Real-Time Reverse Transcription PCR Assay for Detection of Senecavirus A in Swine Vesicular Diagnostic Specimens

Abstract

Senecavirus A (SV-A), formerly, Seneca Valley virus (SVV), has been detected in swine with vesicular lesions and is thought to be associated with swine idiopathic vesicular disease (SIVD), a vesicular disease syndrome that lacks a defined causative agent. The clinical presentation of SIVD resembles that of other more contagious and economically devastating vesicular diseases, such as foot-and-mouth disease (FMD), swine vesicular disease (SVD), and vesicular stomatitis (VS), that typically require immediate rule out diagnostics to lift restrictions on animal quarantine, movement, and trade. This study presents the development of a sensitive, SYBR Green RT-qPCR assay suitable for detection of SV-A in diagnostic swine specimens. After testing 50 pigs with clinical signs consistent with vesicular disease, 44 (88%) were found to be positive for SV-A by RT-qPCR as compared to none from a negative cohort of 35 animals without vesicular disease, indicating that the assay is able to successfully detect the virus in an endemic population. SV-A RNA was also detectable at a low level in sera from a subset of pigs that presented with (18%) or without (6%) vesicular signs. In 2015, there has been an increase in the occurrence of SV-A in the US, and over 200 specimens submitted to our laboratory for vesicular investigation have tested positive for the virus using this method. SV-A RNA was detectable in all common types of vesicular specimens including swabs and tissue from hoof lesions, oral and snout epithelium, oral swabs, scabs, and internal organ tissues such as liver and lymph node. Genome sequencing analysis from recent virus isolates was performed to confirm target amplicon specificity and was aligned to previous isolates.

Detection of the Emerging Picornavirus Senecavirus A in Pigs, Mice, and Houseflies

“…These findings corroborate the results of recent reports that describe the association of SVA with vesicular disease and neonatal mortality in swine (7,9,10). Additionally, results here are consistent with studies that demonstrated the detection of SVA in vesicular fluid, lesion swabs, lesion scrapings, and oral swabs, indicating that these specimens are appropriate for SVA diagnosis (9,20). SVA nucleic acid has also been detected in serum and in various tissues from affected animals (lymph nodes, tonsil, spleen, and lung) (7,20).…”

“…Additionally, results here are consistent with studies that demonstrated the detection of SVA in vesicular fluid, lesion swabs, lesion scrapings, and oral swabs, indicating that these specimens are appropriate for SVA diagnosis (9,20). SVA nucleic acid has also been detected in serum and in various tissues from affected animals (lymph nodes, tonsil, spleen, and lung) (7,20). Notably, piglets that died during SVA outbreaks presented high viral loads in multiple tissues, including brain, liver, lung, heart, kidney, small intestine, and colon (10).…”

Genomic analysis of codon usage shows influence of mutation pressure, natural selection, and host features on Senecavirus A evolution

“…Compared with DNA virus, it's discovered that RNA virus, such as influenza virus, coronaviruses, exists a highly evolution rate [27e30]. Previous studies revealed that the prototype SVA isolate, namely SVV-001, was isolated in 2001 [31], and due to its selective tropism for human tumor cells, the virus was developed as an oncolytic agent [32].However, the basic viral pathogenesis was not detected accurately. In this study, the synonymous codon usage of the SVA was analyzed to better understand the SVA evolution, especially the interplay between the viruses and the immune response [33].…”

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 .…”