Selective Pressure and Evolution of SARS-CoV-2 Lineages BF.7 and BQ.1.1 Circulating in Italy from July to December 2022.

In this work, we studied the selective pressure and evolutionary analysis on the SARS-CoV-2 BF.7 and BQ.1.1 lineages circulating in Italy from July to December 2022. Two different datasets were constructed: the first comprised 694 SARS-CoV-2 BF.7 lineage sequences and the second comprised 734 BQ.1.1 sequences, available in the Italian COVID-19 Genomic (I-Co-Gen) platform and GISAID (last access date 15 December 2022). Alignments were performed with MAFFT v.7 under the Galaxy platform. The HYPHY software was used to study the selective pressure. Four positively selected sites (two in nsp3 and two in the spike) were identified in the BF.7 dataset, and two (one in ORF8 and one in the spike gene) were identified in the BQ.1.1 dataset. Mutation analysis revealed that R408S and N440K are very common in the spike of the BF.7 genomes, as well as L452R among BQ.1.1. N1329D and Q180H in nsp3 were found, respectively, at low and rare frequencies in BF.7, while I121L and I121T were found to be rare in ORF8 for BQ.1.1. The positively selected sites may have been driven by the selection for increased viral fitness, under circumstances of defined selective pressure, as well by host genetic factors.

Persistence of hepatitis E virus (HEV) subtypes 3c and 3e: Long-term cold storage and heat treatments.

Hepatitis E virus (HEV) is the causative agent of foodborne infections occurring in high income countries mainly by consumption of undercooked and raw pork products. The virus is zoonotic with pigs and wild boars as the main reservoirs. Several studies proved the presence of HEV-RNA in pork liver sausages, pâté and other pork by-products. However, the detection of HEV nucleic acids does not necessary correspond to infectious virus and information on the persistence of the virus in the food is still limited. To which extent and how long the virus can survive after conventional industrial and home-made conservation and cooking procedures is largely unknown. In the present study, we investigated the persistence of two subtypes of HEV-3, by measuring the viral RNA on cell supernatant of infected A549 cells, after long-term storage at +4 °C and -20 °C and after heating for short or long-time span. Results confirmed that either low temperature storage (+4 °C) or freezing (-20 °C) do not influence the survival of the virus, and only a moderate reduction of presence of its RNA after 12 weeks at +4 °C was observed. To the other side, heating at 56 °C for long time (1 h) or at higher temperatures (>65 °C) for shorter time inactivated the virus successfully.

Investigation of Salmonella, hepatitis E virus (HEV) and viral indicators of fecal contamination in four Italian pig slaughterhouses, 2021-2022.

In the pork production chain, the control at slaughterhouse aims to ensure safe food thanks to proper hygienic conditions during all steps of the slaughtering. Salmonella is one of the main foodborne pathogens in the EU causing a great number of human cases, and pigs also contribute to its spreading. Pig is the main reservoir of the zoonotic hepatitis E virus (HEV) that can be present in liver, bile, feces and even rarely in blood and muscle. The aim of this study was to assess the presence of both Salmonella and HEV in several points of the slaughtering chain, including pig trucks. Other viruses hosted in the gut flora of pigs and shed in feces were also assayed (porcine adenovirus PAdV, rotavirus, norovirus, and mammalian orthoreovirus MRV). Torque teno sus virus (TTSuV) present in both feces, liver and blood was also considered. Four Italian pig abattoirs were sampled in 12 critical points, 5 of which were the outer surface of carcasses before processing. HEV and rotavirus (RVA) were not detected. Norovirus was detected once. Salmonella was detected in two of the 4 abattoirs: in the two lairage pens, in the site of evisceration and on one carcass, indicating the presence of Salmonella if carcass is improper handled. The sampling sites positive for Salmonella were also positive for PAdV. MRV was detected in 10 swabs, from only two abattoirs, mainly in outer surface of carcasses. TTSuV was also detected in all abattoirs. Our study has revealed a diverse group of viruses, each serving as indicator of either fecal (NoV, RVA, PAdV, MRV) or blood contamination (TTSuV). TTSuV could be relevant as blood contamination indicators, crucial for viruses with a viremic stage, such as HEV. The simultaneous presence of PAdV with Salmonella is relevant, suggesting PAdV as a promising indicator for fecal contamination for both bacterial and viruses. In conclusion, even in the absence of HEV, the widespread presence of Salmonella at various points in the chain, underscores the need for vigilant monitoring and mitigation strategies which could be achieved by testing not only bacteria indicators as expected by current regulation, but also some viruses (PAdV, TTSuV, MRV) which could represent other sources of fecal contamination.

Tracking the Selective Pressure Profile and Gene Flow of SARS-CoV-2 Delta Variant in Italy from April to October 2021 and Frequencies of Key Mutations from Three Representative Italian Regions.

The SARS-CoV-2 Delta variant of concern (VOC) was often associated with serious clinical course of the COVID-19 disease. Herein, we investigated the selective pressure, gene flow and evaluation on the frequencies of mutations causing amino acid substitutions in the Delta variant in three Italian regions. A total of 1500 SARS-CoV-2 Delta genomes, collected in Italy from April to October 2021 were investigated, including a subset of 596 from three Italian regions. The selective pressure and the frequency of amino acid substitutions and the prediction of their possible impact on the stability of the proteins were investigated. Delta variant dataset, in this study, identified 68 sites under positive selection: 16 in the spike (23.5%), 11 in nsp2 (16.2%) and 10 in nsp12 (14.7%) genes. Three of the positive sites in the spike were located in the receptor-binding domain (RBD). In Delta genomes from the three regions, 6 changes were identified as very common (>83.7%), 4 as common (>64.0%), 21 at low frequency (2.1%-25.0%) and 29 rare (≤2.0%). The detection of positive selection on key mutations may represent a model to identify recurrent signature mutations of the virus.

First serological and molecular investigation of hepatitis E virus infection in dromedary camels in Algeria.

Hepatitis E is an acute self-limited or fulminant infection in humans, caused by the hepatitis E virus (HEV). This member of the Hepeviridae family has been identified in a wide range of domestic and wild animals all over the world, with a possible transmission to humans through fecal oral route, direct contact and ingestion of contaminated meat products, making it one of the global zoonotic and public health major concerns. Since there is no monitoring program and a lack of data on HEV in animals in Algeria, the current preliminary survey has been undertaken to elucidate the exposure to the virus in camels at abattoirs of six southern provinces of Algeria. Two-hundred and eight sera/plasma were collected and analyzed (by double antigen sandwich ELISA) for the presence of total anti-HEV antibodies, among which 35.1% were positive, but no HEV RNA could be isolated from them (by two pan-HEV nested RT-PCR and broad range real-time reverse transcription RT-PCR). The univariate analysis showed significant associations (p < 0.05) between HEV seroprevalence and province of origin, age, and sex of camels, whereas the multivariable logistic regression analysis revealed a negative impact of camels' age on it. The obtained results confirm that HEV infection is widespread established in the camelid population of Algeria.

Erinaceus coronavirus persistence in hedgehogs (Erinaceus europaeus) in a non-invasive, in vivo, experimental setting.

In the last 20 years, new zoonotic CoV strains have emerged (SARS-CoV, MERS-CoV, and SARS-CoV-2), and new species have also been reported in animals. In Europe, the Erinaceus coronavirus (EriCoV) was recently described in Erinaceus europaeus. However, information on the prevalence and duration of viral shedding is unknown. In this study, feces samples were collected from 102 European hedgehogs hosted in the Center for the Recovery of Wild Fauna in Rome and analyzed for the presence of EriCoV RNA by Reverse Transcription-PCR. In total, 45 animals (44.1%) resulted positive for EriCoV at the first sampling and 63 (61.7%) animals were positive at the follow-up, which was performed from the 3rd to the 86th day. The duration of fecal virus shedding showed a mean duration of 22.8 days and lasted up to 62 days. Eighteen hedgehogs showed intermittent viral shedding. Phylogenetic analysis showed a correlation with EriCoV strains reported in Germany, the United Kingdom, and northern Italy. None of the EriCoV sequences showed the CD200 ortholog insertion, previously observed in strains isolated in animals from northern Italy. Interestingly, all but one animal revealed the presence in their feces of the same EriCoV sequences, analyzing the short genomic region at 3' spike gene and 5' ORF3a 500bp fragment (100% nt.id.) in both first and follow-up samples. This result suggests that animals were infected with the same strain during their stay at the center. Our results confirm that EriCoV can persist in hedgehogs for a long period, underlining that hedgehogs are an important commensal reservoir for Merbecovirus. A long duration of viral shedding increases the likelihood that the virus will spread in the environment.

Hepatitis E Virus (HEV) in Heavy Pigs in Slaughterhouses of Northern Italy: Investigation of Seroprevalence, Viraemia, and Faecal Shedding.

Hepatitis E virus (HEV) is considered an emerging threat in Europe, owing to the increased number of human cases and the widespread presence of the virus in pigs at farms. Most cases in industrialized countries are caused by the zoonotic HEV-3 genotype. The main transmission route of HEV-3 in Europe is foodborne, through consumption of raw or undercooked liver pork and wild boar meat. Pigs become susceptible to HEV infection after the loss of maternal immunity, and the majority of adult pigs test positive for IgG anti-HEV antibodies. Nonetheless, HEV-infected pigs in terms of liver, faeces, and rarely blood are identified at slaughterhouses. The present study aimed to investigate the prevalence of HEV-positive batches of Italian heavy pigs at slaughterhouses, assessing the presence of animals still shedding HEV upon their arrival at the slaughterhouse by sampling faeces collected from the floor of the trucks used for their transport. The occurrence of viraemic animals and the seroprevalence of anti-HEV antibodies were also assessed. The results obtained indicated the presence of anti-HEV IgM (1.9%), and a high seroprevalence of anti-HEV total antibodies (IgG, IgM, IgA; 89.2%, n = 260). HEV RNA was not detected in either plasma or faecal samples. Nevertheless, seropositive animals were identified in all eight batches investigated, confirming the widespread exposure of pigs to HEV at both individual and farm levels. Future studies are needed to assess the factors associated with the risk of HEV presence on farms, with the aim to prevent virus introduction and spread within farms, thereby eliminating the risk at slaughterhouse.

Cross-sectional study of hepatitis E virus (HEV) circulation in Italian pig farms.

Foodborne transmission is considered the main way of spreading zoonotic hepatitis E virus (HEV) infection in Europe. In recent years, the human cases of hepatitis E in subjects without history of travel in endemic areas have raised, suggesting that domestic HEV transmission is increasing. Pork products with or without liver, are often indicated as the source of many human foodborne HEV cases as well as small outbreaks. Pigs are recognized as the main reservoir of the zoonotic HEV-3 genotype, the most frequently detected in human cases in the EU. In the absence of a harmonized surveillance of HEV circulation, data on prevalence are heterogeneous but confirm a widespread circulation of HEV-3 in pig herds across EU. HEV-3 can pass through the food chain from farm to fork when infected animals are slaughtered. In Italy, several studies reported the circulation of HEV-3 in pig farms, but results are heterogeneous due to different methodologies applied. In the present study, we performed a survey over 51 pig herds belonging to three main types of farms: breeding, fattening and farrow-to-finish. HEV-RNA was analyzed by broad range Real-time RT-PCR on 20 samples for each farm, obtained by pooling together feces from 10 individuals. Overall, HEV RNA was confirmed on 150 fecal pooled samples out of 1,032 (14.5%). At least one positive pooled sample was detected from 18 farms out of 51 tested (35.3%). By lowering the number of infected pigs at primary production, the risk of HEV-3 entering into the food chain can be reduced. Hence, information on HEV circulation in herds is highly relevant for choosing preventive measures and deserves development of a monitoring program and further investigations.

Absence of Hepatitis E Virus (HEV) in Italian Lagomorph Species Sampled between 2019 and 2021.

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.

In Vitro Replication of Swine Hepatitis E Virus (HEV): Production of Cell-Adapted Strains.

The hepatitis E caused by the virus HEV of genotypes HEV-3 and HEV-4 is a zoonotic foodborne disease spread worldwide. HEV is currently classified into eight different genotypes (HEV-1-8). Genotypes HEV-3 and HEV-4 are zoonotic and are further divided into subtypes. Most of the information on HEV replication remains unknown due to the lack of an efficient cell cultivation system. Over the last couple of years, several protocols for HEV cultivation have been developed on different cell lines; even if they were troublesome, long, and scarcely reproducible, they offered the opportunity to study the replicative cycle of the virus. In the present study, we aimed to obtain a protocol ready to use viral stock in serum free medium that can be used with reduced time of growth and without any purification steps. The employed method allowed isolation and cell adaptation of four swine HEV-3 strains, belonging to three different subtypes. Phylogenetic analyses conducted on partial genome sequences of in vitro isolated strains did not reveal any insertion in the hypervariable region (HVR) of the genomes. A limited number of mutations was acquired in the genome during the virus growth in the partial sequences of Methyltransferase (Met) and ORF2 coding genes.

SARS-CoV-2 in a Mink Farm in Italy: Case Description, Molecular and Serological Diagnosis by Comparing Different Tests.

This study described a SARS-CoV-2 infection in minks on an Italian farm. Surveillance was performed based on clinical examination and a collection of 1879 swabs and 74 sera from dead and live animals. The farm was placed under surveillance for 4.5 months, from the end of July 2020, when a man working on the farm tested positive by RT-PCR, till mid-December 2020 when all the animals were sacrificed. Clinical examination revealed no clinical signs or increased mortality rates attributable to SARS-CoV-2, while diagnostic tests detected only four weak PCR-positive samples, but 100% of sera were positive for SARS-CoV-2 anti-S antibodies. The phylogenetic analysis of two SARS-CoV-2 sequences from two minks and the sequence of the worker showed that they belonged to different clades. It could be therefore assumed that two distinct introductions of the virus occurred on the farm, and that the first introduction probably occurred before the start of the surveillance period. From the data collected, and especially from the detection of specific antibodies through the combination of different tests, it can be postulated that syndromic surveillance combined with genome detection by PCR may not be sufficient to achieve a diagnosis in asymptomatic animals. In particular, the serological approach, especially when using tests directed towards the S protein, may be useful for improving the traceability of virus circulation in similar environments.

Dynamic of Hepatitis E Virus (HEV) Shedding in Pigs.

Genotype 3 of hepatitis E virus (HEV-3) is the most common in Europe in both humans and pigs. HEV-3 strains are zoonotic, and foodborne cases associated with consumption of raw and undercooked pork products, mainly liver sausages, have been described. HEV-3 circulates largely in European pig farms, maybe due to its long persistence in the environment. Animals get infected around 3-4 months of age; shortly after, the infection starts to decline up to the age of slaughtering (8-9 months of age in Italy). With the purpose to understand the duration in farmed pigs of the shedding of the virus and its quantity, HEV-RNA detection was performed by Real-time RT-PCR from feces collected individually from two groups of 23 pigs. Sampling was conducted for 4 months shortly before slaughtering age. At 4-months-old, all animals were shedding HEV-3 to high load around 105 genome copies per gram (GC/g). Prevalence was higher in growers than in fatteners, with most of the pigs still positive around 166 days of age. Beyond some difference among individual pigs, the amount of HEV in feces decreased with the age of animals. The longest fattening period should ensure a lower risk of HEV shedder animals at slaughter, reducing the risk of food contamination.

Novel subtypes and unexpected heterogeneity of hepatitis E viral strains in wild boar captured in a small area in Central Italy.

Wild boar is the main sylvatic reservoir of the genotype 3 of hepatitis E virus (HEV). The occurrence of HEV-3 human cases has been linked to the consumption of raw or undercooked pig and wild boar meat and liver. The zoonotic transmission of HEV-3 has been confirmed by sequencing identical or strictly related viral strains in humans, wild boar and derived food. The HEV sequences classified within the HEV-3 genotype are highly variable, and although only one serotype has been identified so far, the observed differences allow for the further classification of the HEV-3 genotype into subtypes, named in alphabetical order. Compared to human and pig strains, an even higher heterogeneity is observed among strains infecting wild boar. In the present study, the genetic variability of eight HEV-3 strains detected in wild boars sampled in a small geographical area in Central Italy (Lazio and Umbria regions) was investigated by full genome sequencing and phylogenetic analysis. The strains were classified within the HEV-3a, HEV-3c, HEV-3f subtypes and within two new recently proposed subtypes. Results demonstrate - despite the relatively small geographic area of origin - an unexpected divergence within HEV-3 strains hosted by the investigated wild boar population and highlights the need for extensive sequencing of HEV in reservoirs to fully understand diversity, geographical distribution and evolution of this group of viruses.

Detection and whole genome sequencing of murine norovirus in animal facility in Italy.

Viruses belonging to the genus Norovirus (NoV) of the family Caliciviridae are the major cause of acute viral gastroenteritis worldwide. NoVs are classified into 10 genogroups (GI-GX), and those belonging to the genogroup GV are able to infect several species of rodents. To evaluate the circulation of MNV among mice housed in an Italian facility, sampling was performed over two separate periods, in 2011, and 3 years later in 2014. During the two samplings, 75 fecal samples were collected from healthy mice housed in the animal facility and subjected to RT-PCR for viral detection. After the analysis, 41/75 animals (54.6%) resulted positive for the presence of MNV in feces. Nucleotide sequencing revealed the presence of two MNV variants co-circulating in both 2011 and 2014. One MNV strain was isolated on RAW264.7 cell line, and subjected to full genome sequencing. Our study showed that the murine noroviruses are widespread in the investigated animal facility, despite guidelines for animal care and maintenance. Full genome sequence analysis of the MNV strain described in this study showed a correlation with other strains circulating in Europe. Understanding the molecular epidemiology of this virus should give insight into its natural history and evolution in mice.

SARS-CoV-2 complete genome sequencing from the Italian Campania region using a highly automated next generation sequencing system.

BACKGROUND: Since the first complete genome sequencing of SARS-CoV-2 in December 2019, more than 550,000 genomes have been submitted into the GISAID database. Sequencing of the SARS-CoV-2 genome might allow identification of variants with increased contagiousness, different clinical patterns and/or different response to vaccines. A highly automated next generation sequencing (NGS)-based method might facilitate an active genomic surveillance of the virus. METHODS: RNA was extracted from 27 nasopharyngeal swabs obtained from citizens of the Italian Campania region in March-April 2020 who tested positive for SARS-CoV-2. Following viral RNA quantification, sequencing was performed using the Ion AmpliSeq SARS-CoV-2 Research Panel on the Genexus Integrated Sequencer, an automated technology for library preparation and sequencing. The SARS-CoV-2 complete genomes were built using the pipeline SARS-CoV-2 RECoVERY (REconstruction of COronaVirus gEnomes & Rapid analYsis) and analysed by IQ-TREE software. RESULTS: The complete genome (100%) of SARS-CoV-2 was successfully obtained for 21/27 samples. In particular, the complete genome was fully sequenced for all 15 samples with high viral titer (> 200 copies/µl), for the two samples with a viral genome copy number < 200 but greater than 20, and for 4/10 samples with a viral load < 20 viral copies. The complete genome sequences classified into the B.1 and B.1.1 SARS-CoV-2 lineages. In comparison to the reference strain Wuhan-Hu-1, 48 total nucleotide variants were observed with 26 non-synonymous substitutions, 18 synonymous and 4 reported in untranslated regions (UTRs). Ten of the 26 non-synonymous variants were observed in ORF1ab, 7 in S, 1 in ORF3a, 2 in M and 6 in N genes. CONCLUSIONS: The Genexus system resulted successful for SARS-CoV-2 complete genome sequencing, also in cases with low viral copies. The use of this highly automated system might facilitate the standardization of SARS-CoV-2 sequencing protocols and make faster the identification of novel variants during the pandemic.

Possible Human-to-Dog Transmission of SARS-CoV-2, Italy, 2020.

We detected severe acute respiratory syndrome coronavirus 2 in an otherwise healthy poodle living with 4 family members who had coronavirus disease. We observed antibodies in serum samples taken from the dog, indicating seroconversion. Full-length genome sequencing showed that the canine and human viruses were identical, suggesting human-to-animal transmission.

Long-term surveillance for hepatitis E virus in an Italian two-site farrow-to-finish swine farm.

In humans, hepatitis E virus (HEV) is responsible for an acute enterically transmitted hepatitis, which can become chronic in immune-compromised patients. Genotypes 3 and 4 (HEV-3 and HEV-4) are zoonotic, and domestic pigs and wild boar are the main reservoirs. The occurrence of autochthonous cases in Europe, which have been increasing over the last 10 years, has been associated with food-borne zoonotic transmission of HEV-3, mainly linked to consumption of undercooked or raw pork products (sausages containing liver) and wild boar meat. Zoonotic HEV-3 strains are widespread on pig farms, but little information is available on the dynamic of HEV-3 infection within farms, among pigs. The aims of this study were to evaluate the prevalence of the infection among pigs of different ages along the production chain by the zoonotic HEVs, and to evaluate how long the virus may persist in the farm environment. The presence of HEV-RNA was investigated by real-time reverse transcription PCR (RT-PCR) in 281 test faecal pools over 19 months (2017-2019) on a two-site farrow-to-finish farm (about 1,000 sows), in Northern Italy. A total of 67/281 test faecal pools (23.8%) resulted positive for the presence of HEV-RNA (site 1: 59/221, 26.7%; site 2: 8/60, 13.3%). Nucleotide sequencing revealed a unique HEV-3 viral variant circulating during 19 months of surveillance. The same HEV-3 strain was detected in the same farm on 2012, indicating the persistence of the same virus over 7 years, and highlighting the role of the environment as a continuous source of infection on pig farms. The results confirmed the circulation of the zoonotic genotype HEV-3 in pigs before slaughtering.

Pilot Investigation on the Presence of Anti-Hepatitis E Virus (HEV) Antibodies in Piglet Processing Fluids.

Identifying Hepatitis E virus (HEV)-positive pig farms is important to implement surveillance programs for this emerging zoonotic agent. The aim of this study was to evaluate the use of serosanguineous fluids obtained as part of castration practice (processing fluids (PFs)) to detect anti-HEV antibodies in newborn piglets. Ninety-five paired serum and PF samples were collected from piglets of 29 different litters and tested with a commercial ELISA kit. A significant positive correlation (Spearman's rho: 0.600; p < 0.01) was found between anti-HEV antibodies in serum and PF samples. In 26 out of 29 litters (89.7%), there was at least one positive piglet in the serum. Sixteen litters out of 29 (55.2%) were also positive in PFs. To simulate the use of PF as pooled samples, the limit of detection of the ELISA was assessed mixing the PF sample with strong, medium, medium-weak and weak ELISA titres with 3, 4, 5 and 6 negative PF samples. Our results suggest that it is still possible to identify a positive PF pool when at least one individual PF sample with medium or strong antibody levels is mixed with 5 or 6 individual negative PF samples. The detection of anti-HEV maternal-derived antibodies in PF confirms a past exposure of sows to the virus. PF may represent a rapid, noninvasive and economical tool to identify HEV-positive farms.

Detection and Characterization of Porcine Sapelovirus in Italian Pig Farms.

Porcine sapelovirus (PSV) belongs to the genus Sapelovirus of the family Picornaviridae. PSV infects pigs asymptomatically, but it can also cause severe neurologic, enteric, and respiratory symptoms or reproductive failure. Sapelovirus infections have been reported worldwide in pigs. The objective of this study was to investigate the presence and the prevalence of PSV in Italian swine farms in animals of different ages to clarify the occurrence of the infection and the genetic characteristics of circulating strains. In the present study, 92 pools of fecal samples, collected from pigs across three farms, were analyzed by Reverse Transcriptase-polymerase Chain Reaction-PCR (RT-PCR). Fecal pools from young growers (63/64) were found positive for Sapelovirus in all farms while detection in sows (4/28) was observed in only one farm. Phylogenetic analyses of the 19 partial capsid protein nucleotide sequences (VP1) (6-7 each farm) enable the classification of the virus sequences into three distinct clades and highlighted the high heterogeneity within one farm. The whole genome sequence obtained from one strain showed the highest correlation with the Italian strain detected in 2015. The study adds novel information about the circulation and heterogeneity of PSV strains in Italy and considering the movement of pigs across Europe would also be informative for other countries.

Molecular Characterization of HEV Genotype 3 in Italy at Human/Animal Interface.

Hepatitis E virus (HEV) is an emerging public health issue in industrialized countries. In the last decade the number of autochthonous human infections has increased in Europe. Genotype 3 (HEV-3) is typically zoonotic, being foodborne the main route of transmission to humans, and is the most frequently detected in Europe in both humans and animals (mainly pigs and wild boars). In Italy, the first autochthonous human case was reported in 1999; since then, HEV-3 has been widely detected in both humans and animals. Despite the zoonotic characteristic of HEV-3 is well established, the correlation between animal and human strains has been poorly investigated in Italy. In the present study, we compared the subtype distribution of HEV-3 in humans and animals (swine and wild boar) in the period 2000-2018 from Italy. The dataset for this analysis included a total of 96 Italian ORF2 sequences (300 nt long), including both NCBI database-derived (n = 64) and recent sequences (2016-2018, n = 32) obtained in this study. The results show that subtype 3f is the most frequent in humans and pigs, followed by the HEV-3e, HEV-3c and other unassignable HEV-3 strains. Diversely, in wild boar a wider group of HEV-3 subtypes have been detected, including HEV-3a, which has also been detected for the first time in a human patient in Central Italy in 2017, and a wide group of unassignable HEV-3 strains. The phylogenetic analysis including, besides Italian strains, also sequences from other countries retrieved from the NCBI database, indicated that human Italian sequences, in particular those of HEV-3f and HEV-3e, form significant clusters mainly with sequences of animal origin from the same country. Nevertheless, for HEV-3c, rarely detected in Italian pigs, human sequences from Italy are more correlated to human sequences from other European countries. Furthermore, clusters of near-identical human strains identified in a short time interval in Lazio Region (Central Italy) can be recognized in the phylogenetic tree, suggesting that multiple infections originating from a common source have occurred, and confirming the importance of sequencing support to HEV surveillance.