Avian reovirus (ARV) causes viral arthritis, chronic respiratory diseases, retarded growth and malabsorption syndrome. However, the precise molecular mechanism remains unclear. Here, we report the host cellular proteins that interact with ARV p17 by yeast two-hybrid screening. In this study, the p17 gene was cloned into pGBKT7 to obtain the bait plasmid pGBKT7-p17. After several rounds of screening of a chicken cDNA library, 43 positive clones were identified as possible host factors that interacted with p17. A BLAST search of the sequences was performed on the NCBI website, which ultimately revealed 19 interacting proteins. Gene ontology enrichment and Kyoto Encyclopedia of Genes and Genome analyses indicated that the acquired proteins were involved in multicellular organismal processes, metabolic processes, and biological regulation. When the subcellular localization of the host protein and ARV p17 protein was investigated, we observed colocalization of p17-GFP with IGF2BP1-RED and PQBP1-RED in the transfected cells but not with FGF1-RED. The direct interaction of ARV p17 protein with IGF2BP1 and PQBP1 was confirmed by coimmunoprecipitation and GST pulldown assays. We used RT-qPCR to assess the expression variation during ARV infection. The results showed that IGF2BP1, PAPSS2, RPL5, NEDD4L, PRPS2 and IFI16 were significantly upregulated, whereas the expression of FGF1, CDH2 and PQBP1 was markedly decreased in DF-1 cells infected with ARV. Finally, we demonstrated that IGF2BP1 had a positive effect on ARV replication, while PQBP1 had the opposite effect. Our findings provide valuable information for better insights into ARV's pathogenesis and the role of the p17 protein in this process.

This study developed a dual-antigen enzyme-linked immunosorbent assay (ELISA) based on σB protein and genotype 5-specific σC protein of avian reovirus (ARV). First, σB and σC proteins were expressed and purified using recombinant technology. Through optimization of coating conditions, the optimal antigen combination was determined to be a mixture of the two proteins at a 1:3 molecular ratio (total concentration: 0.8 μg/mL). Key parameters of the indirect ELISA were optimized via checkerboard titration. Validation confirmed that the dual-antigen ELISA exhibited a sensitivity of 1:3200 against genotype 5 ARV-positive sera, with no cross-reactivity and a coefficient of variation of 2.9–8.6%, demonstrating excellent reproducibility. In application testing, the method specifically detected serum antibodies against genotype 5 ARV variant strains, achieving a 100% positive detection rate in experimental chickens within the first week post-challenge and effectively monitoring dynamic antibody changes in infected flocks. Furthermore, the detection rate for genotype 5-positive serum samples (100%) was significantly higher than that of a commercial kit (75%). This dual-antigen indirect ELISA overcomes the sensitivity limitations associated with conventional genotype 5 ARV detection methods and provides a reliable tool for epidemiological surveillance and infection monitoring.