Unraveling the origin of the Azorean viral nervous necrosis 2024 outbreak through spatiotemporal and time-resolved phylogenetic analysis

In late 2024, a mortality event affecting wild dusky grouper (Epinephelus marginatus) was reported across multiple islands of the Azores archipelago, Portugal. Following the previous report of the initial detection of nervous necrosis virus (NNV) infection, biological material was submitted to the National Reference Laboratory, where the outbreak was confirmed and the virus was comprehensively characterized through molecular approaches. Phylogenetic analyses based on RNA1 and RNA2 genomic segments consistently classified the detected virus within genotype I (RGNNV), the most widespread betanodavirus genotype. Maximum likelihood reconstructions revealed strong clustering of the Azorean sequences within the RGNNV lineage, with high bootstrap support and no evidence of genomic reassortment. To further investigate the origin and spread of the outbreak, a spatiotemporal and time-resolved phylogenetic analysis was conducted using the INSaFLU/Nextstrain framework. This approach identified a well-supported monophyletic cluster corresponding to the Azorean outbreak, suggesting a recent common ancestor and local expansion. Comparative analyses indicated closest genetic similarity to strains previously reported in the Indo-Pacific, supporting the hypothesis of long-distance viral dissemination followed by regional emergence, rather than direct transmission from geographically closer European outbreaks. This study provides the first spatiotemporal reconstruction of an NNV outbreak in the Azores and highlights the potential epidemiological interface between aquaculture-associated viral reservoirs and wild marine populations. These findings underscore the need for enhanced surveillance and integrated management strategies to mitigate the impact of viral diseases on vulnerable marine species.