Inactivation of Human Norovirus in Contaminated Oysters and Clams by High Hydrostatic Pressure

Abstract

cHuman norovirus (NoV) is the most frequent causative agent of food-borne disease associated with shellfish consumption. In this study, the effect of high hydrostatic pressure (HHP) on inactivation of NoV was determined. Genogroup I.1 (GI.1) or genogroup II.4 (GII.4) NoV was inoculated into oyster homogenates and treated at 300 to 600 MPa at 25, 6, and 1°C for 5 min. After HHP, samples were treated with RNase and viral particles were extracted with porcine gastric mucin (PGM)-conjugated magnetic beads (PGM-MBs). Viral RNA was then quantified by real-time reverse transcription (RT)-PCR. Since PGM contains histoblood group-like antigens, which can act as receptors for NoV, deficiency for binding to PGM is an indication of loss of infectivity of NoV. After binding to PGM-MBs, RT-PCR-detectable NoV RNA in oysters was reduced by 0.4 to >4 log 10 by HHP at 300 to 600 MPa. The GI.1 NoV was more resistant to HHP than the GII.4 NoV (P < 0.05). HHP at lower temperatures significantly enhanced the inactivation of NoV in oysters (P < 0.05). Pressure treatment was also conducted for clam homogenates. Treatment at 450 MPa at 1°C achieved a >4 log 10 reduction of GI.1 NoV in both oyster and clam homogenates. It is therefore concluded that HHP could be applied as a potential intervention for inactivating NoV in raw shellfish. The method of pretreatment of samples with RNase, extraction of viral particles using PGM-MB binding, and quantification of viral RNA using RT-PCR can be explored as a practical means of distinguishing between infectious and noninfectious NoV.

Variable High-Pressure-Processing Sensitivities for Genogroup II Human Noroviruses

“…Since the mechanism of viral inactivation by high pressure is the perturbation of the viral capsid and receptorbinding ability, intact viral particles, but not damaged particles, should be able to bind to PGM-MBs. Using this method, Ye et al (27) found that 600 MPa for 5 min at 6°C was required for reduction below the detection limit (Ͼ4.1-log reduction) of the HuNoV GI.1 strain, which seems to be consistent with a recent human volunteer study. Leon et al (22) reported that HPP at 600 MPa and 6°C for 5 min, but not 5-min 400-MPa treatments (at 6°C or 25°C), completely inactivated 4 logs of the GI.1 HuNoV within seeded oysters.…”

“…In contrast, hepatitis A virus (HAV), another picornavirus, was found to be more sensitive to HPP, especially under acidic conditions, with more than a 5-log virus reduction observed after treatment at 400 MPa for 1 min (31). Recently, Li et al (26) and Ye et al (27) found that HuNoV strains from two different genogroups (G1.1 and GII.4) showed different sensitivity to HPP using a PGM-MB binding assay. Specifically, a 3.6-log reduction of GII.4 was observed in oyster homogenate at 400 MPa and 6°C for 5 min, whereas only 1.3 logs of GI.1 lost binding ability under the same conditions.…”

“…Using the PGM-MB assay, it was found that HuNoV GI.1 and GII.4 strains can be reduced by 0.4 to 4 log 10 by HPP at 300 to 600 MPa (27). In addition, a GI.1 virus was found to be more resistant than a GII.4 virus to HPP.…”

“…Based on this theory, a PGM magnetic bead (PGM-MB) binding assay was developed to detect intact HuNoV (25,26). It was found that HuNoVs GI.1 and GII.4 bound efficiently to PGM-MB (26,27). Previously, we found that HPP disrupts the structure of the norovirus capsid without degradation of viral genomic RNA (13).…”

Comprehensive Comparison of Cultivable Norovirus Surrogates in Response to Different Inactivation and Disinfection Treatments

“…Inactivation of surrogates in other foods, such as milk, juice, strawberry puree, and blueberries, will expand the application of HHP pathogen inactivation to a variety of foods (47,48,62). A recent study on the use of HHP on contaminated oysters and clams found that 300 MPa did not inactivate GI and GII noroviruses (63). Several studies on the use of HHP to inactivate surrogate viruses seeded in a variety of nonfood matrices have been published (47,62,(64)(65)(66)(67)(68).…”