Novel goose astrovirus (nGAstV) infection causes visceral gout and high mortality in goslings, but at present, there are no effective vaccines or antiviral agents. Here, we identified a neutralizing nanobody (Nb60) targeting the spike (P2) protein of nGAstV capable of inhibiting viral replication by blocking both viral attachment and entry. For effective in vivo delivery, we designed an mRNA-encoded homo-tandem nanobody (3×Nb60) flanked by an Anser interleukin-2 (IL-2) signal peptide and Anser untranslated regions that exhibited an approximately twofold improvement in neutralization potency compared to the mRNA-encoded Nb60 monomer. The mRNA encoding nanobody was encapsulated with lipid nanoparticles (LNPs) to prolong its half-life in serum by approximately 48 h. In a gosling infection model, the 3×Nb60 mRNA-LNP (2 mg/kg) produced 100% survival for both prophylaxis and therapy, significantly reduced viral shedding and viral loads in tissues, attenuated hepatic and renal pathology, and restored serum uric acid, alanine aminotransferase, and aspartate aminotransferase levels to close to the baseline values. Epitope mapping revealed that Nb60 recognizes a critical conformational epitope on the P2 protein, with residues 529R and 553G being essential for binding. The findings establish an mRNA-encoded nanobody platform as a promising strategy for prophylaxis and therapy in the early stages of nGAstV infection.IMPORTANCENovel goose astrovirus (nGAstV) has caused widespread outbreaks of visceral gout in goslings across China since 2017, leading to substantial economic losses in the poultry industry. The disease predominantly affects goslings under 3 weeks of age, and the short disease course limits the utility of conventional vaccines. This study establishes an mRNA-encoded nanobody (Nb) platform in which avian-specific regulatory elements, including an Anser IL-2 signal peptide and an Anser globin untranslated region, enable efficient expression of a neutralizing Nb60 in avian cells. A homo-tandem design (3×Nb60) further prolongs the in vivo half-life, improves antiviral potency, and provides complete protection in goslings. By offering a species-matched delivery framework, this platform provides a rapid-response strategy for emerging avian viral diseases and may also shed light on the development of mRNA-encoded antibody therapeutics against human enteric viruses.
