The goal of this study was to examine the efficacy of HPP treatments for Norwalk virus inactivation in artificially seeded raw, whole oysters. HPP treatment (600 MPa, 6°C, 5 min) inactivated HuNoV in oysters and prevented infection among challenged volunteers. HuNoV-infected subjects displayed symptoms consistent with the published literature. In addition, 92% of the infected subjects exhibited a unique WBC (granulocyte) shift and this was significantly associated with infection.
To date, this is the first demonstration of HuNoV inactivation by high pressure in a human challenge study. The highest-pressure conditions (600 MPa, 6°C, 5 min), but not the lower-pressure conditions (400 MPa, 6 or 25°C, 5 min), inactivated HuNoV in oysters and prevented HuNoV infection among all of the subjects challenged with HPP-treated oysters. The findings from this study suggest that HuNoV is less sensitive to pressure than animal caliciviruses used in surrogate in vitro
PFU reduction studies. Studies of HPP treatments of surrogate HuNoV have reported that 275 MPa for 5 min inactivated >6 log10
PFU of FCV (11
) and 400 MPa for 5 min at 5°C inactivated 4 log10
PFU of MNV-1 within oysters (25
). Furthermore, research on the mechanism of action of HPP on MNV suggests that HPP may inactivate MNV by disrupting the MNV receptor responsible for MNV binding and cell entry (39
). In contrast, 400 MPa was insufficient to prevent HuNoV infections among our human subjects, which suggests that a 4-log10
genome equivalent reduction of HuNoV was not achieved. These results suggest that the mechanism of HPP inactivation may work differently against HuNoV than against animal caliciviruses and may require higher pressures for HuNoV inactivation.
While the magnitude of infectious HuNoV reduction at 400 MPa could not be directly determined in this study because the outcome of this study was dichotomous (the HPP treatment either inactivated or did not inactivate HuNoV in oysters), a proposed model of the expected log10
reduction by the three HPP treatments is included (see Fig. S2 in the supplemental material). Furthermore, it is unclear whether an intermediate pressure (between 400 and 600 MPa) or other pressure-temperature combinations would sufficiently inactivate HuNoV within oysters and prevent HuNoV infection among subjects. The diverse sensitivities seen with these caliciviruses to HPP treatments may be common within families of viruses. Viruses such as foot-and-mouth disease virus, human rhinovirus, and poliovirus within the family Picornaviridae
demonstrate a wide range of sensitivities to HPP (24
). It is conceivable that different HuNoV genogroups, and perhaps different clusters within a HuNoV genogroup, would exhibit varied sensitivities to HPP (48
Symptoms associated with HuNoV infection from this study were consistent with the published literature (reviewed in reference 56
). The most common symptoms among the infected subjects included nausea, fever, and diarrhea. The duration of symptoms and viral shedding among infected subjects in this study was also consistent with previous studies. Infected subjects experienced symptoms early in the course of infection. These resolved rapidly, followed by an extended period of virus shedding. The longest shedding period observed was through day 34, as detected by RT-PCR in stool samples (). This observation was consistent with a recent clinical trial in which subjects experimentally infected with HuNoV experienced symptomatic illness for 1 to 2 days and shed virus a median of 28 days (range, 13 to 56 days) after challenge (4
An interesting observation in this study was a transient leukocytosis or “left shift” toward granulocyte production exhibited by volunteers during the early stages of HuNoV infection. This left shift was characterized as a percentage of granulocyte WBCs that was greater than 72% of the total WBC count occurring after the challenge day. In this study, 92% of the infected subjects experienced a left shift toward granulocyte production. Other HuNoV challenge and pediatric viral gastroenteritis studies have reported a similar transient leukocytosis in which a granulocyte shift was identified 48 h postinfection and gradually returned to the baseline by day 5 postinfection (6
). These data suggest that left shifts could be used as an adjunctive tool for the clinical diagnosis of norovirus infection in the hospital setting, especially for patients with gastroenteritis.
The practical commercial application of HPP for HuNoV-contaminated shellfish will require treatments to be economical, viable for current commercial units, and acceptable to consumers. The 5-min HPP treatments tested in this study are economical for high-throughput operations. Although the shellfish industry uses HPP at pressures of approximately 300 MPa to facilitate oyster shucking, extend shelf life, and reduce total bacterial counts, including those of Vibrio
), it is not clear whether the higher pressures required, as suggested by our data, to reduce HuNoV contamination in shellfish will be viable for current commercial units. In addition to evaluation of the upper pressure limits of current commercial units, the consumer acceptability of 600-MPa HPP-treated oysters needs to be investigated. In this study, the high HPP pressure of 600 MPa, which was effective at inactivating HuNoV, induced a mildly cooked whitish appearance. While 400-MPa-treated oysters have been shown to be acceptable to consumers (37
), it is uncertain whether an uncooked 600-MPa-treated oyster is also acceptable to consumers. A 600-MPa-treated oyster may be acceptable for consumption if it is subjected to a further processing step such as cooking. Cooking combined with HPP may be a promising strategy because cooked HPP-treated oysters may be organoleptically indistinguishable from cooked non-HPP-treated oysters.
Two limitations of this research were the use of only one HuNoV strain (the prototypical Norwalk virus) and the seeding of oysters with HuNoV via injection instead of natural bioaccumulation. Injection of HuNoV into the oyster digestive tract was the most accurate way to deliver the exact dosage of HuNoV into each oyster, whereas natural bioaccumulation of viruses via natural uptake from HuNoV-seeded seawater would have led to uncertainty regarding the virus levels actually within the shellfish. Two strengths of this research include the randomized, double-blind experimental design and that we directly tested HuNoV instead of using animal caliciviruses as surrogates for HuNoV.
While data from this study suggest a potential intervention to inactivate infectious HuNoV in oysters for the commercial shellfish industry, additional studies are needed to: (i) determine virus reduction levels between 400 and 600 MPa, (ii) examine more closely the effect of temperature on HuNoV inactivation by HPP and consumer acceptability, (iii) evaluate matrix and composition effects such as salinity and pH on virus inactivation, (iv) evaluate the utility of HPP treatment of HuNoV in other foods (e.g., uncooked fruit and vegetable products), (v) determine the mechanism of HPP inactivation for HuNov, and (vi) evaluate the effect of HPP on different strains of HuNoV.