Detecting Senecavirus A in tissues: development of a new diagnostic test at the University of Minnesota

Summary

In Canada and the USA alike, Senecavirus A is a challenge for producers and veterinarians because of its clinical similarity to Food and Mouth Disease (FMD). Indeed, Senecavirus A, is a causative agent of swine vesicular disease with lesions developing on the snout, around the mouth and on the coronary band of the feet. Therefore, being able to differentiate Senecavirus A infections from FMD rapidly is of utmost importance to be able to take the appropriate measures.

In the past months, several diagnostic tests have been developed at the University of Minnesota to detect antibodies against Senecavirus A. The difference between those tests and the in situ hybridization (ISH) described here is that ISH targets the genetic material included in the viral particle and marks it as a red spot as can be seen on the figure below. This advantage of this method is to be able to locate the virus and gives additional information to researcher wanting to study the behavior of Senecavirus A in the body of the pig.

in situ hybridization senecavirus A pigs
Red dots and clusters represent the presence of SVV mRNA within an erosive lesion on the tongue of a pig © 2017 Resende et al.

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.

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Webinar on Senecavirus A from Dr. Sturos tomorrow 03/28 at 4pm

Matt Sturos
Dr. Matt Sturos

Dr. Matt Sturos, diagnostic pathologist at the University of Minnesota, Veterinary Diagnostic Laboratory will be presenting the latest information on Senecavirus A in swine, tomorrow at 4pm in a learning session organized by the Minnesota Veterinary Medical Association (MVMA). Participants can join in person at the MVMA conference room or online via WebEX.

 

More information here

Efficacy of three disinfectants against Senecavirus A on five surfaces and at two temperatures

What disinfectant to use against Senecavirus A? Does it vary based on surface type or temperature? These are the questions that a group of researchers from the University of Minnesota – Veterinary Diagnostic Laboratory are answering in the latest issue of the Journal of Swine Health and Production (JSHAP).

The project tested:
3 disinfectants: Household bleach, a phenolic disinfectant, and a quaternary ammoniun-aldehyde
5 surfaces: aluminium, stainless steel, rubber, cement, and plastic
2 temperatures: 25°C (77F) and 4°C (39F)

singh-et-al-2017-disinfectants-against-senecavirus-a

Summary
Objectives: To evaluate the virucidal efficacy of three commercial disinfectants against Senecavirus A (SVA) on five different surfaces at ~25°C and 4°C.
Materials and methods: Household bleach, a phenolic disinfectant, and a quaternary ammonium-aldehyde disinfectant were tested at manufacturer’s recommended concentrations against a contemporary strain of SVA on aluminum, stainless steel, rubber, cement, and plastic surfaces at ~25°C and 4°C. Virus propagation and titration were performed on swine testicular cells. Viral titers were calculated before and after exposure to the disinfectant being tested.
Results: At ~25°C, household bleach at 1:20 dilution inactivated ≥ 99.99% of the virus within 10 to 15 minutes on aluminum, rubber, and plastic. On stainless steel and cured cement, it inactivated 99.97% and 99.98% of the virus, respectively. At 4°C, bleach inactivated ≥ 99.99% of the virus within 5 to15 minutes on all surfaces except rubber; on rubber, inactivation was 99.91% after 15 minutes. The phenolic disinfectant at the manufacturer’s recommended concentration inactivated only ≤ 82.41% of the virus at either temperature and on any surface, even after a 60-minute contact time. Results for the quaternary ammonium disinfectant were intermediate: 78.12% to 99.81% of the virus was inactivated within 60 minutes at both temperatures and on all surfaces. To detect differences between disinfectants, paired Wilcoxon tests were performed. At 10- and 15-minute time points, efficacies of the three disinfectants differed significantly.
Implications: Significant variation exists in the antiviral efficacies of different disinfectants. Hence, they should be tested against various pathogens before use in the field.

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A new indirect ELISA to identify antibodies against Senecavirus A is now available at the UMN-VDL

dvorak-elisa-senecavirus-a
Today, we are very pleased to report that a new indirect ELISA to identify Senecavirus A antibodies has been validated at the University of Minnesota and is now available for our Veterinary Diagnostic Laboratory clients. This ELISA targets specifically antibodies against Viral Protein 2 (VP2) and has a sensitivity of 94.2% and a specificity of 89.7%. The test does not cross react with antibodies against Foot-and-Mouth Disease allowing for a quick differentiation between a Senecavirus A outbreak and a costly foreign animal disease.
elisa-types
Fig 1: Different ELISA types (Source: nptel.ac.in)
Abstract
Background: Senecavirus A (SVA), a member of the family Picornaviridae, genus Senecavirus, is a recently identified single-stranded RNA virus closely related to members of the Cardiovirus genus. SVA was originally identified as a cell culture contaminant and was not associated with disease until 2007 when it was first observed in pigs with Idiopathic Vesicular Disease (IVD). Vesicular disease is sporadically observed in swine, is not debilitating, but is significant due to its resemblance to foreign animal diseases, such as foot-and-mouth disease (FMD), whose presence would be economically devastating to the United States. IVD disrupts swine production until foreign animal diseases can be ruled out. Identification and characterization of SVA as a cause of IVD will help to quickly rule out infection by foreign animal diseases.
Methods: We have developed and characterized an indirect ELISA assay to specifically identify serum antibodies to SVA. Viral protein 1, 2 and 3 (VP1, VP2, VP3) were expressed, isolated, and purified from E. coli and used to coat plates for an indirect ELISA. Sera from pigs with and without IVD symptoms as well as a time course following animals from an infected farm, were analyzed to determine the antibody responses to VP1, VP2, and VP3.
Results: Antibody responses to VP2 were higher than VP1 and VP3 and showed high affinity binding on an avidity ELISA. ROC analysis of the SVA VP2 ELISA showed a sensitivity of 94.2% and a specificity of 89.7%. Compared to IFA, the quantitative ELISA showed an 89% agreement in negative samples and positive samples from 4–60 days after appearance of clinical signs. Immune sera positive for FMDV, encephalomyocarditis virus, and porcine epidemic diarrhea virus antibodies did not cross-react.
Conclusions: A simple ELISA based on detection of antibodies to SVA VP2 will help to differentially diagnose IVD due to SVA and rule out the presence of economically devastating foreign animal diseases.

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Development and validation of a competitive ELISA as a screening test for Senecavirus A

An article published in the Journal of Veterinary Diagnostic Investigation (JVDI) presents a competitive Enzyme-Linked ImmunoSorbent Assay (cELISA) and a virus neutralization test (VNT), both validated for the screening of Senecavirus A in a research setting, by the National Centre for Foreign Animal disease (NCFAD). The diagnostic specificity and sensitivity were 98.2% and 96.9% for the cELISA, and 99.6% (99.0–99.9%) and 98.2% (95.8–99.4%) for the VNT, respectively.

In Canada and the USA alike, Senecavirus A is a challenge for producers and veterinarians because of its clinical similarity to Food and Mouth Disease (FMD). Indeed, Senecavirus A, is a causative agent of swine vesicular disease with lesions developing on the snout, around the mouth and on the coronary band of the feet. Therefore, being able to differentiate Senecavirus A infections from FMD rapidly is of utmost importance to be able to take the appropriate measures.

The University of Minnesota, Veterinary Diagnostic Laboratory has developed an ImmunoFluorescence Assay (IFA) to detect antibodies against Senecavirus A. This test was used as a reference for the validation of the cELISA and VNT established by Drs. Goolia, Yang, Babiuk, and Nfon from NCFAD in collaboration with Drs. Vannucci and Patnayak from the UMN-VDL.

celisa-vnt-senecavirus-a-vannucci

Abstract: Senecavirus A (SVA; family Picornaviridae) is a nonenveloped, single-stranded RNA virus associated with idiopathic vesicular disease (IVD) in swine. SVA was detected in pigs with IVD in Brazil, United States, Canada, and China in 2015, triggering the need to develop and/or validate serologic assays for SVA. Our objective was to fully validate a previously developed competitive enzyme-linked immunosorbent assay (cELISA) as a screening test for antibodies to SVA. Additional objectives included the development and validation of a virus neutralization test (VNT) as a confirmatory test for SVA antibody detection, and the comparison of the cELISA, VNT, and an existing immunofluorescent antibody test (IFAT) for the detection of SVA antibodies in serial bleeds from SVA outbreaks. The diagnostic specificity and sensitivity were 98.2% (97.2–98.9%) and 96.9% (94.5–98.4%) for the cELISA, and 99.6% (99.0–99.9%) and 98.2% (95.8–99.4%) for the VNT, respectively. There was strong agreement among cELISA, VNT, and IFAT when compared based on kappa coefficient. Based on these performance characteristics, these tests are considered suitable for serologic detection of SVA in pigs.

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Investigation of Senecavirus A pathogenesis in finishing pigs

Dr. Fabio Vannucci, a University of Minnesota swine pathologist and his graduate student Dr. Talita Resende collaborated with a team from South Dakota State University to study the pathogenesis of Senecavirus A in finishing pigs. The results of their experiments were published online a few weeks ago in the Journal of General Virology and the printed version should be following shortly.

The importance of Senecavirus A in swine production resides in a striking resemblance in clinical signs with Food and Mouth Disease. Indeed, Senecavirus A causes vesicular lesions around the mouth and on the feet of pigs.

The collaborative work showed that Senecavirus A viremia occurred between 3 to 10 days post-inoculation (dpi), and that the neutralizing antibody response started 5 dpi. Clinical signs first observed 4dpi, lasted up to 10 days.
This study advances our understanding of Senecavirus A pathogenesis to hopefully be able to better manage it in the future.

vannucci-senecavirusa

Abstract: Senecavirus A (SVA) is an emerging picornavirus that has been recently associated with vesicular disease and neonatal mortality in swine. Many aspects of SVA infection biology and pathogenesis, however, remain unknown. Here the pathogenesis of SVA was investigated in finishing pigs. Animals were inoculated via the oronasal route with a contemporary SVA strain SD15-26 and monitored for clinical signs and lesions associated with SVA infection. Viremia was assessed in serum and virus shedding monitored in oral and nasal secretions and feces by real-time reverse transcriptase PCR (RT-qPCR) and/or virus isolation. Additionally, viral load and tissue distribution were assessed during acute infection and following convalescence from disease. Clinical signs characterized by lethargy and lameness were first observed on day 4 pi and persisted for ~2-10 days. Vesicular lesions were observed on the snout and feet, affecting the coronary bands, dewclaws, interdigital space and heel/sole of SVA-infected animals. A short-term viremia was detected between days 3-10 post-inoculation (pi), whereas virus shedding was detected between days 1-28 pi in oral and nasal secretions and feces. Notably, RT-qPCR and in situ hybridization (ISH) performed on tissues collected on day 38 pi revealed the presence of SVA RNA in the tonsil of all SVA infected animals. Serological responses to SVA were characterized by early neutralizing antibody responses (5 days pi), which coincided with a progressive decrease in the levels of viremia, virus shedding and viral load in tissues. This study provides significant insights on the pathogenesis and infectious dynamics of SVA in swine.

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