Vaccination is a critical strategy for controlling H9N2 avian influenza, a subtype with significant implications for poultry health and public safety. Current vaccines hinder serological differentiation between naturally infected and vaccinated animals, complicating disease surveillance and eradication efforts. Here, we developed a novel H9-subtype differentiating-infected-from-vaccinated-animals (DIVA) vaccine using reverse genetics. The recombinant virus, Re-H9-DIVA-J2, was engineered by replacing the neuraminidase (NA) gene of a clinically isolated H9 strain (A/chicken/Guangdong/J2/2016) with the NA ectodomain from a B/Yamagata-lineage influenza virus (B/Massachusetts/2/2012), while retaining six internal genes from the H1N1 PR8 strain. The chimeric virus exhibited low pathogenicity in chicken embryos, high growth titers (HA≥8 log2), and stable genetic inheritance of the B-type NA marker over 10 passages. Three batches of inactivated vaccines were tested in specific-pathogen-free (SPF) chickens, demonstrating robust immunogenicity with hemagglutination inhibition (HI) antibody titers peaking at 10 log2 by 21 days post-vaccination. Challenge experiments confirmed full clinical protection and reduced viral shedding (above 90 % protection). Critically, sera from Re-H9-DIVA-J2-vaccinated chickens showed no cross-reactivity with A-type N2 protein in immunofluorescence (IFA) and ELISA assays, distinguishing them from sera of wild-type-infected or conventional H9N2-vaccinated animals. This study presents a safe, immunogenic H9 marker vaccine compatible with DIVA diagnostics, offering a promising tool for H9N2 control and eradication.
Keywords: Chimeric NA; DIVA; H9N2; Reverse genetics; Serological differentiation.
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