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We isolated Issyk-Kul virus (ISKV) from a bat sampled from Italy in 2021 and conducted ISKV-specific surveillance in bats collected in Italy during 2017-2023. ISKV circulation among synanthropic and sedentary species of bat, such as Savi's pipistrelle bat (Hypsugo savii) in northern Italy, may have public health implications in this region. [ABSTRACT FROM AUTHOR]

Background: Bats are recognized as reservoir species for multiple viruses. However, little is known on bats' health and mortality. Thus, this study aimed to investigate the main causes of death of bats from Turin province (North-western Italy) and to describe gross and histopathological lesions potentially associated with the presence of selected bat viruses. Results: A total of 71 bats belonging to 9 different species of the families Vespertilionidae and Molossidae were necropsied and samples of the main organs were submitted to histopathological examination. Also, aliquots of the small intestine, liver, spleen, lung, and brain were collected and submitted to biomolecular investigation for the identification of Coronaviridae, Poxviridae, Reoviridae (Mammalian orthoreovirus species), Rhabdoviridae (Vaprio ledantevirus and Lyssavirus species) and Kobuvirus. The majority of bats died from traumatic lesions due to unknown trauma or predation (n = 40/71, 56.3%), followed by emaciation (n = 13/71,18.3%). The main observed gross lesions were patagium and skin lesions (n = 23/71, 32.4%), forelimbs fractures (n = 15/71, 21.1%) and gastric distension (n = 10/71,14.1%). Histologically, the main lesions consisted of lymphoplasmacytic pneumonia (n = 24/71, 33.8%), skin/patagium dermatitis (n = 23/71, 32.4%), liver steatosis and hepatitis (n = 12, 16.9%), and white pulp depletion in the spleen (n = 7/71, 9.8%). Regarding emergent bat viruses, only poxvirus (n = 2, 2.8%) and orthoreovirus (n = 12/71, 16.9%) were detected in a low percentage of bats. Conclusions: Trauma is the main lesion observed in bats collected in Turin province (North-western Italy) associated with forelimb fractures and the detected viral positivity rate seems to suggest that they did not represent a threat for human health. [ABSTRACT FROM AUTHOR]

Simple Summary: This article discusses the isolation of respirovirus 3 from a sample of femoral bone marrow from a wild boar carcass imported from Australia. The complete genome sequence was determined, and based on our results, the wild boar isolate may result from cross-species infection of wild boars with BRV3. The application of NGS techniques has allowed us to investigate and characterise the genome of this strain, which was initially isolated in 2004. Paramyxoviruses are important pathogens affecting various animals, including humans. In this study, we identified a paramyxovirus in 2004 (180608_2004), isolated from a sample of the femoral marrow bone of a wild boar carcass imported from Australia. Antigenic and morphological characteristics indicated that this virus was similar to members of the family Paramyxoviridae. The complete genome phylogenetic analysis grouped this virus into genotype A of bovine parainfluenza virus type 3 (BPIV-3), recently renamed bovine respirovirus type 3 (BRV3), which also includes two swine paramyxoviruses (SPMV)—Texas-81 and ISU-92—isolated from encephalitic pigs in the United States in 1982 and 1992, respectively. The wild boar 180608_2004 strain was more closely related to both the BRV3 shipping fever (SF) strain and the SPMV Texas-81 strain at the nucleotide and amino acid levels than the SPMV ISU-92 strain. The high sequence identity to BRV3 suggested that this virus can be transferred from cattle to wild boars. The potential for cross-species transmission in the Respirovirus genus makes it essential for intensified genomic surveillance. [ABSTRACT FROM AUTHOR]

Serosurveillance among animals, including pets, plays an important role in the current coronavirus disease 2019 (COVID‐19) pandemic, because severe acute respiratory coronavirus 2 (SARS‐CoV‐2) infections in animal populations could result in the establishment of new virus reservoirs. Serological assays that offer the required sensitivity and specificity are essential. In this study, we evaluated the diagnostic performance of three different commercially available immunoassays for the detection of SARS‐CoV‐2 antibodies in pets, namely two ELISA tests for the detection of antibodies against SARS‐CoV‐2 nucleocapsid [ID Screen SARS CoV‐2 double antigen multispecies (Double antigen) and ID Screen® SARS‐CoV‐2‐N IgG indirect ELISA (Indirect)] and one test for the detection of neutralizing antibodies against SARS‐CoV‐2 receptor‐binding‐domain [surrogate virus neutralization test (sVNT)]. The obtained results were compared with those of conventional virus neutralization test (VNT), which was regarded as reference method. A total of 191 serum samples were analysed. Thirteen (6.8%) samples showed VNT‐positive results. The overall sensitivity was higher for sVNT (100%) compared to nucleocapsid‐based ELISA assays (23% for Double antigen and 60% for Indirect). The specificity was 100% for Indirect ELISA and sVNT, when a higher cut‐off (>30%) was used compared to the one previously defined by the manufacturer (>20%), whereas the other test showed lower value (99%). The sVNT test showed the highest accuracy and agreement with VNT, with a perfect agreement when the higher cut‐off was applied. The agreement between each nucleocapsid‐based ELISA test and VNT was 96% for Indirect and 94% for Double antigen. Our findings showed that some commercially available serological tests may lead to a high rate of false‐negative results, highlighting the importance of assays validation for the detection of SARS‐CoV‐2 antibodies in domestic animals. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

In this study, virological surveillance focused on coronaviruses in marmots in the Alpine region in 2022, captured as part of a population control reduction program in the Livigno area. Seventy-six faecal samples were randomly collected from marmots at the time of capture and release and tested for genome detection of pan-coronavirus, pan-pestivirus, canine distemper virus, and influenza A and D virus. Nine faecal samples were positive in the Pan-CoV RT-PCR, while all were negative for the other viruses. Pan-coronavirus positives were further identified using Illumina's complete genome sequencing, which showed the highest homology with Bovine Coronavirus previously detected in roe deer in the Alps. Blood samples (n.35) were collected randomly from animals at release and tested for bovine coronavirus (BCoV) antibodies using competitive ELISA and VNT. Serological analyses revealed that 8/35 sera were positive for BCoV antibodies in both serological tests. This study provides molecular and serological evidence of the presence of BCoV in an alpine marmot population due to a likely spillover event. Marmots share areas and pastures with roe deer and other wild ruminants, and environmental transmission is a concrete possibility. [ABSTRACT FROM AUTHOR]

Simple Summary: Equine influenza (EI) is an acute and highly contagious viral disease of equids characterized by fever and respiratory signs. Ongoing antigenic mutations that are typical of influenza viruses, which may cause a reduction in the effectiveness of vaccines, highlight how crucial both surveillance and virus characterization are for updating vaccine compositions. The aim of this study was to verify the identity of the equine influenza virus strains detected in Italy, especially in the absence of formal active surveillance. Twenty nasal swabs, collected from symptomatic horses located in North and Central Italy between February and April 2019 were positive for influenza A virus (IAV) RRT-PCR. Sequencing identified an isolated strain as H3N8 Florida lineage clade 1 for one sample from Brescia, Lombardy Region. This study is the first report of H3N8 Florida lineage clade 1 circulation in Italy, confirming the value of monitoring for EIV circulating strain in relation to the appropriateness of the vaccine virus composition for maximum efficacy. Background: Equine influenza (EI) is a highly contagious viral disease of equids characterized by pyrexia and respiratory signs. Like other influenza A viruses, antigenic drift or shift could lead to a vaccine-induced immunity breakdown if vaccine strains are not updated. The aim of this study was to genetically characterize EIV strains circulating in Italy, detected in PCR-positive samples collected from suspected cases, especially in the absence of formal active surveillance. Methods: Between February and April 2019, blood samples and nasal swabs collected from each of the 20 symptomatic horses from North and Central Italy were submitted to the National Reference Centre for Equine Diseases in Italy to confirm preliminary analysis performed by other laboratories. Results: None of the sera analysed using haemagglutination inhibition and single radial haemolysis presented a predominant serological reactivity pattern for any antigen employed. All nasal swabs were positive with IAV RRT-PCR. Only one strain, isolated in an embryonated chicken egg from a sample collected from a horse of a stable located in Brescia, Lombardy, was identified as H3N8 Florida lineage clade 1 (FC1). In the constructed phylogenetic trees, this strain is located within the FC1, together with the virus isolated in France in 2018 (MK501761). Conclusions: This study reports the first detection of H3N8 FC1 in Italy, highlighting the importance of monitoring circulating EIV strains to verify the vaccine composition appropriateness for maximum efficacy. [ABSTRACT FROM AUTHOR]

Highlights • Three Alpha-CoV strains were fully sequenced by NGS method. • The Italian strains were classified into two novel Alpha-CoV species. • The phylogenetic analysis on RdRp fragment sequences showed correlation to European strains. Abstract Coronaviruses (CoVs) have been detected worldwide in several bat species, which are considered the main reservoir. The attention to the high diversity of CoVs hosted by bats has increased during the last decade due to the high number of human infections caused by two zoonotic Beta-CoVs, SARS-CoV and MERS-CoV, that cause several respiratory diseases. Among coronaviruses, two Alpha-CoV strains (HuCoV-229E and HuCoV-NL63) cause mild respiratory disease that can change to severe disease in children, elderly and individuals affected by illnesses. Phylogenetic analysis conducted on bat Alpha-CoV strains revealed their evolutive correlation to human strains, suggesting their origin in bats. The genome of CoVs is characterized by a high frequency of mutations and recombination events, increasing their ability to switch hosts and their zoonotic potential. In this study, three strains of Alpha-CoV genera detected in Italian bats (Pipistrellus kuhlii) were fully sequenced by Next Generation Sequencing (NGS) and characterized. The complete genome analysis showed the correlation of the Italians strains with a Chinese strain detected in 2013 and, based on CoV molecular species demarcation, two new Alpha-CoV species were established. The analysis of a fragment of the RNA-dependent RNA polymerase (RdRp) showed the correlation of the Italian strains with CoVs that was only detected in the bat Pipistrellus genera (Pipistrellus kuhlii and Pipistrellus Pipistrellus) in European countries. [ABSTRACT FROM AUTHOR]

Influenza D virus (IDV) was first reported in 2011 in swine in Oklahoma and consequently found in cattle, sheep, and goats across North America and Eurasia. Cattle have been proposed as the natural reservoir. In this study, we developed and validated a MAb‐based competitive ELISA for the detection of antibodies against IDV. Thirty‐one hybridomas specific to IDV were generated using Balb/C mice immunised with purified IDV/Swine/Italy/199724‐3/2015. The specificity of MAbs was determined by comparing their reactivity with the homologous and other influenza A viruses along with additional bovine and swine viruses. A solid‐phase competitive ELISA (IDV‐cELISA) was set up using the partially purified antigen coated to the plate and incubation of two serum dilutions (1/10 and 1/20) followed by addition of a peroxidase‐conjugated MAb as competitor, which had shown wide intratype cross‐reactivity and positivity in HI. To evaluate the diagnostic performances of IDV‐cELISA, we used 884 sera (414 negative and 470 positive) from different species. ROC analyses were performed to enable the selection of best cut‐off value and estimation of diagnostic specificity and sensitivity. The agreement between IDV‐cELISA and HI test was assessed by Cohen's kappa value (κ). The κ analysis showed an almost perfect agreement (κ = 0.93; 95%CI −0.899 to 0.961) between HI test and IDV‐cELISA. ROC analysis showed that IDV‐cELISA was accurate with an area under the curve (AUC) = 0.999, 95% CI 0.993–1.000). A cut‐off value of 65% was selected with Se and Sp values of 99.35 (95% CI 98.1–99.9) and 98.75 (95% CI 97.1–99.6). These results proved excellent diagnostic performances of IDV‐cELISA, which compared to HI presented major advantages, such as suitability for automation, low dependence on individual skills, spectrophotometric reading, and easy interpretation of the results. This assay can be exploited to detect anti‐IDV antibodies in different animal species. [ABSTRACT FROM AUTHOR]

Background: Middle East respiratory syndrome coronavirus (MERS-CoV), which belongs to beta group of coronavirus, can infect multiple host species and causes severe diseases in humans. Multiple surveillance and phylogenetic studies suggest a bat origin. In this study, we describe the detection and full genome characterization of two CoVs closely related to MERS-CoV from two Italian bats, Pipistrellus kuhlii and Hypsugo savii. Methods: Pool of viscera were tested by a pan-coronavirus RT-PCR. Virus isolation was attempted by inoculation in different cell lines. Full genome sequencing was performed using the Ion Torrent platform and phylogenetic trees were performed using IQtree software. Similarity plots of CoV clade c genomes were generated by using SSE v1.2. The three dimensional macromolecular structure (3DMMS) of the receptor binding domain (RBD) in the S protein was predicted by sequence-homology method using the protein data bank (PDB). Results: Both samples resulted positive to the pan-coronavirus RT-PCR (IT-batCoVs) and their genome organization showed identical pattern of MERS CoV. Phylogenetic analysis showed a monophyletic group placed in the Beta2c clade formed by MERS-CoV sequences originating from humans and camels and bat-related sequences from Africa, Italy and China. The comparison of the secondary and 3DMMS of the RBD of IT-batCoVs with MERS, HKU4 and HKU5 bat sequences showed two aa deletions located in a region corresponding to the external subdomain of MERS-RBD in IT-batCoV and HKU5 RBDs. Conclusions: This study reported two beta CoVs closely related to MERS that were obtained from two bats belonging to two commonly recorded species in Italy (P. kuhlii and H. savii). The analysis of the RBD showed similar structure in IT-batCoVs and HKU5 respect to HKU4 sequences. Since the RBD domain of HKU4 but not HKU5 can bind to the human DPP4 receptor for MERS-CoV, it is possible to suggest also for IT-batCoVs the absence of DPP4-binding potential. More surveillance studies are needed to better investigate the potential intermediate hosts that may play a role in the interspecies transmission of known and currently unknown coronaviruses with particular attention to the S protein and the receptor specificity and binding affinity. [ABSTRACT FROM AUTHOR]

Porcine epidemic diarrhea virus (PEDV) has caused economic losses in the Americas, Asia and Europe in recent years. Reliable serological assays are essential for epidemiological studies and vaccine evaluation. The objective of this study was to compare the ability of five enzyme-linked immunosorbent assays (ELISAs) to detect antibodies against different PEDV strains in pig serum. A total of 732 serum samples from North American or European pigs were tested. Samples included experimental samples from pigs infected with classical (G1a PEDV) or variant genogroup 1 PEDV (G1b PEDV), pandemic genogroup 2 PEDV (G2b PEDV) or non-infected controls. Field samples from herds with confirmed or unknown PEDV exposure were also used. Three indirect ELISAs based on G2b antigens (ELISAs 1, 2 and 3), a competitive ELISA based on the G2b antigen (ELISA 4) and a competitive ELISA based on the G1a antigen (ELISA 5) were compared. Overall, the tests had a moderate agreement (κ = 0.61). G1a PEDV infected pigs were earliest detected by ELISA 3, G1b PEDV infected pigs were earliest detected by ELISAs 4 and 5 and the performance of all tests was similar for the G2b PEDV group. ELISA 1 showed the overall lowest detection on experimentally and field derived samples. Diagnostic sensitivity and specificity with a 95% probability interval were estimated to be 68.2% (62.1–74.4%) and 97.5% (95.2–99.0%) for ELISA 1, 73.7% (71.5–79.6%) and 98.4% (96.6–99.5%) for ELISA 2, 86.2% (81.1–90.6%) and 91.6% (87.7–94.8%) for ELISA 3, 78.3% (72.8–83.5%) and 99.7% (98.2–100%) for ELISA 4, and 93.5% (90.3–96.0%) and 91.2% (83.8–97.9%) for ELISA 5. Differences in detection among assays seem to be more related to intrinsic factors of an assay than to the PEDV antigen used. [ABSTRACT FROM AUTHOR]

Mammalian Orthoreoviruses 3 (MRV3) have been described in diarrheic pigs from USA and Asia. We firstly detected MRV3 in Europe (Italy) in piglets showing severe diarrhea associated with Porcine Epidemic Diarrhea. The virus was phylogenetically related to European reoviruses of human and bat origin and to US and Chinese pig MRV3. [ABSTRACT FROM AUTHOR]

A renewed interest in mammalian orthoreoviruses (MRVs) has emerged since new viruses related to bat MRV type 3, detected in Europe, were identified in humans and pigs with gastroenteritis. This study reports the isolation and characterization of a novel reassortant MRV from the lesser horseshoe bat (Rhinolophus hipposideros). The isolate, here designated BatMRV1-IT2011, was first identified by electron microscopy and confirmed using PCR and virus-neutralization tests. The full genome sequence was obtained by next-generation sequencing. Molecular and antigenic characterizations revealed that BatMRV1-IT2011 belonged to serotype 1, which had not previously been identified in bats. Phylogenetic and recombination detection program analyses suggested that BatMRV1-IT2011 was a reassortant strain containing an S1 genome segment similar to those of MRV T1/bovine/Maryland/Clone23/59 and C/bovine/ Indiana/MRV00304/2014, while other segments were more similar to MRVs of different hosts, origins and serotypes. The presence of neutralizing antibodies against MRVs has also been investigated in animals (dogs, pigs, bovines and horses). Preliminary results suggested that MRVs are widespread in animals and that infections containing multiple serotypes, including MRVs of serotype 1 with an S1 gene similar to BatMRV1-IT2011, are common. This paper extends the current knowledge of MRVs and stresses the importance to continue and improve MRV surveillance in bats and other mammals through the development and standardization of specific diagnostic tools. [ABSTRACT FROM AUTHOR]

Bats are natural reservoirs for many mammalian coronaviruses, which have received renewed interest after the discovery of the severe acute respiratory syndrome (SARS) and the Middle East respiratory syndrome (MERS) CoV in humans. This study describes the identification and molecular characterization of alphacoronaviruses and betacoronaviruses in bats in Italy, from 2010 to 2012. Sixty-nine faecal samples and 126 carcasses were tested using pan-coronavirus RT-PCR. Coronavirus RNAs were detected in seven faecal samples and nine carcasses. A phylogenetic analysis of RNA-dependent RNA polymerase sequence fragments aided in identifying two alphacoronaviruses from Kuhl's pipistrelle (Pipistrellus kuhlii), three clade 2b betacoronaviruses from lesser horseshoe bats (Rhinolophus hipposideros), and 10 clade 2c betacoronaviruses from Kuhl's pipistrelle, common noctule (Nyctalus noctula), and Savi's pipistrelle (Hypsugo savii). This study fills a substantive gap in the knowledge on bat-CoV ecology in Italy, and extends the current knowledge on clade 2c betacoronaviruses with new sequences obtained from bats that have not been previously described as hosts of these viruses. [ABSTRACT FROM AUTHOR]

Abstract: Diagnostic and containment measures are essential for the management of avian influenza. In this study, a monoclonal antibody (MAb)-based competitive ELISA for detecting antibodies against H5 avian influenza viruses was developed and validated. Twenty-five anti-H5 MAbs were characterised using competitive, indirect and sandwich ELISAs, immunofluorescence, Western blotting and virus neutralisation and haemagglutination inhibition assays. One MAb (5D8) with wide intra-subtype cross-reactivity was selected and characterised using escape mutant selection. Epitope analysis showed that this MAb recognises a conformational epitope comprising amino acid residues in positions 170, 235 and 240 located in the receptor binding domain. The diagnostic performance of the test was evaluated by ROC analysis using a panel of 950 known sera collected from different avian species, including chickens, turkeys, ducks, pheasants, wild Anseriformes and ostriches. The competitive ELISA had excellent diagnostic performance and discriminatory power with high Se and Sp values (Se: 99.6–95% CI 98.0–100; Sp: 99.4–95% CI 98.5–99.8). In addition to its excellent diagnostic performance, properties of the competitive ELISA, such as high feasibility of testing sera without pre-treatment and potential for automation and instrument-mediated detection, make it ideal for screening samples, confirming positive HI assay results or analysing samples that are difficult to test using the HI assay. [Copyright &y& Elsevier]

Phleboviruses are arboviruses transmitted by sand flies, mosquitoes and ticks. Some sand fly-borne phleboviruses cause illnesses in humans, such as the summer fevers caused by the Sicilian and Naples viruses or meningitis caused by the Toscana virus. Indeed, traces of several phleboviral infections have been serologically detected in domestic animals, but their potential pathogenic role in vertebrates other than humans is still unclear, as is the role of vertebrates as potential reservoirs of these viruses. In this study, we report the results of a serological survey performed on domestic animals sampled in Northern Italy, against four phleboviruses isolated from sand flies in the same area. The sera of 23 dogs, 165 sheep and 23 goats were tested with a virus neutralization assay for Toscana virus, Fermo virus, Ponticelli I virus and Ponticelli III virus. Neutralizing antibodies against one or more phleboviruses were detected in four out of 23 dogs, 31 out of 165 sheep and 12 out of 23 goats. This study shows preliminary evidence for the distribution pattern of phleboviral infections in different animal species, highlighting the potential infection of the Toscana virus in dogs and the Fermo virus in goats. [ABSTRACT FROM AUTHOR]

Over the last few decades, several pestiviruses have been discovered in ruminants, pigs, and, more recently, in non-ungulate hosts. Consequently, the nomenclature and taxonomy of pestiviruses have been updated. The Tunisian sheep-like pestivirus (TSV, Pestivirus N) is an additional ovine pestivirus genetically closely related to classical swine fever virus (CSFV). In this study, during a survey of pestivirus infections in ovine farms in the Lombardy region of Northern Italy, we identified and isolated a pestivirus strain from a sheep that was found to belong to Pestivirus N species based on its genomic nucleotide identity. The sheep itself and its lamb were found to be persistently infected. We performed molecular characterization and phylogenetic analysis of three viral genomic regions (a fragment of 5′-UTR, partial Npro, and the whole E2 region). In conclusion, these results confirmed circulating TSV in Northern Italy after notification in Sicily, Italy, and France. Correlation with Italian, Tunisian, and French strains showed that detection might have resulted from the trading of live animals between countries, which supports the need for health control measures. [ABSTRACT FROM AUTHOR]

Simple Summary: African swine fever (ASF) is a highly lethal viral disease affecting suids and caused by the African Swine Fever Virus (ASFV). ASF was described for the first time in 1921 in Kenya. The ASF genotype I virus was introduced to Europe in 1957, marking the onset of the first European wave. In 2007, ASFV genotype II was detected in Georgia, affecting domestic pigs and wild boars, and it later spread to several European and extra-European countries, including Italy. This report focuses on the strategic challenges encountered in the attempt to eradicate ASFV amongst domestic pigs in the Lombardy region. The joint efforts that were implemented facilitated the eradication of ASF in domestic pigs in just over 1.5 months, representing an example of effective and timely cooperation to mitigate both the spread of the infection and the economic repercussions for the Italian and global pig industries. African swine fever (ASF) is a severe viral disease characterized by high lethality in suids and caused by the African Swine Fever Virus (ASFV). The ASF genotype I virus was introduced to Europe in 1957, marking the onset of the first European epidemic wave. In 2007, ASFV genotype II was detected in Georgia, affecting domestic pigs and wild boars before spreading to various European and extra-European countries, including Italy. The first case of ASFV in Italy was documented on 7 January 2022, in a wild boar in the Piedmont region. Since then, several ASFV-positive wild boar carcasses have been identified in the Piedmont and Liguria regions. By June 2023, ASFV had spread to Lombardy, one of the major pig-producing regions in northern Italy; the virus was first detected in early summer in wild boar carcasses. Two months later, it was diagnosed in a commercial pig farm as a consequence of the disease's spread amongst wild boars and an increase in the viral environmental load. This report aims to describe the features of ASFV domestic pig outbreaks that occurred in the Zinasco municipality (Lombardy) and the joint efforts to mitigate potential direct and indirect economic impacts on the Italian and global pig industry. The epidemiological investigation and the measures implemented, which were all performed according to national and European regulations, as well as exceptional ad hoc measures aimed at protecting the pig industry, are described in order to provide a practical and effective approach to combating ASF. [ABSTRACT FROM AUTHOR]

Simple Summary: Pathogens transmitted to humans and animals by Phlebotomines are relatively neglected, as they cause infectious diseases which represent an underestimated burden in most European countries. Several sand fly species are competent vectors of Leishmaniasis, an endemic disease that has spread widely throughout the Mediterranean region in conjunction with sand flies' movements. In the Lombardy region, information on sand flies is poor and/or outdated. Therefore, the present study was undertaken to preliminarily ascertain the species composition, distribution, and diversity in representative Lombardy localities. The sampling took advantage of regional surveillance plans namely, West Nile virus and leishmaniasis monitoring plans. A focused sampling was also performed in areas identified as favorable for vector presence. Sampling was conducted using CO2–CDC traps conducted every two and/or three weeks. From trapping for the West Nile monitoring plan, 21 out of 44 capture sites were positive for sand flies, while for the leishmaniasis monitoring plan, 11 out of the 40 trapping sites detected sand flies' presence. Specimen identification was conducted by identifying morphological features. Phlebotomus perniciosus was the most abundant species (87.76% of specimens collected). Adequate and well-structured monitoring of sand fly populations is essential to provide information about distribution patterns of vector species present in defined geographical areas, as they could enhance pathogen circulation. This study investigated the species composition and density of sand flies in the Lombardy region (Northern Italy). Sand flies were collected using CDC traps baited with CO2 (CO2–CDC traps) between June and August 2021. A total of 670 sand flies were collected. The specimens were identified as seven species belonging to two genera, Phlebotomus and Sergentomyia, namely, S. minuta, Ph. perniciosus, Ph. perfiliewii, Ph. neglectus, Ph. mascitti, Ph. papatasi, and Ph. ariasi. Phlebotomus perniciosus was the most abundant species (87.76%), followed by Ph. perfiliewii (7.31%), Ph. neglectus (3.13%), S. minuta (0.75%), Ph. mascitti (0.6%), Ph. papatasi (0.3%), and Ph. ariasi, for which only one specimen was identified. Among these identified species, five are considered vectors of Leishmania, which causes cutaneous and visceral leishmaniasis. As vector presence increases the risk of vector-borne leishmaniasis, these results suggest that Northern Italy could be a potential area of pathogen circulation over the next few years. These preliminary results suggest that the risk of borne leishmaniasis is high in this region of Northern Italy. Monitoring the distribution of sand fly species in areas suitable for their persistence is important for control programs aimed at reducing the risk of leishmaniasis infection. [ABSTRACT FROM AUTHOR]

Background: In Italy, malaria was endemic until the 1970s, when it was declared eradicated by WHO. Nowadays, with the persistence of competent mosquito populations, the effect of climate change, and increased possibility of importing malaria parasites from endemic counties due to growing migration, a malaria resurgence in Italy has become more likely. Hence, enhancing the understanding of the current distribution of the Anopheles maculipennis complex and the factors that influence the presence of this malaria vector is crucial, especially in Northern Italy, characterised by a high density of both human population and livestock. Methods: To assess the presence and abundance of malaria vectors, a 4-year field survey in the plain areas of Lombardy and Emilia-Romagna region in Italy was conducted. Every sampling point was characterised in space by the land use in a 500-m radius and in time considering meteorological data collected in the short and long time periods before sampling. We combined the results of a linear regression model with a random forest analysis to understand the relative importance of the investigated niche dimensions in determining Anopheles mosquito presence and abundance. Results: The estimated normalised variable importance indicates that rice fields were the most important land use class explaining the presence of Anopheles, followed by transitional woodlands and shrubland. Farm buildings were the third variable in terms of importance, likely because of the presence of animal shelters, followed by urbanised land. The two most important meteorological variables influencing the abundance of Anopheles in our study area were mean temperature in the 24 h before the sampling date and the sum of degree-days with temperature between 18 °C and 30 °C in the 14 days before the sampling date. Conclusions: The results obtained in this study could be helpful in predicting the risk of autochthonous malaria transmission, based on local information on land cover classes that might facilitate the presence of malaria vectors and presence of short- and medium-term meteorological conditions favourable to mosquito development and activity. The results can support the design of vector control measures through environmental management. [ABSTRACT FROM AUTHOR]

Tick-borne encephalitis was limited to northeast portions of Italy. We report in Lombardy, a populous region in the northwest, a chamois displaying clinical signs of tickborne encephalitis virus that had multiple virus-positive ticks attached, as well as a symptomatic man. Further, we show serologic evidence of viral circulation in the area. [ABSTRACT FROM AUTHOR]

Interest in bat-related viruses has increased considerably during the last decade, leading to the discovery of a rising number of new viruses in several bat species. Poxviridae are a large, diverse family of DNA viruses that can infect a wide range of vertebrates and invertebrates. To date, only a few documented detections of poxviruses have been described in bat populations on three different continents (America, Africa, and Australia). These viruses are phylogenetically dissimilar and have diverse clinical impacts on their hosts. Herein, we report the isolation, nearly complete genome sequencing, and annotation of a novel poxvirus detected from an insectivorous bat (Hypsugo savii) in Northern Italy. The virus is tentatively named Hypsugopoxvirus (HYPV) after the bat species from which it was isolated. The nearly complete genome size is 166,600 nt and it encodes 161 genes. Genome analyses suggest that HYPV belongs to the Chordopoxvirinae subfamily, with the highest nucleotide identity (85%) to Eptesipoxvirus (EPTV) detected from a microbat Eptesicus fuscus in WA, USA, in 2011. To date, HYPV represents the first poxvirus detected in bats in Europe; thus, its viral ecology and disease associations should be investigated further. [ABSTRACT FROM AUTHOR]

Soil contamination constitutes a significant threat to the health of soil ecosystems in terms of complexity, toxicity, and recalcitrance. Among all contaminants, aliphatic petroleum hydrocarbons (APH) are of particular concern due to their abundance and persistence in the environment and the need of remediation technologies to ensure their removal in an environmentally, socially, and economically sustainable way. Soil remediation technologies presently available on the market to tackle soil contamination by petroleum hydrocarbons (PH) include landfilling, physical treatments (e.g., thermal desorption), chemical treatments (e.g., oxidation), and conventional bioremediation. The first two solutions are costly and energy-intensive approaches. Conversely, bioremediation of on-site excavated soil arranged in biopiles is a more sustainable procedure. Biopiles are engineered heaps able to stimulate microbial activity and enhance biodegradation, thus ensuring the removal of organic pollutants. This soil remediation technology is currently the most environmentally friendly solution available on the market, as it is less energy-intensive and has no detrimental impact on biological soil functions. However, its major limitation is its low removal efficiency, especially for long-chain hydrocarbons (LCH), compared to thermal desorption. Nevertheless, the use of fungi for remediation of environmental contaminants retains the benefits of bioremediation treatments, including low economic, social, and environmental costs, while attaining removal efficiencies similar to thermal desorption. Mycoremediation is a widely studied technology at lab scale, but there are few experiences at pilot scale. Several factors may reduce the overall efficiency of on-site mycoremediation biopiles (mycopiles), and the efficiency detected in the bench scale. These factors include the bioavailability of hydrocarbons, the selection of fungal species and bulking agents and their application rate, the interaction between the inoculated fungi and the indigenous microbiota, soil properties and nutrients, and other environmental factors (e.g., humidity, oxygen, and temperature). The identification of these factors at an early stage of biotreatability experiments would allow the application of this on-site technology to be refined and fine-tuned. This review brings together all mycoremediation work applied to aliphatic petroleum hydrocarbons (APH) and identifies the key factors in making mycoremediation effective. It also includes technological advances that reduce the effect of these factors, such as the structure of mycopiles, the application of surfactants, and the control of environmental factors. [ABSTRACT FROM AUTHOR]

Background: Rhabdoviridae is one of the most ecologically diverse families of RNA viruses which can infect a wide range of vertebrates and invertebrates. Bats, among mammals, are pointed to harbor a significantly higher proportion of unknown or emerging viruses with zoonotic potential. Herein, we report the isolation of a novel rhabdovirus, detected in the framework of a virological survey on bats implemented in North Italy. Methods: Virus isolation and identification were performed on samples of 635 bats by using cell cultures, negative staining electron microscopy and PCRs for different viruses. NGS was commonly performed on cell culture supernatants showing cytopathic effect or in case of samples resulted positive by at least one of the PCRs included in the diagnostic protocol. Results: A rhabdovirus was isolated from different organs of a Pipistrellus kuhlii. Virus identification was obtained by electron microscopy and NGS sequencing. The complete genome size was 11,774 nt comprised 5 genes, encoding the canonical rhabdovirus structural proteins, and an additional transcriptional unit (U1) encoding a hypothetical small protein (157aa) (3’-N-P-M-G-U1-L-5′). The genome organization and phylogenetic analysis suggest that the new virus, named Vaprio virus (VAPV), belongs to the recently established genus Ledantevirus (subgroup B) and it is highly divergent to its closest known relative, Le Dantec virus (LDV) (human, 1965 Senegal). A specific RT-PCR amplifying a 350 bp fragment of the ORF 6 gene, encoding for L protein, was developed and used to test retrospectively a subset of 76 bats coming from the same area and period, revealing two more VAPV positive bats. Conclusions: VAPV is a novel isolate of chiropteran rhabdovirus. Genome organization and phylogenetic analyses demonstrated that VAPV should be considered a novel species within the genus Ledantevirus for which viral ecology and disease associations should be investigated. [ABSTRACT FROM AUTHOR]

Correction: After Publication of the article [1], it has been brought to our attention that an author's name has been spelt incorrectly. The correct spelling should be "Massimo Ciccozzi", but it was previously included as "Massimo Cicozzi". The original version has now been revised to reflect this. [ABSTRACT FROM AUTHOR]

Highlights • MRV-3 has been identified from lung tissues of alpine chamois in Italian Alps. • Virus belongs to lineage III clustering with strains from dog, bat and diarrheic pig. • Whole genome sequence highlighted reassortment and lack of host specific barriers. • No respiratory symptoms neither lung macroscopic lesions were observed. • High seroprevalence was observed in chamois population during a five-years survey. Abstract Mammalian orthoreoviruses (MRV) type 3 have been recently identified in human and several animal hosts, highlighting the apparent lack of species barriers. Here we report the identification and genetic characterization of MRVs strains in alpine chamois, one of the most abundant wild ungulate in the Alps. Serological survey was also performed by MRV neutralization test in chamois population during five consecutive years (2008-2012). Three novel MRVs were isolated on cell culture from chamois lung tissues. No respiratory or other clinical symptoms neither lung macroscopic lesions were observed in the chamois population. MRV strains were classified as MRV-3 within the lineage III, based on S1 phylogeny, and were closely related to Italian strains identified in dog, bat and diarrheic pig. The full genome sequence was obtained by next-generation sequencing and phylogenetic analyses showed that other segments were more similar to MRVs of different geographic locations, serotypes and hosts, including human, highlighting genome reassortment and lack of host specific barriers. By using serum neutralization test, a high prevalence of MRV-3 antibodies was observed in chamois population throughout the monitored period, showing an endemic level of infection and suggesting a self-maintenance of MRV and/or a continuous spill-over of infection from other animal species. [ABSTRACT FROM AUTHOR]

West Nile disease is a vector-borne disease caused by West Nile virus (WNV), involving mosquitoes as vectors and birds as maintenance hosts. Humans and other mammals can be infected via mosquito bites, developing symptoms ranging from mild fever to severe neurological infection. Due to the worldwide spread of WNV, human infection risk is high in several countries. Nevertheless, there are still several knowledge gaps regarding WNV dynamics. Several aspects of transmission taking place between birds and mosquitoes, such as the length of the infectious period in birds or mosquito biting rates, are still not fully understood, and precise quantitative estimates are still lacking for the European species involved. This lack of knowledge affects the precision of parameter values when modelling the infection, consequently resulting in a potential impairment of the reliability of model simulations and predictions and in a lack of the overall understanding of WNV spread. Further investigations are thus needed to better understand these aspects, but field studies, especially those involving several wild species, such as in the case of WNV, can be challenging. Thus, it becomes crucial to identify which transmission processes most influence the dynamics of WNV. In the present work, we propose a sensitivity analysis to investigate which of the selected epidemiological parameters of WNV have the largest impact on the spread of the infection. Based on a mathematical model simulating WNV spread into the Lombardy region (northern Italy), the basic reproduction number of the infection was estimated and used to quantify infection spread into mosquitoes and birds. Then, we quantified how variations in four epidemiological parameters representing the duration of the infectious period in birds, the mosquito biting rate on birds, and the competence and susceptibility to infection of different bird species might affect WNV transmission. Our study highlights that knowledge gaps in WNV epidemiology affect the precision in several parameters. Although all investigated parameters affected the spread of WNV and the modelling precision, the duration of the infectious period in birds and mosquito biting rate are the most impactful, pointing out the need of focusing future studies on a better estimate of these parameters at first. In addition, our study suggests that a WNV outbreak is very likely to occur in all areas with suitable temperatures, highlighting the wide area where WNV represents a serious risk for public health. Author summary: Infectious communicable diseases are currently one of the main burdens for human beings and public health. The comprehension of their spread and maintenance is one of the main goals to facilitate their control and eradication, but due to the complexity of their cycles and transmission processes, obtaining this information is often difficult and demanding. The control of vector-borne diseases in particular represents an important and very complex challenge for public health. Mathematical models are suitable tools to investigate disease dynamics and their transmission mechanisms and processes. To build a suitable model that can simulate transmission dynamics, a reliable and precise estimate of parameters for measuring transmission mechanisms is fundamental. We thus propose a sensitivity analysis of four unknown epidemiological parameters (bird recovery rate, mosquito biting rate, avian susceptibility to infection and avian competence to infection) that play a crucial role in driving West Nile virus (WNV) infection to determine which of them have the greatest impact on infection spread. This analysis suggests that the infectious period in birds and mosquito biting rate are the parameters to be prioritised in investigation to increase our ability to model WNV spread. [ABSTRACT FROM AUTHOR]

The number of newly described sandfly-borne phleboviruses has been steadily growing in recent years. Some phleboviruses are human pathogens, but their health relevance is largely uncharacterized. We aimed to investigate the circulation of these viruses in the Emilia-Romagna region where several have already been described. A total of 482 sandflies were collected in a site in Reggio Emilia in 2019 and 2020. Sandflies collected in 2020 were grouped in 21 pools with a maximum of 25 sandflies per pool, submitted to real time PCR, and isolated in Vero cell culture. Complete genome sequencing showed the isolation of a strain of a Ponticelli III virus. This virus, which belongs to the species Adana phlebovirus, differed in the M segment from the Ponticelli I and Ponticelli II viruses. Analysis performed on the genomic segments of the newly isolated virus compared with other phleboviruses highlighted a strong purifying selection in the L segments, and different substitution saturation, highest in the M segments. Future research should address the ecological processes driving the occurrence of these novel phleboviruses and their possible impact on public health. [ABSTRACT FROM AUTHOR]

Porcine epidemic diarrhea virus (PEDV) has caused extensive economic losses to pig producers in many countries. It was recently introduced, for the first time, into North America and outbreaks have occurred again in multiple countries within Europe as well. To assess the properties of various diagnostic assays for the detection of PEDV infection, multiple panels of porcine sera have been shared and tested for the presence of antibodies against PEDV in an inter-laboratory ring trial. Different laboratories have used a variety of “in house” ELISAs and also one commercial assay. The sensitivity and specificity of each assay has been estimated using a Bayesian analysis applied to the ring trial results obtained with the different assays in the absence of a gold standard. Although different characteristics were found, it can be concluded that each of the assays used can detect infection of pigs at a herd level by either the early European strains of PEDV or the recently circulating strains (INDEL and non-INDEL). However, not all the assays seem suitable for demonstrating freedom from disease in a country. The results from individual animals, especially when the infection has occurred within an experimental situation, show more variation. [ABSTRACT FROM AUTHOR]

Porcine circovirus type 2 (PCV2) is associated with a number of diseases and syndromes, collectively referred to as porcine circovirus-associated disease. The main objective of this study was to define some in vitro correlates of protection after injection of inactivated PCV2 vaccines with a defined antigen mass. Twelve pigs were vaccinated with three different doses of inactivated, whole-virus antigen (211-844 ng), while four animals were injected with a commercial vaccine (positive control) and four other pigs were mock-vaccinated with phosphate-buffered saline (PBS) in the same oil emulsion. Four weeks later, they were intranasally challenged with 2 × 105 TCID50 of a PCV2a strain. Antibody was measured in blood and oral fluids by enzyme-linked immunosorbent assay (ELISA) and a neutralization assay. PCV2 was quantified in serum by real-time polymerase chain reaction for ORF2 gene. PCV2-specific cell-mediated responses were investigated by an IFN-γ release assay in whole blood, IFN-γ ELISPOT, and lymphocyte proliferation (Ki-67 and BrDU assays). All the vaccines under study but mock provided complete or incomplete protection from PCV2 infection in terms of post-challenge viremia. Serum antibody titers (ELISA and neutralizing) after vaccination were not correlated with protection, as opposed to the early neutralizing antibody levels of vaccinated pigs at day 7 after infection. Cell-mediated immune parameters showed a good correlation with vaccine efficacy. In particular, the IFN-γ release assay at 3 weeks after vaccination was an effective marker for predicting protection. All control pigs always tested negative in assays of cell-mediated immunity. Our results outline in vitro testing procedures toward reduced animal usage in the control of PCV2 vaccine batches. [ABSTRACT FROM AUTHOR]

SARS‐CoV‐2 positive or seropositive owned cats have been reported worldwide. The detection of seropositive stray cats in the proximity of farms of infected minks, coupled with the demonstration of cat‐to‐cat transmission in experimental settings, raise the question whether stray cats may have an epidemiological role in the COVID‐19 pandemic and may act as sentinel for the circulation of SARS‐CoV‐2. The aim of this study was to evaluate the presence of SARS‐CoV‐2 RNA and anti‐SARS‐CoV‐2 antibodies in free roaming cats belonging to colonies located in an area highly affected by the COVID‐19 pandemic and to correlate the results with the positivity rate in people sharing the same area. Interdigital, cutaneous, oropharyngeal, nasal and rectal swabs, as well as blood samples, were collected from 99 cats living in colonies and admitted to our hospital for neutering. This caseload corresponds to the 24.2% of the feline population living in the 25 sampled colonies and to the 5.6% of all the free‐roaming registered cats. The presence of SARS‐CoV‐2 RNA in swabs was assessed using real time RT‐PCR. Anti‐SARS‐CoV‐2 serum antibodies were assessed using commercially available ELISA kits and confirmed by serum virus neutralization. In people, the SARS‐CoV‐2 positivity rate ranged from 3.0% to 5.1% (mean rate: 4.1%) and the seropositive rate from 12.1% to 16.3% (mean rate: 14.2%). Most of the colonies were in urban areas and resident cats had frequent contacts with external cats or people. A COVID‐19 positive caretaker was found, whereas all the cats were negative for SARS‐CoV‐2 RNA and seronegative. Although the negative results cannot exclude previous infections followed by decrease of antibodies, this study suggests that colony cats do not have an important epidemiological role in SARS‐CoV‐2 transmission dynamics. Further studies on larger caseloads are warranted, also in the light of the emerging new viral variants, on a One Health perspective. [ABSTRACT FROM AUTHOR]

In-vitro viral studies are still fundamental for biomedical research since studying the virus kinetics on cells is crucial for the determination of the biological properties of viruses and for screening the inhibitors of infections. Moreover, testing potential viral contaminants is often mandatory for safety evaluation. Nowadays, viral cytopathic effects are mainly evaluated through end-point assays requiring dye-staining combined with optical evaluation. Recently, optical-based automatized equipment has been marketed, aimed at the real-time screening of cell-layer status and obtaining further insights, which are unavailable with end-point assays. However, these technologies present two huge limitations, namely, high costs and the possibility to study only cytopathic viruses, whose effects lead to plaque formation and layer disruption. Here, we employed poly(3,4-ethylenedioxythiophene):poly(styrene sulfonate) (Pedot:Pss) organic electrochemical transistors (OECTs) for the real-time, electrical monitoring of the infection of cytolytic viruses, i.e., encephalomyocarditis virus (EMCV), and non-cytolytic viruses, i.e., bovine coronavirus (B-CoV), on cells. OECT data on EMCV were validated using a commercially-available optical-based technology, which, however, failed in the B-CoV titration analysis, as expected. The OECTs proved to be reliable, fast, and versatile devices for viral infection monitoring, which could be scaled up at low cost, reducing the operator workload and speeding up in-vitro assays in the biomedical research field. [ABSTRACT FROM AUTHOR]

Several emerging pestiviruses have been reported lately, some of which have proved to cause disease. Recently, a new ovine pestivirus (OVPV), isolated from aborted lambs, with high genetic identity to classical swine fever virus (CSFV), has proved to induce reproductive disorders in pregnant ewes. OVPV also generated strong serological and molecular cross-reaction with CSFV. To assess the capacity of OVPV to infect swine, twelve piglets were infected either by intranasal or intramuscular route. Daily clinical evaluation and weekly samplings were performed to determine pathogenicity, viral replication and excretion and induction of immune response. Five weeks later, two pigs from each group were euthanized and tissue samples were collected to study viral replication and distribution. OVPV generated only mild clinical signs in the piglets, including wasting and polyarthritis. The virus was able to replicate, as shown by the RNA levels found in sera and swabs and persisted in tonsil for at least 5 weeks. Viral replication activated the innate and adaptive immunity, evidenced by the induction of interferon-alpha levels early after infection and cross-neutralizing antibodies against CSFV, including humoural response against CSFV E2 and Erns glycoproteins. Close antigenic relation between OVPV and CSFV genotype 2.3 was detected. To determine the OVPV protection against CSFV, the OVPV-infected pigs were challenged with a highly virulent strain. Strong clinical, virological and immunological protection was generated in the OVPV-infected pigs, in direct contrast with the infection control group. Our findings show, for the first time, the OVPV capacity to infect swine, activate immunity, and the robust protection conferred against CSFV. In addition, their genetic and antigenic similarities, the close relationship between both viruses, suggest their possible coevolution as two branches stemming from a shared origin at the same time in two different hosts. [ABSTRACT FROM AUTHOR]

Epizootic haemorrhagic disease virus (EHDV) is a member of the Orbivirus genus in the Reoviridae family, and it is the etiological agent of an arthropod-transmitted disease that affects domestic and wild ruminants. Due to its significant economic impact, many attempts have been done in order to develop diagnostic immunoassays mainly based on the use of the viral protein 7 (VP7), that is, the immunodominant serogroup-specific antigen. In this work, a recombinant VP7 (recVP7) of EHDV serotype 2 was produced in a baculovirus system, and after purification using ion metal affinity chromatography, we obtained a high yield of recombinant protein characterized by a high degree of purity. We used the purified recVP7 as reagent to develop a competitive enzyme-linked immunoassay (c-ELISA), and we tested the presence of EHDV antibodies in 185 dromedary camel serum samples. The c-ELISA showed good performance parameters in recognising positive sera of naturally EHDV-infected dromedary camels; in particular, our developed test reached 85.7% of sensitivity, 98.1% of specificity, 93% of accuracy, and a high agreement value with results obtained by the commercial ELISA kit (Cohen's kappa value of 0.85) that we adopted as the reference method. This c-ELISA could be a useful screening test to monitor the virus spread in camels that are sentinel animals for endemic areas of disease. [ABSTRACT FROM AUTHOR]

Abstract: The objective of this study was the development and the evaluation of competitive ELISA assays based on monoclonal antibodies for the detection of antibodies specific for neuraminidase type 1 (N1), 2 (N2) and 3 (N3) in avian sera. A total of 1450 sera from different avian species (854 negative, 185 positive to N1, 136 positive to N2, 219 positive to N3 and 56 positive to other N subtypes sera) were analysed in parallel by the three ELISAs. ROC analyses were performed to enable the selection of best cut-off values and estimation of diagnostic specificity and sensitivity. In addition, the correlation between the new developed ELISAs and the neuraminidase inhibition test was evaluated on a limited number of sera. The validation process of the three ELISAs proved excellent diagnostic performances, with very high specificity and sensitivity, ranging from 99.4 to 99.8% and from 97.6 to 100%, respectively in the three assays. The discriminating potential between antibodies elicited against homologous and heterologous N validates the test for use in “DIVA” assays, to distinguish between vaccinated and infected birds. [Copyright &y& Elsevier]

The correct identification of mosquito vectors is often hampered by the presence of morphologically indiscernible sibling species. The Maculipennis complex is one of these groups that include both malaria vectors of primary importance and species of low/negligible epidemiological relevance, of which distribution data in Italy are outdated. Our study was aimed at providing an updated distribution of Maculipennis complex in Northern Italy through the sampling and morphological/molecular identification of specimens from five regions. The most abundant species was Anopheles messeae (2032), followed by Anopheles maculipennis s.s. (418), Anopheles atroparvus (28) and Anopheles melanoon (13). Taking advantage of ITS2 barcoding, we were able to finely characterize tested mosquitoes, classifying all the Anopheles messeae specimens as Anopheles daciae, a taxon with debated rank to which we referred as species inquirenda (sp. inq.). The distribution of species was characterized by Ecological Niche Models (ENMs), fed by recorded points of presence. ENMs provided clues on the ecological preferences of the detected species, with An. daciae sp. inq. linked to stable breeding sites and An. maculipennis s.s. more associated to ephemeral breeding sites. We demonstrate that historical Anopheles malaria vectors are still present in Northern Italy. [ABSTRACT FROM AUTHOR]

Abstract: Porcine epidemic diarrhoea (PED) is a contagious enteric disease of pigs caused by a coronavirus. A double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) based on the use of monoclonal antibodies was developed for the detection of porcine epidemic diarrhoea virus (PEDV). The DAS-ELISA was compared with RT-PCR in the examination of 506 specimens collected during 2006–2007 from pigs originating from different farms located in the Po valley. Both faecal samples obtained directly from the rectum of live animals showing clinical signs and intestinal samples collected from the caecum of deceased pigs were included in the study. The correlation between the two methods was higher when testing faecal samples (K =0.97, 95% CI: 0.94–1.00) than testing intestinal samples (K =0.62, 95% CI: 0.35–0.89). The use of ELISA technology provided an efficient and effective mean of evaluating the presence of coronavirus PED antigen in field samples and indicates that this procedure is a very useful tool in epidemiological studies. [Copyright &y& Elsevier]

Simple Summary: Most of the newly emerging infections arise from animal reservoirs, frequently represented by wildlife species. Western European hedgehogs (Erinaceus europaeus) are mammalian hibernators, mainly nocturnal and insectivorous, living in natural open and green spaces as well as artificial, rural and urban, areas. They are generalist predators of macro-invertebrates, but they may also eat meat, bird eggs and on occasion pet food. These ecological and feeding habits, along with their high population densities, notable synanthropic attitudes, frequent contacts with sympatric wild and domestic species, including humans, implicate the possibility of intra- and interspecies interactions accounting for the possible involvement of E. europaeus in the ecology of several potentially emerging pathogens, including coronaviruses. Using PCR-based and virus isolation methods, we found that 58.3% of 24 hedgehogs' fecal samples were PCR-positive for Erinaceus coronaviruses (EriCoVs). We did not observe any clinical disease related to the EriCoV infection in hedgehogs. However, the high mutation rates characterizing members of the Coronaviridae family and their potential successful interspecies host jumps—as that likely occurred in the Novel coronavirus (2019-nCoV) emergence—should be considered in the management of hedgehogs admitted to multi-species wildlife rehabilitation centers, recommending their return back to the original recovery areas. The Western European Hedgehog (Erinaceus europaeus) is one of the four hedgehog species belonging to the genus Erinaceus. Among them, E. amurensis is extant in East Asia's areas only, whereas E. europaeus, E. roumanicus and E. concolor are mainly found in Europe. E. europaeus is endemically distributed from western to central and southern Europe, including Italy. Western European hedgehogs' ecological and feeding habits, along with their high population densities, notable synanthropic attitudes, frequent contacts with sympatric wild and domestic species, including humans, implicate the possible involvement of E. europaeus in the ecology of potentially emerging viruses, such as coronaviruses, influenza A and influenza D viruses, canine distemper virus, pestiviruses and Aujeszky's disease virus. We examined 24 E. europaeus individuals found injured in urban and rural areas of Northern Italy. Of the 24 fecal samples collected and tested for the above-mentioned pathogens by both PCR-based and virus isolation methods, 14 were found PCR-positive for betacoronaviruses belonging to lineage C and related to the known Erinaceus coronaviruses (EriCoVs), as determined by partial sequencing of the virus genome. Our findings suggest that hedgehogs could be considered natural reservoirs of CoVs, and also act as chronic shedding carriers of these potentially emerging RNA viruses. [ABSTRACT FROM AUTHOR]

Atypical porcine pestivirus (APPV) is a newly recognized member of the Flaviviridae family. This novel porcine pestivirus was first described in 2015 in the USA, where it has been associated with congenital tremor type A-II in new-born piglets. APPV is widely distributed in domestic pigs in Europe and Asia. In this study, a virological survey was performed in Northern Italy to investigate the presence of APPV using molecular methods. Testing of 360 abortion samples from pig herds revealed two APPV strains from distinct provinces in the Lombardy region and testing of 430 wild boar blood samples revealed three strains, one from Lombardy and two from Emilia Romagna. The nucleotide sequencing of a fragment of the nonstructural protein 3-coding region revealed a high similarity to the previously detected European strains (Spanish, German, and Italian) of APPV. [ABSTRACT FROM AUTHOR]

The family Picornaviridae includes important human and animal pathogens that are associated with a wide range of diseases and, in some cases, have zoonotic potential. During epidemiological surveillance of bats, we identified, by next-generation sequencing (NGS) techniques, the presence of picornavirus RNA in a common pipistrelle bat (Pipistrellus pipistrellus). By coupling NGS, primer-walking strategies, and sequence-independent protocols to obtain the sequences of the 5′ and 3′ termini, we reconstructed the genome sequence of picornavirus strain ITA/2017/189/18-155. The genome of the bat picornavirus is 8.2 kb in length and encodes a polyprotein of 2462 amino acids. A comparison of polyprotein sequences revealed that this virus is distantly related (65.1% and 70.9% sequence identity at the nucleotide and amino acid level, respectively) to a bat aichivirus identified in 2010. Phylogenetic analysis showed that this picornavirus clustered closely with members of the genus Kobuvirus, which also includes human and animal aichiviruses. The identification of aichiviruses in several animal hosts is providing hints that will lead to an understanding of their origin and evolutionary patterns. [ABSTRACT FROM AUTHOR]

Abstract The sand flies are the biological vectors of a variety of viruses belonging to the Phlebovirus genus. As several of these viruses, like Toscana virus, are important agents of diseases in humans, the definition the phleboviruses circulating in a particular area is an important health issue. This work presents results obtained between 2013 and 2016, by testing 25,853 field-collected sand flies, sampled at 50 sites in the Northern edge of the Apennine Mountains, in Emilia-Romagna and Lombardy regions (Italy). Isolation of the three reassortant Ponticelli I, II, and III viruses was successful from five pools of sand flies. These results, and phylogenetic analysis made by obtained sequences, suggest the presence of eight different viruses: Toscana virus, Fermo-like virus, Corfou virus, Ponticelli viruses, and two unknown putative viruses. The co-circulation of different phlebovirus reported in this study, indicate a very dynamic and complex situation, which deserves a more detailed investigation to characterize their possible pathogenicity to humans and animals. Highlights • More than 21,000 sand flies, collected in Northern Italy between 2013 and 2016, were tested. • Toscana, Fermo-like, Corfou, Ponticelli, and two unknown viruses were detected. • The three reassortant Ponticelli I, II, and III viruses were isolated. • The potential pathogenicity of these co-circulating viruses must be investigated. [ABSTRACT FROM AUTHOR]

West Nile virus (WNV) and Usutu virus (USUV), genus Flavivirus , are members of the Japanese encephalitis virus antigenic complex, and are maintained primarily in an enzootic cycle between mosquitoes and birds. WNV is zoonotic, and poses a threat to public health, especially in relation to blood transfusion. Serosurveillance of wild birds is suitable for early detection of WNV circulation, although concerns remain to be addressed as regards i) the type of test used, whether ELISA, virus neutralization test (VNT), plaque reduction neutralization test (PRNT), ii) the reagents (antigens, revealing antibodies), iii) the different bird species involved, and iv) potential cross-reactions with other Flaviviruses, such as USUV. The authors developed an indirect IgG ELISA with pan-avian specificity using EDIII protein as antigen and a monoclonal antibody (mAb 1A3) with broad reactivity for avian IgG. A total of 140 serum samples were collected from juvenile European magpies ( Pica pica ) in areas where both WNV and USUV were co-circulating. The samples were then tested using this in-house ELISA and VNT in parallel. Estimation of test accuracy was performed using different Bayesian two latent class models. At a cut-off set at an optical density percentage (OD%) of 15, the ELISA showed a posterior median of diagnostic sensitivity (DSe) of 88% (95%PCI: 73–99%) and a diagnostic specificity (DSp) of 86% (95%PCI: 68–99%). At this cut-off, ELISA and VNT (cut-off 1/10) performances were comparable: DSe = 91% (95%PCI: 79–99%), and DSp = 77% (95%PCI: 59–98%). With the cut-off increased to 30 OD%, the ELISA DSe dropped to 78% (95%PCI: 52–99%), and the DSp rose to 94% (95%PCI: 83–100%). In field conditions, the cut-off that yields the best accuracy for the ELISA appears to correspond to 15 OD%. In areas where other Flaviviruses are circulating, however, it might be appropriate to raise the cut-off to 30 OD% in order to achieve higher specificity and reduce the detection of seropositive birds infected by other Flaviviruses, such as USUV. [ABSTRACT FROM AUTHOR]

In 2011, a group A rotavirus was isolated from the brain of a fox with encephalitis and neurologic signs, detected by rabies surveillance in Italy. Intracerebral inoculation of fox brain homogenates into mice was fatal. Genome sequencing revealed a heterologous rotavirus of avian origin, which could provide a model for investigating rotavirus neurovirulence. [ABSTRACT FROM AUTHOR]

This study evaluated the vector competence of Aedes albopictus in transmitting USUV after oral infection under laboratory conditions. Ae. albopictus showed a low vector competence for USUV, although the positive body sample found with a very high number of viral copies at one week post infection indicates that a replication in the mosquito body can occur, and that USUV can escape the midgut barrier. Field data from an extensive entomological arboviruses surveillance program showed a relevant incidence of Ae. albopictus USUV positive pools in the period 2009–2012 while all pools were negative from 2013 on. No conceivable explanation regarding this field evidence was addressed, suggesting that attention must be paid to the trend of development of this vector-pathogen association, being aware of the potential rapid arbovirus’ adaptation to new vectors, to prevent possible new disease’s emergence. [ABSTRACT FROM AUTHOR]

West Nile virus lineage 2 (WNV-2) was mainly confined to sub-Saharan Africa until the early 2000s, when it was identified for the first time in Central Europe causing outbreaks of human and animal infection. The aim of this study was to reconstruct the origin and dispersion of WNV-2 in Central Europe and Italy on a phylodynamic and phylogeographical basis. To this aim, discrete and continuous space phylogeographical models were applied to a total of 33 newly characterised full-length viral genomes obtained from mosquitoes, birds and humans in Northern Italy in the years 2013–2015 aligned with 64 complete sequences isolated mainly in Europe. The European isolates segregated into two highly significant clades: a small one including three sequences and a large clade including the majority of isolates obtained in Central Europe since 2004. Discrete phylogeographical analysis showed that the most probable location of the root of the largest European clade was in Hungary a mean 12.78 years ago. The European clade bifurcated into two highly supported subclades: one including most of the Central/East European isolates and the other encompassing all of the isolates obtained in Greece. The continuous space phylogeographical analysis of the Italian clade showed that WNV-2 entered Italy in about 2008, probably by crossing the Adriatic sea and reaching a central area of the Po Valley. The epidemic then spread simultaneously eastward, to reach the region of the Po delta in 2013, and westward to the border area between Lombardy and Piedmont in 2014; later, the western strain changed direction southward, and reached the central area of the Po valley once again in 2015. Over a period of about seven years, the virus spread all over an area of northern Italy by following the Po river and its main tributaries. [ABSTRACT FROM AUTHOR]

Usutu virus is an arbovirus closely related to West Nile virus (genus Flavivirus ), which circulates between mosquitoes and wild birds. This virus has been increasingly reported in Europe, raising concerns for its possible pathogenic potential for wild birds and humans. This study reports the whole genome sequences of 15 strains of USUV, isolated between 2010 and 2014 from mosquitoes and wild birds in the course of West Nile virus surveillance in the Emilia-Romagna and Lombardy regions of Northern Italy. Both whole and 656 partial genome sequences, obtained from isolated viruses and field samples (mosquitoes and wild birds), were analyzed to describe the temporal and geographical spread of USUV in the surveyed area. The detected sequences belonged to two groups, with one circulating primarily in the northwestern part of the area and the other in the southeastern part. This pattern is likely the result of different routes of introduction from the North (over the Alps) and from the East, respectively. The phylogenetic analysis of obtained sequences and other European sequences demonstrated that the majority of European strains belonged to one main clade, while less common strains, mainly from Western Europe, fell in other two clades. This analysis strongly suggested an autochthonous evolution process of strains of the main clade from a common ancestor with an estimated time of arrival in Europe at the beginning of the 1990s. In addition to causing mass mortality in wild birds, Usutu virus can infect humans and can sporadically cause disease. These factors and the endemization of the Usutu virus in a large area of Europe, sustained by the obtained data, strongly support the need to adequately survey Usutu virus in areas in which its circulation is detected. [ABSTRACT FROM AUTHOR]

West Nile virus (WNV) is an important zoonotic pathogen maintained in a natural transmission cycle between mosquitoes and birds as reservoir hosts. In dead-end hosts, such as humans, infection may result in fatal neurologic disease translating into disease and death-related suffering and increased health care costs. In humans, WNV may also be transmitted through blood transfusions and organ transplants. WNV is not present in Switzerland yet, but competent vector species (especially Culex pipiens and Aedes japonicus) are prevalent and an introduction of the virus, likely through wild birds, is expected at any time. Therefore, it is important for Switzerland to be prepared and establish a surveillance system for WNV to initiate increased prevention activities, such as the screening of blood and organ donations and public education activities in case virus circulation is detected. The long-term goal of these surveillance measures would be a reduced infection rate in humans resulting in less suffering and reduced health care costs. To provide the basis for a pragmatic and resource-effective WNV surveillance program, this study used aliquots of serum samples of free-range laying hens taken at the abattoir and collected in the frame of the ongoing Swiss Avian Influenza and Newcastle Disease monitoring program for a 2-year period. All 961 aliquots were analyzed using a commercial competitive WNV enzyme-linked immunosorbent assay (ELISA). The study allowed to set up sampling and laboratory routines as a basis for future WNV surveillance activities. At this stage there is no evidence for circulation of WNV in Switzerland. [ABSTRACT FROM AUTHOR]

• Wing shape variation of four anopheles species was describe by UMAP algorithm. • HDBSCAN found 13 morphotypes in An. Daciae sp. inq and 4 in An. maculipennis s.s. • All morphotypes showed a similar pattern of variation in the wing veins. • Shape variability might results from constrains related to wing functionality. Geometric morphometric analysis was combined with two different unsupervised machine learning algorithms, UMAP and HDBSCAN, to visualize morphological differences in wing shape among and within four Anopheles sibling species (An. atroparvus , An. melanoon, An. maculipennis s.s. and An. daciae sp. inq.) of the Maculipennis complex in Northern Italy. Specifically, we evaluated: (1) wing shape variation among and within species; (2) the consistencies between groups of An. maculipennis s.s. and An. daciae sp. inq. identified based on COI sequences and wing shape variability; and (3) the spatial and temporal distribution of different morphotypes. UMAP detected at least 13 main patterns of variation in wing shape among the four analyzed species and mapped intraspecific morphological variations. The relationship between the most abundant COI haplotypes of An. daciae sp. inq. and shape ordination/variation was not significant. However, morphological variation within haplotypes was reported. HDBSCAN also recognized different clusters of morphotypes within An. daciae sp. inq. (12) and An. maculipennis s.s. (4). All morphotypes shared a similar pattern of variation in the subcostal vein, in the anal vein and in the radio-medial cross-vein of the wing. On the contrary, the marginal part of the wings remained unchanged in all clusters of both species. Any spatial-temporal significant difference was observed in the frequency of the identified morphotypes. Our study demonstrated that machine learning algorithms are a useful tool combined with geometric morphometrics and suggest to deepen the analysis of inter and intra specific shape variability to evaluate evolutionary constrains related to wing functionality. [ABSTRACT FROM AUTHOR]

West Nile virus (WNV) is a recently re-emerged health problem in Europe. In Italy, an increasing number of outbreaks of West Nile disease, with occurrences of human cases, have been reported since 2008. This is particularly true in northern Italy, where entomological surveillance systems have been implemented at a regional level. The aim of this study was to use, for the first time, all the entomological data collected in the five regions undergoing surveillance for WNV in northern Italy to characterize the viral circulation (at a spatial and temporal scale), identify potential mosquito vectors, and specify relationships between virus circulation and meteorological conditions. In 2013, 286 sites covering the entire Pianura Padana area were monitored. A total of 757,461 mosquitoes were sampled. Of these, 562,079 were tested by real-time PCR in 9,268 pools, of which 180 (1.9%) were positive for WNV. The largest part of the detected WNV sequences belonged to lineage II, demonstrating that, unlike those in the past, the 2013 outbreak was mainly sustained by this WNV lineage. This surveillance also detected the Usutu virus, a WNV-related flavivirus, in 241 (2.6%) pools. The WNV surveillance systems precisely identified the area affected by the virus and detected the viral circulation approximately two weeks before the occurrence of onset of human cases. Ninety percent of the sampled mosquitoes were Culex pipiens, and 178/180 WNV-positive pools were composed of only this species, suggesting this mosquito is the main WNV vector in northern Italy. A significantly higher abundance of the vector was recorded in the WNV circulation area, which was characterized by warmer and less rainy conditions and greater evapotranspiration compared to the rest of the Pianura Padana, suggesting that areas exposed to these conditions are more suitable for WNV circulation. This observation highlights warmer and less rainy conditions as factors able to enhance WNV circulation and cause virus spillover outside the sylvatic cycle. [ABSTRACT FROM AUTHOR]

The present trial investigated the effect of PCV2 vaccination on viremia, virus shedding and viral load in maternal tissues and foetuses of conventional gilts inseminated with PCV2b-spiked semen. Twelve gilts were randomly divided into two groups of six animals each (vaccinated infected, VI; non-vaccinated infected, NVI). Estrus synchronization was followed by artificial insemination (AI) with a single PCV2 negative semen dose supplemented with 0.2 mL of a PCV2b suspension containing 104.4 TCID50/50 µL (total viral dose: 105 TCID50). Vaginal, nasal and faecal swabs, and blood samples were collected weekly from two days before artificial insemination till the end of the experimental period (55 days post AI; DPAI) and tested by real-time PCR (qPCR) for PCV2; sera were tested for anti- PCV2 antibodies. During necropsy foetal and maternal tissues were collected for qPCR and histopathology. In each of the VI and NVI groups three out of the six gilts were pregnant at 29 DPAI. The VI group showed a significantly lower proportion of PCR-positive swabs: 24.6% VI vs 71.3% NVI. PCV2 clearance was demonstrated by qPCR in lymphoid tissue during the trial in the VI group. Only one foetus was PCV2-positive (in the NVI group) and three amniotic fluids of the NVI group. PCV2 was found in a significantly lower proportion of the placenta of foetuses in the VI group (39%) than the NVI group (77%). The PCV2 vaccine seems to play an active role in reducing virus shedding, tissue viral load and foetoplacental infection. [ABSTRACT FROM AUTHOR]