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

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-log₁₀ 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-log₁₀ 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.     

Molecular Epidemiology of Oyster-Related Human Noroviruses and Their Global Genetic Diversity and Temporal-Geographical Distribution from 1983 to 2014

Noroviruses (NoVs) are a leading cause of epidemic and sporadic cases of acute gastroenteritis worldwide. Oysters are well recognized as the main vectors of environmentally transmitted NoVs, and disease outbreaks linked to oyster consumption have been commonly observed. Here, to quantify the genetic diversity, temporal distribution, and circulation of oyster-related NoVs on a global scale, 1,077 oyster-related NoV sequences deposited from 1983 to 2014 were downloaded from both NCBI GenBank and the NoroNet outbreak database and were then screened for quality control. A total of 665 sequences with reliable information were obtained and were subsequently subjected to genotyping and phylogenetic analyses. The results indicated that the majority of oyster-related NoV sequences were obtained from coastal countries and regions and that the numbers of sequences in these regions were unevenly distributed. Moreover, >80% of human NoV genotypes were detected in oyster samples or oyster-related outbreaks. A higher proportion of genogroup I (GI) (34%) was observed for oyster-related sequences than for non-oyster-related outbreaks, where GII strains dominated with an overwhelming majority of >90%, indicating that the prevalences of GI and GII are different in humans and oysters. In addition, a related convergence of the circulation trend was found between oyster-related NoV sequences and human pandemic outbreaks. This suggests that oysters not only act as a vector of NoV through environmental transmission but also serve as an important reservoir of human NoVs. These results highlight the importance of oysters in the persistence and transmission of human NoVs in the environment and have important implications for the surveillance of human NoVs in oyster samples.     

High-Pressure Inactivation of Hepatitis A Virus within Oysters

Previous results demonstrated that hepatitis A virus (HAV) could be inactivated by high hydrostatic pressure (HHP) (D. H. Kingsley, D. Hoover, E. Papafragkou, and G. P. Richards, J. Food Prot. 65:1605-1609, 2002); however, direct evaluation of HAV inactivation within contaminated oysters was not performed. In this study, we report confirmation that HAV within contaminated shellfish is inactivated by HHP. Shellfish were initially contaminated with HAV by using a flowthrough system. PFU reductions of >1, >2, and >3 log₁₀ were observed for 1-min treatments at 350, 375, and 400 megapascals, respectively, within a temperature range of 8.7 to 10.3°C. Bioconcentration of nearly 6 log₁₀ PFU of HAV per oyster was achieved under simulated natural conditions. These results suggest that HHP treatment of raw shellfish will be a viable strategy for the reduction of infectious HAV.     

Norovirus detection in shellfish using two Real-Time RT-PCR methods

Shellfish are recognized as a potential vehicle of viral diseases. The aim of the present study was to determine the ability of two real-time RT-PCR methods (an in-house method and a commercial kit) for detecting Norovirus (NoV) belonging to genogroups GI and GII in shellfish. The analyses were performed both on a Norovirus Reference Panel (NRP), consisting of synthetic RNA, and on naturally contaminated mussels. For the experiments carried out on the NRP a statistically significant difference (?2=8.03) was shown between the results obtained by the two methods. The in-house real-time RT-PCR allowed the detection of all genotypes belonging to GI and GII, while the commercial kit was not suitable for the detection of the majority of the GI sequences constituting the panel. No significant difference was instead detected in the experiments carried out on shellfish, where the presence of GI was always concomitant with GII. Both methods were suitable for detection of NoV in shellfish, however the in-house real-time RT-PCR method had the advantage of differentiating GI and GII contamination. As regards the shellfish analysed, a considerable frequency of NoV contamination (34.4% of the samples) was detected, with a predominance of NoV GII.     

Bovine Norovirus: Carbohydrate Ligand,Environmental Contamination,and Potential Cross-Species Transmission via Oysters

Noroviruses (NoV) are major agents of acute gastroenteritis in humans and the primary pathogens of shellfish-related outbreaks. Previous studies showed that some human strains bind to oyster tissues through carbohydrate ligands that are similar to their human receptors. Thus, based on presentation of shared norovirus carbohydrate ligands, oysters could selectively concentrate animal strains with increased ability to overcome species barriers. In comparison with human GI and GII strains, bovine GIII NoV strains, although frequently detected in bovine feces and waters of two estuaries of Brittany, were seldom detected in oysters grown in these estuaries. Characterization of the carbohydrate ligand from a new GIII strain indicated recognition of the alpha-galactosidase (α-Gal) epitope not expressed by humans, similar to the GIII.2 Newbury2 strain. This ligand was not detectable on oyster tissues, suggesting that oysters may not be able to accumulate substantial amounts of GIII strains due to the lack of shared carbohydrate ligand and that they should be unable to contribute to select GIII strains with an increased ability to recognize humans.Environmental sources of animal pathogens and, most specifically, of RNA viruses may constitute substantial risk factors for cross-species transmission to humans (14). In this context, noroviruses (NoVs) infecting cattle could be of importance owing to the high densities of cows bred in areas of human activities. The ability of shellfish to concentrate pathogens released in seawater raises questions about the transmission of animal NoVs to humans through oyster consumption, but so far very few studies have compared water and shellfish contamination. One of the first such studies, conducted more than 30 years ago, comparing the presence of enterovirus by cell culture in water and oysters yielded about the same frequency of positive water (59%) and shellfish samples (35%) (12). More recently, phages of Bacteroides fragilis and Salmonella detected in sewage effluents were also detected in receiving waters and oysters (6). Human NoVs were detected in 75% of river water samples and in 60% of oyster beds (38). Only one study reported the detection of porcine norovirus in 15% of shellfish collected from the U.S. market but no information from the surrounding water was available (8).NoVs are small nonenveloped viruses approximately 30 nm in diameter with a positive-sense, single-stranded RNA genome. They belong to the Caliciviridae family, and in humans they are the most frequent cause of diarrhea outbreaks in all age groups (11, 28). They are classified in five genogroups, with human strains belonging to genogroups I, II, and IV, GIII strains infecting cattle, and murine strains classified in GV (45). Recently, two new genogroups (VI and VII) infecting animals have been proposed (29). Based on analysis of the open reading frame 2 (ORF2) sequence encoding the capsid protein, high diversity has been observed, with the result that genogroups have been subdivided into clusters, including up to 19 for GII strains. Porcine NoVs have been classified into three clusters of GII (GII.11, GII.18, and GII.19) while all bovine strains of NoV described so far belong to GIII (25, 29, 41, 45). The first bovine strain, Bo/Newbury2/1976/UK (NB2), was isolated in the United Kingdom from calves with diarrhea (43). Later, another distinct genotype of bovine NoV, Bo/Jena/1978/GER, was identified in Germany (21). These two strains represent the prototypes of the GIII.2 and GIII.1 genotypes, respectively.Although many gaps persist in our understanding of human NoV infections and pathogenesis, recent advances demonstrated a genetically determined host susceptibility based on histo-blood group antigen diversity. Various human NoV strains attach to distinct carbohydrates of the ABH and Lewis histo-blood group family, and evidence accumulated from volunteer studies and outbreaks indicates that binding to these carbohydrates is required for infection (19, 35). In addition, it was recently shown that the prototype bovine GIII.2 strain binds to a related carbohydrate structure which is absent from human tissues (44). Similarly, it was also demonstrated that some strains of either GI or GII specifically attach to oysters tissues through recognition of histo-blood group antigens (HBGAs) (17, 22, 36). This finding could help explain other observations, such as the rapid contamination of oysters, long persistence of viral particles, and, consequently, shellfish-borne outbreaks (3, 16). It additionally suggests that oysters may not merely act as passive filters randomly accumulating virus particles but, instead, may also act as selective filters specifically concentrating strains by recognition of carbohydrate epitopes shared with humans. As shellfish are grown in coastal waters frequently exposed to contamination from bovine in neighboring fields, they may be contaminated by these animal strains. This raises the issue of the potential role of oysters in the emergence of bovine NoVs into the human population.The aim of our study is to provide quantitative data on the presence of GIII NoV strains in comparison with GI and GII strains in bovine feces, rivers, or estuarine waters as well as shellfish from an area of both high cattle density and high-density oyster breeding. The possibility of GIII strain-specific binding to carbohydrate ligands of oyster tissues that may be shared with cows and humans is additionally examined. The results are discussed in the context of the environmental data in order to provide a first appreciation of the risk of GIII NoV transmission to humans through oyster consumption.     

Thermal Inactivation of Enteric Viruses and Bioaccumulation of Enteric Foodborne Viruses in Live Oysters (Crassostrea virginica)

Human enteric viruses are among the main causative agents of shellfish-associated outbreaks. In this study, the kinetics of viral bioaccumulation in live oysters and the heat stabilities of the predominant enteric viruses were determined both in tissue culture and in oyster tissues. A human norovirus (HuNoV) GII.4 strain, HuNoV surrogates (murine norovirus [MNV-1], Tulane virus [TV]), hepatitis A virus (HAV), and human rotavirus (RV) bioaccumulated to high titers within oyster tissues, with different patterns of bioaccumulation for the different viruses. We tested the thermal stability of each virus at 62, 72, and 80°C in culture medium. The viruses can be ranked from the most heat resistant to the least stable as follows: HAV, RV, TV, MNV-1. In addition, we found that oyster tissues provided protection to the viruses during heat treatment. To decipher the mechanism underlying viral inactivation by heat, purified TV was treated at 80°C for increasing time intervals. It was found that the integrity of the viral capsid was disrupted, whereas viral genomic RNA remained intact. Interestingly, heat treatment leading to complete loss of TV infectivity was not sufficient to completely disrupt the receptor binding activity of TV, as determined by the porcine gastric mucin–magnetic bead binding assay. Similarly, HuNoV virus-like particles (VLPs) and a HuNoV GII.4 strain retained some receptor binding ability following heat treatment. Although foodborne viruses have variable heat stability, 80°C for >6 min was sufficient to completely inactivate enteric viruses in oysters, with the exception of HAV.     

Meta-Analysis of the Reduction of Norovirus and Male-Specific Coliphage Concentrations in Wastewater Treatment Plants

Human norovirus (NoV) is the leading cause of foodborne illness in the United States and Canada. Wastewater treatment plant (WWTP) effluents impacting bivalve mollusk-growing areas are potential sources of NoV contamination. We have developed a meta-analysis that evaluates WWTP influent concentrations and log₁₀ reductions of NoV genotype I (NoV GI; in numbers of genome copies per liter [gc/liter]), NoV genotype II (NoV GII; in gc/liter), and male-specific coliphage (MSC; in number of PFU per liter), a proposed viral surrogate for NoV. The meta-analysis included relevant data (2,943 measurements) reported in the scientific literature through September 2013 and previously unpublished surveillance data from the United States and Canada. Model results indicated that the mean WWTP influent concentration of NoV GII (3.9 log₁₀ gc/liter; 95% credible interval [CI], 3.5, 4.3 log₁₀ gc/liter) is larger than the value for NoV GI (1.5 log₁₀ gc/liter; 95% CI, 0.4, 2.4 log₁₀ gc/liter), with large variations occurring from one WWTP to another. For WWTPs with mechanical systems and chlorine disinfection, mean log₁₀ reductions were −2.4 log₁₀ gc/liter (95% CI, −3.9, −1.1 log₁₀ gc/liter) for NoV GI, −2.7 log₁₀ gc/liter (95% CI, −3.6, −1.9 log₁₀ gc/liter) for NoV GII, and −2.9 log₁₀ PFU per liter (95% CI, −3.4, −2.4 log₁₀ PFU per liter) for MSCs. Comparable values for WWTPs with lagoon systems and chlorine disinfection were −1.4 log₁₀ gc/liter (95% CI, −3.3, 0.5 log₁₀ gc/liter) for NoV GI, −1.7 log₁₀ gc/liter (95% CI, −3.1, −0.3 log₁₀ gc/liter) for NoV GII, and −3.6 log₁₀ PFU per liter (95% CI, −4.8, −2.4 PFU per liter) for MSCs. Within WWTPs, correlations exist between mean NoV GI and NoV GII influent concentrations and between the mean log₁₀ reduction in NoV GII and the mean log₁₀ reduction in MSCs.     

Host Genetic Factors Affect Susceptibility to Norovirus Infections in Burkina Faso

Norovirus (NoV) constitutes the second most common viral pathogen causing pediatric diarrhea after rotavirus. In Africa, diarrhea is a major health problem in children, and yet few studies have been performed regarding NoV. The association of histo-blood group antigens (HBGA) and susceptibility to NoV infection is well established in Caucasian populations with non-secretors being resistant to many common NoV strains. No study regarding HBGA and NoV susceptibility has yet been performed in Africa. We collected 309 stool and 208 saliva samples from diarrheal children in Ouagadougou, Burkina Faso; May 2009 to March 2010. NoV was detected using real-time PCR, and genotyped by sequencing. Saliva samples were ABO, Lewis and secretor phenotyped using in house ELISA assays. NoV was detected in 12% (n = 37) of the samples. The genotype diversity was unusually large; overall the 37 positive samples belonged to 14 genotypes. Only children <2 years of age were NoV positive and the GII.4 NoVs were more frequent in the late dry season (Jan-May). NoV infections were observed less in children with the secretor-negative phenotype or blood group A (OR 0.18; p = 0.012 and OR 0.31; p = 0.054; respectively), with two non-secretors infected with genotypes GII.7 and GII.4 respectively. Lewis-negative (Le^a−b−) children, representing 32% of the study population, were susceptible to GII, but were not infected with any NoV GI. GII.4 strains preferentially infected children with blood group B whereas secretor-positive children with blood group O were infected with the largest variety of genotypes. This is the first study identifying host genetic factors associated with susceptibility to NoV in an African population, and suggests that while the non-secretor phenotype provides protection; the Lewis b antigen is not necessary for GII infection.     

Detection and phylogenetic analysis of norovirus in Corbicula fluminea in a freshwater river in Japan

To study the molecular epidemiology of noroviruses (NoVs) in bivalves residing in freshwater rivers, we detected, quantified and phylogenetically analyzed the NoV genome in purified concentrates obtained from the gills and digestive diverticula of Corbicula fluminea in a freshwater river in Gunma Prefecture, Japan. We detected the NoV genome in 35 of the 58 C. fluminea samples. Based on our phylogenetic analysis, the NoV genome detected in the samples was classified into 4 genotypes (GI/1, GI/2, GI/3 and GI/4) in genogroup I and 5 genotypes (GII/3, GII/4, GII/5, GII/8 and GII/12) in genogroup II. The phylogenetic tree showed wide genetic diversity among the genogroups. In addition, more than 10(4) copies of the NoV genome were detected in 2 of 35 samples. These results suggest that the freshwater bivalve C. fluminea is a reservoir for NoVs, similar to seawater bivalves such as oysters.     

Norovirus Distribution within an Estuarine Environment

Human norovirus (NoV) has been studied extensively as an important cause of gastroenteritis outbreaks worldwide. While oysters are a primary vehicle for infection, few studies have examined the wider distribution of NoV in the estuarine environment. Active shellfish-harvesting areas in Georgia were examined for the prevalence, genotype diversity, and concentrations of NoV in a variety of estuarine sample types over the course of 1 year. Of the 225 samples (9 oyster, 72 water, 72 63- to 200-μm plankton, and 72 >200-μm plankton) collected from 12 stations across two estuaries, 21 samples (9.3%) tested positive for NoV. By sample type, 55.0% (5/9) of oysters, 8.3% (6/72) of water samples, 11.1% (8/72) of 63- to 200-μm plankton samples, and 2.8% (2/72) of >200-μm plankton samples were positive for human NoV. The two NoV-positive >200-μm plankton samples, which contained mainly zooplankton, had the greatest quantity of NoV genomes (3.5 × 10¹³ and 1.7 × 10¹⁵ genomes g⁻¹) of any sample tested. The majority, 90.5% (19/21), of the samples tested positive for genogroup I NoV, and only 9.5% (2/21) of the samples tested positive for genogroup II. The high concentrations of NoV in plankton samples compared to water and oyster samples were unexpected and provide new insights into the presence and distribution of human NoV in the water environment.Human norovirus (NoV) is the leading cause of nonbacterial gastroenteritis worldwide (3). The Centers for Disease Control and Prevention (CDC) estimate that 23 million cases of acute gastroenteritis due to NoV occur each year, with symptoms including acute-onset vomiting, watery nonbloody diarrhea with abdominal cramps, and nausea (35). NoV outbreaks are pervasive for many reasons, but particularly because the virus is highly contagious and environmentally hardy (7). Additionally, infected individuals can excrete millions of viral particles in feces, leading to large numbers in sewage (16). Without proper removal or inactivation during wastewater treatment, the viruses can be released into recreational and shellfish-harvesting water bodies. Complete inactivation of NoV during sewage treatment is rare, and even in areas with proper wastewater treatment, contamination of oyster beds has been reported (5, 16, 17, 32, 38). Because bivalve molluscan shellfish are believed to act as filters for viruses and other microbes and because NoV is extremely infectious (as little as one viral particle is required for disease), the disease risk for consumption of raw oysters is high (27, 33, 40).Human NoV genogroup I (GI) and GII have been detected in oyster samples harvested from bays and estuaries worldwide (5, 10, 20). Ueki et al. (42) detected NoV in both shellfish and the surrounding river water in Japan and concluded that NoV contamination was most likely due to sewage and treated wastewater input into the river; however, no study has yet been able to characterize how NoV may be naturally distributed in an estuarine system, including in water, adhered to particles (including plankton), and in shellfish. The limitations are due in part to a lack of adequate detection methods specifically adapted to different environmental-sample types (8). Using a newly developed detection and quantification protocol (21), this study aimed to examine the distribution of NoV genogroups across a range of sample types within an estuarine system with the goal of better characterizing possible circulation of viruses between water, plankton, and oysters.     

Susceptibility of Murine Norovirus and Hepatitis A Virus to Electron Beam Irradiation in Oysters and Quantifying the Reduction in Potential Infection Risks

Consumption of raw oysters is an exposure route for human norovirus (NoV) and hepatitis A virus (HAV). Therefore, efficient postharvest oyster treatment technology is needed to reduce public health risks. This study evaluated the inactivation of HAV and the NoV research surrogate, murine norovirus-1 (MNV-1), in oysters (Crassostrea virginica) by electron beam (E-beam) irradiation. The reduction of potential infection risks was quantified for E-beam irradiation technology employed on raw oysters at various virus contamination levels. The E-beam dose required to reduce the MNV and HAV titer by 90% (D₁₀ value) in whole oysters was 4.05 (standard deviations [SD], ±0.63) and 4.83 (SD, ±0.08) kGy, respectively. Microbial risk assessment suggests that if a typical serving of 12 raw oysters was contaminated with 10⁵ PFU, a 5-kGy treatment would achieve a 12% reduction (from 4.49 out of 10 persons to 3.95 out of 10 persons) in NoV infection and a 16% reduction (from 9.21 out of 10 persons to 7.76 out of 10 persons) in HAV infections. If the serving size contained only 10² PFU of viruses, a 5-kGy treatment would achieve a 26% reduction (2.74 out of 10 persons to 2.03 out of 10 persons) of NoV and 91% reduction (2.1 out of 10 persons to 1.93 out of 100 persons) of HAV infection risks. This study shows that although E-beam processing cannot completely eliminate the risk of viral illness, infection risks can be reduced.