Waterborne Norovirus outbreak at a seaside resort likely originating from municipal water distribution system failure.

In May 2016 a Norovirus (NoV) gastroenteritis outbreak involved a high school class visiting a seaside resort near Taormina (Mascali, Sicily). Twenty-four students and a teacher were affected and 17 of them showed symptoms on the second day of the journey, while the others got ill within the following 2 days. Symptoms included vomiting, diarrhoea and fever, and 12 students required hospitalisation. Stool samples tested positive for NoV genome by Real-Time polymerase chain reaction assay in all 25 symptomatic subjects. The GII.P2/GII.2 NoV genotype was linked to the outbreak by ORF1/ORF2 sequence analysis. The epidemiological features of the outbreak were consistent with food/waterborne followed by person-to-person and/or vomit transmission. Food consumed at a shared lunch on the first day of the trip was associated to illness and drinking un-bottled tap water was also considered as a risk factor. The analysis of water samples revealed the presence of bacterial indicators of faecal contamination in the water used in the resort as well as in other areas of the municipal water network, linking the NoV gastroenteritis outbreak to tap water pollution from sewage leakage. From a single water sample, an amplicon whose sequence corresponded to the capsid genotype recovered from patients could be obtained.

Detection of Human Bocavirus Species 2 and 3 in Bivalve Shellfish in Italy.

Human bocavirus (HBoV) has been shown to be a common cause of respiratory infections and gastroenteritis in children. Recently, HBoVs have been detected in sewage and river waters in Italy and worldwide. However, studies on their presence in other water environments and in bivalve mollusks are not yet available. In this study, 316 bivalve shellfish samples collected in three Italian regions over a 6-year period (2012 to 2017) were analyzed by nested PCR and sequencing using broad-range primer pairs targeting the capsid proteins VP1 and VP2 of HBoV. The virus was detected in 27 samples (8.5% of the total samples), and a statistically significant difference was found within the three regions. A further 13 samples, collected in geographic and temporal proximity to positive samples, were included in the study to assess the spread of HBoV in shellfish production areas at the time of contamination. Twelve of these additional samples were found to be positive for HBoV. All positive samples in this study were characterized as HBoV species 2 (17 samples; 8 different sequences) or species 3 (22 samples; 4 different sequences). This study reports the occurrence of HBoV in bivalve shellfish and shows evidence of considerable spatial spread of the virus throughout shellfish production areas. Further studies are needed to elucidate both the role of HBoV as an agent of gastroenteritis and the risk for foodborne transmission of this virus.IMPORTANCE Human bocavirus is recognized as an important cause of acute respiratory tract infections and has recently been considered an etiological agent of gastroenteritis in the pediatric population. Our findings document that HBoVs are detected in bivalve shellfish with a relevant prevalence and suggest that an assessment of the risk for foodborne transmission of these viruses should be undertaken.

Detection and molecular characterization of betanodaviruses retrieved from bivalve molluscs.

Betanodaviruses are small ssRNA viruses responsible for viral encephalopathy and retinopathy, otherwise known as viral nervous necrosis, in marine fish worldwide. These viruses can be either horizontally or vertically transmitted and have been sporadically detected in invertebrates, which seem to be one of the possible viral sources. Twenty-eight new betanodavirus strains were retrieved in three molluscs species collected from different European countries between 2008 and 2015. The phylogenetic analyses revealed that strains retrieved from bivalve molluscs are closely related to viruses detected in finfish in Southern Europe in the period 2000-2009. Nevertheless, a new betanodavirus strain, markedly different from the other members of the RGNNV genotype, was detected. Such a massive and varied presence of betanodaviruses in bivalve molluscs greatly stresses the risks of transmission previously feared for other invertebrates. Bivalve molluscs reared in the same area as farmed and wild finfish could act as a reservoir of the virus. Furthermore, current European regulations allow relaying activities and the sale of live bivalve molluscs, which could pose a real risk of spreading betanodaviruses across different geographic regions. To our knowledge, this is the first study, which focuses on the detection and genetic characterization of betanodaviruses in bivalve molluscs.

Introduction of Asian canine parvovirus in Europe through dog importation.

Canine parvovirus (CPV) is an important infectious agent of domestic and wild carnivores, responsible for severe and often fatal haemorrhagic gastroenteritis and leukopenia. This paper reports the genomic characterization of a CPV strain collected from a dog recently imported to Italy from Thailand. The virus was detected in all tissue samples collected. The whole genome encompassing the two open reading frames encoding for non-structural (NS1/NS2) and structural (VP1/VP2) proteins was amplified and sequenced. On the basis of genetic analysis of the VP2 gene, the isolate was characterized as CPV-2c, but it presented genetic signatures typical of Asian strains. Sequence analysis revealed the presence of amino acid changes never observed in European CPV-2c strains (NS1: Ile60Val, Tyr544Phe, Glu545Val, Leu630Pro; VP2: Ala5Gly, Phe267Tyr, Tyr324Ile, Gln370Arg). By phylogenetic analysis of full-length VP2 gene, the analysed strain clustered together with Asian viruses. Therefore, a possible introduction of the virus from Asia through the imported dog was suggested, thus confirming the important role of movement of dogs in the global spread of viruses. In addition, full-length genome analysis could help better trace the spread of canine viruses through different continents.

Differentiation and characterization of rat adipose tissue mesenchymal stem cells into endothelial-like cells.

In this study, mesenchymal stem cells were isolated from rat adipose tissue (AD-MSCs) to characterize and differentiate them into endothelial-like cells. AD-MSCs were isolated by mechanical and enzymatic treatments, and their identity was verified by colony-forming units (CFU) test and by differentiation into cells of mesodermal lineages. The endothelial differentiation was induced by plating another aliquot of cells in EGM-2 medium, enriched with specific endothelial growth factors. Five subcultures were performed. The expression of stemness genes (OCT4, SOX2 and NANOG) was investigated. The presence of CD90 and the absence of the CD45 were evaluated by flow cytometry. The endothelial-like cells were characterized by the evaluation of morphological changes and gene expression analysis for endothelial markers (CD31, CD144, CD146). Characterization of AD-MSCs showed their ability to form clones, to differentiate in vitro and the OCT-4, SOX-2, NANOG genes expression. Immunophenotypic characterization showed the CD90 presence and the CD45 absence. The endothelial-like cells showed morphological changes, the expression of CD31, CD144, CD146 genes and the presence of CD31 membrane receptor. Matrigel assay showed their ability to form network and vessels-like structures. This study lays the foundations for future evaluation of the potential AD-MSCs pro-angiogenic and therapeutic role.

Circulation of multiple subtypes of bovine viral diarrhoea virus type 1 with no evidence for HoBi-like pestivirus in cattle herds of southern Italy.

Pestiviruses of cattle include bovine viral diarrhoea 1 (BVDV-1) and 2 (BVDV-2) plus an emerging group, named HoBi-like pestivirus. In the present paper, the results of an epidemiological survey for pestiviruses circulating in cattle in southern Italy are presented. Molecular assays carried out on a total of 924 bovine samples detected 74 BVDV strains, including 73 BVDV-1 and 1 BVDV-2 viruses. Phylogenetic analysis carried out on partial 5'UTR and Npro sequences revealed the presence of 6 different subtypes of BVDV-1 and a single BVDV-2c strain. BVDV-1 displayed a high level of genetic heterogeneity, which can have both prophylactic and diagnostic implications. In addition, the detection of BVDV-2c highlights the need for a continuous surveillance for the emergence of new pestivirus strains in cattle farms in southern Italy.

Evidence for Tunisian-Like Pestiviruses Presence in Small Ruminants in Italy Since 2007.

The genus Pestivirus, which belongs to the Flaviviridae family, includes ssRNA+ viruses responsible for infectious diseases in pigs, cattle, sheep, goats and other domestic and wild ruminants. Like most of the RNA viruses, pestivirus has high genome variability with practical consequences on disease epidemiology, diagnosis and control. In addition to the officially recognized species in the genus Pestivirus, such as BVDV-1, BVDV-2, BDV and CSFV, other pestiviruses have been detected. Furthermore, most of the ruminant pestiviruses show low or absent species specificity observed in serological tests and are able to infect multiple species. Particularly, small ruminants are receptive hosts of the most heterogeneous group of pestiviruses. The aim of this study was to carry out the molecular characterization of pestiviruses isolated from sheep and goats in Sicily, Italy. Phylogenetic analysis of two viral genomic regions (a fragment of 5'-UTR and the whole Npro regions) revealed the presence of different pestivirus genotypes in the analysed goat and sheep herds. Two of five viral isolates were clustered with BVDV-1d viruses, a strain widespread in Italy, but never reported in Sicily. The other three isolates formed a distinct cluster with high similarity to Tunisian isolates, recently proposed as a new pestivirus species. This represents the first evidence for Tunisian-like pestivirus presence in small ruminants in Italy. Furthermore, one of the isolates was collected from a goat, representing the first isolation of Tunisian-like pestivirus from this species.

Hepatitis A and E Viruses in Wastewaters, in River Waters, and in Bivalve Molluscs in Italy.

Several studies have reported the detection of hepatitis A (HAV) and E (HEV) virus in sewage waters, indicating a possibility of contamination of aquatic environments. The objective of the present study was to assess the occurrence of HAV and HEV in different water environments, following the route of contamination from raw sewage through treated effluent to the surface waters receiving wastewater discharges . Bivalve molluscan shellfish samples were also analyzed, as sentinel of marine pollution. Samples were tested by RT-PCR nested type in the VP1/2A junction for HAV, and in the ORF1 and ORF2 regions for HEV. Hepatitis A RNA was detected in 12 water samples: 7/21 (33.3%) raw sewage samples, 3/21 (14.3%) treated sewage samples, and 2/27 (7.4%) river water samples. Five sequences were classified as genotype IA, while the remaining 7 sequences belonged to genotype IB. In bivalves, HAV was detected in 13/56 samples (23.2%), 12 genotype IB and one genotype IA. Whether the presence of HAV in the matrices tested indicates the potential for waterborne and foodborne transmission is unknown, since infectivity of the virus was not demonstrated. HEV was detected in one raw sewage sample and in one river sample, both belonging to genotype 3. Sequences were similar to sequences detected previously in Italy in patients with autochthonous HEV (no travel history) and in animals (swine). To our knowledge, this is the first detection of HEV in river waters in Italy, suggesting that surface water can be a potential source for exposure .

Pandemic influenza A/H1N1 virus in a swine farm house in Sicily, Italy.

This report describes a pandemic A/H1N1 (H1N1 pdm) virus outbreak occurred in December, 2009 in a swine farm used as research facility (Istituto Mediterraneo Trapianti e Terapie ad Alta Specializzazione) for preclinical studies, located in Sicily, Italy. All the 13 pigs of the farm, showed cough, fever, inappetence and weakness. At the same time, an unvaccinated worker of the stabling showed influenza-like symptoms. RNAv extracted from two swabs collected from infected pigs resulted positive by Real Time RT-PCR for Influenza A virus. Furthermore, after growth on embryonated eggs, viral isolates were identified by Real Time RT-PCR specific for H1N1 pdm virus and characterized antigenically. Sequencing of the whole genome was also performed. All sera taken from animals and from the worker were tested by a competitive influenza A ELISA and by the haemoagglutination inhibition test. Serological findings confirmed the circulation of influenza virus H1N1 pdm in pigs and the presence of specific antibodies against H1N1 pdm in human serum. The results of this study seem to support a H1N1 pdm transmission from man to animals showing the importance of serological and virological investigation to control the pig farms and the importance of close cooperation between the different authorities like veterinarian and human public.

Ovine catarrhal fever (Bluetongue): analysis of Culicoides species in seropositive farms.

Bluetongue (BT) is an orbiviral disease of wild and domestic ruminants, mainly sheep. In Sicily, the first Bluetongue outbreak occurred in October 2000; there have been 76 recorded outbreaks so far. The National Surveillance Plan, based on European Union Commission Decision 138/2001/CE, establishes serological and entomological surveys. This plan consists of controls of seronegative cattle, called 'sentry' as indicators for the presence and circulation of virus in defined areas. To check the seroconversions, the regional territory has been subdivided in 400 km(2) areas including 58 seronegative cattle, periodically checked by serological tests. All positive sera have been tested to detect the specific serotype by the National Reference Centre for Exotic Diseases (CESME) at the Istituto Zooprofilattico Sperimentale Abruzzo e Molise in Teramo (IZS Teramo). Moreover, entomological surveillance has been implemented in seropositive herds, to investigate the presence of insect vectors belonging to Culicoides genus. The goal of the present communication is to report on the different species of Culicoides found in the farms with Bluetongue virus and to investigate on the probable role of new competent vectors. This paper concerns data analysis of 581 light-trap catches collected in 321 farms from 2003 to 2008. We observed that 82% of checked farms were positive for Culicoides spp., and only 10% of the farms were positive for Culicoides imicola.

Identification of a novel bluetongue virus vector species of Culicoides in Sicily.

The vectors of bluetongue virus are certain species of Culicoides biting midges, and in the Mediterranean area Culicoides imicola has long been considered to be the only field vector. In Sicily an entomological and serological surveillance programme has been in operation since the autumn of 2000, which has shown that the prevalence and abundance of C. imicola is lower than in many other Italian regions. Moreover, in 2002, there were outbreaks of bluetongue in the absence of C. imicola, and in these regions bluetongue viral RNA was detected by means of a nested reverse-transcriptase PCR in wild-caught, non-blood-engorged, parous Culicoides pulicaris. Furthermore, bluetongue virus serotype 2 was isolated on five occasions from extracts of non-blood-engorged parous C. pulicaris by using embryonated hens eggs and BHK-21 cells as assay systems. These findings suggest that in parts of Italy and possibly in other areas of Europe, where C. imicola is absent or rare, C. pulicaris may act as a fully competent vector of bluetongue virus.