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Human noroviruses (HuNov) are the major cause of foodborne illness globally. Several HuNoV outbreaks have been linked to contaminated ready-to-eat seaweed products. Standard protocols such as the ISO 15216 show limited efficiency in extracting foodborne viruses from seaweed products. Therefore, we evaluated the efficiency of an extraction protocol based on porcine gastric mucin conjugated magnetic beads (PGM-MBs) to recover HuNoVs from Wakame seaweed salad. Compared to other HuNoV extraction methods, the PGM-MB method was more efficient. We then aimed to further improve this protocol by modifying several factors such as the buffers, pH, bead concentration, centrifugation and incubation time. The optimized PGM-MB method yielded 19 ± 3% and 17 ± 4% recovery, for HuNoV GI and GII, respectively. The limit of detection (LOD95) for Wakame seaweed salad was 131 and 56 genomic equivalents per 25 g for HuNoV GI and GII. Although some variability in recovery efficiency was observed between the PGM sources, the optimized PGM-MB protocol effectively extracts HuNoVs from Wakame seaweed salads of various brands and other commodities such as dates, green onions, and salted seaweed. These results support the implementation of the optimized PGM-MB method as a viable alternative for HuNoV surveillance in complex food matrices.

Human noroviruses (HuNoVs) are the leading cause of non-bacterial gastroenteritis worldwide, exhibiting extensive genetic diversity driven by recombination and mutation. The norovirus RNA-dependent RNA polymerase (RdRp), encoded by the NS7 gene, is the main driver of virus evolution by determining the mutation and recombination rates, and impacting viral fitness. Herein, we conducted spatiotemporal phylodynamic analysis on 1,094 complete RdRp amino acid sequences from genogroups GI and GII collected between 1972 and 2024 to investigate molecular evolution, amino acid variation, evolutionary rates, and selection pressures. Time-scaled phylogenetic analysis revealed that GI polymerases diverged into three major lineages since the year ~ 1630, while GII polymerases formed four lineages, including a distinct cluster for P16. Genotype distribution was dominated by GII.P16 (43%) and GII.P31 (20%), both historically associated with GII.4 capsids. Amino acid substitutions were rare to non-existent within conserved motifs (A–G), indicating strong structural and functional constraints. Evolutionary rate estimates showed GII polymerases evolve faster (mean 1.5 × 10⁻³ substitutions/site/year) than GI (mean 3.8 × 10⁻⁴ substitutions/site/year), though overall RdRp evolves more slowly than the major capsid protein VP1. Selection analyses demonstrated pervasive purifying selection, with limited evidence of positive selection at codons outside of the conserved motifs. These findings highlight the high conservation of RdRp, its slower evolutionary dynamics compared to capsid gene, and its role in norovirus diversification, supporting its relevance as a target for antiviral development.

Foodborne viruses such as human norovirus (HuNoV) and hepatitis A virus (HAV) are the major causes of foodborne illnesses worldwide. These viruses have a low infectious dose and are persistent in the environment and food for weeks. Ready-to-eat (RTE) low moisture foods (LMFs) undergo minimal pathogen reduction processes. In recent years, multiple foodborne HAV outbreaks involving hundreds of individuals were associated with the consumption of dates, indicating that they could be important vehicles for foodborne infection. There is no standard method for the extraction and detection of foodborne viruses from dates, but herein we have compared the efficiency of three different protocols based on the ISO 15216 method in the extraction of murine norovirus (MNV) from whole Medjool dates and successfully employed the best performing method in the extraction of HAV, HuNoV GI, and GII and determined the limit of detection (LOD95) of 61, 148, and 184 genomic equivalent (gEq) per 25 g, respectively. Finally, we tested the adopted method on various varieties of dates including pitted ones and reported the detection of HuNoV GI and GII from four naturally contaminated date varieties. This ISO 15216 protocol could be employed for surveillance purposes and outbreak management related to dates.

Human coronaviruses, including SARS-CoV-2, are known to spread mainly via close contact and respiratory droplets. However, other potential means of transmission may be present. Fomite-mediated transmission occurs when viruses are deposited onto a surface and then transfer to a subsequent individual. Surfaces can become contaminated directly from respiratory droplets or from a contaminated hand. Due to mask mandates in many countries around the world, the former is less likely. Hands can become contaminated if respiratory droplets are deposited on them (i.e., coughing or sneezing) or through contact with fecal material where human coronaviruses (HCoVs) can be shed. The focus of this paper is on whether human coronaviruses can transfer efficiently from contaminated hands to food or food contact surfaces. The surfaces chosen were: stainless steel, plastic, cucumber and apple. Transfer was first tested with cellular maintenance media and three viruses: two human coronaviruses, 229E and OC43, and murine norovirus-1, as a surrogate for human norovirus. There was no transfer for either of the human coronaviruses to any of the surfaces. Murine norovirus-1 did transfer to stainless steel, cucumber and apple, with transfer efficiencies of 9.19%, 5.95% and 0.329%, respectively. Human coronavirus OC43 transfer was then tested in the presence of fecal material, and transfer was observed for stainless steel (0.52%), cucumber (19.82%) and apple (15.51%) but not plastic. This study indicates that human coronaviruses do not transfer effectively from contaminated hands to contact surfaces without the presence of fecal material.

The involvement of the gastrointestinal (GI) tract in severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection has been reported in multiple studies. Since it has been demonstrated that human intestinal epithelial cells support productive viral replication and that a substantial portion of infected individuals shed the virus in feces, the possibility of fecal–oral and fecal-respiratory modes of transmission have been proposed for SARS-CoV-2. In order to establish viral replication in the intestine, enteric viruses need to retain their infectivity in often low pH gastric fluids, and in intestinal fluids, which contain digestive enzymes and bile salts. In this study, we examined whether human coronaviruses OC43 (HCoV-OC43) can remain infectious in simulated GI fluids that models human fasting-state and fed-state, in the presence or absence of food. We demonstrated that except for fasting-state gastric fluid (pH 1.6), the virus can remain infectious in all other gastrointestinal fluids for 1 h. Furthermore, we demonstrated that presence of food could significantly improve viral survival in gastric fluids. Therefore, this study provides evidence that ingestion with food could protect the virus against inactivation by the GI fluids.

Enteric viruses, such as human norovirus (NoV) and hepatitis A virus (HAV), are the major causes of foodborne illnesses worldwide. These viruses have low infectious dose, and may remain infectious for weeks in the environment and food. Limited information is available regarding viral survival and transmission in low-moisture foods (LMF). LMFs are generally considered as ready-to-eat products, which undergo no or minimal pathogen reduction steps. However, numerous foodborne viral outbreaks associated with LMFs have been reported in recent years. The objective of this study was to examine the survival of foodborne viruses in LMFs during 4-week storage at ambient temperature and to evaluate the efficacy of advanced oxidative process (AOP) treatment in the inactivation of these viruses. For this purpose, select LMFs such as pistachios, chocolate, and cereal were inoculated with HAV and the norovirus surrogates, murine norovirus (MNV) and feline calicivirus (FCV), then viral survival on these food matrices was measured over a four-week incubation at ambient temperature, by both plaque assay and droplet-digital RT-PCR (ddRT-PCR) using the modified ISO-15216 method as well as the magnetic bead assay for viral recovery. We observed an approximately 0.5 log reduction in viral genome copies, and 1 log reduction in viral infectivity for all three tested viruses following storage of select inoculated LMFs for 4 weeks. Therefore, the present study shows that the examined foodborne viruses can persist for a long time in LMFs. Next, we examined the inactivation efficacy of AOP treatment, which combines UV-C, ozone, and hydrogen peroxide vapor, and observed that while approximately 100% (4 log) inactivation can be achieved for FCV, and MNV in chocolate, the inactivation efficiency diminishes to approximately 90% (1 log) in pistachios and 70% (< 1 log) in cereal. AOP treatment could therefore be a good candidate for risk reduction of foodborne viruses from certain LMFs depending on the food matrix and surface of treatment.

We tested liver samples from 372 Norway rats (Rattus norvegicus) from southern Ontario, Canada, during 2018–2021 to investigate presence of hepatitis E virus infection. Overall, 21 (5.6%) rats tested positive for the virus. Sequence analysis demonstrated all infections to be rat hepatitis E virus (Rocahepevirus ratti genotype C1).

Infection with the hepatitis E virus (HEV) is very common worldwide. HEV causes acute viral hepatitis with approximately 20 million cases per year. While HEV genotypes 1 and 2 cause large waterborne and foodborne outbreaks with a significant mortality in developing countries, genotypes 3 and 4 are more prevalent in developed countries with transmission being mostly zoonotic. In North America and Europe, HEV has been increasingly detected in swine, and exposure to pigs and pork products is considered to be the primary source of infection. Therefore we set out to investigate the prevalence of HEV in retail pork products available in Canada, by screening meal-size portions of pork pâtés, raw pork sausages, and raw pork livers. The presence of the HEV genomes was determined by RT-PCR and viral RNA was quantified by digital droplet PCR. Overall, HEV was detected in 47% of the sampled pork pâtés and 10.5% of the sampled raw pork livers, but not in the sampled pork sausages, and sequencing confirmed that all HEV strains belonged to genotype 3. Further phylogenetic analysis revealed that except for one isolate that clusters with subtype 3d, all isolates belong to subtype 3a. Amino acid variations between the isolates were also observed in the sequenced capsid region. In conclusion, the prevalence of HEV in pâtés and raw pork livers observed in this study is in agreement with the current HEV distribution in pork products reported in other developed countries.

Background Human norovirus is the leading cause of viral gastroenteritis globally, and the GII.4 has been the most predominant genotype for decades. This genotype has numerous variants that have caused repeated epidemics worldwide. However, the molecular evolutionary signatures among the GII.4 variants have not been elucidated throughout the viral genome. Method A metagenomic, next-generation sequencing method, based on Illumina RNA-Seq, was applied to determine norovirus sequences from clinical samples. Results Herein, the obtained deep-sequencing data was employed to analyze full-genomic sequences from GII.4 variants prevailing in Canada from 2012 to 2016. Phylogenetic analysis demonstrated that the majority of these sequences belong to New Orleans 2009 and Sydney 2012 strains, and a recombinant sequence was also identified. Genome-wide similarity analyses implied that while the capsid gene is highly diverse among the isolates, the viral protease and polymerase genes remain relatively conserved. Numerous amino acid substitutions were observed at each putative antigenic epitope of the VP1 protein, whereas few amino acid changes were identified in the polymerase protein. Co-infection with other enteric RNA viruses was investigated and the astrovirus genome was identified in one of the samples. Conclusions Overall this study demonstrated the application of whole genome sequencing as an important tool in molecular characterization of noroviruses.

Foodborne viruses such as norovirus and hepatitis A virus cause frequent outbreaks associated with the consumption of raw or undercooked oysters. Viral particles are bioaccumulated in the oyster’s digestive glands, making RNA extraction and RT-PCR detection difficult due to the complex nature of the food matrix and the presence of RT-PCR inhibitors. Herein, we have developed a viral RNA extraction protocol from raw oysters using murine norovirus (MNV) as a surrogate for human noroviruses. The method combines lysis in Tri-Reagent reagent, followed by RNA extraction using Direct-Zol purification columns and lithium chloride precipitation. Viral load quantification was performed by both qRT-PCR and droplet-digital RT-PCR. We have demonstrated that this method can efficiently remove RT-PCR inhibitors, and is sensitive enough to reliably detect viral contamination at 25 PFU/0.2 g. We have also compared the efficiency of this method with the ISO 15216-1:2017 method and Method E developed by Quang and colleagues, and observed significantly higher efficiency compared with the ISO 15216-1 method and comparable efficiency with Method E, with less steps, and shorter hands-on time.