A Gnotobiotic Pig Model for Determining Human Norovirus Inactivation by High-Pressure Processing

Abstract

e Human norovirus (NoV) is responsible for over 90% of outbreaks of acute nonbacterial gastroenteritis worldwide and accounts for 60% of cases of foodborne illness in the United States. Currently, the infectivity of human NoVs is poorly understood due to the lack of a cell culture system. In this study, we determined the survival of a human NoV genogroup II, genotype 4 (GII.4) strain in seeded oyster homogenates after high-pressure processing (HPP) using a novel receptor binding assay and a gnotobiotic pig model. Pressure conditions of 350 MPa at 0°C for 2 min led to a 3.7-log 10 reduction in the number of viral RNA copies in oysters, as measured by the porcine gastric mucin-conjugated magnetic bead (PGM-MB) binding assay and real-time RT-PCR, whereas pressure conditions of 350 MPa at 35°C for 2 min achieved only a 1-log 10 reduction in the number of RNA copies. Newborn gnotobiotic piglets orally fed oyster homogenate treated at 350 MPa and 0°C for 2 min did not have viral RNA shedding in feces, histologic lesions, or viral replication in the small intestine. In contrast, gnotobiotic piglets fed oysters treated at 350 MPa and 35°C for 2 min had high levels of viral shedding in feces and exhibited significant histologic lesions and viral replication in the small intestine. Collectively, these data demonstrate that (i) human NoV survival estimated by an in vitro PGM-MB virus binding assay is consistent with the infectivity determined by an in vivo gnotobiotic piglet model and (ii) HPP is capable of inactivating a human NoV GII.4 strain at commercially acceptable pressure levels. Human norovirus (NoV), a member of the Caliciviridae family, is responsible for over 90% of the outbreaks of acute nonbacterial gastroenteritis worldwide and accounts for more than 60% of the cases of foodborne illness in the United States (1, 2). It is estimated that 48 million individuals, or about 17% of the U.S. population, are sickened each year, leading to approximately 128,000 hospitalizations and 3,000 fatalities in the United States (2). Human NoV is transmitted primarily through the fecal-oral route either by direct person-to-person contact or by fecally contaminated food or water. Human NoV is highly contagious and stable, and only a few virus particles are thought to be sufficient to cause an infection (3, 4). Outbreaks frequently occur in restaurants, hotels, day care centers, schools, nursing homes, cruise ships, swimming pools, hospitals, and military installations. Despite the significant economic impact and high morbidity caused by human NoV, no vaccines or antiviral drugs with activity against this virus are currently available (5, 6). This is due in large part to the lack of a cell culture system and a small-animal model for human NoV (6, 7). As a consequence, the survival of human NoV is poorly understood.A major high-risk food for human NoV contamination is seafood, particularly bivalves, such as oysters, mussels, and clams (8-11). These animals are filter feeders and can readily bioaccumulate human NoV in their tissue...

Persistence and Elimination of Human Norovirus in Food and on Food Contact Surfaces: A Critical Review

“…This is in agreement with the findings of Leon et al (44) and indicates that the PGM-MB binding assay may provide information on the potential infectivity of NoV detectable by RT-PCR. A recent study by Lou et al (56) showed that HPP treatment of NoV-contaminated oysters, which were subsequently fed to gnotobiotic piglets, gave infectivity results consistent with those predicted from the PGM-MB binding assay. Ye et al (110) refined this detection system by incorporating treatment with RNase prior to binding to the mucin, to remove RNA from damaged virus capsids.…”

“…This work is undergoing validation to ascertain its efficacy and could provide a better indication for the presence of infectious NoV particles using RT-qPCR. The PGM-MB binding assay gave good predictive ability to differentiate infectious viruses when compared to an infectivity study of NoV infection in a gnotobiotic pig model (56). Capsid integrity has also been evaluated using propidium monoazide intercalation and RNase treatment (21), virus binding to Caco-2 cells followed by long-range RT-qPCR (47), and other techniques.…”

Research Progress in Viral Inactivation Utilizing Human Norovirus Surrogates

“…Reducing the treatment temperature generally ended up enhancing MNV inactivation, as reducing the temperature from 20 to 0 • C increased viral reduction by nearly 4 log 10 when a 350 MPa 2 min treatment was applied (Huang et al, 2014), similar to the observations for human norovirus strains (Lou et al, 2015b. Additionally, introducing water to the samples prior to treatment also significantly enhanced reduction, achieving additional 1-2 log reduction over the "dry" sample.…”

“…However, when the pressure was increased above 800 MPa complete reduction of VLP integrity was observed in 15 min or less . Interestingly, further human norovirus work for which infectivity was determined using a gnotobiotic pig model demonstrated the efficacy of HPP with treatments as low as 350 MPa for 2 min so long as treatments were at 0 • C (Lou et al, 2015b).…”

Evaluation of High-Pressure Processing in Inactivation of the Hepatitis E Virus

“…HEV response to 500 MPa for 15 min has been previously investigated with RT-qPCR viability markers (PMAxx and platinum chloride, PtCl4-RT-qPCR), but was only able to report the presence of intact viral particles post treatment (43). The inactivation by HPP of other foodborne viruses, including norovirus, Hepatitis A virus (HAV), and surrogates for foodborne viruses has been investigated (26, 44-46). The sensitivity of specific viruses to high-pressure treatment can vary significantly.…”