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Simple Summary: Wild boars and feral pigs are underinvestigated hosts for influenza A viruses (IAVs). This study confirmed and evaluated viral circulation in the Emilia-Romagna wild boar population between 2017 and 2022. Samples were collected at post mortems and screened for IAVs; 0.37% of the tested animals provided positive results. Positive samples were subtyped, isolated, and genotyped via full-genome sequencing. The results highlight the co-circulation of the same viral genotypes in overlapping years in both pigs and wild boars in the same geographical area. Considering the role of domestic and wild Sus scrofa species in the IAVs' ecology, surveillance against these viruses in the wild boar population needs to be implemented. A systematic surveillance against influenza A viruses (IAVs) in the Suidae population is essential, considering their role as IAV mixing vessels. However, the viral circulation in wild Sus scrofa species is poorly investigated in comparison to the knowledge of IAV infection dynamics in domestic pigs. This study investigated the circulation and the genetic diversity of wild boars' IAVs detected in the Emilia-Romagna region (2017–2022). A total of 4605 lung samples were screened via an M gene real-time RT-PCR for SwIAV; positive samples were subtyped by multiplex RT-PCR, and viral isolation was attempted. Isolated strains (3 out of the 17 positives) were fully sequenced to evaluate viral genotypic diversity. H1N1 was the most frequently detected subtype, with identification of H1pdm09N1 and H1avN1. Whole-genome phylogenetic analysis revealed SwIAVs belonging to different genotypes, with different genetic combinations, and highlighted the simultaneous circulation of the same genotypes in both pigs and wild boars, supporting the hypothesis of SwIAV spillover events at the wildlife–livestock interface. This study represents an update on the wild boar SwIAV Italian situation, and the strains' complete genome analysis showed an evolving and interesting situation that deserves further investigation. [ABSTRACT FROM AUTHOR]

Abstract Background Diagnosis of West Nile virus (WNV) infections is often difficult due to the extensive antigenic cross-reactivity among flaviviruses, especially in geographic regions where two or more of these viruses are present causing sequential infections. The purpose of this study was to characterize a panel of monoclonal antibodies (MAbs) produced against WNV to verify their applicability in WNV diagnosis and in mapping epitope targets of neutralizing MAbs. Methods Six MAbs were produced and characterized by isotyping, virus-neutralization, western blotting and MAb-epitope competition. The MAb reactivity against various WNVs belonging to lineage 1 and 2 and other related flaviviruses was also evaluated. The molecular basis of epitopes recognized by neutralizing MAbs was defined through the selection and sequencing of MAb escape mutants. Competitive binding assays between MAbs and experimental equine and chicken sera were designed to identify specific MAb reaction to epitopes with high immunogenicity. Results All MAbs showed stronger reactivity with all WNVs tested and good competition for antigen binding in ELISA tests with WNV-positive equine and chicken sera. Four MAbs (3B2, 3D6, 4D3, 1C3) resulted specific for WNV, while two MAbs (2A8, 4G9) showed cross-reaction with Usutu virus. Three MAbs (3B2, 3D6, 4D3) showed neutralizing activity. Sequence analysis of 3B2 and 3D6 escape mutants showed an amino acid change at E307 (Lys → Glu) in the E protein gene, whereas 4D3 variants identified mutations encoding amino acid changed at E276 (Ser → Ile) or E278 (Thr → Ile). 3B2 and 3D6 mapped to a region on the lateral surface of domain III of E protein, which is known to be a specific and strong neutralizing epitope for WNV, while MAb 4D3 recognized a novel specific neutralizing epitope on domain II of E protein that has not previously been described with WNV MAbs. Conclusions MAbs generated in this study can be applied to various analytical methods for virological and serological WNV diagnosis. A novel WNV-specific and neutralizing MAb (4D3) directed against the unknown epitope on domain II of E protein can be useful to better understand the role of E protein epitopes involved in the mechanism of WNV neutralization.

Abstract A fatal Encephalomyocarditis virus (EMCV) infection epidemic involving fifteen primates occurred between October 2006 and February 2007 at the Natura Viva Zoo. This large open-field zoo park located near Lake Garda in Northern Italy hosts one thousand animals belonging to one hundred and fifty different species, including various lemur species. This lemur collection is the most relevant and rich in Italy. A second outbreak between September and November 2008 involved three lemurs. In all cases, the clinical signs were sudden deaths generally without any evident symptoms or only with mild unspecific clinical signs. Gross pathologic changes were characterized by myocarditis (diffuse or focal pallor of the myocardium), pulmonary congestion, emphysema, oedema and thoracic fluid. The EMCV was isolated and recognized as the causative agent of both outbreaks. The first outbreak in particular was associated with a rodent plague, confirming that rats are an important risk factor for the occurrence of the EMCV infection.

Highlights • The emergence of HP isolates represents a main concern for the PRRS control. • The PRRSV-1 DV vaccine provided partial protection against the HP-PRRSV-1 PR40 strain. • Cellular immunity was recalled in vaccinated-PR40 infected pigs after infection. • Vaccinated pigs mounted a rapid and boosted VN-Ab response after challenge. • The vaccine induced adaptive immunity which counteracted the heterologous HP strain. Abstract PRRS is one of the main viral diseases in pig production, causing huge economic losses to the swine industry worldwide. The virus shows an intrinsic genomic instability and is able to change continuously, with the emergence of new strains, with different pathogenicity patterns. Commercially available vaccines only partially prevent or counteract the disease and the correlated losses. Moreover, the emergence of highly virulent and pathogenetic isolates represents a particular concern for PRRS control and diagnosis. The purpose of this study was to evaluate the efficacy of a modified-live virus (MLV) PRRSV-1 commercial vaccine in reducing the severity of the disease and minimizing losses upon challenge with a highly pathogenic PRRSV-1.1 Italian isolate (PRRSV-1_PR40/2014). Four different groups were compared: C (unvaccinated-uninfected), VAC-C (vaccinated-uninfected), PR40 (unvaccinated-infected) and VAC-PR40 (vaccinated-infected). The tested vaccine provided partial, but statistically significant clinical, virological and pathological protection after challenge under experimental conditions. In particular, vaccinated animals showed reduced viremia in terms of duration and magnitude, reduced respiratory signs and pathological lesions. Vaccination was able to trigger adaptive immunity able to respond efficiently also against the HP PR40 isolate. Vaccinated animals showed higher average daily weight gain, even during the viremic period, compared to non-vaccinated challenged pigs. [ABSTRACT FROM AUTHOR]

Highly pathogenic (HP) PRRSV isolates have been discovered within both PRRSV-1 and PRRSV-2 genotypes and investigated in recent years especially for their ability to cause extremely severe disease in conventional pig herds. The exacerbation of general and respiratory clinical signs has been attributed not only to an efficient replication (virulence) but also to the ability to dysregulate viral recognition and induce mechanisms of immune evasion or immune enhancement of humoral and cellular anti-viral responses differently from non-HP PRRSV isolates in terms of intensity and temporal onset. Thus, the understanding of the immunopathogenesis of HP PRRSV is a major concern for the study of virus biology and development of efficacious vaccines. The present study aims at addressing the modulation of relevant immune cell subsets by flow cytometry in the blood of 4-week-old pigs experimentally infected with the recently discovered PR40/2014 HP PRRSV-1.1 strain phenotypically characterized in Canelli et al. (2017) compared to pigs infected with a non-HP PRRSV isolate (PR11/2014) and uninfected controls. PR40 infected animals showed an early and marked reduction of pro-inflammatory CD172α+ CD14+CD16+ and CD14+CD163+ monocytes and TCRγδ+CD8α+/CD8α- lymphocytes when pigs were most infected, possibly due to a recruitment sustaining an acute inflammatory response in target tissues. The prolonged increased CD3+CD16+ NKT cell levels may sustain peripheral inflammation and/or the anti-viral response. The late reduction (potential depletion) of γ/δ T lymphocytes and CD3+CD4+CD8α- naïve Th lymphocytes paralleled with the delayed increase of CD3+CD4+CD8α+ memory and CD3+CD4-CD8α/β + cytotoxic T lymphocytes. In addition, PR40 infection showed an early depletion of activated CD4+CD25+ T lymphocytes and Tregs together with an intense and lasting depletion of CD21+ B lymphocytes. Overall, these features demonstrate that the more severe clinical signs observed upon infection with the HP PR40 strain are sustained by remarkable changes in the peripheral blood distribution of immune cells and provide further insights into the immune regulation/immunopathogenesis induced by PRRSV-1 subtype 1 European isolates. [ABSTRACT FROM AUTHOR]

Highly pathogenic (HP) isolates of the PRRS virus started emerging in North America and Asia in the late 1990s. More recently, they have emerged in Europe. These isolates are characterized by high viral loads, severe general clinical signs and high mortality, in sows, weaners and growers. Their genome shows a discontinuous aminoacids deletion in the non-structural protein 2 (NSP2). The present study was aimed at characterizing the clinical, pathological and immunological features of a highly pathogenetic, Italian PRRSV-1 subtype 1 isolate (PRRSV1_PR40/2014), following experimental infection in conventional 4-weeks-old pigs. The PRRSV1_PR40/2014 infected group showed severe clinical signs (high fever and dispnoea). Pathological lesions, including severe lymphocytopenia in bronchial lymph-nodes and thymus were also recorded. Higher serum PRRSV genome copies and lower virus neutralizing antibody titer were observed in the PR40 group, when compared to the group infected with a conventional PRRSV strain. The genetic analysis of the strain, and the phenotypic features observed in the field and reproduced in the experimental study, confirmed the high pathogenicity of the Italian PRRSV-1 subtype 1 PR40 isolate. [ABSTRACT FROM AUTHOR]

Background: This study was aimed at evaluating the clinical protection, the level of Porcine circovirus type 2 (PCV2) viremia and the immune response (antibodies and IFN-γ secreting cells (SC)) in piglets derived from PCV2 vaccinated sows and themselves vaccinated against PCV2 at different age, namely at 4, 6 and 8 weeks. The cohort study has been carried out over three subsequent production cycles (replicates). At the start/enrolment, 46 gilts were considered at first mating, bled and vaccinated. At the first, second and third farrowing, dams were bled and re-vaccinated at the subsequent mating after weaning piglets. Overall 400 piglets at each farrowing (first, second and third) were randomly allocated in three different groups (100 piglets/group) based on the timing of vaccination (4, 6 or 8 weeks of age). A fourth group was kept non-vaccinated (controls). Piglets were vaccinated intramuscularly with one dose (2 mL) of a commercial PCV2a-based subunit vaccine (Porcilis® PCV). Twenty animals per group were bled at weaning and from vaccination to slaughter every 4 weeks for the detection of PCV2 viremia, humoral and cell-mediated immune responses. Clinical signs and individual treatments (morbidity), mortality, and body weight of all piglets were recorded. Results: All vaccination schemes (4, 6 and 8 weeks of age) were able to induce an antibody response and IFN-γ SC. The highest clinical and virological protection sustained by immune reactivity was observed in pigs vaccinated at 6 weeks of age. Overall, repeated PCV2 vaccination in sows at mating and the subsequent higher levels of maternally derived antibodies did not significantly interfere with the induction of both humoral and cell-mediated immunity in their piglets after vaccination. Conclusions: The combination of vaccination in sows at mating and in piglets at 6 weeks of age was more effective for controlling PCV2 natural infection, than other vaccination schemas, thus sustaining that some interference of MDA with the induction of an efficient immune response could be considered. In conclusion, optimal vaccination strategy needs to balance the levels of passive immunity, the management practices and timing of infection. [ABSTRACT FROM AUTHOR]

Avian rotaviruses are still largely undefined despite being widespread in several avian species and despite the economic impact of rotavirus (RV) enteritis in poultry flocks. In this study, the presence of different avian RV groups was investigated in commercial poultry flocks reared in Northern and Central Italy and with a history of enteric diseases. Faeces or intestinal contents from different avian species previously found to contain RV particles by electron microscopy (EM) were analysed by both RNA-polyacrylamide gel electrophoresis and reverse transcription-polymerase chain reaction specific for groups A, D, F and G RVs. Group D avian RV was detected in 107 of 117 samples tested (91.5%), whereas groups A, F and G avian RVs were present in 70 (59%), 61 (52.1%) and 31 (26.5%) samples, respectively. Multiple presence of different RV groups was detected in 83% of samples. This study provides novel data on the prevalence of genetically different avian RVs in Italian poultry flocks. This information is useful to elucidate the epidemiology of avian RVs circulating in Italy. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

A fatal Encephalomyocarditis virus (EMCV) infection epidemic involving fifteen primates occurred between October 2006 and February 2007 at the Natura Viva Zoo. This large open-field zoo park located near Lake Garda in Northern Italy hosts one thousand animals belonging to one hundred and fifty different species, including various lemur species. This lemur collection is the most relevant and rich in Italy. A second outbreak between September and November 2008 involved three lemurs. In all cases, the clinical signs were sudden deaths generally without any evident symptoms or only with mild unspecific clinical signs. Gross pathologic changes were characterized by myocarditis (diffuse or focal pallor of the myocardium), pulmonary congestion, emphysema, oedema and thoracic fluid. The EMCV was isolated and recognized as the causative agent of both outbreaks. The first outbreak in particular was associated with a rodent plague, confirming that rats are an important risk factor for the occurrence of the EMCV infection. [ABSTRACT FROM AUTHOR]

• The impact of PRRSV strains of different virulence on thymic macrophages was examined. • Animals died 10–14 days after PR40 or PR11 infection showed the most severe lesions. • The number of N-protein+, CD172a+, CD163+ and BA4D5+ cells increased at 10–14dpi. • No marked differences were observed between the PR11 and PR40 strains in this study. • Vaccination restrained virus spread and lesions in thymus of PR40-infected animals. The emergence of "highly pathogenic" isolates of porcine reproductive and respiratory syndrome virus (HP-PRRSV) has raised new concerns about PRRS control. Cells from the porcine monocyte-macrophage lineage represent the target for this virus, which replicates mainly in the lung, and especially in HP-PRRSV strains, also in lymphoid organs, such as the thymus. This study aimed at evaluating the impact of two PRRSV strains of different virulence on thymic macrophages as well as after heterologous vaccination. After experimental infection with PR11 and PR40 PRRSV1 subtype 1 strains (low and high virulent, respectively) samples from thymus were analysed by histopathology and immunohistochemistry for PRRSV N protein, TUNEL, CD172a, CD163, CD107a and BA4D5 expression. Mortality was similar in both infected groups, but lung lesions and thymus atrophy were more intense in PR40 group. Animals died at 10–14 dpi after PR11 or PR40 infection showed the most severe histopathological lesions, with a strong inflammatory response of the stroma and extensive cell death phenomena in the cortex. These animals presented an increase in the number of N protein, CD172a, CD163 and BA4D5 positive cells in the stroma and the cortex together with a decrease in the number of CD107a positive cells. Our results highlight the recruitment of macrophages in the thymus, the increase in the expression of CD163 and the regulation of the host cytotoxic activity by macrophages. However, no marked differences were observed between PR11- and PR40-infected animals. Heterologous vaccination restrained virus spread and lesions extent in the thymus of PR40-infected animals. [ABSTRACT FROM AUTHOR]

Highlights • IFN-activated macrophages differentiate PRRSV strains with varying virulence. • A collection of European PRRSV-1 strains were classified in this in vitro model. • Only the PRRSV-1 strain PR40 triggered significant release of TNF and IL-1β. Abstract The recent emergence of highly pathogenic porcine reproductive and respiratory syndrome virus 1 (PRRSV-1) strains has caused severe economic losses. The biological elements defining virulence and pathogenicity are still unclear. In vitro characteristics using natural target cells of PRRSV provide important information to understand the basis of virulence at the cellular level, and provide a mean to reduce animal experimentations to achieve this goal. Here, we compared PRRSV strains from two geographically different regions, with varying in vivo characteristics, in terms of their interactions with monocyte-derived macrophages (MDMs). The strains included Lena and BOR59 from Belarus, and ILI6 from Russia, as well as PR11 and PR40, both from Italy. As a reference, we used a cell culture-adapted version of Lelystad, LVP. MDMs were pre-treated with IFNγ, IL-4 or IFNβ, in order to understand responses in polarized and antiviral MDMs. In general, independent of the geographical origin, the strains with high virulence infected a higher percentage of MDMs and replicated to higher titers. These virulence-dependent differences were most pronounced when the MDMs had been treated with IFNβ. Differentiation between intermediate and low virulent PRRSV was difficult, due to variations between different experiments, but LVP differed clearly from all field strains. IFNα and IL-10 were not detected in any experiment, but PR40 induced TNF and IL-1β. Taken together, these results validate the MDM model to understand pathogenicity factors of PRRSV and confirm the importance of the escape from type I and II IFN-mediated effects for PRRSV virulence. [ABSTRACT FROM AUTHOR]

Background and Aim: Seasonal changes, especially temperature and photoperiod, are well-known determining factors of swine reproductive capacity, but the underlying mechanisms remain unknown. This study aimed to investigate the effect of age and seasonal variations on boar seminal plasma steroids (dehydroepiandrosterone [DHEA], cortisol [CORT], and testosterone [TEST]) over 1 year. Materials and Methods: Four commercial hybrid adult boars (Large White × Duroc), aged between 12 and 44 months, were repeatedly evaluated at the Department of Veterinary Medical Sciences of the University of Bologna. Daily temperature and light hours relating to the collection date were considered for each observation within the four astronomical seasons: Winter, spring, summer, and autumn. Hormones were quantified using radioimmunoassay. The association between seasonal factors and hormone concentrations was evaluated using linear regression models. Univariate models were estimated for each hormone to assess the influence of the independent variables; two multivariate models were assessed to evaluate the effect of temperature and daylight hours, including boar and season factors. Results: Age significantly affected all analyzed hormones (CORT p < 0.0001; DHEA p < 0.0001; and TEST p < 0.0001). The highest average levels were found for each hormone during summertime, suggesting a positive correlation between steroid concentrations with temperature and light hours. Conclusion: The results of this study support the hypothesis that the increase in external temperature and light hours is somehow associated with higher levels of steroid concentrations in the seminal plasma of in-housed boars. These findings may help further investigate seasonal fluctuations in reproductive outcomes, which are well-known for porcine species. [ABSTRACT FROM AUTHOR]

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