Validation of an RNAscope assay for the detection of avian influenza A virus

Abstract

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 ρc = 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 ( p ≤ 0.05). Overall, our results indicate that RNAscope ISH is a suitable and sensitive tool for in situ detection of AIAV in FFPE tissues.

Pathological investigation of high pathogenicity avian influenza H5N8 in captive houbara bustards (Chlamydotis undulata), the United Arab Emirates 2020

“…Nevertheless, Gaide et al . 28 successfully detected AIVs by RNAscope ISH and IHC in FFPE blocks stored at room temperature between 2009 and 2022, with no detrimental effects on final results.…”

“…Negative controls included sections incubated without the primary antibody or with another monoclonal antibody belonging to the same isotype (IgG2). The RNAscope ISH assay relied on a custom-designed probe (V-H5N8-M1M2) targeting the well-conserved influenza matrix protein genes (M gene) 1 and 2 of HPAIV (H5N8) Clade 2.3.4.4b (A/chicken/France/20P016448/2020) following the RNAscope® 2.5 HD Assay RED (Advanced Cell Diagnostics, Hayward, CA) 27 , 28 . A probe targeting the dihydrodipicolinate reductase gene from the Bacillus subtilis strain SMY, served as negative control, while sections of AIV positive tissues were used as positive controls.…”

High pathogenicity avian influenza A (H5N1) clade 2.3.4.4b virus infection in a captive Tibetan black bear ( Ursus thibetanus ): investigations based on paraffin-embedded tissues, France, 2022

“…To determine the presence of avian influenza A virus RNA and assess its distribution within the bear tissue sections, RNAscope ISH was performed as previously described ( 29 ). Briefly, we used probes targeting M1 and M2 genes (V-InfluenzaA-H5N8-M2M1 probe), H5 HA gene (V-InfluenzaA-H5N8-HA-O1 probe) of clade 2.3.4.4b HPAIV H5, and an RNAscope 2.5 high-definition red assay, according to the manufacturer’s instructions, including mild pretreatment conditions (15-min incubation with protease digestion for antigenic retrieval) and hematoxylin counterstaining.…”

High pathogenicity avian influenza A (H5N1) clade 2.3.4.4b virus infection in a captive Tibetan black bear (Ursus thibetanus): investigations based on paraffin-embedded tissues, France, 2022

“…To determine the presence of Avian Influenza A Virus RNA and assess its distribution within the bear tissue sections, RNAscope in situ hybridization (RNAscope ISH) was performed as previously described [23]. Briefly, we used probes targeting M1 and M2 genes (V-InfluenzaA-H5N8-M2M1 probe), H5 hemagglutinin gene (V-InfluenzaA-H5N8-HA-O1 probe) of clade 2.3.4.4b HPAIV H5 and a RNAscope 2.5 high-definition red assay, according to the manufacturer’s instructions, including mild pretreatment conditions (15 min incubation with protease digestion for antigenic retrieval) and hematoxylin counterstaining.…”