Porcine teschoviruses (PTVs) are swine-specific picornaviruses associated with neurological and systemic disease, ranging from severe paralytic Teschen disease to milder forms of porcine encephalomyelitis, known as Talfan disease. Despite their global distribution and veterinary relevance, complete genomic information and molecular tools for PTVs are not available. Here, we report the first complete genome sequences of PTV-A11 (strain Dresden) and a recent PTV-A field isolate (strain Gi2020_1). Both genomes, each approximately 7.2 kb in size, include a previously uncharacterized 5'-terminal region upstream of the poly(C) tract (S-segment), thereby elucidating the complete genomic architecture of PTVs for the first time. The conserved 117-118 nucleotide S-segment closes a long-standing gap in teschovirus genomics and is essential for viral replication. Using reverse genetics, we established infectious molecular clones of both strains that recapitulate the properties of their parental strains. These systems enabled functional analyses of viral gene products, including the demonstration that the leader protein is dispensable for genome replication and morphogenesis but contributes to efficient virus growth. In addition, we developed a replication-competent subgenomic replicon and engineered fluorescent reporter viruses, including a stable mCherry-expressing virus that supports robust infection analysis and allows quantitative protein expression measurements. Together, these findings define the complete PTV genome organization and provide a versatile molecular toolbox for studying teschovirus replication, pathogenesis, and control.IMPORTANCETeschen disease was once a devastating neurological disease of swine caused by highly virulent porcine teschovirus strains (PTVs) but has become rare following their eradication. Current control strategies base on hygiene measures and herd-specific vaccination providing limited protection and leaving swine populations vulnerable to the (re-)emergence of neuroinvasive PTVs. Less virulent strains remain endemic worldwide and continue to impair animal health, welfare, and production efficiency. Progress toward broadly effective vaccines and antivirals has been constrained by incomplete knowledge of the PTV genome. Here, we identify and functionally define the previously unrecognized S-segment that completes the 5' UTR of PTVs. We further established infectious cDNA clones and developed subgenomic replicons, leaderless viruses, and fluorescent reporter viruses. Our tools enable direct genetic manipulation of PTVs. They provide a platform for mechanistic studies of viral replication, attenuation, and antigen design supporting rational development of vaccines and antiviral strategies against emerging PTVs.

Bovine viral diarrhea (BVD) is a major cause of economic losses in the global cattle industry, particularly in countries characterized by intensive livestock production systems. Pestivirus tauri, formerly known as Bovine viral diarrhea virus type 2 (BVDV-2), is the current taxonomic designation according to the International Committee on Taxonomy of Viruses (ICTV). Between 2005 and 2018, Pestivirus tauri was detected in cattle herds in mainland Italy, particularly in the Lombardy region. Four viral strains were successfully isolated in cell cultures and subjected to whole-genome sequencing. Phylogenetic reconstruction placed all Italian isolates within the Pestivirus tauri subgenotype c, a lineage encompassing strains reported in Asia, Europe and the United States. Consistently, comparative sequence identity analyses indicated the highest similarity with the Parker strain (USA, 1991) and the Potsdam 1600 strain (Germany, 2000). These results contribute to a more detailed understanding of Pestivirus tauri genomic architecture and evolutionary dynamics, providing a valuable resource for comparative genomic studies. Such data are crucial for exploring viral diversity and evolution, optimizing the design of diagnostic primers and probes, and advancing insights into the molecular epidemiology of Pestivirus.

The genus Pestivirus includes several species responsible for economically important diseases in domestic and wild animals. In 2017, a novel ovine pestivirus (ovIT PeV) was isolated from aborted lamb fetuses and molecular analysis showed a genetic distinction from known Pestivirus species with the closest phylogenetic relationship to classical swine fever virus (CSFV, Pestivirus suis). This close similarity is concerning because CSF is a notifiable disease to the World Organization for Animal Health (WOAH) and is subject to eradication campaigns. Moreover, ovIT PeV can infect pigs and induce an early strong immune response that can cause false-positive results in CSF antibody ELISAs used for CSF surveillance. Our study aimed to develop an indirect ELISA (iELISA) to serologically distinguish ovIT PeV from CSFV in pigs. Hence, we expressed the recombinant E2 glycoproteins of ovIT PeV and CSFV (strain Diepholz) in mammalian cells. Two E2 iELISAs were evaluated using a panel of CSFV, ovIT PeV and negative pig sera. Each serum showed higher antibody titres in the iELISA with the homologous antigen rather than the heterologous one. This study highlighted the effectiveness of using virus-specific antigens and comparing antibody titres when assessing infections with serologically cross-reactive viruses.

Thanks to its ethological and physiological characteristics, the hedgehog is a synanthropic species of particular importance for the maintenance and possible spread of pathogens, some of which are zoonotic. Among these, we can include the mammalian orthoreovirus (MRV), which is characterized by respiratory, gastrointestinal, and neurological symptoms in both animals and humans. MRV is characterized by a high capacity for genetic reassortment and intragenic rearrangement, and the ability to infect a wide range of mammals. This work aims to investigate the presence of MRVs and its genomic characterization in hedgehogs. During the two-year period from 2022 to 2023, the intestine and lungs were collected from 293 hedgehogs and subjected to real-time PCR to detect the L1 gene. Positive samples were subjected to a typing RT-PCR targeting a portion of the S1 gene and then to sequencing. A total of 38 hedgehogs tested positive by real-time PCR (p = 13%). Typing RT-PCR demonstrated the positivity of 25 samples for serotype 3. Four samples, representative of the main groups recognized during the phylogenetic analysis, underwent whole genome sequencing, revealing the presence of reassortment phenomena between strains related to bats, chamois, and human MRVs.

Porcine Respirovirus 1 (PRV1) is an enveloped, single-stranded, negative-sense RNA virus belonging to the genus Respirovirus within the Paramyxoviridae family. Since its first detection in China in 2013, PRV1 has been identified in several American and European countries. Although its pathogenicity is uncertain, recent studies have suggested that it may play a role in the Porcine Respiratory Disease Complex (PRDC) because of its capacity to replicate in the upper and lower respiratory tracts. This study aimed to determine the spread of PRV1 in Northern Italy and the phylogeny of the isolates. Therefore, PRV1 was investigated using real-time RT-PCR in 902 samples collected from September 2022 to September 2023 from pigs with respiratory symptoms in North Italy. Fourteen (1.55%) samples tested as PRV1-positive. The full-length fusion (F) gene, which codifies for a major surface protein, was amplified and used for phylogenetic analysis to help carry out molecular epidemiological studies on this virus. In addition, swine influenza virus (SIV) and porcine reproductive and respiratory syndrome virus (PRRSV) infections were detected in most of the PRV1-positive samples. In conclusion, we report the detection of PRV1 in Italy and discuss its potential role as a co-factor in causing the Porcine Respiratory Disease Complex.

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.

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.

Intro: Coronaviruses infect humans and a wide range of wild and domestic animals. Some CoVs could be zoonotic, being able to mutate, crossing the species barrier and infecting humans (e.g. SARS-CoV and MERS-CoV). Since the emergence of SARS-CoV-2, several studies were carried out to ascertain the susceptibility of both domestic and wild animals to SARS-CoV-2. However, information on some species is lacking, and for others only RDB-ACE receptor affinity studies have been carried out. Considering the high densities of Marmota marmota in the alpine environment, where livestock and recreational activities are commonly present, this study aims to investigate the presence and characterization of CoVs in this species. Methods: During provincial relocation plan carried out in 2021 and 2022, 170 alpine marmots were captured in municipality of Livigno in Sondrio province (North-Italy) for decreasing animal density and, after a quarantine period, they were released in other alpine places. Faecal samples were collected from each animal and then subjected to RNA extraction and nested RT-PCR pan-Coronavirus and real time RT-PCR for SARS-CoV-2. PCR positive samples for pan-CoV were then sequenced. Findings: The pan-Coronavirus RT-PCR detected CoVs in seven marmots. The CoV sequence originating from one marmot sampled in 2021 had 97% affinity to strains isolated in lagomorphs. The other six sequences from 2022 were highly correlate with Bovine Beta-CoVs. This could be explained by the fact that marmots share alpine pastures with these species; in fact, the trapping area in 2022 represented grazing and forage production areas. All samples tested for SARS-CoV-2 resulted negative. Conclusion: Despite the absence of zoonotic coronaviruses, marmots show high plasticity in harbouring CoVs of sympatric species. For this reason, and considering the affinity of their ACE-receptor demonstrated for SARS-CoV, it would be worthwhile to increase surveillance for CoVs in this species.

Intro: Several rodents, including mice and the brown rat, are synanthropic animals usually found in rural and urban environments in contact with other animals and humans. Rodents are natural reservoirs of infectious agents and could harbour a plethora of zoonotic pathogens of public health importance. Taking advantage of a parallel study on presence and distribution of Hantaviruses, we aimed to investigate the occurrence in mice of other viruses with zoonotic or economic impact. Methods: From May to July 2022, 41 mice (Mus domesticus) were captured and killed by using baited snap traps in 13 selected cattle, goat and poultry farms located in the Piedmont region. Gut and lung samples were homogenised and tested by PCR methods for pan-Coronavirus (CoV) and SARS-CoV-2, pan-Pestivirus, Mammalian orthoreoviruses, Canine Distemper virus (CDV), Flaviviruses, Influenza A (IAV) and D (IDV) viruses. Findings: All captured animals did not present at necropsy lesions related to infectious diseases. Virological investigations detected the presence of CoV in six mice. By sequencing Rodent CoVs was identified in two samples (four more pending). Mammalian orthoreovirus was detected in nine animals and typing and characterization are in progress. One mouse, captured in a bovine farm, tested slightly positive for IDV and confirmation of positivity is in progress by complete sequencing with NGS approach. All samples were negative for Flaviviruses, IAV, CDV, pan-Pestivirus and SARS-CoV-2. Conclusion: Rodents are well adapted to a wide range of habitats, including peri-urban and rural environments, where they benefit from human activities. These results, although preliminary, underline the importance of enhancing surveillance in rodents in anthropized areas to better assess the presence of zoonotic agents and the potential risk of transmission.

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.

In Italy, the West Nile Virus surveillance plan considers a multidisciplinary approach to identify the presence of the virus in the environment (entomological, ornithological, and equine surveillance) and to determine the risk of infections through potentially infected donors (blood and organ donors). The costs associated with the surveillance program for the Lombardy Region between 2014 and 2018 were estimated. The costs of the program were compared with a scenario in which the program was not implemented, requiring individual blood donation nucleic acid amplification tests (NAT) to detect the presence of WNV in human samples throughout the seasonal period of vector presence. Considering the five-year period, the application of the environmental/veterinary surveillance program allowed a reduction in costs incurred in the Lombardy Region of 7.7 million EUR. An integrated surveillance system, including birds, mosquito vectors, and dead-end hosts such as horses and humans, can prevent viral transmission to the human population, as well as anticipate the detection of WNV using NAT in blood and organ donors. The surveillance program within a One Health context has given the possibility to both document the expansion of the endemic area of WNV in northern Italy and avoid most of the NAT-related costs.

Canine distemper virus (CDV) is a fatal, highly contagious disease found in wild and domestic carnivores. Several outbreaks have occurred in wildlife in Italy in recent years. This study aims to detect CDV in wildlife following the increasing mortality of foxes (Vulpes vulpes) in the Emilia-Romagna region (northern Italy) observed in 2021. Sixty-seven foxes and one badger (Meles meles) were subjected to necropsy followed by histological examination and were analyzed with molecular techniques to detect the presence of CDV. Of the tested animals, 16% (nine foxes and one badger) were positive for CDV. Phylogenetic analysis showed two different lineages based on complete H gene sequences. The Europe/South America-1 lineage was detected in one fox from Modena, which resembled the CDV variant associated with a previous outbreak in northern Italy in 2018, while the European Wildlife lineage was detected in animals from the Rimini province. Amino acid analysis highlighted a Y549H mutation in all sequences collected, which is commonly associated with increased virulence.

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.

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.

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.

Canine distemper (CD) is a fatal, highly contagious disease of wild and domestic carnivores. In the Alpine territory, several outbreaks have occurred in the past few decades within wild populations. This study investigated the presence of canine distemper virus (CDV) infections in wild carnivores in Lombardy, relating to the different circulating genotypes. From 2018 to 2020, foxes, badgers, and martens collected during passive surveillance were subjected to necropsy and histological examination, showing classical signs and microscopic lesions related to CDV. Pools of viscera from each animal were analysed by molecular methods and immunoelectron microscopy. Total prevalences of 39.7%, 52.6%, and 14.3% were recorded in foxes, badgers, and stone martens, respectively. A phylogenetic analysis showed that the sequences obtained belonged to the European 1 lineage and were divided into two different clades (a and b) according to the geographical conformation of alpine valleys included in the study. Clade a was related to the European outbreaks originating from Germany in 2006–2010, while clade b was closely related to the CDV sequences originating from northeastern Italy during the 2011–2018 epidemic wave. Our results suggest that CDV is currently well adapted to wild carnivores, mostly circulating with subclinical manifestations and without severe impact on the dynamics of these populations.

Wild birds play an important role in the circulation and spread of pathogens that are potentially zoonotic or of high economic impact on zootechnical production. They include, for example, West Nile virus (WNV), Usutu virus (USUV), avian influenza virus (AIV), and Newcastle disease virus (NDV), which, despite having mostly an asymptomatic course in wild birds, have a strong impact on public health and zootechnical production. This study investigated the presence of these viruses in several wild bird species from North Italy during the biennium 2019–2020. Wild birds derived from 76 different species belonging to 20 orders. Out of 679 birds, 27 were positive for WNV (lineage 2) with a prevalence of 4%; all birds were negative for USUV; one gull was positive for H13N6 influenza virus, and 12 samples were positive for NDV with a prevalence of 2%. Despite the low prevalence observed, the analyses performed on these species provide further data, allowing a better understanding of the diffusion and evolution of diseases of both economic and zoonotic importance.

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.

This study describes an Aujeszky's disease case in an adult male red fox found in an urban area in Central Italy, that exhibited a fatal infection with neurological lesions, but neither itching nor skin lesions. Diagnostic examinations included histology, and parasitological, bacteriological and virological analyses. Detection of parasitic enteric pathogens, bacteria, E. coli, Leptospira spp., rabies, canine distemper virus, parvovirus, hepatitis E virus and pseudorabies virus (PrV) was performed. Results showed the presence of a gE-deleted PrVthat was closely related to the NIA-3 strain but differed from the PrV strains currently circulating in wild boars and domestic pigs in Italy. All the results led to the conclusion that the fox suffered from Aujeszky's disease caused by a gE-deleted PrV strain closely related to a vaccine strain. The epidemiological link between the PrV vaccine strain and fox infection remains unclear. It could involve vaccinated pigs as a primary source of infection by direct or indirect contact with the red fox or less likely it could be related to improper use of the vaccine in the fox.

This study shows the origin and the pathogenic role of a novel ovine pestivirus (OVPV) isolated in 2017 in Italy, as a pathogenic agent causing severe abortions after infection in pregnant ewes and high capacity for virus trans-placental transmission as well as the birth of lambs suffering OVPV-persistent infection. The OVPV infection induced early antibody response detected by the specific ELISA against classical swine fever virus (CSFV), another important virus affecting swine. The neutralizing antibody response were similar against CSFV strains from genotype 2 and the OVPV. These viruses showed high identity in the B/C domain of the E2-glycoprotein. Close molecular diagnostics cross-reactivity between CSFV and OVPV was found and a new OVPV molecular assay was developed. The phylodynamic analysis showed that CSFV seems to have emerged as the result of an inter-species jump of Tunisian sheep virus (TSV) from sheep to pigs. The OVPV and the CSFV share the TSV as a common ancestor, emerging around 300 years ago. This suggests that the differentiation of TSV into two dangerous new viruses for animal health (CSFV and OVPV) was likely favored by human intervention for the close housing of multiple species for intensive livestock production.