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Introduction: In Italy, despite the existence of an epidemiological surveillance system for Arboviruses, managed in a One Health perspective, the implementation of genomic characterisation remains fragmented and no systematic genomic surveillance initiative integrating human and animal data has been established. The purpose of this study is to investigate the contribution of Whole Genome Sequencing (WGS) in the integrated genomic characterization of West Nile virus in target avian reservoirs, mosquito vectors and infected humans in Romagna (northeastern Italy). Methods: The study was conducted in Romagna, northeastern Italy, between May and October 2023 involving patients with encephalitis/meningitis and healthy blood donors who tested positive for WNV during screening programmes. Mosquitoes were sampled with attractive traps, and resident and migratory birds were collected as of active or passive surveillance. Samples were screened with WNV-specific PCR (Polymerase Chain Reaction) assays, and positive samples were subjected to WGS by tagmentation and hybrid-capture-mediated target enrichment (Viral Surveillance Panel, Illumina). Assemblies and consensus sequences were obtained with Bowtie2 v.2.4.1 and BCFtools. Maximum likelihood trees for viral phylogenetic inference were constructed with IQ-TREE 2.0.3 with automatic selection of the best nucleotide substitution model, 1000 ultrafast bootstrap replicates and midpoint rooted. Results: Eleven WNV lineage 2 sequences were obtained (4 meningitis cases, 3 blood donors, 3 Culex pipiens and 1 Pica pica). Genome sequencing yielded 98.8-100% coverage. Coding sequences maintained high nucleotide and deduced aminoacid conservation, with an average identity of 99.82% and 99.92%. Mutations of virulent clade 2d were identified, affecting infection rates in avian hosts and mosquito vectors and neuroinvasiveness in humans. Obtained sequences clustered within the Central-Southern European clade with robust bootstrap support, forming a distinct cluster with other viruses isolated in Italy from 2021 to 2023. Discussion: Our study, albeit with preliminary and limited results, provides an insight into integrated genomic characterisation of WNV strains circulating in the study area, where WNV is endemic and which is considered a high-transmission-risk hotspot due to its geoclimatic characteristics and its proximity with one of the main stop-overs and breeding ground for migratory birds in Central and Southern Europe (Delta of Po river). Continued genomic surveillance, throughout future seasons, will support the tracking of arboviruses genetic diversity, evolution, emergence of new variants or introduction of genetically diverse strains. Conclusion: Our approach suggests the importance of adopting an interdisciplinary multistakeholder strategy to integrate the steps from clinical diagnosis to entomological and ornithological monitoring, up to epidemiological and genomic surveillance to create an operational process involving human and animal health suitable for the genomic characterisation of both autochthonous and imported Arboviruses. Engaging both human and animal health sectors, will facilitate a comprehensive characterisation of endemic and emerging Arboviruses to mitigate their impact on public health.

Introduction: Viral inactivation constitutes the basis of many immunoprophylaxis strategies, alongside subunit and, more recently, nucleic-acid-based vaccines. The growing and continuous demand for vaccine doses has stimulated research in the field, boosting the development of new viral inactivation techniques, like high hydrostatic pressure inactivation (HHP), already used in the food industry for controlling microbial contaminations. Studying the efficacy of this method to inactivate viruses may offer new possibilities. Methods: HHP-mediated inactivation of SARS-CoV-2 B.1 and BQ.1.1 lineages ultracentrifugation-purified viral isolates was carried out with a high-pressure system (Avure Technologies Inc.). Three different pressures, maintained for 5 minutes, were tested: 400, 500 and 600 MPa. Virus infectivity reduction was assessed on Vero E6 cell culture. The impact of the inactivation process on virion ultrastructure and on viral proteins’ antigenicity was evaluated by negative staining Transmission Electron Microscopy (TEM) and western blot. Immunogenicity was measured in a murine model (CD1 IGS mice) to assess its capacity to induce B- and T-cell-mediated responses at different time points after immunisation (ELISA, seroneutralisation and T-SPOT assays). Results: Inactivation assessment on Vero E6 cell culture highlighted that both variants were completely inactivated at 500 and 600 MPa, while virus isolates treated at 400 MPa retained infectivity, although replicating with slower kinetics. Even at the highest tested pressure, the viral particles were morphologically identifiable. Inactivation from 500 MPa nonetheless resulted in alterations of the outer surface, appearing smoother due to spikes damage. In agreement with evidence from TEM examination, while the ability of the spikes to bind antibodies was reduced compared with the non-HHP-inactivated control, other structural proteins, i.e., nucleocapsid and membrane, were not damaged. Inactivated viruses were thus capable to induce both an antibody and cell-mediated response in the murine model. Discussion: Preliminary results show HHP efficacy in producing viral inactivates that can efficiently be used as immunogens in murine animal models. The process has so far been validated against SARS-CoV-2, but can universally be used for other emerging and re-emerging viruses with a high impact on human and animal health. The same inactivation process is currently being evaluated for the inactivation of other viruses with high health impact, with promising pilot data. Conclusion: The development of a HHP viral inactivation method allowing the cost-effective production of large quantities of inactivated viral suspension (∼ 5€/litre) might represent an important turning point with a high intrinsic potential of clinical translation in the immunoprophylactic field for the development of new low-cost thermostable vaccines. In fact, not requiring a cold chain for distribution, they are suitable for use also in LMICs (Low- and Middle-Income Countries), which have historically been plagued by substantial disparities when it comes to public health policies, bridging the gap of vaccine access, distribution, and uptake.