Ligilactobacillus salivarius inhibits GII.4[P16] human norovirus replication in zebrafish larvae

Human gut bacteria can modulate human norovirus (HuNoV) infection, underscoring the need to characterize virus-bacterium interactions. This study evaluated the effect of Ligilactobacillus salivarius on HuNoV GII.4[P16] replication using a zebrafish larva model. A GII.4[P16]-positive stool specimen was resuspended in phosphate-buffered saline (PBS), centrifuged, and the supernatant stored at -80°C. L. salivarius was grown overnight in de Man, Rogosa, and Sharpe broth, from which bacterial cells and cell-free supernatant (CFS) were collected. The overnight broth (OB), cells, and CFS were each mixed with virus suspension at a 1:1 ratio, and 3 nL were microinjected into 3-day post-fertilization larvae; controls received PBS with virus. Daily until 4 days post-infection (dpi), 2 samples of 10 pooled larvae were collected, and RNA was extracted and quantified by droplet digital PCR. Two-way analysis of variance showed that treatment and time significantly influenced viral RNA levels (P < 0.001), with a marginal interaction (P = 0.051) indicating consistent inhibitory trends across the study period. CFS-injected larvae showed replication comparable to controls (P > 0.05), peaking at 5.31 log RNA copies/larva at 1 dpi, whereas cell-containing treatments (cells and OB) significantly reduced replication (P < 0.01) throughout the 4-day period. An in vitro binding assay showed that 67% of the virus inoculum physically bound to L. salivarius. In a further comparison, both live and heat-killed bacteria reduced replication initially; live L. salivarius significantly inhibited replication at 3 dpi (P < 0.05). These findings support the hypothesis that L. salivarius physically binds to HuNoV GII.4[P16] and inhibits viral replication, highlighting its potential as a probiotic intervention for HuNoV infection.IMPORTANCENorovirus remains the leading cause of acute gastroenteritis worldwide, responsible for nearly 700 million cases and substantial economic burden annually, yet no vaccine or antiviral therapy currently exists. This study's findings are significant because they identify a specific probiotic strain, Ligilactobacillus salivarius, capable of directly binding to and inhibiting replication of a clinically relevant norovirus genotype (GII.4[P16]), which is responsible for many recent outbreaks globally. By demonstrating that live bacterial cells-not just secreted metabolites-drive this antiviral effect, the study points toward a mechanistic, cell-mediated mode of action that could inform targeted probiotic formulations. The use of a zebrafish larva model also offers a valuable, scalable in vivo system for studying norovirus-microbiome interactions, addressing a major bottleneck in norovirus research caused by the lack of robust cell culture systems. Together, these findings advance understanding of bacterium-virus interactions and support further development of probiotic-based strategies for preventing or reducing norovirus infection severity.