Non-thermal processing technologies and viral viability assays: Key approaches for studying human norovirus (HuNoV) in foods

Human noroviruses (HuNoVs) are the leading cause of foodborne and waterborne viral gastroenteritis globally. To mitigate the risks posed by this enteric virus, it is essential to identify effective non-thermal technologies for inactivating HuNoV, alongside reliable methods to assess viral infectivity. This dual approach, combining viral inactivation technologies with infectivity assays, will help the food industry and regulatory authorities in ensuring public health. The aim of this PhD thesis is to evaluate the efficacy of two non-thermal technologies, High Pressure Processing (HPP) and Plasma-Activated Seawater (PASW), for inactivating HuNoV and its surrogates in high-risk food items such as berries and shellfish. The study employs RT-qPCR and viability RT-qPCR assays, in addition to traditional and advanced cell culture techniques such as the novel Human Intestinal Enteroid (HIE) system, to assess HuNoV viral inactivation and infectivity.Human noroviruses (HuNoVs) are the leading cause of foodborne and waterborne viral gastroenteritis globally. To mitigate the risks posed by this enteric virus, it is essential to identify effective non-thermal technologies for inactivating HuNoV, alongside reliable methods to assess viral infectivity. This dual approach, combining viral inactivation technologies with infectivity assays, will help the food industry and regulatory authorities in ensuring public health. The aim of this PhD thesis is to evaluate the efficacy of two non-thermal technologies, High Pressure Processing (HPP) and Plasma-Activated Seawater (PASW), for inactivating HuNoV and its surrogates in high-risk food items such as berries and shellfish. The study employs RT-qPCR and viability RT-qPCR assays, in addition to traditional and advanced cell culture techniques such as the novel Human Intestinal Enteroid (HIE) system, to assess HuNoV viral inactivation and infectivity

C12 | Studio e valutazioni sull’inattivazione in vitro di escherichia coli utilizzando la tecnologia della plasma activated fog

Scopo. Gli alimenti ready to eat (RTE) sono ad elevato rischio microbiologico per la possibile presenza di agenti patogeni, responsabili di infezioni/tossinfezioni, come Escherichia coli, Salmonella spp., Listeria monocytogenes e virus a trasmissione alimentare; La produzione di RTE è in espansione per la praticità di utilizzo offerta al consumatore e le Aziende necessitano di tecnologie innovative per ridurre il rischio microbiologico. Tra le tecnologie non-termiche emergenti per la decontaminazione degli alimenti, l’acqua attivata via plasma (Plasma Activated Water, PAW) si configura come un’alternativa sostenibile ed efficace per il controllo del rischio microbiologico. L’efficacia antimicrobica della PAW è da attribuire a molecole reattive dell’ossigeno e dell’azoto (Reactive Oxygen and Nitrogen Species, RONS), che si generano durante il trattamento; la tecnologia della nebbia attivata via Plasma (Plasma Activated Fog, PAF) non si limita alla semplice nebulizzazione della PAW in ambiente confinato, ma impiega i RONS prodotti dalla scarica elettrica, ad elevate concentrazioni e dispersi in un aerosol, per la decontaminazione microbica. In questo studio è stata indagata l’efficacia della PAF per l’inattivazione in vitro di E. coli. Metodi. È stata utilizzata una sorgente di plasma, alimentata da aria ambiente e operante a pressione atmosferica con differenti livelli di energia, allo scopo di valutare quali fossero i parametri più adatti alla decontaminazione in vitro di E. coli; sono state utilizzate due tipologie di PAF: PAF_A con un livello di energia medio di 40 mJ e PAF_B con un livello di energia medio di 12 mJ. Il ceppo di E. coli CECT 9198 a 3 differenti concentrazioni (102, 103 e 104 UFC/100µL) è stato distribuito uniformemente fino a completo adsorbimento su Piastre di Plate Count Agar (PCA, Oxoid). Successivamente le piastre sono state sottoposte all’azione di PAF per 3’ e quindi incubate per 24 h alla temperatura di 37 °C. La concentrazione quali-quantitativa dei prodotti ottenuti con l’attivazione al plasma delle 2 tipologie di PAF utilizzate per lo studio (RONS), è stata monitorata con l’impiego di kit commerciali. Tutte le analisi sono state condotte in duplicato. Risultati. Il trattamento con PAF_A ha dimostrato un’efficacia antimicrobica significativamente superiore rispetto a PAF_B. Con PAF_A si è ottenuta una riduzione di 2 log UFC/mL di E. coli in tutte le diluizioni prese in esame, mentre con PAF_B si è ottenuta una minore riduzione, (circa 1.4 log UFC/mL). Questi risultati sono strettamente correlati alla quantità di RONS ((ioni nitrito (NO2-), ioni nitrato (NO3-) e di perossido d’idrogeno (H2O2)) riscontrati nelle due diverse PAF. In particolare, nella PAF_A sono stati misurati 135 mg/L, 1,00 mg/L, e 1,14 mg/L di NO3- , NO2- e H2O2, rispettivamente. Conclusioni. La PAF ha consentito una buona inattivazione della crescita di E. coli su terreno PCA alle diverse concentrazioni prese in esame, esprimendo un’efficacia proporzionale all’energia utilizzata nel trattamento e relativa presenza di RONS. I risultati ottenuti permettono di asserire che l’uso della tecnologia PAF, può essere considerata un innovativo e promettente approccio alla decontaminazione microbica degli alimenti RTE. Pertanto in futuro le indagini saranno rivolte alla valutazione dell’attività antimicrobica di PAF su altri patogeni con indagini eseguite direttamente sulle matrici alimentari a rischio

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

Activación por plasma frío de agua de mar para el control de norovirus humanos y patógenos bacterianos durante la purificación de moluscos bivalvos

El plasma frío representa una tecnología prometedora para el control de patógenos en una plétora de aplicaciones en agricultura y alimentación. Los moluscos bivalvos pueden representar un problema de salud pública ya que en ocasiones su consumo se ha asociado a brotes virales y bacterianos. Por lo tanto, la regulación europea exige una etapa de depuración para los moluscos bivalvos dependiendo de la calidad de las aguas de la zona producción. Este estudio ha investigado la activación por plasma frío de agua de mar (PASWs), su composición reactiva de especies de oxígeno y nitrógeno (RONS), y la capacidad de inactivar patógenos víricos y bacterianos de transmisión alimentaria asociados a moluscos bivalvos. La inactivación de los bacteriófagos F-específicos (MS2) y somáticos (φ174), así como la de los modelos de norovirus humano (norovirus murino, MNV y virus de Tulane, TV) y norovirus humano (HuNoV GII.4) se determinaron utilizando recuentos de placas y ensayos de infectividad en cultivo celular, incluyendo el nuevo modelo de enteroides intestinales humanos (HIE) para HuNoV. Además, se caracterizó la cinética de inactivación de Escherichia coli, Salmonella spp. y Vibrio parahaemolyticus. Los resultados mostraron la completa inactivación de fagos (6-8 log), los modelos (5-6 log), HuNoV (6 log) y patógenos bacterianos (6-7 log) durante las primeras 24 h, preservando la viabilidad de los mejillones. El análisis de los RONS permitió relacionar los nitritos (NO2-) con la inactivación microbiana. El estudio ha demostrado que el uso de PASW representa una tecnología adecuada para depurar eficazmente los moluscos bivalvos y así controlar el riesgo microbiológico asociado a su contaminación viral o bacteriana.Los autores agradecen a RENASCO S.R.L. por proporcionar el agua activada por plasma. También se agradece Mare Gioioso SRL (Bari, Italia) por proporcionar las muestras de agua de mar depuradas

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

Estudio de inactivación de norovirus humano en puré de fresas mediante enteroides intestinales humanos y modelización predictiva

El consumo de fresas contaminadas por norovirus humano (HuNoV) ha sido asociado a brotes de gastroenteritis aguda. Los tratamientos no térmicos, como el procesado mediante altas presiones hidrostáticas (HPP), representan tecnologías capaces de inactivar microrganismos patógenos y, además, preservar las propiedades organolépticas de las fresas y de sus productos derivados. El estudio se ha centrado en evaluar el efecto de HPP modulando los parámetros operativos de presión (350-450 MPa) y tiempo (0-10 min) en puré de fresa inoculado con HuNoV genogrupo I (GI.3[P13]) y II (GII.4 Sydney [P16]), junto con norovirus murino (MNV) y el virus Tulane (TV) empleados como modelos cultivables. La inactivación viral se evaluó mediante (i) cultivo celular empleando el nuevo modelo de enteroides intestinales humanos (HIE) para HuNoV, y las líneas celulares RAW 264.7 y LCC-MK2 para los modelos cultivables; (ii) RT-qPCR y (iii) RT-qPCR de viabilidad con tres agentes intercalantes (CL, PMAxx y PtCl4). Los resultados de infectividad mostraron que el TV es más resistente que el MNV a los tratamientos de HPP, como confirmó la modelización de la cinética de inactivación. Además, el HuNoV GI.3 [P13] resultó más resistente que el GII.4 Sydney [P16]. Por lo tanto, TV, pero no MNV, imita los patrones de inactivación de HuNoV GII.4 Sydney [P16]. Por lo tanto, el estudio ha demostrado que un tratamiento a presión de 450 MPa superior a 5 min es suficiente para controlar la contaminación vírica en puré de fresas. También, se demostró que la RT-qPCR de viabilidad es un método mejorado con respecto a la RT-qPCR para estimar la inactivación viral en condiciones extremas (450 MPa), pero no en los tratamientos a menor presión. Finalmente, se ha demostrado que el modelo HIE proporciona información robusta sobre la inactivación de patógenos víricos humanos como el HuNoV. El modelo HIE por lo tanto puede ser utilizado para validar la eficacia de los tratamientos de inactivación y así garantizar la protección de la salud pública.Los autores quieren agradecer al Prof. Javier Buesa (Departamento de Microbiología, Facultad de Medicina, Universidad de Valencia, Valencia, España) por facilitar muestras fecales positivas para norovirus humano

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

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