A novel diagnostic platform for in situ detection and subtyping of Rotaviruses and Influenza A in pigs

In situ hybridization (ISH) is a nucleic acid-based method that allows the detection of a particular RNA or DNA sequence within the tissue sections. A novel ISH RNA-based chromogenic technique (RNAScope) describes single-molecule visualization through the use of hybridization-based signal amplification system. These characteristics make this platform a promising diagnostic approach, especially by improving sensitivities issues faced by classical ISH techniques. The objective of this study was to evaluate the use RNA-ISH technique in a duplex assay for simultaneously detection Rotaviruses Groups A (RVA), B (RVB) and C (RVC), and Influenza A H1 and H3 genes.

Probes targeting specific genomic regions of the RVA, RVB and RVC (VP6 gene), and Influenza A (H1 and H3 genes) were developed based on validated PCR primers currently used at the veterinary diagnostic laboratory in University of Minnesota (MNVDL). Formalin-fixed paraffin embedded tissues were selected based on PCR results. Duplex assays were development with probes for Rotavirus group A and B, Rotavirus A and C, and Influenza H1 and H3. Hybridization signal was detected as green and red colorimetric staining followed by counterstaining with hematoxylin.

Rotaviruses. A total of 30 samples of small intestines were evaluated. Four samples were positive by PCR for the three Rotavirus group with Ct ranged from 22 to 27 for RVA, from 27 to 30 for RVB and from 23 to 28 for RVC. One sample was positive for RVB (Ct 30) and RVC (Ct 23). Two samples were positive for RVA (Ct 20 and 22). Twenty-three samples were positive for RVA and RVC with Ct ranged from 16 to 28 for RVA and from 21 to 35 for RVC. Twelve samples negative by PCR for all three rotaviruses groups were used as controls.

Influenza A. A total of 16 lung tissues were evaluated. Eight were positive for both subtype H1 and H3 by PCR with CT values ranged from 15 to 33. Four were positive for H1 and four positives for H3. Five samples known as negative for Influenza A were used as controls. There were no cross-reactions among the probes. ISH-RNA was able detected Ct value up to 31 in the intestinal epithelium for Rotavirus and up to 30 in the respiratory epithelium for Influenza A.

The designed probes were successfully able to differentially detected and in situ subtyped RVA, RVB, RVC and Influenza A H1 and H3. The lack of non-specific staining in the negative controls demonstrated the 100% specificity. In the samples evaluated, ISH-RNA sensitivity was higher than usually has been reported by IHC. The amplification steps applied in this platform may be critical for improving the ISH sensitivity.

Disclosure of Interest: None Declared.