Hepatitis E virus (HEV) causes viral hepatitis, posing a risk to vulnerable populations. Although swine are established reservoirs, the involvement of other animal species remains undefined. This study investigated 301 livers from naturally deceased European hedgehogs collected from wildlife centres (northern Italy), to determine their role in HEV transmission, spread and epidemiology, considering their proximity to urban areas. No positive samples were found by Real-time PCR, suggesting that hedgehogs play a marginal role in HEV epidemiology. Further studies are required to investigate other potential wild reservoirs to gain insight into HEV transmission dynamics, given the global impact of HEV-related diseases.

Wastewater-based environmental surveillance enables the monitoring of SARS-CoV-2 dynamics within populations, offering critical epidemiological insights. Numerous workflows for tracking SARS-CoV-2 have been developed globally, underscoring the need for interlaboratory comparisons to ensure data consistency and comparability. An inter-calibration test was conducted among laboratories within the network monitoring SARS-CoV-2 in wastewater samples across the Lombardy region (Italy). The test aimed to evaluate data reliability and identify potential sources of variability using robust statistical approaches. Three wastewater samples were analyzed in parallel by four laboratories using identical pre-analytical (PEG-8000-based centrifugation) and analytical processes (qPCR targeting N1/N3 and Orf-1ab). A two-way ANOVA framework within Generalized Linear Models was applied, and multiple pairwise comparisons among laboratories were performed using the Bonferroni post hoc test. The statistical analysis revealed that the primary source of variability in the results was associated with the analytical phase. This variability was likely influenced by differences in the standard curves used by the laboratories to quantify SARS-CoV-2 concentrations, as well as the size of the wastewater treatment plants. The findings of this study highlight the importance of interlaboratory testing in verifying the consistency of analytical determinations and in identifying the key sources of variation.

The genus Enterovirus (EV) includes small ubiquitous viruses with a single-stranded positive-RNA genome. Most EV infections are asymptomatic, but pathogenic EVs are responsible for a wide range of diseases and occasionally epidemics. The aim of the work was to monitor the distribution of EVs present in wastewater collected during the 2 year study period (2022-2023). Three hundred ninety-nine samples were received from three wastewater treatment plants of the Brescia, Cremona, and Bergamo provinces, Lombardy, Italy. One-step real-time reverse transcription-PCR screening showed positivity in 107 samples. Of these, 67 were successfully genotyped: 8 by Sanger sequencing only, 7 by next-generation sequencing metabarcoding only, and 52 by both methods. The most detected species was Enterovirus B, followed by Enterovirus A, Enterovirus C, and Enterovirus D. As many as 26 different types of non-polio EVs were identified, of which the most frequently detected were two Enterovirus B: echovirus 11 (E11) and coxsackievirus B5 (CVB5). Among Enterovirus A, the predominant types were CVA16 and CVA6. As for Enterovirus C, the types with the highest frequency were CVA13 and EV-C99; in addition, CVA24, which has been associated with outbreaks of acute hemorrhagic conjunctivitis, was found in two samples from Verziano (Brescia) and one from Bergamo. The Enterovirus D species was represented only by type EV-D68, which has recently been related to outbreaks in Europe and the United States. This study reveals a wide EV presence in urban wastewater in the considered area.IMPORTANCEWastewater-based epidemiology uses urban wastewater as a source of dynamic observation for monitoring the circulation of pathogens. A major strength of this surveillance approach is its ability to detect both symptomatic and asymptomatic infections. Through molecular characterization, it was possible to identify uncommon EVs that may lead to serious complications. Furthermore, next-generation sequencing metabarcoding allowed the identification of multiple EV types within a single sample. Wastewater monitoring could therefore be further leveraged as a complementary tool to support the monitoring of severe non-polio EV-related diseases.

Abstract Background Hepatitis E virus (HEV) is the agent of hepatitis E, an emerging public-health infection with an increasing incidence in Europe. Due to the apparent lack of species barriers, HEV is considered a zoonotic agent, with swine as the main reservoir. This study investigated HEV prevalence in humans and domestic pigs in Northern Italy, a highly anthropic area with the highest pig farm density, to gain insights into the risk of HEV transmission from swine to humans. Methods A total of 508 blood samples were collected from blood donors (341 males and 167 females; from 18 to 66 years old) and from 1619 pigs in 40 different farms. Serological analyses were performed with ELISA to detect anti-HEV antibodies. In addition, 120 faecal pool samples collected from the pig farms with the highest seroprevalence and 69 salami produced in the same area were analysed to detect viral genome by Real-time PCR. Results In humans, HEV seroprevalence was 4.9% (CI95% 0.03-0.07); positive donors were mainly males, with seroprevalence increasing up to 12% (CI95% 0.09-0.15) in the 46-55 age group. Seroprevalence in swine was 53.00% (CI95% 0.50-0.55), with HEV detected in only 1% (CI95% 0.001-0.045) faeces. All food samples tested negative. Conclusions Even if the seroprevalence in pigs highlighted that HEV was actively circulating in the farms, the low seroprevalence in humans, the scarce HEV detection in swine faeces, and the absence in food products suggested that HEV transmission via faecal-oral or foodborne routes is unlikely, probably due to the implementation of severe biosecurity measures and high standards of hygiene in swine farms, and the continuous assessment of the critical points in the pig supply chain (from farm to fork). The serological surveillance of humans and the monitoring of farmed animals are needed to support the competent authorities in timely managing public health issues, improving human health and veterinary safety from a One Health perspective. Key messages • In the human-swine interface is important to monitor HEV presence. • The One Health approach is essential to control zoonotic diseases.

Over the last few decades, tick-borne encephalitis (TBE) has become a growing public health problem in Europe. The tick-borne encephalitis virus (TBEV) is a zoonotic virus that affects the central nervous system (CNS). TBEV has been detected in 27 European countries, and the rise in TBE cases is mainly due to environmental and ecological factors, and factors that increase the risk of human exposure to infected ticks. The infection via the alimentary route is the second most common means of TBEV transmission to humans. Raw milk from infected goats, sheep, or cows has been identified as a source of human food-borne infections. This study aims to gather new information on the prevalence of tick-borne encephalitis virus (TBEV) in raw goat’s and cow’s milk and related raw products in the Lombard Alps (Italy). This is important due to the close proximity of Lombardy to the Triveneto region, where TBE is endemic, and southern Switzerland, where numerous TBEV-positive mammals have been found. Throughout 2023, a passive monitoring plan was implemented on samples delivered for TBEV analyses from the Alpine pastures. In total, 248 specimens including raw milk, raw milk cheese, and butter were tested. This is the first monitoring of food at risk of TBEV transmission in a non-endemic region with evidence of TBEV circulation. Despite testing a wide range of dairy products, no sample tested positive for RNA-TBEV by real-time RT-PCR. Preliminary results suggest that raw milk and raw dairy products do not pose a significant risk of TBEV transmission to humans in the territory of Lombardy.

In June 2022, a gastroenteritis outbreak occurred in a town in Northern Italy, possibly associated with the ingestion of norovirus from public drinking water. Noroviruses are highly infectious RNA viruses, with high stability in the environment. They are the primary cause of non-bacterial gastroenteritis worldwide, and despite the fact that the disease is mainly self-limiting, norovirus infection can lead to severe illness in the immunocompromised, the elderly and children. Immediately after the notification of the suspected norovirus outbreak, faecal specimens were collected from hospitalised patients, and water samples were collected from public drinking fountains in the affected area, to confirm the presence of norovirus. Norovirus was detected in 80% (95% CI 0.58-0.91) of the faecal specimens, and in 50% (95% CI 0.28-0.72) of the water samples using RT (reverse transcription) Real-time PCR. The identification of GII genotype in all samples confirmed public drinking water as the source of norovirus contamination. In addition, in one faeces and one water sample, the co-presence of genotypes GI and GII was detected. The strains were typed by sequencing, with most of them belonging to the genotype GII.3. Immediately after the confirmation of norovirus contamination in public drinking water, the local competent authorities applied safety measures, resulting in a decline in number of cases. Moreover, after the application of disinfection protocols in the water plant, the sampling was repeated with negative results for norovirus in the affected area. However, positive samples were found in the neighbouring area (prevalence 10.00%, 95% CI 0.02-0.40) and in the water spring (prevalence 50.00%, 95% CI 0.21-0.78), suggesting norovirus persistence and spread from the water source. The prompt identification of the source of contamination, and collaboration with the local authorities guided the implementation of proper procedures to control viral spread, resulting in the successful control of the outbreak.

Abstract Background Wastewaters surveillance has proven to be a valuable tool in detecting the spread of SARS-CoV-2 and other pathogens. Enterovirus (EV) and Human bocavirus (HBoV) are viruses that can infect humans, causing mild to severe pathologies transmitted also through the fecal-oral route. EVs are ssRNA viruses, grouped among four taxonomic species EV-A to D. HBoV is ssDNA virus mainly affects infants. Genus Bocavirus includes 4 genotypes: HBoV1 to 4, detected mainly in feces. EVs and HBoVs infections are often not notified, causing loss of data on the actual incidence. The aim of this study was to investigate the presence and diffusion of EVs and HBoVs in wastewaters. Methods 200 wastewater samples were collected during 2022, at the inlet of wastewater treatment plants in three cities of the Lombardy Region (i.e.: Brescia, Bergamo, and Cremona). EVs and HBoVs were detected by RT Real-time PCR and Real-time PCR respectively using specific primers for target region of the viral genome. Positive samples were genotyped by Sanger sequencing. Prevalence was calculated with the Blaker's method. Results Overall EV and HBoV prevalences were 20.5% (CI95% 15.5-26.2%) and 37% (CI95%.30.6-43:9%), respectively. EV was mainly detected in the mild season with a prevalence of 68.3% (CI95% 53.0-80.4%). Sequence analysis reported that EV-B species was the most prevalent in this study (9 samples). HBoVs positive samples’ prevalence was higher in cold season (71.6% CI95% 60.5-80.6%), than mild season. HBoV2 showed higher prevalence (85.6% CI95% 75.6-92.0%) than HBoV3. Conclusions This study provided insight into the seasonal spread of EVs and HBoVs circulating and can contribute to understand the disease burden. Wastewaters surveillance provides a wide and more complete epidemiological data than the clinical one of a single person, rather providing information of a community of individuals making the data very valuable to the competent authorities. Key messages • Wastewaters surveillance is a useful tool for investigating the spread and circulation of viruses in population. • EVs and HBoVS are widespread in Northen Italy population.

Abstract Background Sars-CoV2 pandemic has shown the importance of wastewater surveillance to monitor virus diffusion and its environmental permanence; WHO experts highlighted the importance of wastewater controls as early detection system to individuate Sars-CoV2 and other viruses which could potentially spread to the population. The aim of this work was to investigate the presence of Sars-CoV2 in wastewater from Brescia, Cremona and Bergamo cities during 2021-2023 years after pandemic event. Methods From the end of 2021 until now, 341 wastewater samples were collected and analyzed at Food Control department to detect Sars-CoV2. After the inactivation at 56 °C for 30 minutes, samples were centrifugated and extracted by eGENE-UP® system (bioMérieux). Virus detection was based on three genes amplification: ORF, N1 and N3. Quantification in genome copies/µL (cg/µL) was performed. Results Almost all samples were positive for virus presence; in particular ORF gene was detected in 94% of samples (IC95% 91,6 - 96,7%); the other genes N1 and N3 were detected respectively in 91% (IC95% 87,2 - 93,6) and 98% (IC95% 95 - 99%). In only three samples all genes simultaneously were not detected. ORF and N1 were quantified between 103 and 104 cg/µL without differences among seasons. N3 was quantified at higher values than the other genes (104 in 57% of samples; IC95% 51,4 - 62,2%). Conclusions The pandemic has run out with the safety measures applied, but wastewater surveillance has highlighted virus circulation, even if at lower, constant levels than in pandemic period. Virus quantity in wastewater followed and sometime preceded epidemic trend. Wastewater control could support competent authorities in epidemic event management. Environmental surveillance, as already demonstrated during pandemic period, could represent an alert system for Sars-Cov2 and oral fecal route virus spreading Key messages • Wastewater surveillance is an important tool to control Sars-CoV2 mutation and spreading. • Environmental viruses’ surveillance can support competent authorities in decisions regarding public health.

Norovirus (NoV) is an enteric virus with foodborne transmission. Bivalve shellfish are a main source of infections and outbreaks. In Italy a voluntary based monitoring plan to check the safety of bivalve shellfish was set up at provincial level. This study describes the occurrence and distribution of NoV in the Northern Adriatic Sea and in the Ligurian Sea. From October 2018 to September 2020, 807 bivalve shellfish samples (n = 205 oysters, n = 182 mussels, n = 348 clams, n = 72 other bivalve shellfish) were tested by One-Step Retrotranscription Real-time polymerase chain reaction for NoV GI and GII and quantified according to the ISO 15216-2:2013 and ISO 15216-1:2017. Positive samples were further analyzed to determine genotype by sequencing of the ORF1/ORF2 junction of the viral genome. A total of 126 samples were positive for NoV, mussels, and oysters had the highest probability of being positive and positive samples were found mainly in the colder season. Of these samples, 46% were NoV GII, 13% NoV GI, and 40% carried both genogroups. Thirty-seven samples were typeable (GI n = 12 and GII n = 25) with GI samples belonging to four genotypes and GII samples belonging to five genotypes. GII.3 genotype was the most prevalent, followed by GII.4, particularly Sydney 2012 subtype, a leading cause of infections worldwide, was found in three oysters' and three clams' samples. The phylogenetic analysis revealed a high heterogeneity among the species that are scattered in several clusters. Considering the low infectious dose the overall presence of NoV in edible shellfish, particular those to be eaten raw or undercooked, is moderately high. The presence of genotypes frequently involved in human infections strengthens the need for ongoing monitoring, which should be extended at national level.

The zoonotic hepatitis E virus genotype 3 (HEV-3) causes most autochthonous human hepatitis E cases in Europe, which are due to the consumption of raw or undercooked food products of animal origin. Pigs and wild boars are considered the main reservoirs of this genotype, while rabbits are the reservoir of a distinct phylogenetic group named HEV-3ra, which is classified within the HEV-3 genotype but in a separate clade. Evidence for the zoonotic potential of HEV-3ra was suggested by its detection in immunocompromised patients in several European countries. HEV-3ra infection was found in farmed and feral rabbit populations worldwide and its circulation was reported in a few European countries, including Italy. Furthermore, Italy is one of the major rabbit meat producers and consumers across Europe, but only a few studies investigated the presence of HEV in this reservoir. The aim of this study was to assess the presence of HEV in 328 Italian hares and 59 farmed rabbits collected in 3 Italian macro-areas (North, North-Central, and South-Central), between 2019 and 2021. For this purpose, liver samples were used to detect HEV RNA using broad-range real-time RT-PCR and nested RT-PCR. Using 28 liver transudates from hares, the ELISA test for anti-HEV IgG detection was also performed. Neither HEV RNA nor anti-HEV antibodies were detected. Further studies will be conducted to assess the HEV presence in Italian lagomorphs to establish the role of this host and the possible risk of transmission for workers with occupational exposure, to pet owners and via food.

Torque teno sus virus (TTSuV) is a non-enveloped circular ssDNA virus which frequently infects swine and has been associated with hepatic, respiratory, and autoimmune disorders. TTSuV’s pathogenic role is still uncertain, and clear data in the literature on virus reservoirs are lacking. The aims of this study were to investigate the presence of potentially zoonotic TTSuV in wild animals in Northern Italy and to evaluate their role as reservoirs. Liver samples were collected between 2016 and 2020 during four hunting seasons from wild boars (Sus scrofa), red deer (Cervus elaphus), roe deer (Capreolus capreolus), and chamois (Rupicapra rupicapra). Samples originated from areas in Northern Italy characterized by different traits, i.e., mountains and flatland with, respectively low and high farm density and anthropization. Viral identification was carried out by end-point PCR with specific primers for TTSuV1a and TTSuVk2a species. TTSuV prevalence in wild boars was higher in the mountains than in the flatland (prevalence of 6.2% and 2.3%, respectively). In wild ruminants only TTSuVk2a was detected (with a prevalence of 9.4%). Our findings shed light on the occurrence and distribution of TTSuV in some wild animal species, investigating their possible role as reservoirs.

In 2019, SARS-CoV-2 was identified as the cause of an easily transmissible disease that was declared as a world pandemic. Foodborne transmission was never reported. However, early studies suggested that food could be involved in SARS-CoV-2 entry in the human gastrointestinal tract leading to possible infection, and highlighting the importance of further studies to inspect possible issues linked to food consumption. In this perspective, this work aimed at monitoring SARS-CoV-2 presence in some food and mains water samples in Northern Italy during the COVID-19 pandemic (2020–2022). A total of 1806 foods, 112 mains water samples, and 580 swabs on meat and dairy product surfaces were analyzed for SARS-CoV-2 RNA detection by Real-time PCR. All the analyzed samples were negative to viral RNA detection with the exception of one vegetable sample. Even if data on foodborne coronavirus transmission suggested a limited importance of this pathway, the impact of the current pandemic in Northern Italy deserved a rigorous investigation to rule out such possibility. Indeed, gaining insight on all SARS-CoV-2 possible transmission pathways, including the foodborne route, seemed of interest to maintain consumers’ confidence and trust in food safety, and for the effective management of the current, and future, possible pandemics.

Background SARS CoV2 pandemic has generated interest in research of virus spread sources, apart from human contagion. In particular different studies are performed to understand surfaces involvement; during the epidemic event, different types of studies on surfaces have been performed. However, there is little information regarding the presence of the virus in food; wastewater contamination and poor hygiene practices could be the sources of viral contamination in handled food products. The aim of this work was to analyze different types of food for Sars CoV 2 presence. Methods 390 samples of molluscs, 353 vegetables, 10 meat, 72 water, 169 berries, 16 seafood products and ready-to-eat dishes were analysed (2020-2021). Samples were selected from those provided for routine analysis during the pandemic period. The preparation was done based on ISO 15216-2 indications, followed by extraction with Nuclisense®Minimag® system; real-time PCR was applied to detect the Sars CoV2 virus Open Reading Frame 1ab, with two different master mixes. Results All samples tested were negative for ORF1ab detection. Many hypothesis can be made about negative Results: it could be possible that contamination levels in water, or on surfaces in contact with food, are too low to generate a detection signal; again, handling practices may be sufficient to avoid the transmission of the virus to food. Otherwise, the selected genetic target and the Real-time PCR performed, could have been not so efficient in detecting virus as expected. Conclusions In this preliminary work, food does not seem to be a substrate for Sars CoV2 contamination; other food categories or transformation/production processes could be investigated to confirm the obtained results. Also, different targets and amplification systems would have to be evaluated to verify the rule of foodstuffs as Sars CoV2 vehicles. Key messages Primary production and manual processed food don't seem to be at risk for SARS CoV2 contamination. Food has not been identified as a source of Sars CoV2 spreading.

Mammalian orthoreoviruses (MRVs) are emerging infectious agents that may affect wild animals. MRVs are usually associated with asymptomatic or mild respiratory and enteric infections. However, severe clinical manifestations have been occasionally reported in human and animal hosts. An insight into their circulation is essential to minimize the risk of diffusion to farmed animals and possibly to humans. The aim of this study was to investigate the presence of likely zoonotic MRVs in wild ungulates. Liver samples were collected from wild boar, red deer, roe deer, and chamois. Samples originated from two areas (Sondrio and Parma provinces) in Northern Italy with different environmental characteristics. MRV detection was carried out by PCR; confirmation by sequencing and typing for MRV type 3, which has been frequently associated with disease in pigs, were carried out for positive samples. MRV prevalence was as high as 45.3% in wild boars and 40.6% in red deer in the Sondrio area, with lower prevalence in the Parma area (15.4% in wild boars). Our findings shed light on MRV occurrence and distribution in some wild species and posed the issue of their possible role as reservoir.