A novel RNA-based in situ hybridization to detect Seneca Valley virus in neonatal piglets and sows affected with vesicular disease

Abstract

Seneca Valley virus (SVV) is the causative agent of an emerging vesicular disease in swine, which is clinically indistinguishable from other vesicular diseases such as foot-and-mouth disease. In addition, SVV has been associated with neonatal mortality in piglets. While a commercial SVV qRT-PCR is available, commercial antibodies are lacking to diagnose SVV infections by immunohistochemistry (IHC). Thus, a novel in situ hybridization technique—RNAscope (ISH) was developed to detect SVVRNA in infected tissues. From a total of 78 samples evaluated, 30 were positive by qRT-PCR and ISH-RNA, including vesicular lesions of affected sows, ulcerative lesions in the tongue of piglets and various other tissues with no evidence of histological lesions. Nineteen samples were negative for SVV by qRT-PCR and ISH-RNA. The Ct values of the qRT-PCR from ISH-RNA positive tissues varied from 12.0 to 32.6 (5.12 x 106 to 5.31 RNA copies/g, respectively). The ISH-RNA technique is an important tool in diagnosing and investigating the pathogenesis of SVV and other emerging pathogens.

Persistent Infection and Transmission of Senecavirus A from Carrier Sows to Contact Piglets

“…Swabs (oral, nasal, and rectal/fecal) and blood samples (serum and whole heparinized blood) were collected during the acute phase (days 0, 1, 3, 5, 7, 14, 21, 28, and 35 p.i.) and chronic/persistent phase (days 46,47,48,49,50,51,52,53,54,55,56,57,58,59, and 60 p.i.) of the experiment.…”

“…Serum and peripheral blood mononuclear cell (PBMCs) were processed and stored as previously described (14,20). Additionally, oro-fecal swabs (oral swab followed by rectal swab) were collected from piglets born to sows in G4 on days 46,47,48,49,50,51,52,53,54,55,56, 57, 58, and 59 p.i. One animal of each group (G1, G2, G3, and G4) was euthanized on day 44 p.i., to confirm the presence of SVA in the tonsil.…”

“…Formalin-fixed tissue sections (tonsil) were used to perform in situ hybridization (ISH) for SVA RNA (RNAScope). The ISH was performed as previously described (53). The ISH probe utilized was specific for the viral genome of Seneca Valley virus (nt 301 to 1345 of the VP1 gene; GenBank accession number EU271758.1) (Advanced Cell Diagnostics, Inc.) (53).…”

Reverse transcriptase droplet digital PCR to identify the emerging vesicular virus Senecavirus A in biological samples

“…Molecular methods such as conventional PCR, real‐time PCR, in situ hybridization technique‐RNAscope, nested‐PCR and massive parallel sequencing have been already available to be used in the detection of viral RNA of SVA (Bracht, O'Hearn, Fabian, Barrette, & Sayed, ; Dallagnol, Otonel, Leme, Alfieri, & Alfieri, ; Feronato et al., ; Fowler et al., ; Laguardia‐Nascimento et al., ; Pinheiro‐de‐Oliveira et al., ; Resende, Marthaler, & Vannucci, ; Vannucci et al., ). However, reverse transcriptase followed by droplet digital PCR (RT‐ddPCR) standardized in the present study offers a new methodology for detection and absolute quantification for Senecavirus A .…”

“…RT-ddPCR one-step 0.185 TCID50 0. (Bracht, O'Hearn, Fabian, Barrette, & Sayed, 2016;Dallagnol, Otonel, Leme, Alfieri, & Alfieri, 2017;Feronato et al, 2018;Fowler et al, 2017;Laguardia-Nascimento et al, 2016;Pinheiro-de-Oliveira et al, 2018;Resende, Marthaler, & Vannucci, 2017;Vannucci et al, 2015). However, reverse transcriptase followed by droplet digital PCR (RT-ddPCR) standardized in the present study offers a new methodology for detection and absolute quantification for Senecavirus A.…”