Macromolecular nanocellulose carrier with MSRV-specific nanobody for targeted antiviral therapy in aquaculture

Micropterus salmoides rhabdovirus (MSRV) causes recurring outbreaks in largemouth bass aquaculture, resulting in substantial economic losses. We developed a targeted nanoparticle delivery system to enhance antiviral efficacy. An anti-MSRV nanobody (NbY1) was isolated from phage display and confirmed to specifically bind MSRV via ELISA and immunofluorescence. Carboxylated bacterial nanocellulose (BNC-COOH) was synthesized through acid hydrolysis and TEMPO-mediated oxidation, yielding rod-like nanofibers with average dimensions of 59.5 nm diameter and 403.1 nm length. Fluorescence imaging confirmed tissue penetration of BNC-COOH in fish during immersion exposure. Arctigenin (Arg), which exhibits anti-MSRV activity, was functionalized to preserve its antiviral properties while enabling conjugation. The final BNC-Arg-NbY1 nanoparticles were assembled via amide condensation (average size 81.58 ± 4.9 nm; Arg loading 9.75%; NbY1 content 19.06%). The system showed good biocompatibility at concentrations up to 200 mg/L over 14 days. In vitro studies demonstrated that BNC-Arg-NbY1 enhanced cellular targeting and reduced viral cytopathic effects compared to free NbY1. In vivo immersion delivery resulted in significantly higher tissue accumulation in liver, kidney, and intestine versus BNC-Arg alone (p < 0.05). At equivalent Arg doses (1.25-2.5 mg/L), BNC-Arg-NbY1 exceeded the antiviral potency of free Arg. Notably, at 32 mg/L, the nanoparticle system reduced viral loads and improved survival to 55%, outperforming BNC-Arg (40%) and free Arg (30%). This work demonstrates enhanced targeted delivery for aquaculture antiviral applications.