Norovirus occurrence and tissue localization in farmed Mediterranean mussels (Mytilus galloprovincialis) from Sardinia, Italy.

Norovirus (NoV) is the leading cause of foodborne gastroenteritis worldwide, with bivalve molluscs representing a major transmission vehicle. This study investigated the occurrence, temporal variability, and tissue localization of NoV in farmed Mytilus galloprovincialis from Sardinia (Italy), integrating long-term surveillance data with experimental histological analyses. A total of 592 mussel samples collected between 2015 and 2025 within official monitoring activities were analyzed by RT-qPCR according to ISO 15216. Overall, NoV RNA was detected in 15.2% of samples. Due to uneven sampling outside the active surveillance period, statistical analyses were restricted to 2015-2018, during which both year and season significantly influenced NoV detection (p < 0.0001), with higher odds observed in 2016 and during spring, and reduced probability in summer. Due to uneven sampling outside the active surveillance period, statistical analyses were restricted to 2015-2018, during which both year and season significantly influenced NoV detection (p < 0.0001), with higher odds observed in 2016 and during spring, and reduced probability in summer. To investigate tissue-specific viral localization, mussels were experimentally exposed under controlled laboratory conditions and analyzed by immunohistochemistry. Norovirus antigens were detected intracellularly within digestive epithelial cells 24 h post-exposure, with additional focal detection in the intestine and gill epithelium, supporting a multistep uptake and retention process associated with filter feeding. Rather than challenging existing detection standards, these findings provide in situ confirmation of tissue-associated viral persistence in M. galloprovincialis and offer mechanistic support for current monitoring approaches. Overall, the integration of epidemiological and histological evidence improves understanding of norovirus persistence in Mediterranean mussel production systems and highlights implications for seasonally informed surveillance and food safety risk management.