Development and application of a VNAR-based detection nanobody for avian influenza virus H9N2

The H9N2 subtype avian influenza virus, as a mutant strain of low-pathogenic avian influenza (LPAI), has augmented its transmission ability and pathogenicity via genetic evolution. At present, it has incurred the most substantial economic losses to the global poultry industry, particularly in Asia where it is extensively prevalent. To tackle this challenge, it is essential to devise effective prevention and control strategies for the H9N2 subtype. Among these strategies, vaccine development and highly sensitive diagnostic techniques are the primary countermeasures. Although more than twenty different vaccines and diagnostic kits for various types of avian influenza are currently available on the market, existing detection methods lack sufficient specificity for avian influenza virus subtypes. Precise subtype differentiation still depends on polymerase chain reaction (PCR)-based methods, which necessitate specialized equipment and are rarely accessible in on-site settings such as poultry farms. Our laboratory has developed specific single-domain antibodies (VNAR) derived from sharks against the H9N2 subtype of the avian influenza virus. The recombinant anti-H9N2 VNAR demonstrates high specificity for hemagglutinin (HA) binding activity and can specifically recognize, bind to, and neutralize the H9N2 subtype avian influenza virus on the surface of Madin-Darby canine kidney (MDCK) cells. Owing to their low molecular weight, excellent stability, and enhanced tissue penetration, these specific VNAR molecules possess extremely high application potential, rendering them innovative candidate drugs for the diagnosis and prevention of H9N2 infections.