Previously, nanobodies (Nbs) targeting ORF2 protein of novel goose astrovirus (nGAstV) were identified and tested for their specificity and binding affinity to nGAstV. However, their ability to neutralize and prevent the viral infection in goslings remained unexplored, especially those fused with Fc domain of chicken IgG (cFc). In the present study, using nGAstV ORF2 as target antigen, Nb-positive clones from already developed Nb phage library reacting with nGAstV were obtained through three rounds of panning and enrichment. Four Nbs based on sequence variation and solubility were selected, fused with cFc, and genetically engineered to produce chimeric nanobodies. The specific reactivity of these Nbs-Fc with nGAstV was assessed by IFA and WB analysis, the affinity and IC₅₀ of Nbs-Fc were determined by bio-layer Interferometry tests and iELISA, respectively. And the neutralizing activity of one nanobody with good biological activity (Nb9-2-cFc) was assessed by cell infection and in vivo antiviral assays in goslings. All the four Nbs-Fc were successfully expressed and confirmed through SDS-PAGE. These Nbs exhibited strong binding affinity to nGAstV, as demonstrated by IFA and WB analysis. Among these, Nb9-2-cFc showed the highest affinity (K_d=59.6 pM) and neutralization activity (IC₅₀=15.75ng/mL) against nGAstV. Further, In vitro assays revealed that this Nb could reduce virus proliferation by blocking the binding of ORF2 to its receptor. In vivo evaluation of prophylactic activity in goslings revealed that Nb9-2-cFc provided significant protection against nGAstV infection by reducing viral load and histopathological damage in target organs. These findings highlight Nb9-2-cFc as a promising candidate for emergency prevention and treatment against nGAstV infection in goslings.

To Cite This Article: Ge Y, Yang J, Liu X, An F, Cui Z and Zeng Q, 2025. Single-domain antibody fused with chicken IgG Fc-fragment protects goslings against novel goose astrovirus (nGAstV). Pak Vet J, 45(1): 257-267. http://dx.doi.org/10.29261/pakvetj/2025.120