Recombinant DIVA Peptide Fusion Protein (DPFP) Differentiates H7N9 Infected from Vaccinated Chickens

The H7N9 subtype avian influenza virus (AIV) poses a significant and ongoing threat to the poultry industry, particularly in China. Effective control strategies rely on both vaccination and robust surveillance. A critical challenge in vaccinated poultry populations is the inability of standard serological tests to distinguish between animals that have been vaccinated and those that have been naturally infected with the virus. This gap necessitates the development of Differentiate Infected from Vaccinated Animals (DIVA) assays, which are crucial for accurate disease monitoring and outbreak management, preventing unnecessary culling and facilitating targeted interventions.

Researchers developed a recombinant diagnostic antigen, the DIVA peptide fusion protein (DPFP), for H7N9 AIV. This protein, comprising E. coli thioredoxin A, the H7N9 DIVA peptide, and a His affinity tag, was expressed in a soluble form in E.coli. Its diagnostic potential was evaluated using immunoblotting and enzyme-linked immunosorbent assay (ELISA). The ELISA was optimized to differentiate H7N9 virus-infected chicken serum from H7N9 vaccination serum. A specific OD450 cut-off value was established to define positive and negative results for H7N9 DIVA diagnosis.

The recombinant DPFP was efficiently expressed in E.coli as a soluble protein, indicating a viable production method. Immunoblotting analysis confirmed that the DPFP was specifically recognized by serum from H7N9 virus-infected chickens but not by serum from H7N9 vaccinated chickens, demonstrating its differential diagnostic capability. Subsequently, an ELISA based on the DPFP was successfully established and optimized. This ELISA effectively differentiated between H7N9 virus-infected and vaccinated chickens. > An OD450 value of 0.5 was precisely defined as the cut-off for H7N9 DIVA diagnosis, providing a clear threshold for interpretation. This study highlights the utility of prokaryotic expression systems for producing diagnostic antigens capable of discriminating H7N9 virus exposure from vaccination.