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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: Toscana virus (TOSV, Phlebovirus genus) is a neurotropic sandfly-transmitted virus whose distribution encompasses various countries within the Mediterranean basin. TOSV infection remains a non-notifiable disease in Europe with no official surveillance plans in place despite being considered a significant public health concern in Southern Mediterranean countries. In order to mitigate the impact of this virus on human health, it is crucial to gain a deeper understanding beyond the anecdotal and fragmented evidence that currently exists. The aim of this study is to develop an amplicon-based whole genome sequencing method suitable for the genomic characterization of TOSV. Methods: Primers were designed on TOSV lineage A S, M and L segments reference sequences (GenBank: GCA_031497085.1) using Primal Scheme web tool to generate 400 bp tiled amplicons. To guarantee coverage and sensitivity, a multi-sequence alignment of GenBank-available complete sequences was used to identify mismatches and introduce degenerations in relevant ambiguous sites. Two-pool multiplex amplicon-based library preparation was carried out with Illumina Microbial Amplicon Prep (iMAP, Illumina) and custom designed primers. Two viral isolates on Vero E6 cells and three clinical samples of patients presenting with meningitis were sequenced. De novo assembly was performed with BaseSpace DRAGEN Targeted Microbial (version 1.3.1). Results: A set of 45 oligonucleotide primer pairs (26 for segment L, 13 for segment M and 6 for segment S) that amplify overlapping segments spanning TOSV genome was generated. The primers sets were subsequently compared to an alignment of 8 sequences for segment L, 16 for segment M and 32 for segment S to introduce degenerations. Genome sequencing yielded a mean 98.2% coverage for high-viral-titre propagates on Vero E6 cell culture. Two human samples (1 urine and 1 cerebrospinal fluid) with Ct (Cycle threshold) values of 32 and 33 yielded to sequences with coverage of 91.3% and 80.3%. Another urine sample with Ct = 35 yielded to 47% of mean coverage. Discussion: Our study, albeit with preliminary and limited but very promising results for propagates and samples with relatively high Ct values, proposes a new whole genome amplicon-based sequencing method for genomic characterization of TOSV one of the most neglected and least monitored arboviruses. Alongside an increasingly necessary systematic epidemiological monitoring of TOSV infections, WGS may be instrumental to unravel its genomic makeup and to gain insights into its biology and transmission dynamics. Conclusion: Taking advantage and making the most of the sequencing infrastructures, bioinformatics pipelines, and data-sharing platforms, established during the COVID-19 pandemic, it will make possible to establish an integrated surveillance led by sentinel territorial network to targeting TOSV and other endemic and emerging Arboviruses. This knowledge can inform the development of new diagnostics, therapeutics, and preventive measures, particularly in TOSV high risk regions.

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.