Validation of an RNAscope Assay for the Detection of Avian Influenza A Virus

Highly pathogenic avian influenza (HPAI) is an acute viral disease associated with high mortality and great economic losses. Immunohistochemistry (IHC) is a common diagnostic and research tool for the demonstration of avian influenza A virus (AIAV) antigens within affected tissues, supporting etiologic diagnosis and assessing viral distribution in both naturally and experimentally infected birds. RNAscope in situ hybridization (ISH) has been used successfully for the identification of a variety of viral nucleic acids within histologic samples. We validated RNAscope ISH for the detection of AIAV in formalin-fixed, paraffin-embedded (FFPE) tissues. RNAscope ISH targeting the AIAV matrix gene and anti-IAV nucleoprotein IHC were performed on 61 FFPE tissue sections obtained from 3 AIAV-negative, 16 H5 HPAIAV, and 1 low pathogenicity AIAV naturally infected birds, including 7 species sampled between 2009 and 2022. All AIAV-negative birds were confirmed negative by both techniques. All AIAVs were detected successfully by both techniques in all selected tissues and species. Subsequently, H-score comparison was assessed through computer-assisted quantitative analysis on a tissue microarray comprised of 132 tissue cores from 9 HPAIAV-infected domestic ducks. Pearson correlation of r = 0.95 (0.94-0.97), Lin concordance coefficient of ρ = 0.91 (0.88-0.93), and Bland-Altman analysis indicated high correlation and moderate concordance between the 2 techniques. H-score values were significantly higher with RNAscope ISH compared to IHC for brain, lung, and pancreatic tissues ( ≤ 0.05). Overall, our results indicate that RNAscope ISH is a suitable and sensitive tool for in situ detection of AIAV in FFPE tissues.