Challenges for estimating human norovirus infectivity by viability RT-qPCR as compared to replication in human intestinal enteroids

Viability RT-qPCR, a molecular detection method combining viability marker pre-treatment with RT-qPCR, has been proposed to infer infectivity of viruses which is particularly relevant for non-culturable viruses or sophisticated cell culture systems. Being human noroviruses (HuNoV) most frequently associated with foodborne outbreaks, this study compared different viability techniques and infectivity in human intestinal enteroids (HIE) to ultimately determine whether the molecular approaches could serve as rapid assays to predict HuNoV inac-tivation in high-risk food. To this end, the performance of three viability RT-qPCR assays with different inter-calating markers ((Viability PCR Crosslinker Kit (CL), propidium monoazide (PMAxxTM), and platinum chloride (PtCl4)) in estimating survival of HuNoV exposed to thermal and high pressure (HPP) treatments was compared to replication tested in the HIE cell culture model. A nearly full-length genomic molecular assay coupled with PMAxxTM to infer HuNoV thermal inactivation was also assessed. The experimental design included HuNoV genogroup I.3 [P13], GII.4 Sydney [P16], GII.6 [P7], along with Tulane virus (TV) serving as surrogate. Finally, viability RT-qPCR was tested in HPP-treated strawberry puree, selected as a food matrix with high viral contamination risk. PMAxxTM and CL performed evenly, while PtCl4 affected HuNoV infectivity. Taking all experimental data together, viability RT-qPCR was demonstrated to be an improved method over direct RT-qPCR to estimate viral inactivation at extreme thermal (95 degrees C) and HPP (450 MPa) exposures, but not under milder conditions as amplification signals were detected. Despite its complexity and limitations, the HIE demonstrated a more robust model than viability RT-qPCR to assess HuNoV infectivity

Evaluation of hepatitis E virus RNA persistence in experimentally contaminated cured pork liver sausages

Hepatitis E is a disease sustained by RNA viruses, which have four different genotypes, all of which are responsible for acute forms of hepatitis. Genotypes 1 and 2 infect only humans, causing epidemics mainly transmitted by contaminated water, while genotypes 3 and 4 are zoonotic, and the infection is linked to the consumption of raw or undercooked meat or meat products. Hepatitis E virus (HEV) genotypes 3 and 4 have been detected in domestic Suidae, considered the asymptomatic reservoir of HEV, and in wild animals such as wild boar and deer. Despite scientific studies that have highlighted the presence of HEV in cured meat products, such as pork liver sausages, the viral persistence in the different production steps of curing has not been evaluated. Therefore, this study aimed to evaluate the persistence of HEV genotype 3 during the different curing and storage times of experimentally contaminated pork liver sausages using biomolecular methods. The sausages tested positive at all curing and storage times. This study confirms the potential risk attributed to pork liver sausages in HEV transmission. However, to guarantee an efficient risk assessment, future studies will be performed to correlate the presence of HEV RNA with infectious viral particles

Leveraging Plasma-Activated Seawater for the Control of Human Norovirus and Bacterial Pathogens in Shellfish Depuration

Cold plasma is a promising alternative for water treatment owing to pathogen control and a plethora of issues in the agriculture and food sectors. Shellfish pose a serious risk to public health and are linked to large viral and bacterial outbreaks. Hence, current European regulations mandate a depuration step for shellfish on the basis of their geographical growth area. This study investigated the inactivation of relevant viral and bacterial pathogens of three plasma-activated seawaters (PASWs), and their reactive oxygen and nitrogen species (RONS) composition, as being primarily responsible for microbial inactivation. Specifically, F-specific (MS2) and somatic (φ174) bacteriophage, cultivable surrogate (murine norovirus, MNV, and Tulane virus, TV), and human norovirus (HuNoV GII.4) inactivation was determined using plaque counts and infectivity assays, including the novel human intestinal enteroid (HIE) model for HuNoV. Moreover, the kinetic decay of Escherichia coli, Salmonella spp., and Vibrio parahaemolyticus was characterized. The results showed the complete inactivation of phages (6–8 log), surrogates (5–6 log), HuNoV (6 log), and bacterial (6–7 log) pathogens within 24 h while preventing cytotoxicity effects and preserving mussel viability. Nitrites (NO2−) were found to be mostly correlated with microbial decay. This research shows that PASWs are a suitable option to depurate bivalve mollusks and control the biohazard risk linked to their microbiological contamination, either viral or bacterial

Epidemiological and genetic evaluation of HEV in swine slaughtered in Sicily region (Italy)

: Hepatitis E virus (HEV) is the etiological agent of acute viral hepatitis, a disease transmitted by the oral-faecal route. In Europe, zoonotic transmission of HEV-3 genotype is associated with the consumption of raw or slightly cooked meat of pigs and wild boars that are considered the main reservoirs. This work aims to assess the occurrence of swines' HEV RNA liver samples and rectal swabs slaughtered in Sicily using biomolecular methods. HEV-RNA was detected in 17.5 % (21/120) liver samples analyzed and in 3.7 % (3/81) rectal swabs examined. All positive samples were predicted as genotype 3 and subtype 3c (75 %). These data suggest a potential HEV transmission to humans through close contact with pig breeders, veterinarians, slaughterhouse personnel, and pork meat product consumption. Moreover, there are few scientific data evaluating the HEV spread around pigs and humans in Sicily. Therefore, further studies are necessary to correlate humans with swine serotypes and to assess the HEV presence and persistence in food and the risk during the slaughtering process. These surveys allow to clarify the role of the swine species as a potential source of infection for other domestic or wild animals and humans and to establish possible control measures throughout the food chain

Nosema apis and Nosema ceranae in Beehives of the Apulian Region of Italy: How Citizen Science Can Support Scientific Research

Nosema apis and Nosema ceranae are two well-known pathogens affecting the health of honeybees. To help understand how honeybee colonies are affected by these pathogens, the aim of this study was to analyze the impact of Nosema ceranae and Nosema apis in hives in the Apulian Region through a Citizen Science approach. First, a form about the health status of the beehives was filled out by beekeepers. After an inspection visit to confirm the signs observed by beekeepers, adult honeybee samples collected from beehives in four Apulian provinces (Taranto, Bari, Foggia and Brindisi) were subjected to light microscopy investigation for the detection of Nosema spp. spores and to molecular analysis using species-specific primers for the discrimination of Nosema apis spores from those of Nosema ceranae. Among the forty-eight samples, thirty-six tested positive for Nosema ceranae, and one sample tested positive for Nosema apis. The forms filled out by beekeepers revealed that only 5/36 beehives that tested positive for Nosema ceranae showed signs of depopulation and reduced honey production, while 19/36 had only low honey yield. This study provides data on Nosema apis and Nosema ceranae prevalence in Italy and correlates the presence of these intestinal pathogens with the most important problems encountered by local beekeepers. © 2024 by the authors

Occurrence of hepatitis E virus (HEV) in Calabrian wild boars

Hepatitis E virus (HEV) is an emerging pathogen in industrialized countries. HEV infections in humans are mainly related to the HEV-3 genotype, predominant in Europe and widespread in wild boars' food products. However, there are little relevant data around HEV prevalence in wild boars, although they are considered the main HEV reservoir and used for typical food products such as liver sausages. Our study aimed to assess HEV occurrence and genetic variability in Calabrian wild boars hunted in the central and ionic area of Catanzaro's province. A total of 86 wild boar liver samples were analyzed showing an overall HEV RNA prevalence of 26.7% (23/86). All positive samples were characterized molecularly as genotype 3 and predicted as HEV-3c subtype despite the shortness of fragment employed for the molecular analysis. This data is in line with previous studies conducted in Europe highlighting the public health concern of these results. Biomolecular methods performed in our study detected only the HEV RNA positivity of analyzed samples without information about the virus viability. Consequently, it is not possible to fully estimate the risk related to the consumption of wild boar's liver sausages or wild boar meat products. Our results highlight the need for further studies in order to investigate the virus viability and to link wild boar's meat consumption with HEV human seroprevalence in Italian regions (Abruzzo, Lazio, Campania and Calabria) where typical wild boar's products are consumed. In this way, the Competent Authorities could perform a complete risk assessment, implement risk management and establish proper measures to ensure the public health and prevent relative human disease.: Hepatitis E virus (HEV) is an emerging pathogen in industrialized countries. HEV infections in humans are mainly related to the HEV-3 genotype, predominant in Europe and widespread in wild boars' food products. However, there are little relevant data around HEV prevalence in wild boars, although they are considered the main HEV reservoir and used for typical food products such as liver sausages. Our study aimed to assess HEV occurrence and genetic variability in Calabrian wild boars hunted in the central and ionic area of Catanzaro's province. A total of 86 wild boar liver samples were analyzed showing an overall HEV RNA prevalence of 26.7% (23/86). All positive samples were characterized molecularly as genotype 3 and predicted as HEV-3c subtype despite the shortness of fragment employed for the molecular analysis. This data is in line with previous studies conducted in Europe highlighting the public health concern of these results. Biomolecular methods performed in our study detected only the HEV RNA positivity of analyzed samples without information about the virus viability. Consequently, it is not possible to fully estimate the risk related to the consumption of wild boar's liver sausages or wild boar meat products. Our results highlight the need for further studies in order to investigate the virus viability and to link wild boar's meat consumption with HEV human seroprevalence in Italian regions (Abruzzo, Lazio, Campania and Calabria) where typical wild boar's products are consumed. In this way, the Competent Authorities could perform a complete risk assessment, implement risk management and establish proper measures to ensure the public health and prevent relative human disease

Survival of a SARS-CoV-2 Surrogate on Flow-Pack Polyethylene and Polystyrene Food Trays at Refrigeration and Room Temperature Conditions

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the etiological agent of the current pandemic referred to as coronavirus disease 2019, is spread by direct and indirect transmission between humans, including contact with contaminated surfaces, frozen food, packaging materials, and storage environments. Food contamination may occur in the “farm-to- table” lifecycle through contact with food handlers and environments. In the present study, the survival of a SARS-CoV-2 surrogate (feline coronavirus (FCoV)) at room temperature and refrigeration conditions for different time intervals on two types packaging widely used packaging, namely flow-pack polyethylene and polystyrene food trays, was investigated. FCoV was stable on the flow-pack polyethylene for 48 h and 120 h at room temperature and 4 °C, respectively, while it persisted on polystyrene food trays for 36 h at room temperature and for 120 h at +4 °C. The results of our study highlight the possible implications of food packaging in the spread of SARS-CoV-2 during the current pandemic

Survival modelling of infectious human norovirus and surrogates for high-pressure inactivation in strawberry puree

Berries contaminated with human norovirus (HuNoV) have been frequently identified as a cause of foodborne gastroenteritis. To prevent virus transmission while preserving sensory and quality parameters, non-thermal treatments, such as high-pressure processing (HPP), can be applied to the berries and products thereof. Here, strawberry purees contaminated with HuNoV genogroup I (GI.3[P13]) and II (GII.4 Sydney [P16]), along with murine norovirus (MNV) and Tulane virus (TV) serving as surrogates, were exposed to HPP at several pressure-time combinations. Virus inactivation was assessed by cell culture, including the novel human intestinal enteroids (HIE) model for HuNoVs. The infectivity results showed TV more resistant than MNV to HPP, as also confirmed by kinetic mathematical modelling. Results indicated that a holding pressure of 450 MPa and an exposure time of ≥5 min are reliable operational conditions for HPP process to successfully control viral contamination. In addition, the inactivation models deduced from MNV and TV viruses were challenged with experimental HuNoV GII.4 infectivity resulting in bias factors <1 for all treatment conditions. This finding validates the proposed models for the conservative estimation of HuNoV inactivation. Our work offers a blueprint for moving forward with inactivation studies using the HIE system, which provides useful practical information on optimum treatments for the best public health outcomes.This research was supported by “PREVISION” (PID2019-105509RJ-I00, MCIN/AEI /10.13039/501100011033) project funded to WR. AM is supported by a predoctoral research grant funded by University of Bari “A. Moro” (Italy). IF is supported by a postdoctoral contract grant for the requalification of the Spanish university system from the Ministry of Universities of the Government of Spain, financed by the European Union (NextGeneration EU) (MS21–006). IATA-CSIC is a Centre of Excellence Severo Ochoa (CEX2021–001189-S MCIN/AEI / 10.13039/ 501100011033).With funding from the Spanish government through the ‘Severo Ochoa Centre of Excellence’ accreditation (CEX 2021-001189-S)Peer reviewe

The EU Interreg Project “ADRINET”: Assessment of Well-Known and Emerging Pollutants in Seafood and Their Potential Effects for Food Safety

Anthropogenic activities lead to the spread of chemicals and biological materials, including plastic waste, toxic metals, and pharmaceuticals, of which the impact on the Mediterranean Sea is of high concern. In this context, the EU Interreg Italy-Albania-Montenegro Project "ADRINET (Adriatic Network for Marine Ecosystem) _244" (2018-2020) arises. It aims to carry out biomonitoring campaigns in the main commercial interest of fish and cephalopod species, such as Sparus aurata, Dicentrarchus labrax, Sepia spp., and Loligo spp. sampled in three different subregions of the Mediterranean Sea. The presence of the main environmental contaminants, such as cadmium, microplastics, and antibiotics was investigated in these seafood samples. Contamination by cadmium and antibiotics in the seafood investigated in our study was negligible. However, a high value of microplastics was detected in the stomach and gut of Sparus aurata and Dicentrarchus labrax. Overall, even though the presence of microplastics needs to be investigated by further studies, the results confirmed that the environmental conditions of the three bays investigated by the ADRINET project partners (Italy, Albania, Montenegro) are positive and not affected by intensive anthropogenic activity

Human intestinal enteroids platform to assess the infectivity of gastroenteritis viruses in wastewater

Fecal-orally transmitted gastroenteritis viruses, particularly human noroviruses (HuNoVs), are a public health concern. Viral transmission risk through contaminated water results underexplored as they have remained largely unculturable until recently and the robust measuring of gastroenteritis viruses infectivity in a single cell line is challenging. This study primarily aimed to test the feasibility of the human intestinal enteroids (HIE) model to demonstrate the infectivity of multiple gastroenteritis viruses in wastewater. Initially, key factors affecting viral replication in HIE model were assessed, and results demonstrated that the reagent-assisted disruption of 3D HIE represents an efficient alternative to syringe pass-through, and the filtering of HuNoV stool suspensions could be avoided. Moreover, comparable replication yields of clinical strains of HuNoV genogroup I (GI), HuNoV GII, rotavirus (RV), astrovirus (HAstV), and adenoviruses (HAdV) were obtained in single and multiple co-infections. Then, the optimized HIE model was used to demonstrate the infectivity of multiple naturally occurring gastroenteritis viruses from wastewater. Thus, a total of 28 wastewater samples were subjected to (RT)-qPCR for each virus, with subsequent testing on HIE. Among these, 16 samples (57 %) showed replication of HuNoVs (n = 3), RV (n = 5), HAstV (n = 8), and/or HAdV (n = 5). Three samples showed HuNoV replication, and sequences assigned to HuNoV GI.3[P13] and HuNoV GII.4[P16] genotypes. Concurrent replication of multiple gastroenteritis viruses occurred in 4 wastewater samples. By comparing wastewater concentrate and HIE supernatant sequences, diverse HAstV and HAdV genotypes were identified in 4 samples. In summary, we successfully employed HIE to demonstrate the presence of multiple infectious human gastroenteritis viruses, including HuNoV, in naturally contaminated wastewater samples