The H9N2 avian influenza virus (AIV) remains a serious threat to the global poultry industry and public health. The hemagglutinin (HA) protein is an essential protective antigen of AIVs and a major target of neutralizing antibodies and vaccines. Therefore, in this study, we used rice-derived HA protein as an immunogen to generate monoclonal antibodies (mAbs) and screened them using an immunoperoxidase monolayer assay and indirect enzyme-linked immunosorbent assay. Eight mAbs reacted well with the recombinant H9N2 AIV and HA protein, four of which exhibited potent inhibitory activity against hemagglutination, while three showed remarkable neutralization capacities. Western blotting confirmed that two mAbs bound to the HA protein. Linear epitopes were identified using the mAbs; a novel linear epitope, ⁴⁸⁰HKCDDQCM⁴⁸⁷, was identified. Structural analysis revealed that the novel linear epitope is located at the C-terminus of HA2 near the disulfide bond-linked HA1 and HA2. Alignment of the amino acid sequences showed that the epitope was highly conserved among multiple H9N2 AIV strains. The results of this study provide novel insights for refining vaccine and diagnostic strategies and expand our understanding of the immune response against AIV.

The H9N2 avian influenza virus (AIV) is difficult to prevent and control because of its low pathogenicity and frequent mutation. In a previous study, the HA (hemagglutinin) protein of H9N2 was expressed in a rice endosperm reactor and prepared into a subunit vaccine to immunize chickens and mice, both of which exhibited a good immunity effect. The results of the intermediate tests of the transgenic strains (AIV-1 and AIV-3) showed that the HA gene can be stably expressed. Agronomic traits, such as plant height and number of grains, were significantly optimized in the transgenic strains. Moreover, no exogenous HA genes were found in the leaves of the weeds, and it was initially determined that there was no risk of gene drift. This study provides key technical support for the commercialization of plant subunit vaccines for avian influenza viruses.

Based on a previously validated head-to-tail dimer vaccine model, we designed a dimeric form of the Newcastle disease virus (NDV) HN protein expressed in rice endosperm (designated as Osr2HN) and initially characterized its molecular expression profile. Previous immunization studies in chickens demonstrated that two doses (0.5 μg) or a single dose (5 μg) of Osr2HN provided complete protection against viral challenge. To facilitate its commercialization, two transgenic rice lines (HN-1 and HN-2) were propagated for three generations to systematically evaluate their molecular characteristics, genetic stability and environmental safety. Insertion site analysis, combined with PCR, qRT-PCR and western blotting, confirmed that the exogenous HN gene was stably integrated into the nuclear genome without sequence variations. The transgenic lines exhibited germination rates, growth cycles and 12 agronomic traits comparable to those of the wild-type TP309, with the exception of increased grain chalkiness in HN-2. No horizontal transfer of the HN gene to weed species was detected, and pollen viability remained unchanged. Field-based biodiversity analysis revealed no adverse effects of the HN gene on pest or weed communities. Collectively, these findings from comprehensive molecular analyses and field evaluations confirm the genetic stability, agronomic performance, and environmental safety of Osr2HN-transgenic rice, providing essential data to support its commercialization as a plant-derived vaccine platform.