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The first report of African swine fever virus (ASFV) genotype II in Italy in 2022 marked the beginning of a significant invasion in at least eight Italian regions with different infection clusters. In this study, we used the multi-gene approach to investigate the epidemiological associations between ASFV strains causing cases and outbreaks in wild boar and pigs in Italy from January 2022 to the end of 2023. Our results confirm that all the tested ASFV-positive Italian samples belonged to genotype II and show high homology with genotype II ASFV sequences previously collected in Eurasian countries. Molecular characterization revealed the presence of four genetic groups in Italy. The majority of African swine fever (ASF) samples analyzed in the current study (72%) belonged to genetic group 3, which was the most representative in Europe. The results also provide evidence of the prevalence of genetic group 19 (15.9%). In addition, we identified new putative genetic groups, genetic group 25 (9.1%) and genetic group 26 (3.0%), which have never been described before. This is the first detailed report on the molecular characterization of more than 130 ASFV strains circulating in Italy.

African swine fever virus (ASFV) causes a devastating disease affecting domestic and wild pigs. ASF was first introduced in Sardinia in 1978 and until 2019 only genotype I isolates were identified. A remarkable genetic stability of Sardinian ASFV isolates was described, nevertheless in 2019 two wild boar isolates with a sustained genomic deletion (4342 base pairs) were identified (7303WB/19, 7212WB/19). In this study, we therefore performed in vitro experiments with monocyte-derived macrophages (moMФ) to unravel the phenotypic characteristics of these deleted viruses. Both 7303WB/19 and 7212WB/19 presented a lower growth kinetic in moMФ compared to virulent Sardinian 26544/OG10, using either a high (1) or a low (0.01) multiplicity of infection (MOI). In addition, flow cytometric analysis showed that both 7303WB/19 and 7212WB/19 presented lower intracellular levels of both early and late ASFV proteins. We subsequently investigated whether deleted virus variants were previously circulating in wild boars in Sardinia. In the four years preceding the last genotype I isolation (February 2015-January 2019), other eight wild boar isolates were collected, all belonging to p72 genotype I, B602L subgroup X, but none of them presented a sustained genomic deletion. Overall, we observed the deleted virus isolates in Sardinia only in 2019, at the end of a strong eradication campaign, and our data suggest that it might possess an attenuated phenotype in vivo. A better understanding of ASFV evolution in endemic territories might contribute to development of effective control measures against ASF., (© 2024. The Author(s).)

African swine fever (ASF) is a disease that is a growing threat to the global swine industry. Regulations and restrictions are placed on swine movement to limit the spread of the virus. However, these are costly and time-consuming. Therefore, this study aimed to determine if high-pressure processing (HPP) sanitization techniques would be effective against the ASF virus. Here, it was hypothesized that HPP could inactivate or reduce ASF virus infectivity in tissue homogenates. To test this hypothesis, 30 aliquots of each homogenate (spleen, kidney, loin) were challenge-infected with the Turin/83 strain of ASF, at a 10 7.20 median hemadsorption dose (HAD) 50 /mL. Subsequently, eight aliquots of each homogenate were treated with 600 millipascal (600 MPa) HPP for 3, 5, and 7 min. Six untreated aliquots were used as the controls. Virological results showed a reduction in the viral titer of more than 7-log. These results support the validity of the study hypothesis since HPP treatment was effective in inactivating ASFV in artificially prepared samples. Overall, this study suggests the need for further investigation of other ASFV-contaminated meat products., Competing Interests: The authors declare no conflicts of interest.

African swine fever (ASF) is a contagious disease of wild boar and domestic pigs notifiable to the World Organisation for Animal Health due to its high socio-economic impact. ASF is caused by the complex ASF virus (ASFV), and it can present different clinical manifestations that can be confused with other diseases; for this reason, laboratory testing is necessary for the proper diagnosis of clinically suspected animals. Despite the efforts put into it over decades, no treatment or safe vaccine is globally available, and disease control is based on early diagnosis and the implementation of strict biosecurity measures. In this context, rapid tests have the potential to accelerate and facilitate the identification of infected animals by giving fast on-site results. In this work, we improved the available point-of-care assays for the diagnosis of the disease by the development of a more specific antigen test and a more sensitive antibody test. This antibody detection test allowed for the earlier detection of infected animals than two commercial indirect ELISAs (statistically significant). Moreover, we developed a combined dual rapid test, unifying, in the same cassette, an antigen detection strip and an antibody detection strip. In this study, we confirmed that this combo approach is a useful tool for implementing rapid tests in the field since it increases the percentage of positive samples detected, even when PCR turns negative, while maintaining a good specificity.

Seronegative latent carriers (SNLCs) are animals that carry the virus without detectable antibodies and pose a risk for disease transmission and diagnostic challenges, suggesting the importance of consideration of marker vaccines in managing them. Therefore, in this study, we evaluated two modified live infectious bovine rhinotracheitis (IBR) marker vaccines (single and double deletions) for their ability to generate SNLC calves. These vaccines were administered to four groups (n = 3 in each group) of three-month-old calves in the presence or absence of passive immunity. Three hundred days after the first vaccination and after confirming the IBR seronegativity of all animals, dexamethasone was administered intravenously for five consecutive days. Only animals immunized with the modified live IBR marker vaccine (single deletion) in the absence of passive immunity exhibited a more enduring immune response than those vaccinated in the presence of passive immunity. Moreover, the administration of a modified live IBR marker vaccine (double deletion) to calves with passive immunity generated SNLC. These findings underscore the potential of live IBR marker vaccine (double-deletions) to aid serological diagnostic tools and develop vaccination protocols in achieving the desired immune response, particularly in the context of latent carrier status, offering valuable insights into optimizing vaccination strategies for effective IBR control.

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.

Paslahepevirus balayani (hepatitis E virus [HEV]) is the causative agent of hepatitis E, a worldwide zoonosis involving a wide range of hosts among domestic and wild animals. This species is characterized by a great genomic heterogeneity and includes eight genotypes, HEV-1 to HEV-8. The HEV-3 genotype is one of the most common types circulating in Italy in humans and Suidae. Although domestic and wild Sus scrofa and deer (Cervidae) are recognized as the main reservoirs of HEV, several other wild species are potential carriers. A total of 228 liver samples were collected from nonungulate wild animals, found dead, in the framework of the regional passive surveillance program in Umbria and Marche regions (central Italy) during 2018-20. These were tested using real-time reverse-transcriptase PCR (RT-PCR) for detection of RNA of HEV-1 to HEV-4 and confirmed by nested RT-PCR assay. One of the 11 samples collected from crested porcupines (Hystrix cristata) tested positive for the presence of HEV RNA; all other samples were negative. Sequence analysis based on the full-length genome revealed that this isolate, 49434/UM/2018 (accession no. OL658617), belongs to the HEV-3e subtype. These findings suggest a potential role of crested porcupines as a carrier of HEV infection., (© Wildlife Disease Association 2023.)

We report here the whole-genome sequence of the African swine fever virus (ASFV) genotype II, strain 20355/RM/2022_Italy, identified in a wild boar in the city of Rome (Lazio region, Italy) in April 2022., Competing Interests: The authors declare no conflict of interest.

Background: African Swine Fever (ASF) is an infectious disease that affects domestic pig and wild boar populations. The ASF Virus (ASFV) has a genome characterized by a very complex DNA (170-193 kb) that encodes for more than 200 different proteins. Among these, the highly immunogenic phosphoprotein p30 plays a fundamental role in the induction of specific antibodies. To date, the lack of a vaccine against the disease requires continuous studies to improve knowledge about the virus and the development of new tests in addition to virological ones., Aim: The aim of this work was to produce specific monoclonal antibodies (mAbs) against the p30 protein of ASFV, which could find useful applications in routine diagnostics and the implementation of new diagnostic tools., Methods: ASFV p30 encoding gene was amplified and used for the generation of the recombinant baculovirus by transfection of the Sf21 insect cells. The recombinant protein was analyzed by immunofluorescence assay, purified, and used for mice Balb-c immunization. The hybridomas obtained were cultured and screened, using an indirect Enzyme-linked Immunosorbent Assay (iELISA), in order to select clones that secrete the mAbs of interest., Results: The expression of recombinant p30 protein was assessed using direct immunofluorescence. The purified p30 protein fractions were analyzed by Coomassie gels staining confirming the presence of bands with a molecular weight of 30 kDa and used for the immunization of Balb-c mice. Six clones of pure hybridomas secreting the specific mAbs against recombinant p30 were obtained and tested in iELISA. The mAbs were also characterized by Western blot and immunofluorescence assay. The best results were obtained with the anti-p30 mAb 2B8E10 clone which showed high reactivity with both recombinant and viral p30 protein, respectively., Conclusion: In this work, a recombinant p30 protein produced in an insect cell system was purified and used to immunize Balb-c mice. Six anti-p30 mAbs-secreting hybridomas clone cells were obtained. These mAbs displayed high reactivity against the recombinant protein, but only 2B8E10 mAb showed excellent functionality against the p30 protein produced by ASFV. These results open the possibility to develop different diagnostic assays., Competing Interests: The Authors declare that there is no conflict of interest.

African swine fever (ASF) is responsible for important socio-economic effects in the global pig industry, especially for countries with large-scale piggery sectors. In January 2022, the African swine fever virus (ASFV) genotype II was identified in a wild boar population in mainland Italy (Piedmont region). This study describes the molecular characterization, by Sanger and next-generation sequencing (NGS), of the first index case 632/AL/2022 and of another isolate (2802/AL/2022) reported in the same month, in close proximity to the first, following multiple ASF outbreaks. Phylogenetic analysis based on the B646L gene and NGS clustered the isolates 632/AL/2022 and 2802/AL/2022 within the wide and most homogeneous p72 genotype II that includes viruses from European and Asian countries. The consensus sequence obtained from the ASFV 2802/AL/2022 isolate was 190,598 nucleotides in length and had a mean GC content of 38.38%. At the whole-genome level, ASF isolate 2802/AL/2022 showed a close genetic correlation with the other representative ASFV genotype II strains isolated between April 2007 and January 2022 from wild and domestic pigs in Eastern/Central European (EU) and Asian countries. CVR subtyping clustered the two Italian ASFV strains within the major CVR variant circulating since the first virus introduction in Georgia in 2007. Intergenic region I73R-I329L subtyping placed the Italian ASFV isolates within the variant identical to the strains frequently identified among wild boars and domestic pigs. Presently, given the high sequence similarity, it is impossible to trace the precise geographic origin of the virus at a country level. Moreover, the full-length sequences available in the NCBI are not completely representative of all affected territories.

The zoonotic hepatitis E virus genotype 3 (HEV-3) causes most autochthonous human hepatitis E cases in Europe, which are due to the consumption of raw or undercooked food products of animal origin. Pigs and wild boars are considered the main reservoirs of this genotype, while rabbits are the reservoir of a distinct phylogenetic group named HEV-3ra, which is classified within the HEV-3 genotype but in a separate clade. Evidence for the zoonotic potential of HEV-3ra was suggested by its detection in immunocompromised patients in several European countries. HEV-3ra infection was found in farmed and feral rabbit populations worldwide and its circulation was reported in a few European countries, including Italy. Furthermore, Italy is one of the major rabbit meat producers and consumers across Europe, but only a few studies investigated the presence of HEV in this reservoir. The aim of this study was to assess the presence of HEV in 328 Italian hares and 59 farmed rabbits collected in 3 Italian macro-areas (North, North-Central, and South-Central), between 2019 and 2021. For this purpose, liver samples were used to detect HEV RNA using broad-range real-time RT-PCR and nested RT-PCR. Using 28 liver transudates from hares, the ELISA test for anti-HEV IgG detection was also performed. Neither HEV RNA nor anti-HEV antibodies were detected. Further studies will be conducted to assess the HEV presence in Italian lagomorphs to establish the role of this host and the possible risk of transmission for workers with occupational exposure, to pet owners and via food., Competing Interests: The authors declare no conflict of interest.

Wild boar is the main sylvatic reservoir of the genotype 3 of hepatitis E virus (HEV). The occurrence of HEV-3 human cases has been linked to the consumption of raw or undercooked pig and wild boar meat and liver. The zoonotic transmission of HEV-3 has been confirmed by sequencing identical or strictly related viral strains in humans, wild boar and derived food. The HEV sequences classified within the HEV-3 genotype are highly variable, and although only one serotype has been identified so far, the observed differences allow for the further classification of the HEV-3 genotype into subtypes, named in alphabetical order. Compared to human and pig strains, an even higher heterogeneity is observed among strains infecting wild boar. In the present study, the genetic variability of eight HEV-3 strains detected in wild boars sampled in a small geographical area in Central Italy (Lazio and Umbria regions) was investigated by full genome sequencing and phylogenetic analysis. The strains were classified within the HEV-3a, HEV-3c, HEV-3f subtypes and within two new recently proposed subtypes. Results demonstrate - despite the relatively small geographic area of origin - an unexpected divergence within HEV-3 strains hosted by the investigated wild boar population and highlights the need for extensive sequencing of HEV in reservoirs to fully understand diversity, geographical distribution and evolution of this group of viruses., (© 2022 Wiley-VCH GmbH.)

Three commercially available infectious bovine rhinotracheitis (IBR) live marker vaccines were evaluated for their ability to provide clinical protection to vaccinated calves against wild-type (wt) Bovine alphaherpesvirus-1 (BoHV-1) challenge and their possible effect on wt BoHV-1 latency reactivation following the challenge. On 35 post-vaccination days (PVDs), all animals were challenged with wt BoHV-1. Only the calves in the control group developed severe forms of IBR. The reactivation of latent BoHV-1 was induced by dexamethasone (DMS) treatment on 28 post-challenge days (PCDs). All animals showed IBR clinical signs on three post-DMS treatment days (PDTDs). On PVD 14, all vaccinated animals developed neutralizing antibodies (NAs), whereas in control animals, the NAs appeared post-challenge. The positivity for glycoprotein-B (gB) was detected using real-time polymerase chain reactions in all animals from PCDs 1 to 7. In contrast, the gB-positivity was observed in the immunized calves from PDTDs 3 to 10. Positive expression of gD and gE was observed in nasal swabs of all calves on PDTD 7. These findings suggested that the IBR marker vaccines evaluated in this study protected against wt BoHV-1-induced disease but not against wt BoHV-1-induced latency reactivation, indicating the necessity of developing new products to protect animals from wt BoHV-1-induced latency.

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.

Since the very beginning of the COVID-19 pandemic, SARS-CoV-2 detection has been described in several animal species. A total of 625 outbreaks in animals have been reported globally, affecting 17 species in 32 countries and the human source of infection has been recognized including pet owners, zookeepers, and farmers. In this report, we describe the case of a paucisymptomatic dog in Italy infected with SARS-CoV-2 from a household with three confirmed human cases of COVID-19 living in Pesaro (Marche region, Italy). The dog showed high viral RNA titers in the nasal and oropharyngeal swabs. In the nasal swab, SARS-CoV-2 RNA lasted for a least a week. By sequencing, the strain was assigned to the AY.23 lineage (PANGO), one of the sub-lineages of the major SARS-CoV-2 Delta variant of concern (VOC). Although we did not process the swabs of the three human cases, we strongly suspect a human origin for the dog infection. In this regard, AY.23 sequences, although never released thus far in the Marche region, were detected in the neighboring regions. Our findings highlight once more the need for a One Health approach for SARS-CoV-2 surveillance, management, and control, thus preventing viral spillover from animals to humans.

Small ruminant lentiviruses (SRLVs) represent a very heterogeneous group of ss-RNA viruses that infect sheep and goats worldwide. They cause important, deleterious effects on animal production and limit the animal trade. SRLVs show a high genetic variability due to high mutation rate and frequent recombination events. Indeed, five genotypes (A-E) and several subtypes have been detected. The aim of this work was to genetically characterize SRLVs circulating in central Italy. On this basis, a phylogenetic study on the gag-pol genetic region of 133 sheep, collected from 19 naturally infected flocks, was conducted. In addition, to evaluate the frequency of mutation and the selective pressure on this region, a WebLogo 3 analysis was performed, and the dN/dS ratio was computed. The results showed that 26 samples out of 133 were clustered in genotype A and 106 samples belonged to genotype B, as follows: A9 ( n = 8), A11 ( n = 10), A24 ( n = 7), B1 ( n = 2), B2 ( n = 59), and B3 ( n = 45). No recombination events were found. Mutations were localized mainly in the VR-2 region, and the dN/dS ratio of 0.028 indicated the existence of purifying selection. Since the genetic diversity of SRLVs could make serological identification difficult, it is important to perform molecular characterization to ensure a more reliable diagnosis, to maintain flock health status, and for the application of local and national control programs.

Small ruminant lentiviruses (SRLV) are viruses that retro-transcribe RNA to DNA and show high rates of genetic variability. SRLV affect animals with strains specific for each host species (sheep or goats), resulting in a series of clinical manifestations depending on the virulence of the strain, the host's genetic background and farm production system. The aim of this work was to present an up-to-date overview of the genomic epidemiology and genetic diversity of SRLV in Italy over time (1998-2019). In this study, we investigated 219 SRLV samples collected from 17 different Italian regions in 178 geographically distinct herds by CEREL. Our genetic study was based on partial sequencing of the gag-pol gene (800 bp) and phylogenetic analysis. We identified new subtypes with high heterogeneity, new clusters and recombinant forms. The genetic diversity of Italian SRLV strains may have diagnostic and immunological implications that affect the performance of diagnostic tools. Therefore, it is extremely important to increase the control of genomic variants to improve the control measures.

Bovine Pestivirus heterogeneity is a major challenge for vaccines against bovine viral diarrhea (BVD). In breeding herds, fetal protection is a high priority issue. To some degree, fetal infections in vaccinated heifers have been attributed to the antigenic diversity of bovine Pestiviruses. The purpose of this study was to assess fetal protection against a divergent bovine Pestivirus (Hobi-like Pestivirus, HoBiPeV) with a commercially available modified live vaccine (MLV) claiming fetal protection against BVDV 1 and BVDV 2 up to one year after the first inoculation. Five vaccinated and four unvaccinated heifers were challenged by intranasal inoculation with the HoBiPeV Italy-1/10-1 strain between 82 and 89 days after insemination, i.e. between 4 and 6 months after vaccination. At challenge, neutralizing antibody titers to HoBiPeV in vaccinated heifers were low or even undetectable. Of the four unvaccinated heifers, one control animal aborted (fetus not available) and the remaining three gave birth to HoBiPeV positive calves. Among the heifers of the vaccinated group, one aborted the fetus in the sixth month of pregnancy, which tested Pestivirus negative, while three others gave birth to healthy, HoBiPeV negative calves; the remaining heifer delivered one HoBiPeV positive calf. The results suggest that the BVDV vaccine might be able to elicit a partial fetal protection against HobiPeV, even in absence of a strong specific antibody response., (Copyright © 2021. Published by Elsevier B.V.)

In the last decade in Europe, the number of autochthonous cases of hepatitis E has significantly increased. Most of the cases arise from foodborne infections caused by the zoonotic hepatitis E virus (HEV) genotypes HEV-3 and HEV-4. Several human cases have been linked to consumption of raw or undercooked animal products of both pork (liver sausages) and wild boar meat. In this study, the occurrence of HEV infection was investigated in 611 livers and 88 paired lungs from wild boars collected during the hunting seasons of 2016-2020 in the Umbria-Marche Apennines (Central Italy). Using real-time reverse transcription polymerase chain reaction, 15 liver samples (2.45%) and one lung sample were found to be positive for HEV RNA. The phylogenetic tree built on the partial ORF2 gene revealed that the detected HEV strains belonged to HEV-3f (n = 5), HEV-3e (n = 1) and HEV-3c (n = 1) subtypes. Interestingly, 8 strains were genetically placed in a different cluster, further away from all other subtypes. To corroborate this finding, four complete genomes were obtained by next generation sequencing. The full genome of the HEV strains clustered together with another wild boar strain previously detected in Southern Italy in 2015 but the strains were divergent from all the HEV-3 strains classified in any subtype defined so far. Thus, these strains represent a novel subtype that might have originated in Italy, which we have tentatively named HEV-3n., (© 2020 Wiley-VCH GmbH.)

Maedi-visna virus (MVV) and caprine arthritis encephalitis virus (CAEV), referred to as small ruminant lentiviruses (SRLVs), belong to the genus Lentivirus of the Retroviridae family. SRLVs infect both sheep and goats, causing significant economic losses and animal welfare damage. Recent findings suggest an association between serological status and allelic variants of different genes such as TMEM154 , TLR9 , MYD88 and CCR5 . The aim of this work was to investigate the role of specific polymorphisms of these genes in SRLVs infection in some sheep flocks in Italy. In addition to those already known, novel variants in the TMEM154 (P7H, I74V, I105V) gene were detected in this study. The risk of infection was determined finding an association between the serological status and polymorphisms P7H, E35K, N70I, I74V, I105V of TMEM154 , R447Q, A462S and G520R in TLR9 gene, H176H* and K190K* in MYD88 genes, while no statistical association was observed for the 4-bp deletion of the CCR5 gene. Since no vaccines or treatments have been developed, a genetically based approach could be an innovative strategy to prevent and to control SRLVs infection. Our findings are an important starting point in order to define the genetic resistance profile towards SRLVs infection.

Border disease virus (BDV) belongs to the genus Pestivirus of the family Flaviviridae . Interspecies transmission of BDV between sheep, cattle, and pigs occurs regularly, sometimes making diagnosis a challenge. BDV can yield substantial economic losses, including prenatal and postnatal infections in lambs, which are the primary source of infection and maintenance of the virus in the population. Since BDV is antigenically and genetically related to bovine viral diarrhea virus (BVDV), it might pose a significant risk to cattle, influencing BVDV eradication campaigns. Similarly, the presence of BDV in swine herds due to pestivirus spillover between small ruminants and pigs might cause uncertainty in classical swine fever virus (CSFV) diagnostics. Therefore, knowledge of BDV epidemiology in different geographical regions will help prevent its spread and optimize control measures. Previous epidemiological studies have shown that various BDV genotypes are predominant in different countries. This review provides an overview of the spread of BDV world-wide in different host species.

Recent studies have explored the seropositivity of Bovine alphaherpesvirus 1 (BoHV-1) in water buffaloes, suggesting the urgency for developing strategies to eradicate the virus involving both cattle and water buffaloes. However, in Europe, the glycoprotein E (gE) deleted marker vaccines against BoHV-1 are commercially available only for the cattle industry. This study, for the first time, evaluated the safety and efficacy of a commercial inactivated gE-deleted marker vaccine in water buffalo. Five animals devoid of BoHV-1-neutralizing antibodies were vaccinated via intramuscular route. Five additional animals served as an unvaccinated control group. Sixty days after the first immunization, all animals were experimentally infected with a virulent BoHV-1via intranasal route. A detectable BoHV-1-humoral immune response was observed in the vaccinated group on post-vaccination day 30, whereas the antibodies appeared on post-challenge day 10 in the control group. Moreover, the vaccinated animals neither show viral shedding nor clinical signs compared to the control upon challenge. However, post-challenge, the BoHV-1-specific humoral and cell-mediated immune responses were significantly more increased in vaccinated animals than the control animals. Overall, the present study provides evidence of both the safety and efficacy of an inactivated gE-deleted marker vaccine against BoHV-1 in water buffaloes.

Background: Pigs are considered the main reservoir of genotypes 3 and 4 of hepatitis E virus (HEV), which is the major cause of acute hepatitis of viral origin in humans worldwide. An increasing number of autochthonous HEV infections have been observed in recent years in industrialized countries, most likely as a result of zoonotic transmission through the consumption of raw or undercooked meat products., Methods: Two hundred and thirty-three blood and liver samples were collected at four different local slaughterhouses from domestic pigs bred in Abruzzo, a region of south-central Italy, where there is the highest human seroprevalence to HEV compared with the rest of Italy. An indirect enzyme-linked immunosorbent assay kit was used for detecting anti-HEV IgG in the sera, while the presence of HEV RNA was investigated by performing a real-time reverse transcriptase-polymerase chain reaction (RT-PCR)., Results: Between 87.3% and 100% of swine serum samples collected in different slaughterhouses of Abruzzo were positive for anti-HEV antibodies. Conversely, none of the liver samples collected from the same animals were positive for HEV by real-time RT-PCR., Conclusions: The hypothesis of foodborne zoonotic transmission from local pigs as responsible for the hyperendemic status of Abruzzo cannot be corroborated. However, the high seroprevalence observed in pigs indicates that HEV is highly circulating in these territories. We propose to further investigate the role of wild fauna and trade in carrier pigs, and the maintenance of HEV virulence in the environment and meat supply chain to shed light on the possible sources of human infection and the degree of occupational risk., (© The Author(s) 2020. Published by Oxford University Press on behalf of the European Public Health Association. All rights reserved.)

To gain further insight into the genomic features of border disease virus (BDV), we determined the nearly complete genome sequence of isolate TO/121/04 from an aborted ovine fetus. Its genome contains a single open reading frame (ORF), which comprises 11,681 nucleotides encoding a polyprotein of 3893 amino acids. Phylogenetic analysis of the near full-length genome sequence showed that the BDV isolate differed significantly from all ovine pestiviruses identified so far, thus re-affirming the presence in Italy of this novel genetic group, termed BDV-7.

Bovine respiratory syncytial virus (BRSV) is an economically significant pathogen in cattle production worldwide. Usually, it is detected in outbreaks of respiratory disease, most often during the winter period. During the middle of October 2018, a serious outbreak of respiratory disease occurred in a cattle farm comprising about 300 heads located in Central Italy. The herd was affected by a severe flu-like syndrome unresponsive to any antibiotic treatment. Within 3 weeks, 39 adult animals died, and 12 abortions occurred. Direct and indirect laboratory tests were performed to detect the main pathogens causing the respiratory disease of the affected cattle. The results of laboratory investigations provided evidence of an acute and severe BRSV syndrome characterized by unusual mortality. In order to investigate the molecular underpinnings of this syndrome, phylogenetic analysis of the BRSV strain detected from the outbreak was carried out. The sequence analysis showed that the strain was genetically divergent from BRSV strains previously identified in Italy, as it showed high sequence similarity of more than 97% with strains isolated during a major BRSV epizootic that occurred in Sweden, Norway and Denmark during 2010-2011. The infection of the herd in Italy with this BRSV strain was likely due to the introduction of animals imported into Italy from abroad., (© 2020 The Authors. Veterinary Medicine and Science Published by John Wiley & Sons Ltd.)

In this study, we demonstrated for the first time in Italy, the serological cross-reactivity between Bovine alphaherpesvirus 2 (BoHV-2) and Bovine alphaherpesvirus 1 (BoHV-1). Five months after arriving at a performance test station in Central Italy, a 6-month-old calf, which was part of a group of 57 animals, tested positive for BoHV-1 in a commercial gB-ELISA test. It was immediately transferred to the quarantine unit and subjected to clinical observation and serological and virological investigations. During this period, the calf showed no clinical signs. The results from laboratory investigations demonstrated the presence of antibodies via competitive glycoprotein B (gB) ELISAs, indirect BoHV-1 ELISAs, and indirect BoHV-2 ELISAs. Furthermore, the plaque reduction assay provided evidence for the presence of antibodies only for BoHV-2, whereas the virus neutralization test showed negative results for both BoHV-1 and BoHV-5. These findings strongly suggest the occurrence of a serological cross-reactivity between BoHV-2 and BoHV-1. Interference of BoHV-2 antibodies in serological BoHV-1 diagnostics should be considered during routine IBR tests, especially when animals are kept in a performance test station., (Copyright © 2020 Petrini, König, Righi, Iscaro, Pierini, Casciari, Pellegrini, Gobbi, Giammarioli and De Mia.)

African swine fever virus (ASFV), the cause of a devastating disease affecting domestic and wild pigs, has been present in Sardinia since 1978. In the framework of the regional ASF eradication plan, 4484 illegal pigs were culled between December 2017 and February 2020. The highest disease prevalence was observed in the municipality with the highest free-ranging pig density, and culling actions drastically reduced ASFV circulation among these animals. ASFV-antibody were detected in 36.7% of tested animals, which were apparently healthy, thus, the circulation of low-virulence ASFV isolates was hypothesized. ASFV genome was detected in 53 out of 2726 tested animals, and virus isolation was achieved in two distinct culling actions. Two ASFV haemadsorbing strains were isolated from antibody-positive apparently healthy pigs: 55234/18 and 103917/18. Typing analysis revealed that both isolates belong to p72 genotype I, B602L subgroup X; phylogenetic analysis based on whole genome sequencing data showed that they were closely related to Sardinian ASFV strains collected since 2010, especially 22653/Ca/2014. Our data suggested the absence of immune-escaped ASFV variants circulating among free-ranging pigs, indicating that other elements contributed to virus circulation among these animals. Understanding factors behind disease persistence in endemic settings might contribute to developing effective countermeasures against this disease.

African swine fever (ASF) is a highly contagious and lethal viral disease of pigs and wild boars, which is enzootic in many African countries and on the Italian island of Sardinia, where it has been present since 1978. Previous genetic analyses of Sardinian ASF virus (ASFV) isolates have revealed that they all belong to p72 genotype I, with only minor sequence variations. However, these studies examined only a few selected genes. To distinguish between these closely related isolates and better investigate ASFV evolution in Sardinia, we sequenced the complete genomes of 12 Sardinian ASFV isolates collected between 1978 and 2012, and compared them with 47/Ss/2008 and 26544/OG10. Most of the observed changes occurred in a time-dependent manner; however, their biological significance remains unclear. As a whole, our results demonstrate the remarkable genetic stability of these strains, supporting a single-source introduction of the virus., (© 2020 Blackwell Verlag GmbH.)

The genus Pestivirus comprises globally distributed members of the family Flaviviridae, which cause severe losses in livestock. The most common species of the genus are bovine viral diarrhoea virus type 1 (BVDV-1) and type 2 (BVDV-2), classical swine fever virus (CSFV) and border disease virus (BDV). Recently, a novel ovine pestivirus was repeatedly detected in aborted lamb foetuses on a farm located in the Brescia Province (Italy). Complete genome characterization of this isolate showed that it was highly divergent from known pestivirus species and that it was genetically closely related to CSFV. The aim of this study was to determine the serological relatedness between the identified novel pestivirus and BVDV, BDV and CSFV selected strains for which homologous serum was available, by antigenic characterization performed using cross-neutralization assays. The serological relatedness was expressed as the coefficient of antigenic similarity (R). Both field and specific antisera raised against the ovine pestivirus neutralized the CSFV reference strain Diepholz with titres significantly higher than those specific for the BDV and BVDV strains. Furthermore, the calculated R values clearly indicated that the novel ovine pestivirus is antigenically more related to CSFV than to ruminant pestiviruses, in agreement with the results of the genomic analysis. This would have severe consequences on CSFV serology in the event of a switch to porcine hosts with implications for CSFV surveillance and porcine health management., (© 2020 Blackwell Verlag GmbH.)

Disease caused by bovine leukemia virus (BLV) results in significant economic losses to the livestock industry. To date, there is only one report describing the strains found in Italy. BLV strains (n = 24), collected between 2012 and 2016 from four different Italian regions, were genetically analyzed by direct sequencing of a portion of the BLV env gene, and the sequences were compared with those in the GenBank database. The Italian BLVs clustered into genotypes G2, G4, G6, G7, and G8, revealing a high level of BLV genetic heterogeneity in Italy. This study provides a basis for further investigations into the evolutionary relationship between BLV strains.

A number of animal diseases can be transmitted to pigs via pork meat and pork products imported from infected areas. Therefore, feeding of swill to pigs is regulated or prohibited in many swine-rearing countries. African swine fever is one of the major porcine diseases recognized as significant in this transmission pathway. Assessment of disease risks associated with pork products requires knowledge about the viral load in the original material and for how long infectious virus can be recovered from the resulting product. In this work, we present the effect of the dry curing process on the inactivation of African swine fever virus (ASFV) in three different Italian dry-cured meat products prepared from experimentally infected pigs slaughtered at the peak of viremia. The meat products were processed using commercial methods and industrial procedures currently being followed in Italy. Samples collected at predetermined intervals during processing were analyzed for virus survival by virus isolation and animal inoculation. ASFV was detected by in vivo experiments for up to 18, 60, and 83 days of curing in Italian salami, pork belly, and loin, respectively. These data provide valuable information for the pork processing industry when planning the export of these products., (Copyright © 2018 Elsevier B.V. All rights reserved.)

The complete genome sequences of both biotypes of a pair of bovine viral diarrhea viruses isolated from a bovid affected by mucosal disease were determined by next generation sequencing. The cytopathic virus possessed a 423-base insertion derived from bovine poly ubiquitin in the NS2/3 coding region and one nucleotide change. Both biotypes showed an additional glycosylation site in their N-terminus.

African swine fever (ASF) virus is a DNA virus responsible for a severe haemorrhagic fever in pigs, which (still in the absence of vaccination strategies) results in high mortality rates. Herein, we present a biosensor-based method for the detection of ASF viral DNA in the blood of pigs. The biosensor exploits a single-strand DNA probe with locked nucleic acid nucleotides (LNA) substitutions as the complementary recognition element for the conserved region of vp72 gene of ASF virus. The biosensor was calibrated using qPCR-quantified ASF viral DNA extracted from the blood of pigs experimentally infected with the virulent Italian isolate 49/08, genotype I. Globally, the proposed biosensor showed good sensitivity and specificity, with the limits of detection (LOD) and quantification (LOQ) being 178 and 245 copies/μL of genomic ASF viral DNA, respectively. The reversible nature of the interaction between the DNA/LNA probe and the target DNA sequence granted multiple rapid analyses, with up to 40 analyses per single surface possible, and a single test requiring approximately 5 min. When applied to non-amplified DNA extracts from the blood of field-infected pigs, the assay discriminated between ASFV-infected and ASFV non-infected animals, and allowed the rapid quantification of ASF viral DNA, with values falling in the range 373-1058 copies/μL of genomic ASFV DNA. In this range, excellent correlation was observed between the results of this biosensor and OIE-approved qPCR. This method represents a promising screening assay for preliminary ASF diagnosis, having the major advantages in the relative rapidity, ease-of-use, the reusability of the sensing surface, and low cost per single test., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Bovine respiratory syncytial virus (BRSV) is circulating in cattle in Europe. Although vaccination helps control the disease, its prevalence within and among herds remains high. Previous genetic characterization studies revealed a strict geographic correlation between viral variants; on the other hand, they showed the emergence of new variants in northern Europe. Few studies have described BRSV distribution, and little is known about the genetic features of BRSV strains circulating in Italy. We studied sample-positive tests for BRSV, and sequenced the coding regions of the G and N proteins to determine the presence of divergent variants. Two different sets of sequences were found, including in samples from animals from vaccinated herds. The 2 groups of sequences correspond to 2 time periods and suggest an active role of herd immunity in preventing the spread of infection. Our findings that different strains of BRSV are circulating in Italy and that the virus is evolving rapidly highlight the importance of updating vaccination strategies.

To gain further insight into the genomic features of bovine viral diarrhea virus 1 (BVDV-1) subtypes, we sequenced the complete genome of the BVDV-1 isolate VE/245/12. This is an uncommon subtype that was isolated from a persistently infected animal. Here, we report the complete genome sequence, consisting of 12,295 nucleotides (nt) with an open reading frame of 11,694 nt encoding 3,898 amino acids. Phylogenetic analysis of the full-length genome, 5'-UTR, and N pro region confirmed that the BVDV-1 isolate differed significantly from all of the bovine pestiviruses identified so far, providing evidence for the presence of a distinct novel genetic group.

A single-step TaqMan® RT-qPCR was developed for the detection of bovine viral diarrhea virus type 1 (BVDV-1), an important pathogen of cattle worldwide. The assay was based on conserved 5'UTR sequences of Italian BVDV-1 isolates. In order to establish a diagnostic protocol which simplifies sample collection and processing, the assay was tested on a variety of biological specimens collected from persistently infected calves. The samples analyzed included PBMCs, plasma, dry blood, ear notch and hair bulb. Time and costs required for the analysis of each type of specimen were compared. The RT-qPCR, whose lower limit of detection was 100 copies of viral RNA (1 TCID50), correctly identified all PI animals, irrespective of the type of specimen. The highest copy numbers were obtained from the RNAs extracted from PBMCs, ear notches and hair bulbs. Hair bulb-supernatants directly used as a template allowed identification of all PI animals. In conclusion, based on time and cost evaluation, the most effective and efficient protocol was the one based on the direct analysis of hair bulb-supernatants, avoiding the RNA extraction step.

We report here the full-length sequence of the bovine viral diarrhea virus (BVDV) strain UM/103/04, isolated from a persistently infected cow in Italy. It belongs to the subgenotype 1g, which is described as a sporadic subgenotype in Italy. This is the first report of a complete genome sequence of BVDV-1g., (Copyright © 2017 Bazzucchi et al.)

We evaluated the use of oral fluid as an alternative to serum samples for Classical swine fever virus (CSFV) detection. Individual oral fluid and serum samples were collected at different times post-infection from pigs that were experimentally inoculated with CSFV Alfort 187 strain. We found no evidence of CSFV neutralizing antibodies in swine oral fluid samples under our experimental conditions. In contrast, real-time reverse transcription-polymerase chain reaction could detect CSFV nucleic acid from the oral fluid as early as 8 d postinfection, which also coincided with the time of initial detection in blood samples. The probability of CSFV detection in oral fluid was identical or even higher than in the corresponding blood sample. Our results support the feasibility of using this sampling method for CSFV genome detection, which may represent an additional cost-effective tool for CSF control.

We sequenced the complete genome of bovine viral diarrhea virus (BVDV) strain UM/126/07. It belongs to subgenotype 1h. The complete genome is composed of 12,196 nucleotides organized as one open reading frame encoding 3,898 amino acids. This is the first report of a full-length sequence of BVDV-1h., (Copyright © 2017 Bazzucchi et al.)

Recent studies have suggested that certain classes of endogenous retroviruses (ERVs) may be present in cattle. The aim of this study was increase the scope of knowledge regarding bovine ERVs. The ovine ERV β1 pro/pol sequence was used to design a primer set for polymerase chain reaction (PCR) amplification of a similar sequence in the bovine genome. Through phylogenetic and bioinformatic analysis of the PCR product sequence together with its flanking region, a sequence 8107 bp in length was characterized. This sequence had a typical 5'-LTR-gag-pro-pol-env-LTR-3' organization, and phylogenetic investigation defined it as a bovine ERV β1. Thus, we were able to identify a novel bovine endogenous retrovirus element.

Border disease virus (BDV) belongs to the Pestivirus genus in the family Flaviviridae. Genetic analyses of pestiviruses that have been isolated from sheep and goat have led to the proposal that BDV isolates can be phylogenetically segregated into at least seven clusters, subtypes BDV-1 to BDV-7. In order to investigate the genetic heterogeneity of small ruminant pestivirus isolates in Italy, a selection of 5'-UTR sequences from isolates that were collected from clinical specimens between 2002 and 2014 was analysed. Phylogenetic reconstructions indicated that the BDV-positive samples clustered within the BDV-1, BDV-3, BDV-5, and BDV-7 groups. These results suggested high genetic diversity within the Italian BDV field isolates. The phylogenetic analysis indicated the first evidence of BDV-1 and BDV-5 circulation in Italy. The marked diversity of the pestivirus isolates might reflect the sheep trade with foreign countries.

Sequence-based genotyping was recently used to distinguish between the BVDV-1 and BVDV-2 species of the bovine viral diarrhoea virus (BVDV). Quite recently, a new putative species, BVDV-3, was also detected. The phylogenetic analysis of the 5'-untranslated region (UTR) and Npro region has revealed at least 17 distinct subtypes for BVDV-1 to date. The aim of this study was to further investigate the genetic heterogeneity of BVDV-1 in Italy, by analysing 173 virus sequences from isolates collected over an 18-year period (1997-2014). Viral RNA was extracted from the original biological samples identified as BVDV-1-positive. Reverse transcription (RT) and polymerase chain reaction (PCR) assays targeting a 288-base pair (bp) region of the 5'-UTR and a 428-bp region encoding the autoprotease Npro were performed, and the RT-PCR products were sequenced. The phylogenetic analysis of the 5'-UTR and Npro sequences re-confirmed the circulation of ten out of eleven subtypes previously discovered in Italy. Interestingly, four isolates differed significantly from all of the bovine pestiviruses identified so far, thereby providing evidence for the circulation of three novel subtypes that have not been documented so far. The growing number of reports on BVDV-1 heterogeneity, including the recent findings reported herein, raises concern related to the emergence and spread of new BVDV variants, with possible implications for animal health and disease control. This global issue needs to be addressed with the highest priority.

Oral vaccination against classical swine fever (CSF) is a potent tool to control disease outbreaks in wild boar. So far, vaccination campaigns have been carried out using live attenuated vaccines that do not allow serological differentiation of infected from vaccinated animals (DIVA). Although this drawback is acceptable for wild boar, the use of marker vaccines would facilitate studies on disease and vaccination dynamics. Recently, the CSF marker vaccine candidate CP7_E2alf was assessed for oral immunization under laboratory conditions. Promising results prompted efforts to study the vaccine candidate under field conditions and in bait formulation. In this context, two oral vaccination campaigns were carried out with CP7_E2alf bait vaccines in two areas called 'faunistic-hunting farms' in the region of Umbria, Italy. One campaign was conducted using single vaccination, the second with the routinely employed double vaccination strategy. Both campaigns were carried out before concerted hunting actions were performed. Bait uptake, vaccine virus detection and antibody responses were assessed along with inspections upon gutting. As a comparator, seven wild boar were hand-fed with baits under laboratory conditions. In the field, bait uptake ranged from 63.7% to 98.7%, whereas antibody prevalence reached only 33.3-35.1%. The marker serology showed a strong influence of sample quality on the test outcome with a total of 85% of samples being classified correctly. Vaccine virus was not detectable. Under hand feeding conditions, six out of seven wild boar took up at least one bait, and five of them showed detectable antibody levels seven weeks after vaccination. These results were supplemented by stability tests. Appropriate stability of vaccine virus was shown both under field and laboratory conditions. In total, most results were in line with our expectations. However, optimization of the DIVA assay has to be attempted in the future., (Published by Elsevier Ltd.)

Genetic typing of bovine viral diarrhea virus (BVDV) has distinguished BVDV-1 and BVDV-2 species and an emerging putative third species (HoBi-like virus), recently detected in southern Italy, signaling the occurrence of natural infection in Europe. Recognizing the need to update the data on BVDV genetic variability in Italy for mounting local and European alerts, a wide collection of 5' UTR sequences (n = 371) was selected to identify the frequency of genotypes and subtypes at the herd level. BVDV-1 had the highest frequency, followed by sporadic BVDV-2. No novel HoBi-like viruses were identified. Four distribution patterns of BVDV-1 subtypes were observed: highly prevalent subtypes with a wide temporal-spatial distribution (1b and 1e), low prevalent subtypes with a widespread geographic distribution (1a, 1d, 1g, 1h, and 1k) or a restricted geographic distribution (1f), and sporadic subtypes detected only in single herds (1c, 1j, and 1l). BVDV-1c, k, and l are reported for the first time in Italy. A unique genetic variant was detected in the majority of herds, but cocirculation of genetic variants was also observed. Northern Italy ranked first for BVDV introduction, prevalence, and dispersion. Nevertheless, the presence of sporadic variants in other restricted areas suggests the risk of different routes of BVDV introduction.

Caprine arthritis/encephalitis (CAE) of goats and occasionally sheep are persistent virus infections caused by a lentivirus (CAEV). This viral infection results in arthritis in adult animals and encephalitis in kids. Prognosis for the encephalitic form is normally poor, with substantial economic loss for the farm. In this context an early/fast laboratory diagnosis for CAEV infection could be useful for effective prophylactic action. In this work we performed a quantitative real time PCR designed on the CAEV env gene to detect/quantify in goat/sheep samples, viral RNA or proviral DNA forms of CAEV. This procedure was validated in 15 sheep, experimentally infected with CAEV or with a highly correlated lentivirus (visna maedi, MVV); in addition, a total of 37 clinical goat specimens recruited in CAEV positive herds were analyzed and compared using serological analysis (Elisa and AGID). All samples infected with MVV resulted negative. In sheep experimentally infected with CAEV, proviral DNA was detectable 15 days post infection, whereas the serological methods revealed an indicative positivity after 40-60 days.This method showed a sensitivity of 10(2) env fragments/PCR) with a linear dynamic range of quantitation from 10(3) to 10(7)env fragments/PCR; the R2 correlation coefficient was 0.98. All subjects with a clinical diagnosis for Caprine Arthritis-Encephalitis (CAE) resulted CAEV DNA positive.

Three discrete regions of the African swine fever virus (ASFV) were analysed in the genomes of a wide range of isolates collected from wild and domestic pigs in Sardinia, over a 31-year period (1978-2009). The analysis was conducted by genotyping based on sequence data from three single copy ASF genes. The E183L gene encoding the structural protein p54 and part of the gene encoding the p72 protein were used to delineate genotypes, before intra-genotypic resolution of viral relationships by analysis of tetramer amino acid repeats within the hypervariable central variable region (CVR) of the B602L gene. The data revealed that these isolates did not show significant variation in their p72 and p54 sequence when compared between different isolates showing a remarkable genetic stability of these genome regions. In particular, the phylogeny revealed that all the Sardinian isolates belong to the same largest and most homogeneous p72 genotype I together with viruses from Europe, South America, the Caribbean and West Africa, and p54 genotype Ia which comprises viruses from Europe and America. The analysis of B602L gene revealed a minor difference in the number of tetramer repeats, placing the Sardinian isolates into two clusters, accordingly to their temporal distribution, namely sub-group III and sub-group X, this latter showing a deletion of 12 tetramer repeats located in the centre of the array. The genetic variation of this fragment suggests that one sub-group could be derived from the other supporting the hypothesis of a single introduction of ASFV in Sardinia.

Border disease virus belongs to the Pestivirus genus, within the family Flaviviridae. Genetic analysis of pestiviruses isolated from sheep in continental Europe have led to the proposal that BDV isolates are segregated into at least seven clusters. In 2005 the molecular analysis of an Italian caprine BDV strain provided evidence for the presence of an atypical pestivirus, which may represent the first member of a putative novel pestivirus sub-group. To further build on this study, ovine pestivirus strains were isolated from small ruminant flocks and characterized both genetically and antigenically. A defined section of the 5'UTR and the complete N(pro) coding region were amplified and used for phylogenetic analysis. This revealed that these pestiviruses belong to the BDV species but differed significantly from all previously described ovine pestiviruses providing evidence for the presence of a novel genetic group. Four of the five isolates were also typed antigenically with a panel of pestivirus specific mAbs directed against NS2/3, E(rns) and E2 proteins. The four isolates reacted with a distinct set of mAbs, in particular against the BDV-E2 and the BDV-E(rns) epitopes. The isolates were greatly reactive for E(rns) and NS2/3 mAbs, which are otherwise typical for BVDV-2, and one E2 mAb that typically stains BVDV-1. The Italian pestiviruses analysed in this study, according to their antigenic and genetic properties, clustered into a novel phylogenetic group, that we propose to term BDV-7., (Copyright © 2010 Elsevier B.V. All rights reserved.)