Human norovirus remains the most prevalent foodborne pathogen, resulting in 58% of all foodborne illnesses in the United States, annually. Due to lack of successful cultivation techniques for this virus, research on intervention strategies and disinfection practices to combat this pathogen is still largely underreported. The research performed in this dissertation determined the efficacy of electron beam (e-beam) irradiation and sodium hypochlorite sanitizers at inactivating a human norovirus surrogate (murine norovirus 1, MNV-1) and compared the rates of inactivation against that of an enveloped virus (vesicular stomatitis virus, VSV). This research also attempted to determine the mechanism of viral inactivation for e-beam and sodium hypochlorite.
In Chapter 2, we evaluated the efficacy of e-beam at inactivating MNV-1 inoculated to liquid model systems (phosphate buffered saline, PBS; Dulbecco’s Modified Eagle Medium, DMEM) and fresh produce (shredded cabbage, cut strawberries). MNV-1 proved to be resistant to irradiation in both liquid and food samples. In PBS and DMEM, a dose of 2 kGy provided a less than 1 log reduction of MNV-1. At doses of 4, 6, 8, 10, and 12 kGy, viral reduction in PBS ranged from 2.37 to 6.40 logs, and 1.40 to 3.59 logs in DMEM. At 4 kGy (the maximum irradiation dose approved by the FDA for fresh produce), MNV-1 inoculated to shredded cabbage only experienced a 1 log reduction, and less than 1 log reduction in cut strawberries. Even at 12 kGy, MNV-1 titers were reduced by 3 and 2 logs in cabbage and strawberries, respectively. These results suggest that complex liquid media and the food matrix may protect MNV-1 from irradiation, and that viruses tend to be more resistant to irradiation than bacteria due to their small size and highly stable viral capsid. E-beam does not appear to be a feasible processing technology to inactivate foodborne viruses in food products.
Chapter 3 compares e-beam’s ability to inactivate the nonenveloped MNV-1 versus the enveloped VSV inoculated into PBS and DMEM. Samples were treated with e-beam doses of 0, 4, 8, 16, 24, and 30 kGy. We also attempted to determine e-beam’s mechanism of viral inactivation using transmission electron microscopy (TEM), sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), Western blotting, and reverse transcription polymerase chain reaction (RT-PCR). The mechanism of viral inactivation has been demonstrated with gamma irradiation, but no published study to date has evaluated if e-beam would have a similar mechanism. MNV-1 required 24 kGy in PBS and 30 kGy in DMEM for complete inactivation, while VSV was completely inactivated using 16 kGy for both media. TEM analysis demonstrated that increasing doses of e-beam disrupted the structure of the virions. SDS-PAGE and Western blotting analysis found that irradiation can also degrade viral proteins, though these proteins can remain antigenic in the presence of specific antibodies. Finally, using RT-PCR, irradiation was found to also degrade viral genomic RNA. As expected, the mechanism of inactivation of e-beam is similar to that of gamma irradiation.
Chapter 4 compared the rates of inactivation of MNV-1 and VSV subjected to varying concentrations of sodium hypochlorite (5, 10, 20, 50, 100, 200, 400, 800, and 1,000 ppm) and exposure times (0, 0.5, 1.0, 5.0, 10, and 30 minutes). We also attempted to determine hypochlorite’s mechanism of viral inactivation. As expected, MNV-1 was much more resistant to chlorine, being able to withstand 400 ppm of chlorine for up to 10 minutes (0.46 log PFU/ml of virus remaining). VSV was much more susceptible to chlorine, requiring treatment of at least 10 ppm for 10 minutes for complete viral inactivation. Purified MNV-1 treated with 200 ppm hypochlorite for 1.0, 5.0, and 10 minutes exhibited slight damage to the viral capsid protein, but no physical damage to the virus particle or degradation of the viral RNA was observed. Purified VSV treated at 10 ppm for 1.0, 5.0, and 10 minutes did not exhibit any significant changes in virion structure, viral proteins, or viral RNA. Sodium hypochlorite sanitizer concentrations used in the food industry may not be sufficient to reduce contaminating norovirus to safe levels (since less than 10 particles is sufficient to cause illness). Also, the sanitizer’s mechanism of viral inactivation remains inconclusive.