Optimization of the replication of hepatitis E virus genotype 3 in vitro.

AIMS: Hepatitis E virus (HEV) is responsible for ∼20 million human infections worldwide every year. The genotypes HEV-3 and HEV-4 are zoonotic and are responsible for most of the autochthonous HEV cases in high-income countries. There are several cell culture systems that allow for propagation of different HEV genotypes in vitro. One of these systems uses human lung carcinoma cells (A549), and was further optimized for propagation of HEV-3 47832c strain. In this study, we investigated the effect of different media supplements as well as microRNA-122 (miR-122) on improving the replication of HEV-3 47832c in A549 cells. METHODS AND RESULTS: We observed that supplementation of maintenance media with 5% fetal bovine serum was sufficient for efficient replication of HEV-3, and verified the positive effect of media supplementation with Amphotericin B, MgCl2, and dimethyl sulfoxide on replication of HEV-3. We have also demonstrated that adding miR-122 mimics to the culture media does not have any significant effect on the replication of HEV-3 47832c. CONCLUSIONS: Herein, we detected over a 6-fold increase in HEV-3 replication in A549/D3 cells by adding all three supplements: Amphotericin B, MgCl2, and dimethyl sulfoxide to the culture media, while demonstrating that miR-122 might not play a key role in replication of HEV-3 47832c.

The molecular epidemiology of hepatitis E virus genotype 3 in Canada.

Autochthonous hepatitis E virus (HEV) infection is increasingly reported in industrialized countries and is mostly associated with zoonotic HEV genotype 3 (HEV-3). In this study, we examined the molecular epidemiology of 63 human clinical HEV-3 isolates in Canada between 2014 and 2022. Fifty-five samples were IgM positive, 45 samples were IgG positive and 44 were IgM and IgG positive. The majority of the isolates belong to the subtypes 3a, 3b, and 3j, with high sequence homology to Canadian swine and pork isolates. There were a few isolates that clustered with subtypes 3c, 3e, 3f, 3h, and 3g, and an isolate from chronic infection with a rabbit strain (3ra). Previous studies have demonstrated that the isolates from pork products and swine from Canada belong to subtypes 3a and 3b, therefore, domestic swine HEV is likely responsible for the majority of clinical HEV cases in Canada and further support the hypothesis that swine serve as the main reservoirs for HEV-3 infections. Understanding the associated risk of zoonotic HEV infection requires the establishment of sustainable surveillance strategies at the interface between humans, animals, and the environment within a One-Health framework.

Examining the efficiency of porcine gastric mucin-coated magnetic beads in extraction of noroviruses from frozen berries.

Human norovirus is the leading cause of foodborne gastroenteritis worldwide. Due to the low infectious dose of noroviruses, sensitive methodologies are required to detect and characterize small numbers of viral particles that are found in contaminated foods. The ISO 15216 method, which is internationally recognized for detection of foodborne viruses from high-risk food commodities, is based on viral precipitation, followed by RNA extraction and identification of the viral genome by RT-PCR. Although the ISO 15216 method is efficient, it is time consuming and tedious, does not report on the viral infectivity, and is sensitive to the presence of RT-PCR inhibitors. Norovirus capture by the porcine gastric mucin conjugated magnetic beads (PGM-MB) was developed as an alternative virus recovery method. It relies on the integrity of the viral capsid being able to bind to PGM. PGM contains a variety of histo-blood group antigens (HBGAs) that act as norovirus receptors. Therefore, the PGM-MB method allows for extraction of noroviruses, with potentially intact viral capsids, from complex food matrices. The viral genome can then be released through heat-shock of the captured virus. For this reason, we performed a parallel comparison between the ISO 15216 method and the PGM-MB method in isolation and quantification of noroviruses from frozen raspberries. We have demonstrated that the efficiency of the PGM-MB method in extraction of murine norovirus (MNV) and human norovirus GII.4 from raspberries is equal or better than the ISO 15216 method, while the PGM-MB has fewer steps and shorter turnaround time. Moreover, the PGM-MB method is more efficient in removing the inhibitors prior to RT-PCR analysis.

Assessing the efficacy of different bead-based assays in capturing hepatitis E virus.

Hepatitis E virus (HEV) generally causes acute liver infection in humans and its transmission could be waterborne, foodborne, bloodborne, or zoonotic. To date, there is no standard method for the detection of HEV from food and environmental samples. Herein, we explored the possibility of using magnetic beads for the capture and detection of HEV. For this purpose, we employed Dynabeads M-270 Epoxy magnetic beads, coated with different monoclonal antibodies (mAbs) against HEV capsid protein, and the Nanotrap Microbiome A Particle magnetic beads, which are coated with chemical affinity baits, to capture HEV-3 particles in suspension. Viral RNA was extracted by heat-shock or QIAamp viral RNA kit and subjected to quantification using digital-droplet RT-PCR (ddRT-PCR). We demonstrated that the mAb-coupled Dynabeads and the Nanotrap particles, both were able to successfully capture HEV-3. The latter, however had lower limit of detection (<140gc compared with <1400 gc) and significantly higher extraction efficiency in comparison to the mAb-coupled Dynabeads (41.1% vs 8.8%). We have also observed that viral RNA extraction by heat-shock is less efficient compared to using highly denaturing reagents in QIAmp viral RNA extraction kit. As such, magnetic beads have the potential to be used to capture HEV virions for research and surveillance purposes.

Examining the Effect of Organic Acids on Inactivation of Hepatitis E Virus.

ABSTRACT: Infection with hepatitis E virus genotype 3 (HEV-3) is an emerging cause of illness in developed countries. In North America and Europe, HEV-3 has been increasingly detected in swine, and exposure to pigs and pork products is considered the primary source of infection. We have previously demonstrated the prevalence of the HEV-3 genome in commercial pork products in Canada. In this study, we investigated the application of citric acid and acetic acid to inactivate HEV-3 on food and on food contact surfaces. For this purpose, plastic, stainless steel, and pork pâté surfaces were inoculated with HEV-3 and were treated with acetic acid or citric acid at 1, 3, or 5%. The infectivity of posttreatment viral particles was determined by cell culture. A greater than 2-log reduction in viral infectivity was observed on plastic and stainless steel treated with the organic acids, but the treatment was less effective on HEV infectivity on pork pâté (average reductions of 0.47 log citric acid and 0.63 log acetic acid). Therefore, we conclude that citric acid and acetic acid have potential application to control HEV-3 on food contact surfaces but are not suitable for food.

Efficacy of washing produce in removing human coronavirus OC43 and murine norovirus.

AIMS: Fresh produce is often a vehicle for the transmission of foodborne pathogens such as human norovirus. Thus, it is recommended to wash the surface of produce before consumption, and one of the most common ways to wash produce is by rinsing under running tap water. This study determined the effectiveness of removal of human coronavirus-OC43 (HCoV-OC43), as a surrogate for severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) and murine norovirus-1 (MNV-1), as a surrogate for human norovirus, from contaminated lettuce, apples and cucumbers. METHODS AND RESULTS: The produce surfaces were artificially inoculated in conjunction with faecal material to represent natural contamination. Rinsing under tap water for 10 s at 40 ml/s removed 1.94 ± 0.44, 1.42 ± 0.00 and 1.42 ± 0.42 log of HCoV-OC43 from apple, cucumber and lettuce respectively. The same washing technique removed 1.77 ± 0.17, 1.42 ± 0.07 and 1.79 ± 0.14 log of MNV-1 from apple, cucumber and lettuce respectively. This washing technique was effective at reducing a significant amount of viral contamination, however, it was not enough to eliminate the entire contamination. There was no significant difference in the reduction of viral load between the two viruses, nor between the three surfaces tested in this study. CONCLUSIONS: Our data suggest that washing under tap water would be an efficient way of reducing the risk of foodborne viral transmission only if the level of contamination is less than 2 log PFU. SIGNIFICANCE AND IMPACT OF STUDY: This study demonstrates that running tap water was effective at reducing the amount of infectious HCoV-OC43 and MNV on produce surfaces, and washing produce continues to be an important task to perform prior to consumption to avoid infection by foodborne viruses, particularly for foods which are eaten raw.

Protective Effect of Food Against Inactivation of Human Coronavirus OC43 by Gastrointestinal Fluids.

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.

Human Coronaviruses Do Not Transfer Efficiently between Surfaces in the Absence of Organic Materials.

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.

Examining the persistence of human Coronavirus 229E on fresh produce.

Human coronaviruses (HCoVs) are mainly associated with respiratory infections. However, there is evidence that highly pathogenic HCoVs, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and Middle East Respiratory Syndrome (MERS-CoV), infect the gastrointestinal (GI) tract and are shed in the fecal matter of the infected individuals. These observations have raised questions regarding the possibility of fecal-oral route as well as foodborne transmission of SARS-CoV-2 and MERS-CoV. Studies regarding the survival of HCoVs on inanimate surfaces demonstrate that these viruses can remain infectious for hours to days, however, there is limited data regarding the viral survival on fresh produce, which is usually consumed raw or with minimal heat processing. To address this knowledge gap, we examined the persistence of HCoV-229E, as a surrogate for highly pathogenic HCoVs, on the surface of commonly consumed fresh produce, including: apples, tomatoes, cucumbers and lettuce. Herein, we demonstrated that viral infectivity declines within a few hours post-inoculation (p.i) on apples and tomatoes, and no infectious virus was detected at 24h p.i, while the virus persists in infectious form for 72h p.i on cucumbers and lettuce. The stability of viral RNA was examined by droplet-digital RT-PCR (ddRT-PCR), and it was observed that there is no considerable reduction in viral RNA within 72h p.i.

Survival and Inactivation by Advanced Oxidative Process of Foodborne Viruses in Model Low-Moisture Foods.

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.

Genomic analysis of human noroviruses using combined Illumina-Nanopore data.

Whole-genome sequence analysis of noroviruses is routinely performed by employing a metagenomic approach. While this methodology has several advantages, such as allowing for the examination of co-infection, it has some limitations, such as the requirement of high viral load to achieve full-length or near full-length genomic sequences. In this study, we used a pre-amplification step to obtain full-length genomic amplicons from 39 Canadian GII isolates, followed by deep sequencing on Illumina and Oxford Nanopore platforms. This approach significantly reduced the required viral titre to obtain full-genome coverage. Herein, we compared the coverage and sequences obtained by both platforms and provided an in-depth genomic analysis of the obtained sequences, including the presence of single-nucleotide variants and recombination events.

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

Hepatitis E virus (HEV) causes acute hepatitis with approximately 20 million cases per year globally. Based on genetic diversity, HEV is classified into different genotypes, with genotype 3 (HEV-3) being most prevalent in Europe and North America. The transmission of HEV-3 has been shown to be zoonotic and mainly associated with the consumption of raw or undercooked pork products. Herein, we investigated the efficacy of high-pressure processing (HPP) in inactivation of HEV-3 using a cell culture system. HPP has been indicated as a promising non-thermal pathogen inactivation strategy for treatment of certain high-risk food commodities, without any noticeable changes in their nature. For this purpose, we treated HEV-3 in media with different conditions of HPP: 400 MPa for 1 and 5 min, as well as 600 MPa for 1 and 5 min, at ambient temperature. All four HPP treatments of HEV in media were observed to result in a 2-log reduction in HEV load, as determined by the amounts of extracellular HEV RNA produced at 14-day post-infection, using the A549/D3 cell culture system. However, application of the same treatments to artificially contaminated pork pâté resulted in 0.5 log reduction in viral load. These results indicate that the efficacy of HPP treatment in the inactivation of HEV-3 is matrix-dependent, and independent of maximum pressure between 400 and 600 MPa and hold time between 1 and 5 min. Based on the obtained results, although the HPP treatment of pork pâté reduces the HEV-3 load, it might not be sufficient to fully mitigate the risk.

Evaluation of Bead-Based Assays for the Isolation of Foodborne Viruses from Low-Moisture Foods.

ABSTRACT: Foodborne viruses such as norovirus and hepatitis A virus (HAV) are highly transmissible, persistent in the environment, and resistant to many conventional inactivation methods. Foods can become contaminated with these viruses either at the source of harvest or during food handling and processing. Multiple lines of evidence suggest that foodborne viruses can survive desiccation and dry conditions. Several foodborne virus outbreaks have been linked to low-moisture foods (LMFs), indicating that these foods can be vehicles of virus transmission. However, the efficiencies of common virus extraction methodologies have not been examined with LMFs. We adapted the International Organization for Standardization (ISO) 15216-1:2017 method for virus recovery for use with chocolate, pistachios, and cornflakes. We also developed a magnetic bead assay for the recovery of HAV from LMFs and used the porcine gastric mucin-coated magnetic beads (PGM-MBs) to extract norovirus surrogates, feline calicivirus (FCV), and murine norovirus (MNV) from the same LMFs. The efficiency of virus recovery using the bead-based assay was then compared with that of the ISO 15216-1:2017 method. In chocolate and pistachios, the recovery rates with the PGM-MB method were 5.6- and 21.3-fold higher, respectively, for FCV and 1.65- and 18-fold higher, respectively, for MNV than those with the ISO 15216-1:2017 method. However, the PGM-MB method failed to recover MNV and FCV from cornflakes. The recovery rates for HAV in chocolate, pistachios, and corn flakes with the magnetic bead method were 11.5-, 3-, and 5.6-fold higher, respectively, than those with the ISO 15216-1:2017 method. Thus, depending upon the food matrix and the target virus, the bead-based assays can be used to efficiently and rapidly extract viruses from LMFs.

Evaluation of porcine gastric mucin assay for detection and quantification of human norovirus in fresh herbs and leafy vegetables.

Leafy vegetables and fresh herbs are important parts of a healthy diet, however, they can be common vehicles of norovirus (NoV) infection and lead to serious health and economic concerns globally. NoV is highly infectious and persistent in the food and the environment, while being resistant to conventional food decontamination practices. Herbs and leafy greens are often consumed raw, and if contaminated with NoV, they may cause illness. Consequently, for outbreak prevention and surveillance purposes, sensitive and rapid methods are required to detect the presence of infectious NoV in naturally contaminated produce during its shelf life. Herein, we compared the extraction efficiency of the ISO/TS 15216-1:2017 method with the porcine gastric mucin coated magnetic beads (PGM-MB) assay, combined with heat-denaturation for RNA extraction, for detection of human NoV in artificially contaminated fresh green seaweed, basil, mint, and baby spinach. Droplet-digital RT-PCR was used to quantify the extracted genome by both methods. Our data demonstrated that while the PGM-MB assay takes considerably less time, it yields significantly higher recovery rates compared with the ISO/TS 15216-1:2017. Furthermore, since this method has the ability to be adapted in high-throughput and automated systems, it can be further modified to be employed by the food industry to reduce the number of NoV illnesses and outbreaks at the source of distribution.

Genetic characterization of norovirus GII.4 variants circulating in Canada using a metagenomic technique.

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.

Prevalence and Molecular Characterization of the Hepatitis E Virus in Retail Pork Products Marketed in Canada.

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.

Heat Inactivation of Hepatitis A Virus in Shellfish Using Steam.

Shellfish are an important cause of foodborne viral illness. Consumer-friendly cooking recommendations for shellfish could improve food safety and decrease the risk for infection from contaminated products. Thermal inactivation parameters were established for hepatitis A virus (HAV) in mussels and validated with cooking experiments. Steaming for only 2-5 min was not sufficient to inactivate HAV in mussels in all layers of a steamer. Steaming mussels for 6 min was sufficient to inactivate HAV in all layers. These cooking guidelines produce shellfish with a reduced risk for foodborne virus transmission.

Experiments with the site frequency spectrum.

Evaluating the likelihood function of parameters in highly-structured population genetic models from extant deoxyribonucleic acid (DNA) sequences is computationally prohibitive. In such cases, one may approximately infer the parameters from summary statistics of the data such as the site-frequency-spectrum (SFS) or its linear combinations. Such methods are known as approximate likelihood or Bayesian computations. Using a controlled lumped Markov chain and computational commutative algebraic methods, we compute the exact likelihood of the SFS and many classical linear combinations of it at a non-recombining locus that is neutrally evolving under the infinitely-many-sites mutation model. Using a partially ordered graph of coalescent experiments around the SFS, we provide a decision-theoretic framework for approximate sufficiency. We also extend a family of classical hypothesis tests of standard neutrality at a non-recombining locus based on the SFS to a more powerful version that conditions on the topological information provided by the SFS.

Rates of Hospitalization for Ambulatory Care Sensitive Conditions in the Medicare+Choice Population.

This article evaluates the feasibility of developing hospitalization rates for ambulatory care sensitive conditions (ACSCs) for the Medicare+Choice (M+C) population. M+C inpatient encounter data were used to calculate 15 ACSC rates. We found the initial reporting year of M+C inpatient encounter data had no apparent volume or diagnosis-based biases and over 90 percent of M+C organizations had sufficient enrollment to produce statistically reliable rates. Further, our study results support the premise that ACSCs could be used as sentinel events for potentially vulnerable populations; the oldest old and the disabled experienced statistically significant higher rates of ACSC admissions than younger Medicare beneficiaries.