Detection of serum antibodies to hepatitis E virus in domestic pigs in Italy using a recombinant swine HEV capsid protein

Background: The hepatitis E virus (HEV) has been detected in both humans and animals, particularly pigs, worldwide. Several evidences, including human infection following consumption of raw contaminated meat, suggest a zoonotic transmission of HEV. In Italy, large circulation of genotype 3 HEV has been reported in swine, and recent studies have confirmed the involvement of this genotype in autochthonous human cases. Result: In this study 111 sera collected from healthy pigs in two Italian regions were tested for anti-HEV IgG antibodies. For specific HEV antibody detection in swine, we developed ELISA and Western blotting methods, using a truncated capsid (ORF2) protein lacking the first 111 amino acids of a swine HEV genotype 3 strain. The ORF2-based ELISA revealed anti-HEV antibodies in 104 out of 111 pigs compared with 102 detected with a commercial ELISA kit. A lower number of sera reacted with the recombinant ORF2 protein in a Western blotting format (81/111). Using a Latent class analysis (LCA), the estimated sensitivities for ELISA-ORF2 and ELISA-kit tests were 0.961 and 0.936, respectively, whereas specificities were 0.599 and 0.475. The estimated sensitivity of Western blotting was 0.775, and the specificity was 0.944. Conclusions: The overall results confirm the high prevalence of HEV seropositive healthy pigs in Italy. Through comparisons with a commercial ELISA test, the swine genotype 3 HEV antigen produced in this study was proven suitable to detect anti-HEV antibodies in pig sera by both ELISA and Western Blotting

Looking for the Original Materials and Evidence of Restoration at the Vault of the San Panfilo Church in Tornimparte (AQ)

This paper reports the investigation of six microsamples collected from the vault of the San Panfilo Church in Tornimparte (AQ). The aim was to detect the composition of the pigments and protective/varnishes, and to investigate the executive technique, the conservation state, and the evidence of the restoration works carried out in the past. Six microsamples were analyzed by optical microscopy, scanning electron microscopy coupled with energy-dispersive spectroscopy (EDS), X-ray fluorescence (XRF), and infrared and Raman spectroscopy. The investigations were carried out within the framework of the Tornimparte project "Archeometric investigation of the pictorial cycle of Saturnino Gatti in Tornimparte (AQ, Italy)" sponsored in 2021 by the Italian Association of Archeometry (AIAr)

Laser Controlled Synthesis of Noble Metal Nanoparticle Arrays for Low Concentration Molecule Recognition

Nanostructured gold and silver thin films were grown by pulsed laser deposition. Performing the process in an ambient gas (Ar) leads to the nucleation and growth of nanoparticles in the ablation plasma and their self-organization on the substrate. The dependence of surface nanostructuring of the films on the deposition parameters is discussed considering in particular the number of laser pulses and the ambient gas nature and pressure. The performance of the deposited thin films as substrates for surface-enhanced Raman spectroscopy (SERS) was tested against the detection of molecules at a low concentration. Taking Raman maps on micrometer-sized areas, the spatial homogeneity of the substrates with respect to the SERS signal was tested

Non-Invasive Investigation of Pigments of Wall Painting in S. Maria Delle Palate di Tusa (Messina, Italy)

The characterization of materials used in the archaeological field needs an experimental approach in order to avoid the destruction or perturbation of artworks. In order to afford this purpose, a multi-analytical spectroscopic approach is regularly used. We combined non-invasive analysis by using handheld spectroscopic instrumentations (mainly XRF and Raman spectrometers) in order to characterize the wall painting preserved in the church of S. Maria delle Palate at Halaesa Arconidea archeological site (Tusa, Messina, Italy). The aim of the work is the characterization of the nature of pigments used for the realization of the wall painting. The wall painting, probably representing St. Francis in the act of receiving the stigmata, has been subject to cleaning and restoration. Thanks to use of in situ measurement, we have identified hematite and goethite for the red and yellow respectively, and lazurite for the blue. In addition, some relevant information about the black pigment, the technique used for the realization and the conservation state were also obtained. The results obtained during the diagnostic campaign have been a support for the work of restorers. For the first time, the wall painting has been studied, increasing the knowledge of Halaesa Arconidea archaeological site

Viral and antibody HEV prevalence in swine at slaughterhouse in Italy

Hepatitis E is an acute disease of humans caused by a small RNA virus, Hepatitis E virus (HEV). In recent years, an increasing number of autochthonous human infections have been reported in industrialized countries. Genotype 3 is the main HEV type circulating in swine, and is also reported in sporadic cases of hepatitis E in humans worldwide. To date one serotype has been described. We have conducted a survey to detect antibodies against HEV in 48 swine at a slaughterhouse in Northern Italy, using ELISA test. Mean seroprevalence in the studied animal group was 87.0%. Bile, liver and feces from the 48 animals were also collected, and HEV RNA was detected by nested reverse transcription-polymerase chain reaction, amplifying a fragment of the ORF2. HEV genome was most frequently detected in bile samples (51.1%), followed by feces (33.3%) and liver (20.8%). Thirty-one out of 48 studied pigs (64.6%) were positive for HEV RNA in at least one sample. Overall, HEV RNA was found at a statistically higher rate in the 3\u20134-month-old than in 9\u201310-month-old animals (95.0% vs. 42.9%). Genetic characterization of swine strains identified was performed by sequencing and database alignment. Phylogenetic analysis on the nucleotide sequences from 14 positive PCR products indicated that all strains belonged to genotype 3, clustering in two branches subtypes g3c and g3f

Identificazione di ceppi di Sapovirus e Norovirus in suini asintomatici in allevamenti dell’Emilia Romagna

Norovirus e sapovirus sono due membri della famiglia Caliciviridae. Essi presentano un genoma a RNA monocatenario e sebbene l'organizzazione genomica vari tra i due generi, entrambi codificano per le proteine non strutturali (inclusa la RNA polimerasi RNA dipendente), per la proteina capsidica principale e per una proteina le cui funzioni restano sconosciute. I NoV e SaV sono la seconda causa di ricovero ospedaliero per gastroenterite (GE) pediatrica e i NoV da soli sono responsabili di oltre il 50% delle epidemie di GE nell'adulto. NoV e SaV infettano anche animali domestici e da reddito, e in particolare suini e bovini sono potenziali serbatoi di infezione per l'uomo. Durante il primo semestre del 2006 e del 2008, 201 campioni fecali sono stati prelevati da altrettanti suini sani provenienti da 23 allevamenti localizzati nella regione Emilia-Romagna. I campioni fecali sono stati analizzati per la ricerca di norovirus e sapovirus mediante un saggio di trascrizione inversa e PCR utilizzando la coppia di primer p289-p290, amplificando una regione conservata all'interno dell'RNA polimerasi RNA dipendente (RdRP)). Il metodo permette di individuare sia ceppi di sapovirus (PEC) che di norovirus suini. L'RNA proveniente da tredici campioni fecali suini è risultato positivo al test di RT-PCR, mostrando una banda di DNA delle dimensioni attese (≈319bp per norovirus e 319bp per PEC). Per confermare i risultati ottenuti e caratterizzare i ceppi coinvolti, sei campioni positivi sono stati sequenziati. L'analisi delle sequenze ottenute ha confermato che cinque ceppi appartenevno al genere sapovirus e hanno mostrato un'identità di sequenza nucleotidica tra il 79% e l'85% con altri ceppi suini descritti in Europa. Uno dei ceppi sequenziati è invece risultato appartenere ai norovirus, in particolare al tipo GII.18 che sebbene appartenente allo stesso genogruppo dei ceppi umani di norovirus è un genotipo comune, ad oggi, per i soli ceppi suini. Lo studio condotto, se pur preliminare, ha mostrato una prevalenza del 13% (tra NoV e SaV) in suini sani. Nel suino sembra più comune la presenza di sapovirus. L'analisi di sequenza ha mostrato che i ceppi di PEC e NoV individuati sono correlati e vicini geneticamente ai ceppi umani. Sebbene non dimostrata, la possibile origine zoonotica non può essere esclusa. Ulteriori studi saranno condotti per caratterizzare i ceppi identificati, consentendo di avere un quadro filogenetico più completo

Antigenicity of a recombinant capsid protein from an Italian swine HEV strain

Hepatitis E virus (HEV) is a single-stranded, positive-sense RNA virus. The genome contains three ORFs, of which ORF2 encodes the major structural capsid protein PORF2. HEV is responsible for sporadic infections as well as large epidemics of acute viral hepatitis in developing countries, and is emerging in industrialized areas where it accounts for an increasing number of sporadic cases. Recent observations of indigenous cases in developed regions suggest that pigs may act as a zoonotic source and reservoir of infection. This hypothesis is also supported by the high genetic similarity between human and swine HEV strains circulating in the same geographical areas. In 2006, we investigated the presence of HEV in swine farms in Northern Italy. The results of molecular diagnosis indicated a wide presence of genotype 3 HEV strains, the genotype predominating in pigs in industrialized areas. We expressed a 111-aa deletion fragment of the capsid protein PORF2 of an Italian swine HEV strain in a recombinant baculovirus system using Sf9 insect cells. The 55 kDa recombinant PORF2 did not self-assemble as virus-like particles in insect cells, but was produced at high levels in the cytoplasm and supernatant of cells, as demonstrated by immunocytochemical (ICC) staining and by Western blotting (WB) with immune animal sera. The PORF2 protein was used to produce hyperimmune sera and a panel of monoclonal antibodies (MAbs), which were selected by ELISA. The results showed that the expressed HEV protein is immunogenic and is specifically recognized by MAbs using different assay, including WB and ICC. Further study are being conducted to characterize the MAbs and PORF2

Detection of Hepatitis E virus in pork liver sausages

Hepatitis E is an infectious disease caused by a small RNA virus (Hepatitis E Virus, HEV), which can cause acute hepatitis in humans. Four mammalian HEV genotypes are known, among which g3 and g4 are considered zoonotic. In Italy, g3 strains have been detected in swine, in both farms and slaughterhouses. Food-borne transmission of HEV was reported frequently in sporadic and clustered human cases associate to consumption of raw meat or liver of boar, swine and deer. In this study, we investigated the presence of HEV in liver sausage, which is often consumed raw or undercooked in Italy. Methods: In 2012, 8 packages of pork liver both fresh and dry sausages were bought at a butcher shop in Rome. Each sausages (n\ub0 29) were chopped in three slices (250 mg each, obtaining of 45 and 42 slices for fresh and dry liver sausages) and spiked with Murine Norovirus (MNV-1). After RNA extraction, samples were analyzed by RT-qPCR for MNV detection (extraction process control). Samples confirmed positive for MNV were then analyzed for HEV and Porcine Adenovirus (PAdV, as index virus of swine fecal contamination) by RT-qPCR and Real-Time PCR, respectively. No process control signal was obtained from 19 of 42 dry liver sausages tested. Conversely, the HEV genome was detected in both raw (10 out of 45 slices, 22.2%) and dry (1/23 slices, 4.3%) liver sausages by RTqPCR. RNAs resulting positive for the process control MNV-1 were further analyzed by conventional RT-PCR and PCR assays for HEV and PAdV, respectively. All slices were negative for PAdV, suggesting the absence of swine fecal contamination. On the contrary, three out of 11 RT-qPCR HEV-positive slices of sausages were also positive by at least one of the two conventional RT-PCR performed, which amplify the MTase and RdRp. Sequences were correlated to genotype 3 HEV strains detected in swine in Italy. Since different sequenced regions are currently available from human and swine Italian strains, few conclusions can be drawn on possible correlation of the HEV from sausage characterized with human strains. Although HEV infectivity was not confirmed, this pilot study fosters more investigations on HEV presence in pork-derived food, to assess the possible risk for the consumers

Cloning and expression of a swine Hepatitis E virus capsid protein in insect cells

Hepatitis E virus (HEV) is the causative agent of hepatitis E, and is a positive sense single stranded RNAvirus. The HEV genome is approximately 7.2 kb and contains three open reading frames. ORF2 encodes the major capsid protein. HEV causes large epidemics in developing countries, and is emerging in inchstrialized areas, which accounts for an increasing number of sporadic cases. Genetic similarity between human and swine HEV strains from the same area suggests zoonotic transmission, which might play an important role in spreading infection in Europe and other industrialized areas. Recently, hepatitis E cases have been linked to eating raw or undercooked meat from deer, wild boar or pigs. In 2006, we evaluated the presenceof HEV in swine farms in Northern Italy. The results of molecular diagnosis indicated a wide presence of genotype 3 HEV strains, predominating in pigs worldwide. Viral RNAs from positive samples were used to obtain full-length ORF2 fragments. Adeletion fragment lacking the first 111 aa at the N-terminal portion of the capsid protein was cloned in the baculovirus system. The sequence of the recombinant construct confirmed the proper frame of ORF2 and the genotype the Italian strain, presenting an 85% identity with the genotype 3 prototype swine HEV strain described by Meng et al. in the US in 1997. The bacmide with the HEVA1110RF2 was transfected into Sf9 and High Tn5 insect cells, and the BacHEVA lllORF2virus stock obtained was used to express the HEV capsid protein. Similar protein expression was obtained in Sf9 and Tn5 cells under serum-free culture conditions. One protein band of 55 kDa was detected by western blotting using a pig serum in either cell lysate and supernatant. Self-assembly of capsid protein into virus-like particles was investigated by electron microscopy

Identificazione del virus dell'HEV in suini al macello

L'epatite E costituisce un grave problema di Sanit\ue0 Pubblica in molti Paesi in via di sviluppo e una possibile emergenza nei Paesi industrializzati, dove vengono descritti un numero crescente di casi sporadici. L'agente eziologico (Hepatitis E Virus, HEV) \ue8 un RNA virus privo di envelope, e classificato nel genere hepevirus. Il virus causa vaste epidemie nei Paesi in via di sviluppo, dove la trasmissione \ue8 legata alle scarse condizioni igieniche ed \ue8 veicolato dal consumo di acqua contaminata. Nei Paesi industrializzati, i casi clinici di epatite E sono prevalentemente sporadici e sono in crescita i casi autoctoni in soggetti senza anamnesi di viaggio all'estero. Alcuni di questi risultano in relazione con il contatto diretto o indiretto con suini, cinghiali o cervi infetti. Attualmente, infatti, HEV \ue8 considerato un agente di zoonosi emergente e il suino \ue8 ritenuto un possibile serbatoio del virus. In Giappone, casi di epatite E sono stati associati all'ingestione di carne o organi crudi di suino, cinghiale e cervo. Recentemente la presenza del virus \ue8 stata rilevata anche in fegati al commercio. In Europa \ue8 stata dimostrata la presenza e la diffusione dell'infezione in allevamenti suini in numerosi Paesi, compresa l'Italia. Il presente lavoro ha avuto come obiettivo la valutazione della presenza di HEV in suini adulti al momento della macellazione. A tale scopo, sono stati raccolti campioni di feci, bile e sangue da 48 animali macellati in uno stabilimento del Nord Italia, e giudicati sani sia pre-mortem sia all'esame ispettivo. La presenza di RNA virale di HEV nei diversi campioni \ue8 stata valutata mediante un saggio di ReverseRiverse-Transcription-PCR (RT-PCR) utilizzando i primer A1-S1 che amplificano una regione conservata all'interno della regione capsidica. Il 47% degli animali \ue8 risultato positivo al test molecolare, e per 18 animali sono risultati positivi i campioni fecali e in sei casi i campioni di bile. Per confermare i risultati ottenuti, i campioni positivi sono stati sottoposti a sequenziamento nucleotidico e l'analisi delle sequenze ottenute ha confermato la presenza di genoma di HEV, in particolare corrispondente a ceppi appartenenti al genotipo G3, gi\ue0 identificato nel suino e nell'uomo nei Paesi sviluppati