Divergent minute virus of canines strains identified in illegally imported puppies in Italy.

Minute virus of canines (MVC) belongs to the family Parvoviridae, genus Bocaparvovirus, and has been mainly described during enteritis episodes in young dogs. This study reports the characterization of four divergent MVC strains detected between 2012 and 2018, three of which were from dogs illegally imported into Italy, most probably from Eastern Europe, that cluster together phylogenetically but share low genetic similarity with the fourth MVC from an autochthonous dog and other available MVC sequences. Our data indicate that the introduction of genetically distinct MVC strains occurred through the illegal movement of dogs from a geographic area where a distinct MVC lineage was most likely circulating. Enforced surveillance of MVC in the dog population of Eastern Europe and its neighboring countries may shed light on, and eventually trace back to, illegal animal movements.

First identification of mammalian orthoreovirus type 3 in diarrheic pigs in Europe.

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.

Biological Containment for African Swine Fever (ASF) Laboratories and Animal Facilities: The Italian Challenge in Bridging the Present Regulatory Gap and Enhancing Biosafety and Biosecurity Measures.

The African Swine Fever Virus (ASFV) is a DNA virus of the Asfarviridae family, Asfivirus genus. It is responsible for massive losses in pig populations and drastic direct and indirect economic impacts. The ever-growing handling of ASFV pathological material in laboratories, necessary for either diagnostic or research activities, requires particular attention to avoid accidental virus release from laboratories and its detrimental economic and environmental effects. Recently, the Commission Delegated Regulation (EU) 2020/689 of 17 December 2019 repealed the Commission Decision of 26 May 2003 reporting an ASF diagnostic manual (2003/422/EC) with the minimum and supplementary requirements for ASF laboratories. This decision generated a regulatory gap that has not been addressed yet. This paper aims to describe the Italian National Reference Laboratory (NRL) efforts to develop an effective and reliable biological containment tool for ASF laboratories and animal facilities. The tool consists of comprehensive and harmonized structural and procedural requirements for ASF laboratories and animal facilities that have been developed based on both current and repealed legislation, further entailing a risk assessment and internal audit as indispensable tools to design, adjust, and improve biological containment measures.

Circulation of small ruminant lentivirus in endangered goat and sheep breeds of Southern Italy.

According to the Domestic Animal Diversity Information System (DAD-IS) of the FAO, Italy has one of the largest numbers of local small ruminant breeds among European countries. In Southern Italy, namely the Campania Region, Bagnolese and Laticauda sheep breeds and Cilentana goat breeds are considered endangered according to the DAD-IS. Conservation of endangered animal breeds is a goal of the European Union (EU). However, the role of infectious diseases as risk factors for endangered breeds has rarely been considered. Small ruminant lentiviruses (SRLV) infect sheep and goats, causing slow-progressive, persistent, and debilitating diseases that can lead to animal death and productivity loss. In this study, we investigated the presence of SRLV in Bagnolese, Laticauda, and Cilentana breeds using a commercial ELISA in parallel with an in-house ELISA. The results of the two tests were in good agreement (Cohen Kappa 0.84, 95 % CI = 0.76-0.93). Discrepancies between the two tests were resolved using western blotting. In total, 430 samples were tested (248 Bagnolese, 125 Laticauda, and 57 Cilentana). The apparent prevalence rates were 12.5 %, 6.4 %, and 1.7 % in Bagnolese, Laticauda, and Cilentana, respectively. In the molecular analysis of 11 proviral partial sequences, subtypes B2 and A24 were identified in two Bagnolese herds. Owing to the beneficial role of sheep and goat breeding in marginal areas, it is important to screen the entire population and implement control/eradication of SRLV infections in conjunction with each conservation program.

Swine Norovirus: Past, Present, and Future.

Norovirus, an ssRNA + virus of the family Caliciviridae, is a leading disease burden in humans worldwide, causing an estimated 600 million cases of acute gastroenteritis every year. Since the discovery of norovirus in the faeces of swine in Japan in the 1990s, swine norovirus has been reported in several countries on several continents. The identification of the human-associated GII.4 genotype in swine has raised questions about this animal species as a reservoir of norovirus with zoonotic potential, even if species-specific P-types are usually detected in swine. This review summarises the available data regarding the geographic distribution of norovirus in swine, the years of detection, the genotype characterisation, and the prevalence in specific production groups. Furthermore, we discuss the major bottlenecks for the detection and characterisation of swine noroviruses.

Disinfectants against African Swine Fever: An Updated Review.

African Swine Fever (ASF), a hemorrhagic disease with a high mortality rate in suids, is transmitted via direct and indirect contact with infectious animals and contaminated fomites, respectively. ASF reached Europe in 2014, affecting 14 of the 27 EU countries including, recently, the Italian peninsula. The fast and unprecedented spread of ASF in the EU has highlighted gaps in knowledge regarding transmission mechanisms. Fomites, such as contaminated clothing and footwear, farming tools, equipment and vehicles have been widely reported in the spread of ASF. The absence of available vaccines renders biosecurity measures, cleaning and disinfection procedures an essential control tool, to a greater degree than the others, for the prevention of primary and secondary introductions of ASF in pig farms. In this review, available data on the virucidal activity of chemical compounds as disinfectants against the ASF virus (ASFV) are summarized together with laboratory methods adopted to assess the virucidal activity.

Molecular Investigation of Recent Canine Parvovirus-2 (CPV-2) in Italy Revealed Distinct Clustering.

Canine parvovirus Type 2 (CPV-2) is a worldwide distributed virus considered the major cause of viral gastroenteritis in dogs. Studies on Italian CPV-2 are restricted to viruses circulating until 2017. Only one study provided more updated information on CPV-2 but was limited to the Sicily region. No information regarding the circulation and genetic characteristics of CPV-2 in Northeast Italy has been made available since 2015. The present study investigated the genetic characteristics of CPV-2 circulating in the dog population of Northeast Italy between 2013 and 2019. The VP2 gene of 67 CPV-2 was sequenced, and phylogenetic analysis was performed to identify patterns of distribution. Phylogenetic and molecular analysis highlighted unique characteristics of Northeast Italian CPV-2 and interestingly depicted typical genetic clustering of the Italian CPV-2 strains, showing the existence of distinct CPV-2 genetic groups. Such analysis provided insights into the origin of some Italian CPV-2 genetic clusters, revealing potential introductions from East European countries and the spread of CPV-2 from South/Central to North Italy. This is the first report that describes the genetic characteristics of recent Italian CPV-2. Tracking the genetic characteristics of CPV-2 nationally and globally may have impact on understanding the evolution and distribution of CPV-2, in particular in light of the current humanitarian emergency involving Ukraine, with the massive and uncontrolled movement of people and pet animals.

Carnivore protoparvovirus 1 (CPV-2 and FPV) Circulating in Wild Carnivores and in Puppies Illegally Imported into North-Eastern Italy.

The illegal trade of animals poses several health issues to the global community, among which are the underestimated risk for spillover infection and the potential for an epizootic in both wildlife and domestic naïve populations. We herein describe the genetic and antigenic characterization of viruses of the specie Carnivore protoparvovirus 1 detected at high prevalence in puppies illegally introduced in North Eastern Italy and compared them with those circulating in wild carnivores from the same area. We found evidence of a wide diversity of canine parvoviruses (CPV-2) belonging to different antigenic types in illegally imported pups. In wildlife, we found a high circulation of feline parvovirus (FPV) in golden jackals and badgers, whereas CPV-2 was observed in one wolf only. Although supporting a possible spillover event, the low representation of wolf samples in the present study prevented us from inferring the origin, prevalence and viral diversity of the viruses circulating in this species. Therefore, we suggest performing more thorough investigations before excluding endemic CPV-2 circulation in this species.

Genetic Variability among Swine Influenza Viruses in Italy: Data Analysis of the Period 2017-2020.

Swine play an important role in the ecology of influenza A viruses (IAVs), acting as mixing vessels. Swine (sw) IAVs of H1N1 (including H1N1pdm09), H3N2, and H1N2 subtypes are enzootic in pigs globally, with different geographic distributions. This study investigated the genetic diversity of swIAVs detected during passive surveillance of pig farms in Northern Italy between 2017 and 2020. A total of 672 samples, IAV-positive according to RT-PCR, were subtyped by multiplex RT-PCR. A selection of strains was fully sequenced. High genotypic diversity was detected among the H1N1 and H1N2 strains, while the H3N2 strains showed a stable genetic pattern. The hemagglutinin of the H1Nx swIAVs belonged to HA-1A, HA-1B, and HA-1C lineages. Increasing variability was found in HA-1C strains with the circulation of HA-1C.2, HA-1C.2.1 and HA-1C.2.2 sublineages. Amino acid deletions in the HA-1C receptor binding site were observed and antigenic drift was confirmed. HA-1B strains were mostly represented by the Δ146-147 Italian lineage HA-1B.1.2.2, in combination with the 1990s human-derived NA gene. One antigenic variant cluster in HA-1A strains was identified in 2020. SwIAV circulation in pigs must be monitored continuously since the IAVs' evolution could generate strains with zoonotic potential.

A proof-of-principle study to identify suitable vaccine seed candidates to combat introductions of Eurasian lineage H5 and H7 subtype avian influenza viruses.

Vaccination against avian influenza (AI) is now included amongst the prevention and control measures recommended by international animal health organizations to combat the disease in poultry. For optimal control of human influenza infections, the antigenic variability within subtypes requires the annual update of seed strains for inclusion in vaccines. The decisions taken are based on serological cross-reactivity of viral strains measured by haemagglutination inhibition (HI) tests. The reason for this is to ensure that the vaccine contains strains that are related antigenically to the current circulating field strain as field viruses evolve or are substituted by variants of distinct antigenicity. Such an annual approach is not viable economically for the poultry industry. In the current study, we have applied a similar HI-based approach to demonstrate, as proof of principle, that cross-reactive strains can be identified. Applying the same approach used by the World Health Organization to investigate antigenic differences among human influenza viruses, we assessed the serological cross-reactivity of a selection of natural H5 and H7 subtype viruses. Analysing HI data, we have identified strains that are cross-reactive and may have the potential to act as seed viruses for future vaccine development. This study should be considered a starting point for a more informed approach to the selection of seed strains for the development of avian influenza vaccines against field infections caused by viruses of H5 and H7 subtypes.

Interface between Bats and Pigs in Heavy Pig Production.

Bats are often claimed to be a major source for future viral epidemics, as they are associated with several viruses with zoonotic potential. Here we describe the presence and biodiversity of bats associated with intensive pig farms devoted to the production of heavy pigs in northern Italy. Since chiropters or signs of their presence were not found within animal shelters in our study area, we suggest that fecal viruses with high environmental resistance have the highest likelihood for spillover through indirect transmission. In turn, we investigated the circulation of mammalian orthoreoviruses (MRVs), coronaviruses (CoVs) and astroviruses (AstVs) in pigs and bats sharing the same environment. Results of our preliminary study did not show any bat virus in pigs suggesting that spillover from these animals is rare. However, several AstVs, CoVs and MRVs circulated undetected in pigs. Among those, one MRV was a reassortant strain carrying viral genes likely acquired from bats. On the other hand, we found a swine AstV and a MRV strain carrying swine genes in bat guano, indicating that viral exchange at the bat-pig interface might occur more frequently from pigs to bats rather than the other way around. Considering the indoor farming system as the most common system in the European Union (EU), preventive measures should focus on biosecurity rather than displacement of bats, which are protected throughout the EU and provide critical ecosystem services for rural settings.

Unexpected Genetic Diversity of Two Novel Swine MRVs in Italy.

Mammalian Orthoreoviruses (MRV) are segmented dsRNA viruses in the family Reoviridae. MRVs infect mammals and cause asymptomatic respiratory, gastro-enteric and, rarely, encephalic infections. MRVs are divided into at least three serotypes: MRV1, MRV2 and MRV3. In Europe, swine MRV (swMRV) was first isolated in Austria in 1998 and subsequently reported more than fifteen years later in Italy. In the present study, we characterized two novel reassortant swMRVs identified in one same Italian farm over two years. The two viruses shared the same genetic backbone but showed evidence of reassortment in the S1, S4, M2 segments and were therefore classified into two serotypes: MRV3 in 2016 and MRV2 in 2018. A genetic relation to pig, bat and human MRVs and other unknown sources was identified. A considerable genetic diversity was observed in the Italian MRV3 and MRV2 compared to other available swMRVs. The S1 protein presented unique amino acid signatures in both swMRVs, with unexpected frequencies for MRV2. The remaining genes formed distinct and novel genetic groups that revealed a geographically related evolution of swMRVs in Italy. This is the first report of the complete molecular characterization of novel reassortant swMRVs in Italy and Europe, which suggests a greater genetic diversity of swMRVs never identified before.

Identification of two divergent swine Noroviruses detected at the slaughterhouse in North East Italy.

Norovirus (NoV) has emerged as one of the major causative agents of non-bacterial, food- and water-borne gastroenteritis in humans, with the main genogroup involved in human outbreaks (GII), which has been detected worldwide in different animal species including swine. A four-month investigation at the slaughterhouse aiming to examine the presence of NoV in the swine in North-Eastern Italy, enabled the detection of two divergent Noroviruses (NoVs) (GII.P11) in two swine farms. This represents the first study in the swine population of North-Eastern Italy, which has paved the way for future integrated virological and epidemiological investigations on swine NoVs.

Replication of Influenza D Viruses of Bovine and Swine Origin in Ovine Respiratory Explants and Their Attachment to the Respiratory Tract of Bovine, Sheep, Goat, Horse, and Swine.

Bovine is considered the main reservoir of influenza D virus (IDV), however, low levels of seropositivity in other farmed species suggest a wide range of potential hosts. Nevertheless, it is not clear whether this scenario is the result of rare spillover events upon contact with bovines, or a lack of adaptation of IDV to these hosts. Among these species, sheep represents a crucial component of the rural economy in many developing countries, but little is known about its role in the ecology of the disease. To evaluate the susceptibility of sheep to IDV viruses of different origin, we used ovine respiratory tissues as an ex vivo model and investigated the infective phenotype of two IDV strains isolated from either bovine (IDV-BOV) or swine (IDV-SW). For translatability purposes, we included a parainfluenza type 3 virus, as positive control, given its known respiratory tropism in sheep. We performed a timed evaluation of the viral infectivity, cell tropism and the associated histopathology, by means of tissue culture infectious dose assays on supernatants and histological/immunohistochemical analyses on explanted tissues, respectively. To further investigate differences in the phenotype of these two strains and to identify the potential targets of replication in the most commonly land-based farmed mammalian species, we carried out virus binding assays on histological sections of the respiratory tract of bovine, caprine, ovine, horse and swine. Our results demonstrated that IDV successfully replicates in nasal, tracheal and lung ovine tissues, suggesting a moderate susceptibility of this species to IDV infection. Interestingly, despite the high genetic identity of these strains, IDV- BOV consistently replicated to higher titers than IDV-SW in all respiratory tracts, suggesting IDV viruses might display considerable levels of variability in their phenotype when crossing the species barrier. Virus binding assays confirmed a superior affinity of the IDV viruses for the bovine upper respiratory tract, and a preference for the pharyngeal epithelium of small ruminants, indicating possible targets to improve the sensitivity of virological sampling for diagnostic and post-mortem purposes. Further pathogenesis and cross-species transmission studies will be necessary to elucidate the ecology of IDV and eventually allow the design of cost-effective surveillance strategies.

Avian influenza viruses in poultry products: a review.

The extensive circulation of the H5N1 highly pathogenic avian influenza virus, and the human health threat that it poses, has raised concerns over the food safety implications of this virus infecting poultry. In addition, among the most important risk factors for the possible emergence of avian influenza in the European Union and the United States, the European and Food Safety Agency and the US Department of Agriculture's Animal and Plant Health Inspection Service, respectively, have identified legal and illegal importations of infected poultry commodities. The present paper reviews existing knowledge on the presence of viable avian influenza viruses in poultry commodities.

Unexpected heat resistance of Italian low-pathogenicity and high-pathogenicity avian influenza A viruses of H7 subtype to prolonged exposure at 37 degrees C.

The increased attention of the international community to the occurrence of repeated outbreaks of avian influenza infections worldwide has highlighted several knowledge gaps in the field. Among these, within the scope of the European Union-funded project Fluresist, we addressed the resistance of selected H7 subtype strains at 37 degrees C. In general terms, resistance was high, although some strains were more resistant than others, remaining viable after 15 days at 37 degrees C. These results should be considered when designing guidelines for outbreak management.

First whole genome characterization of porcine astrovirus detected in swine faeces in Italy.

Porcine astroviruses (PoAstV) are found in the gastrointestinal tract of healthy and diseased pigs worldwide. However, their role in causing enteric disease in pigs and other animals has not been elucidated. In the present report, we describe for the first time in Italy, the identification and genetic characterization, through whole genome sequencing, of a PoAstV2 in pigs in Northeast Italy in 2015. This instance is the first detection of PoAstV2 in pigs in Italy. The phylogenetic analysis of the complete ORF2 segment highlights the high similarity of this virus to those circulating that same year in Japan. There are very few full astrovirus genomes available, and the present data represent an important contribution towards a better understanding of the characteristics and evolution of these viruses.

Pathogenicity of a QX strain of infectious bronchitis virus in specific pathogen free and commercial broiler chickens, and evaluation of protection induced by a vaccination programme based on the Ma5 and 4/91 serotypes.

The aims of this study were firstly to evaluate the pathogenicity of an Italian isolate of the QX strain of infectious bronchitis (IB) virus using 1-day-old female specific pathogen free chicks (layer type) and 1-day-old female commercial broiler type chickens, and secondly to assess the level of protection induced in these birds by a vaccination programme including the IB Massachusetts and 4/91 serotype live attenuated vaccines. Unvaccinated birds showed clinical signs of varying severity, predominantly affecting the upper respiratory tract. Vaccinated birds appeared healthy, with the exception of a very mild conjunctivitis affecting a limited number of the broilers. Vaccination fully protected specific pathogen free birds, since no histopathological lesions were observed, nor was virus detected following challenge. In broilers, replication of the challenge virus was not prevented but was significantly reduced. This study confirms that vaccination at 1 day old and at 14 days of age using the Ma5 and 4/91 IB vaccines may be instrumental in reducing the economic impact of QX IB virus infections in layer and broiler farms.

Identification and genetic characterization of bovine enterovirus by combination of two next generation sequencing platforms.

Prompt and accurate diagnosis is warranted for infectious diseases of domestic animals which may have a significant impact on animal production or clinical practice. In this study, the identification and genetic characterization of a bovine enterovirus (BEV) strain isolated from a calf with diarrhea, are described. Two different next generation sequencing platforms were employed. Shotgun metagenomic accomplished by MinION sequencing (Oxford Nanopore Technologies) allowed the identification of BEV RNA from a cell-culture isolate. BEV was then confirmed by a specific real time RT-PCR assay. To achieve the whole genome of this isolate, sequence reads obtained by MinION were coupled with those originating from NextSeq500 (Illumina). Genomic relatedness and phylogeny with extant BEV strains is also reported. Overall, this manuscript highlights the use of the portable MinION sequence technology as a tool for support diagnostics in veterinary practice.