West Nile virus lineage 2 (WNV-2) is a growing public health concern in Europe causing West Nile fever or West Nile neuroinvasive disease (WNND) with substantial morbidity and mortality; however, genomic data from southern Italy are limited despite recent expansion of autochthonous transmission. The aim of the study was to characterize the phylogenetic and molecular features of the WNV-2 strain responsible for the first autochthonous human infection reported in Calabria (2023), and two more additional WNND cases detected in 2024. Full WNV-2 genomes were generated from the three cases. Phylogenetic analysis was performed using all publicly available WNV sequences up to September 2025. Amino acid changes in the polyprotein were compared with known WNV-2 lineage and sub-lineage signatures. The three sequences formed a monophyletic group within sub-lineage WNV-2a, clustering with strains circulating in Central-South-Eastern Europe and showing closest affinity to Hungarian sequences. Non-synonymous substitutions characteristic of the Hungary 578/10 strain (NS2B-119I, NS4B-14G, NS4B-49A, and NS5-298A) were identified and were absent from Central-Northern-Western European and previously reported Italian sequences. Additional substitutions (E-159T, E-399R, and NS3-249P) corresponded to signatures from a fatal WNV-2 infection in a Great Grey Owl in Slovakia. Our study provides the first report of Central-South-Eastern European WNV-2 circulation outside Eastern Europe, supporting its likely spread through the Balkans into Italy by 2022. These findings underscore the rapid spread of WNV-2 in newly affected areas and highlight the critical need for sustained molecular surveillance.

Oropouche virus (OROV) has re-emerged as a significant public health threat during the 2023–2025 outbreak, characterized by an unprecedented geographic expansion beyond endemic regions. The virus has been detected in various regions, including the Caribbean, as well as in Europe and the United States, among travelers from Central and South America. This epidemic was associated with the expansion of a novel reassortant strain, which may exhibit enhanced viral fitness and immune evasion properties. Although orthobunyaviruses are known to counteract interferon responses, the specific strategy adopted by OROV remains incompletely understood. In this study, we compared the in-vitro behavior of the prototype strain BeAn19991 and a 2024 isolate representing the reassortant virus responsible for the 2023–2025 epidemic (ISCDC-2024), focusing on innate immune response. We examined the sensitivity of the two strains to IFN-β antiviral activity and their ability to induce type I IFN in fibroblast. Furthermore, we characterized the innate immune response triggered by in-vitro infection of PBMCs from healthy donors. Although the two viral strains exhibited a similar plateau in viral RNA accumulation and comparable sensitivity to exogenous IFN-β, the IFN-β induction in fibroblasts was delayed, with higher levels induced by ISCDC-2024 than by BeAn19991. Moreover, the two strains displayed distinct innate immune profiles in PBMCs: BeAn19991 predominantly induced a robust antiviral IFN-driven response, whereas ISCDC-2024 infection was associated with an IL-6-dominated inflammatory pattern. Overall, these findings highlight distinct phenotypic features of the contemporary reassortant isolate and suggest strain-specific differences in the modulation of innate immunity, potentially reflecting distinct immune evasion strategies. If confirmed, this hypothesis could have significant implications for infection transmission, viral persistence and disease severity, highlighting the need for continuous surveillance and functional characterization of emerging OROV strains to drive public health interventions and antiviral countermeasures.

Objective: West Nile virus lineage 2 (WNV-2) is a growing public health concern in Europe causing West Nile fever or West Nile neuroinvasive disease (WNND) with substantial morbidity and mortality; however, genomic data from Southern Italy are limited despite recent expansion of autochthonous transmission. The aim of the study was to characterise the phylogenetic and molecular features of the WNV-2 strain responsible for the first autochthonous human infection reported in Calabria (2023), and two more additional WNND cases detected in 2024. Methods: Full WNV-2 genomes were generated from the three cases. Phylogenetic analysis was performed using all publicly available WNV sequences up to September 2025. Amino-acid changes in the polyprotein were compared with known WNV-2 lineage and sub-lineage signatures. Results: The three sequences formed a monophyletic group within sub-lineage WNV-2a, clustering with strains circulating in Central-South-Eastern Europe and showing closest affinity to Hungarian sequences. Non-synonymous substitutions characteristic of the Hungary 578/10 ancestor (NS2B-119I, NS4B-14G, NS4B-49A and NS5-298A), were identified and absent from Central-Northern-Western European and previously reported Italian sequences. Additional substitutions (E-159T, E-399R and NS3-249P) corresponded to signatures from a fatal WNV-2 infection in a Great Grey owl in Slovakia. Conclusions: Our study provides the first report of Central-South-Eastern European WNV-2 circulation outside Eastern Europe supporting its likely spread through the Balkans into Italy by 2022. These findings underscore the rapid spread of WNV-2 in newly affected areas and highlight the critical need for sustained molecular surveillance.