antibody against VSV G protein.

Fig. 6.

Gamma irradiation damages MNV-1 and VSV. Purified MNV-1 and VSV were irradiated at doses of 2.8, 5.6, and 22.4 kGy. Treated and untreated virus particles were negatively stained with 1% ammonium molybdate and visualized by transmission electron microscopy. (A) Untreated MNV-1 virion; (B) MNV-1 particles treated with 2.8 kGy; (C) MNV-1 particles treated with 5.6 kGy; (D) MNV-1 particles treated with 22.4 kGy; (E) untreated VSV virion; (F) VSV particles treated with 2.8 kGy; (G) VSV particles treated with 5.6 kGy; (H) VSV particles treated with 22.4 kGy.

Fig. 7.

RT-PCR analysis of MNV-1 and VSV after gamma irradiation. (A) Detection of the VP1 gene from MNV-1 irradiated with 2.8 and 5.6 kGy. Viral genomic RNA was extracted from either treated or untreated MNV-1. The VP1 gene of MNV-1 was amplified by one-step RT-PCR, and PCR products were visualized on 1% agarose gel electrophoresis. (B) Detection of the VP1 gene from MNV-1 irradiated with 22.4 kGy. (C) Detection of the N gene from VSV irradiated with 2.8 and 5.6 kGy. Viral genomic RNA was extracted from either treated or untreated VSV. The VSV N gene was amplified by one-step RT-PCR. (D) Detection of the N gene from VSV irradiated with 22.4 kGy.

Fig. 8.

Gamma irradiation damages human norovirus VLPs. Human norovirus VLPs were expressed and purified from insect cells using a baculovirus expression system. The VLPs were irradiated at three doses, 2.8, 5.6, and 22.4 kGy. Treated and untreated VLPs were negatively stained with 1% ammonium molybdate and visualized by transmission electron microscopy. (A) Untreated human norovirus VLPs; (B) VLPs treated with 2.8 kGy; (C) VLPs treated with 5.6 kGy; (D) VLPs treated with 22.4 kGy.

Fig. 9.

Gamma irradiation degrades the capsid protein of human norovirus. (A) Visualization of human norovirus capsid protein by 12% SDS-PAGE. The purified VLPs were irradiated with 2.8, 5.6, and 22.4 kGy. Total viral proteins were analyzed by 12% SDS-PAGE, followed by Coomassie staining. VP1 = human norovirus capsid protein; cVP1 = cleaved VP1 protein. (B) Western blot analysis of human norovirus VP1 protein. Samples identical to those shown in panel A were separated by SDS-PAGE and subjected to Western blotting using a polyclonal antibody against VP1 protein. (C) Comparison of the stability levels of the capsid proteins of MNV-1 and human norovirus after exposure to gamma irradiation. Two micrograms of MNV-1 and human norovirus VLPs was treated with 2.8, 5.6, and 22.4 kGy. Total proteins were separated by SDS-PAGE, followed by Coomassie staining. The remaining proteins from gamma irradiation were quantified by ImageQuant TL software. Data points were averages of three replicates.