Research Results

SHIC – Final Research Grant Report

Project #: 15-195

Principal Investigator: Pablo Pineyro, DVM PhD (pablop@iastate.edu) and Luis Gimenez-Lirola, PhD (luisggl@iastate.edu)

Institution: Iowa State University

Industry Summary:

The specific aims of this proposal are to develop a set of diagnostic tools that allows direct detection of SVA.

The first objective was to develop and evaluate a SVA immunofluorescence assay (IFA) for the detection virus in cell culture. The development of this technique has a tremendous impact for confirmation of virus isolation. Once SVA is isolated from clinical samples, direct IFA is the technique of choice to confirm the presence of the virus. Thus, we were able to stain infected cells having a more objective method to confirm infection. In addition, this method, compared to a PCR assay, allows us to identify presence of viable virus. For this specific objective we developed reagents that were not commercially available and now are not only available for ISU Veterinary Diagnostic Laboratory’s diagnostic use, but for researchers and other diagnostic laboratories as well.

Our second objective was to develop a technique that allows identification of the virus in clinical specimens fixed in formalin. Since SVA vesicular lesions are non-specific, this technique is important to detect the virus in lesions and differentiate SVA from other potential causes of vesicular disease. We generated two different antibody reagents that can be used to detect SVA in sections of skin with vesicular lesions. These two antibodies were not commercially available and are now not only available for ISU diagnostic laboratory but for researchers and other diagnostic laboratories.

The third objective was to develop a technique that allows visualization of viral genetic material in clinical specimens. This technique uses fluorescent molecular probes that target two different regions of the virus. In order to reduce the effort and cost involved in fluorescent detection, we further evaluated this probe for detection of SVA with light microscopy. This technique will allow efficient detection of SVA in lesions without the burden of expensive fluorescent scopes. The benefit of molecular detection of SVA in tissues over viral detection by PCR is that can we can also demonstrate viral location in tissues, which will help to understand where and how long the virus can persists in tissues.

In conclusion, we successfully developed a set of reagents that can be used in different diagnostic techniques for virus identification in tissue. These techniques will have a great impact on SVA diagnosis in cases of vesicular disease, providing and supporting the differential diagnosis with other causes of vesicular disease such as foot and mouth disease.

Final Report: Duration of Senecavirus A Shedding From Clinically Affected and Non-affected Sows and Piglets After a Breeding Herd Infection

Project #: 15-206

Principal Investigator: Dr. Chris Rademacher

Institution: Iowa State University

Industry Summary:

This study was designed to evaluate the length of shedding of Senecavirus A (SVA) from a sow farm undergoing an outbreak of SVA in the fall of 2015. Goals were to evaluate the SVA shedding patterns of sows and piglets by PCR and Virus Isolation. In addition, the information obtained regarding SVA shedding pattern should provide some guidance on how long sow herds should be closed to minimize the risk of transmitting the virus to other herds or end point sow cull markets. Tonsil, rectal swabs, and serum were collected from sows and their piglets for 6 consecutive weeks. In sows, PCR results indicated that SVA RNA was detected at low levels out to 6 weeks post outbreak in tonsil and rectal swabs, while detectable levels of SVA RNA in serum were only observed for 3 weeks post outbreak. There was no viable virus isolated from any sow samples. In piglets, PCR results indicated that Senecavirus RNA was detected at low levels (20-40% positive) out to 3 weeks post outbreak in tonsil, rectal swabs, and serum. SVA was isolated in <10% of piglets during weeks 1 and 2 post outbreak, but all were negative by the third week. These findings may suggest that SVA is most likely a short-term risk to other herds and the risk of transmitting Senecavirus A may be lower after 30 days.

Characterization of Seneca Valley Virus Circulating in the US and in Brazil

Project #: 15-192

Principal Investigator: Diego G. Diel1

Co-Investigators: Travis Clement1, Eric Nelson1, Jane Hennings1, Steven Lawson1, Luizinho Caron2, Rejane Schaefer2

Institution: 1South Dakota State University, 2EMBRAPA Swine and Poultry

Industry Summary:

Senecavirus A (SVA) or Seneca Valley virus (SVV) is a picornavirus that was originally identified as a cell culture contaminant in the US in 2002. Subsequent sequencing of unidentified picornaviruses viruses isolated from pigs with a variety of clinical presentations revealed the presence of SVV in the US swine population since 1988. In the past ten years, scattered reports have described the association of SVV with cases of swine idiopathic vesicular disease (SIVD) in New Zealand, Australia, Canada, and the US. Most importantly, since November 2014 there have been increased reports of SVV associated with vesicular disease in swine in Brazil and since July 2015 in the US. The significance of this newly emerging virus lies on its association with vesicular lesions that are indistinguishable from those observed in other high consequence foreign animal diseases (FAD) of swine (i.e foot-and-mouth disease virus, FMDV). Thus, any evidence of vesicular disease in pigs requires a complete diagnostic investigation to rule out the possibility of a FAD. In spite of being present in the US since late 1980’s, there is very limited information on SVA epidemiology. Most importantly, the prevalence of SVV infection and the genetic diversity of viral strains currently circulating in the field remain largely unknown.

Final Report: Development of Reagents and Serological Assays for Seneca Valley Virus

Project #: SHIC #SA1600754; NPB project #15-188 SHIC

Principal Investigator: Steven Lawson

Co-Investigators: E. Nelson, D. Diel, A. Singrey, T. Clement, J. Christopher-Hennings

Institution:South Dakota State University

Industry Summary:

The overall objective of this proposal was to develop and validate diagnostic reagents and tests for Senecavirus A (SVA) antigen and antibody detection. The Specific objectives include:

  1. The development of specific expressed protein and antibody reagents for diagnostic assay development and confirmation of virus isolation attempts, including reagents for immunohistochemistry (IHC), fluorescent antibody (FA) staining and development of serological and antigen capture assays.
  2. The development and validation of first generation serological assays for detection of antibody responses to SVA. These assays included an indirect ELISA, fluorescent microsphere immunoassay (FMIA) and a fluorescent focus neutralization (FFN) assay.

Interim Report: Development of Direct Detection Methods for in situ Diagnostic of Seneca A Virus

Project #: 15-195

Principal Investigator: Pablo Pineyro, DVM PhD and Luis Gimenez-Lirola, PhD

Institution: Iowa State University

Industry Summary:

The specific aims of this proposal are to develop a set of direct diagnostic tools that allows direct detection of SV-A in situ.

Interim Report: Pineyro in situ Diagnostic of Seneca A Virus

Project #: 15-195

Principal Investigator: Pablo Pineyro, DVM PhD and Luis Gimenez-Lirola, PhD

Institution: Iowa State University

Industry Summary:

The specific aims of this proposal are to develop a set of direct diagnostic tools that allows direct detection of SV-A in diagnostic tissues.

A. Development of SVA immunofluorescence assay (IFA) for the detection viral antigen in infected cell culture (Completed).
This objective has been completed on time. The main idea was to develop a tool that allows us to confirm the virus in cell cultures. The development of this technique has a tremendous impact for confirmation of virus isolation. Once SVA is isolated from clinical samples direct IFA is the technique of choice to confirm the presence of the virus. Basically, we will be able to stain infected cell having a more objective way to confirm infection through viral staining with specific antibodies. As compared to a PCR assay, this allows us to identify presence of LIVE virus.
For this specific objective we developed reagents that were not commercially available and now are not only available for ISU Veterinary Diagnostic Laboratory’s diagnostic use, but for researchers and other diagnostic laboratories as well.

B. Development of SVA-IHC for detection of viral antigen in clinical specimens (Currently under development).
This objective has been partially achieved. We proposed to develop a technique that allows us to detect SVA in fixed tissues. Since SVA vesicular lesions are non-specific, this technique will allow us detect the virus in lesions and allows us to differentiate SVA from other potential causes of vesicular disease. In order to achieve this goal, we proposed to generate two different types of antibodies (polyclonal and monoclonal). The development of a polyclonal is complete and already evaluated with excellent results. We are able to detect SVA in section of skin with vesicle. Polyclonal antibodies are easier and faster to produce, but they have less specificity than monoclonal antibodies; thus, they may allow for some cross-reaction and can be more difficult to interpret.

In order to provide a more refined diagnostic tool we also proposed to develop a monoclonal antibody. This technique is still under development. The development of this reagent is done in mice and takes approximately 3 months to complete the process, including multiple steps that cannot be accelerated: the mouse must produce sufficient antibodies to test, and as this is an immune response, takes time to build. Unfortunately, our first attempt provided poor quality antibodies therefore we are repeating the production of new candidate monoclonal antibodies to achieve maximal applicability. We are in the process of screening and evaluating the new SVA-Mab.

For this specific objective we developed reagents that were not commercially available and now are not only available for ISU diagnostic laboratory but for researchers and other diagnostic laboratories.

C. Development of SVA in situ hybridization (fluorescent and/or chromogenic) for direct visualization of viral nucleic acid in clinical specimens (Completed).
This objective has been completed on time. We evaluated a set genetic probes that allows us to confirm the presence of the virus in tissues based on the presence of genetic material. We evaluated two set of fluorescent probes targeting different regions of the virus. One of them (VP1) showed to be adequate to detect SVA genetic material in tissues. In order to reduce the effort and cost involved in fluorescent detection, we further evaluated this probe for detection of SVA detection material with light microscopy. SVA VP1 probe was shown to be efficient detecting viral genetic material with light microcopy. This technique will allow us to detect SVA efficiently in lesions without the burden of expensive fluorescent scopes. The benefit of this over PCR is that it will allow us to detect the exact location of virus, which will help to understand where and how long the virus persists in tissue.

Final Report: SHIC Emerging Disease Fact Sheet

Project #: 15-181

Principal Investigator: James Roth

Objective:

Perform a literature review for each of the disease listed—encephalomyocarditis virus (EMCV), filoviruses: African (e.g., Ebola) and Reston species, Getah virus (GETV), hepatitis E virus (HEV), influenza C (IVC) and D (IVD) viruses, Japanese encephalitis virus (JEV), Menangle virus (MenPV), Nipah virus (NiV), porcine adenovirus (PAdV), porcine astrovirus (PAstV), porcine cytomegalovirus (PCMV), porcine kobuvirus (PKoV), porcine rubulavirus (“blue eye”, PoRV), porcine sapelovirus (PSV), porcine sapovirus (PSaV), porcine teschovirus (PTV), porcine torovirus (ToV), pseudorabies virus (PRV), Sendai virus (SeV), Seneca Valley virus (SVV, also known as Senecavirus A), swine papillomavirus (SPV), swine pox virus (SwPV), vesicular exanthema of swine virus (VESV), and vesicular stomatitis virus (VSV). Develop a literature review and a one-to-two page overview for each of the diseases listed that includes etiology; cleaning and disinfection; epidemiology; transmission; pathogenesis, clinical signs, and postmortem lesions associated with infection in swine; diagnostic tests; immunity; prevention and control; and gaps in preparedness.

Develop a document, including a summary matrix, with information on available diagnostic tests for each of the transboundary production diseases listed in objective, as well as gaps in diagnostic preparedness.

Final Report: Expedited Look Into the Prevalence of Senecavirus A in U.S.

Project #: 15-185

Principal Investigator: Main R, Rossow S, Gauger P, Harmon K, Marthaler D, Vannucci F, Zhang J.

Objective:

Expeditiously obtain some insight to better understanding the prevalence of Senecavirus A (Seneca Valley Virus) currently (8/24/2015 – 9/01/2015) circulating in U.S. swine herds that are not known to be exhibiting clinical signs of acute lameness accompanied by the presence of vesicular lesions on the snout, coronary band, and/or hoof.

Final Report: Evaluation of Disinfectants Against Seneca Valley Virus

Project #: 15-187

Principal Investigator: Goyal, Sagar M.

Objective:

The overall objective is to evaluate the efficacy of certain disinfectants on the inactivation of Seneca Valley Virus (SVV) applied to various surfaces including cured cement, aluminum, stainless steel, and plastic and rubber boots at two different temperatures (40C and ~250C).

Final Report: Seneca Valley Virus Outbreak Investigations

Project #: Systematic epidemiological investigations of cases of Senecavirus A in United States swine breeding herds

Principal Investigator: Holtkamp, Derald, et al.

Objective:

The objectives of this project were to enhance the industry’s knowledge of Senecavirus A’s (SVA) spread and prevention by investigating new cases in a timely, efficient, and uniform manner and to determine the most common gaps in biosecurity that may have led to the introduction of SVA in farms we investigated.

Interim Report: Seneca Valley Virus Genetic Diversity Project Summary

Project #: 15-193

Principal Investigator: Diel, Diego G.

Objective:

To determine the complete genome sequence of SVA strains currently circulating in the United States and in Brazil and to compare SVA complete genome sequences and to identify genetic signatures that might affect the specificity of SVA diagnostic tests.

Final Report: Seneca Valley Virus Shedding Pattern on One Sow Farm in Minnesota

Project #: 15-199

Principal Investigator: Tousignant, Steve, et al.

Objective:

The objectives of this study are to first identify an affected case herd, then conduct an epidemiological investigation and social network analysis, as well as perform longitudinal sample collection on the sow farm to asses shedding patterns of sows, gilt pens and suckling piglets, and develop an archive of samples to be made available for future diagnostic investigations.