Validation of a Real-Time Reverse Transcription PCR Assay for Detection of Porcine Kobuvirus (PKV) in Porcine Diagnostic Samples
Project #: 17-144
Principal Investigators: Dr. Phil Gauger DVM, PhD, Associate Professor; Dr. Karen Harmon, PhD, Clinical Associate Professor
Institution: Iowa State University
Porcine kobuvirus (PKV) is an enteric virus detected in swine feces that emerged in the pig population during the previous two decades. Kobuviruses are members of the family Picornaviridae, which is a different virus family compared to PEDV, PDCoV and TGEV or Rotavirus. The first PKV was detected in Europe in 2008 and since have been identified in domestic swine in China, Thailand, Japan, Korea and the United States (US). Porcine kobuviruses have been associated with clinical diarrhea in some swine populations; however, PKV is also detected in feces from healthy swine lacking clinical signs. A study conducted in the US detected PKV in similar numbers of affected and non-affected swine.
The objective of this research was to validate a real-time reverse transcriptase PCR (rRT-PCR) that would detect US strains of PKV in feces, fecal swabs and oral fluids collected from swine. Sequencing assays were developed and validated based on the genes specific to US strains of PKV. In addition, the assay was evaluated in China using clinical samples that contained PKV strains specific to that region.
Porcine feces, fecal swabs and oral fluids were collected from cases submitted to the Iowa State University Veterinary Diagnostic Laboratory (ISU VDL). There were 1,845 samples collected at the ISU VDL and evaluated by the rRT-PCR including 738 oral fluids (OF), 579 feces, and 528 fecal swabs. Approximately 85.8% (633/738) of the oral fluids, 54.2% (314/579) of the feces and 71.2% (376/528) of the fecal swabs were considered positive for porcine kobuvirus. Sequencing confirmed the detection of PKV on positive samples. Feces and tissue homogenates from 112 porcine clinical samples were evaluated with the ISU VDL rRT-PCR in China. There were 23 PKV positive samples using the ISU VDL test confirmed by sequencing indicating the ISU VDL test has the ability to detect Chinese strains of PKV.
Collectively, PKV RNA is present and can be detected in porcine fecal samples and oral fluids using an diagnostic tests validated at the ISU VDL. Validated samples at this time is limited to oral fluids, fecal swabs and feces. Sequencing is available and can be used for monitoring different strains of PKV in swine populations. The ISU VDL diagnostic test also successfully detected strains of PKV from other regions suggesting the emergence of PKV from different geographic regions are detectable using this test. Overall, the large number of positive samples suggest PKV is widespread in US swine and further research is needed to learn if pigs with or without diarrhea are infected with PKV or if different strains of the virus are more likely to cause diarrhea in swine.
Contact information: Dr. Phil Gauger, Iowa State University Veterinary Diagnostic Laboratory. 515-294-1950; email@example.com
Final Report: Description of Biosecurity Aspects of Herds With Low or High PRRS Incidence and Comparison Within and Between Production Systems
Project #: 16-273
Principal Investigator: Daniel Linhares
Co Investigators: Gustavo Silva, Kimberlee Baker, Derald Holtkamp, Bob Morrison
Institution: Iowa State University, University of Minnesota
Porcine reproductive and respiratory syndrome (PRRS) compromises the health of millions of pigs and costs the industry $664 million annually. Thus, swine producers adopt biosecurity measures with intent to decrease the frequency of PRRS outbreaks. There is a critical need to better understand the effects of biosecurity aspects on frequency of PRRS outbreaks in breeding herds.
Therefore, the objective of this study was to describe key differences in the biosecurity aspects of breeding herds with relative low PRRS incidence, compared to those with relatively high PRRS incidence.
This study included herds from 14 production systems in the US. Within each production system herd selection was completed by ranking production system’s herds based on the number of PRRS outbreaks since 2013 and then randomly selecting 3 farms from the 25th and 75th percentiles. The farms from 25th percentile were defined as ‘low incidence’, and farms in the 75th percentile defined as ‘high incidence’. The biosecurity aspects of each breeding herd were assessed using a 346 questions biosecurity survey that contained multiple choice and short answer questions about herd demographics, swine density, PRRS outbreak history, frequency of risk events, and biosecurity practices related to swine transport, people movement, carcass disposal, supply deliveries, and other risk events. Statistical methods were used to determine which biosecurity aspects were significantly different between the low and high PRRS incidence farms.
Fourteen herd sets (84 herds) were enrolled in the study representing 13 states. The average herd size was 3,453 breeding females (range: 543-7,200) for the low PRRS incidence group and 4,099 breeding females (range: 1,000-10,852) for the high PRRS incidence group. Four general areas of biosecurity separated the low and high PRRS incidence farms: (1) monthly event frequency, (2) downtime requirements, (3) swine density, and (4) operational connections to other swine sites.
Rendering was the most significant difference between the groups: 64.3% of high PRRS incidence herds used rendering compared to 31% of low PRRS incidence herds. Mean monthly rendering frequency was 12.7 for high PRRS incidence herds and 5.7 for low PRRS incidence herds. High PRRS incidence farms had a higher monthly frequency of visits from visitors (range: 1-24) than low PRRS incidence farms (range: 0.5-8). Low PRRS incidence farms had significantly longer downtime requirements for visitors and manure removal personnel than high PRRS incidence farms. High PRRS incidence farms were located in areas with significantly higher densities of wean-to-finish swine. Interestingly, a higher number of boars and finishing pigs within a 3-mile radius were significantly associated with a low PRRS incidence. This may be accounted for, in part, by the higher level of biosecurity practiced at boar studs. Operational connections to other swine sites were also important as several operational connection related variables were associated with high PRRS incidence.
These observations will enable the swine industry to more effectively allocate resources to specific aspects of biosecurity which may help reduce the animal welfare and economic impacts of PRRS in the future. Our group will continue to develop biosecurity scores that correlate (help to explain) the frequency of outbreaks. In a nutshell, this study demonstrated the importance of number of events on the biosecurity risk. In other words, we encourage producers to evaluate possibility of reducing the number of pig animal movements (e.g. reducing number of weaning events per month), and number of people entry in the farm (e.g. reducing number of re-entry events).
Also, there was a significant variation in number of PRRS outbreaks in breeding herds. The risk of PRRS exposure can be measured using ‘biosecurity scores’ derived from questionnaires. Benchmarking the scores, and simple outcomes such as number of pig movements, and number of people entry/re-entry per 1,000 sows may be a great tool for managers and producers to identify opportunities to reduce the vulnerability of their swine operations.
Development of Sensitive and Reliable Diagnostic Assay to Detect Atypical Procine Pestivirus (APPV) in Swine
Project #: 16-256
Principal Investigator: Lalitha Peddireddi
Institution: Kansas State Veterinary Diagnostic Laboratory, Kansas State University
Atypical Porcine Pestivirus (APPV) is reported as an etiological agent for type A-II congenital tremors in newborn piglets. Since the first report of APPV from US in 2015, there have been several reports of this virus from around the world. APPV strains reported thus far from different parts of the world exhibit significant genetic diversity (7-17%). Currently used PCR-based APPV detection methods were developed based on limited sequencing information available at the time of their design and are primarily used for research purposes. Therefore, a well validated, highly sensitive and reliable diagnostic assay, developed based on the most up-to-date sequence information, is critical for effective detection of all the genetic variants of APPV. The main aim of this study is to develop a real-time RT-PCR (qRT-PCR) assay, capable of detecting all currently known genetically divergent APPV strains, and fully validate its use in diagnosing APPV infections in the US swine herds. To achieve the main goal of this project, our objectives included, compiling all newly available APPV sequences, generating more complete genome sequences by sequencing of at least 20 APPV positive clinical samples, from different geographical regions, and use the most updated sequence information to design and validate a new qRT-PCR assay. At the time of our initial assay design, there were a total of 7 published full genome sequences available and our sequencing efforts resulted in two complete genomes out of 20 APPV positive clinical samples. So, our initial assay design targeting a highly conserved region in NA5B gene was based of 9 full genomes and 56 partial sequences. After the availability of 4 additional full genome APPV sequences from China, we noted primer-template mismatches within the NS5B target primers as the China strains exhibited high sequence variability (~17%) compared to other APPV strains reported to date. To overcome potential limitations of this assays ability to detect highly divergent China strains, we have modified our assay design to include a second assay targeting a highly conserved region in NS3 gene as an additional target. So, our final assay is a triplex assay with two APPV target regions (NS3 and NS5B) and host 18S rRNA gene target to serve as internal control to monitor nucleic acid extraction efficiency and to eliminate potential false negatives. Analytical and diagnostic validation of triplex qRT-PCR assay including in vitro transcribed RNA, synthetic target sequences representing divergent China strains, APPV positive and negative samples from experimental infection studies and/or obtained from other laboratories, and clinical samples submitted to KSVDL demonstrated high sensitivity and specificity of the assay. Phylogenetic analysis of 35 partial NS5B sequences in this study, obtained from clinical samples submitted from different states, indicate high genetic diversity (~83%-100% sequence identity) of APPV within the US. This information also supports the ability of our triplex qRT-PCR assay developed in this study to detect genetic variants of APPV currently being circulated within the US swine herds.
Lalitha Peddireddi, DVM, PhD, Director of Molecular Diagnostic Service, Kansas State Veterinary Diagnostic Laboratory, Kansas State University. 785-532-5661; firstname.lastname@example.org.
Atypical Porcine Pestivirus (APPV), a highly divergent newly identified pestivirus, is reported as the etiologic agent for type AII congenital tremors in newborn piglets. Since the first report of this virus in the US swine herds in 2015, APPV has been reported in several countries around the world. Sequence analysis suggests high genetic variation (7-17%) among all currently known APPV strains reported from different parts of the world. Therefore, a sensitive and reliable PCR-based diagnostic test is critical for accurate detection of APPV. The main objective of this study is to develop a quantitative real-time RT-PCR (qRT-PCR) assay using all available and newly generated sequence information, for reliable detection of all currently known APPV strains. In this study, we have developed a triplex qRT-PCR assay, using all available sequences from GenBank as well as sequences newly generated from diagnostic samples. The triplex qRT-PCR assay included two APPV target regions (NS5B and NS3), to enhance assay coverage to detect highly divergent China strains reported recently, and host 18S rRNA target to serve as an internal control to monitor and eliminate any false negatives. Individual qRT-PCR assays for each target (NS3, NS5B and 18S rRNA) were optimized separately and then combined into a duplex (NS3+18S and NS5B+18S) and triplex (NS3+ NS5B+18S) assays. Analytical and diagnostic validation of singleplex, duplex and triplex assays were performed using in vitro transcribed RNA, synthetic target sequences representing divergent China strains, APPV-positive and -negative samples from experimental infection studies and clinical samples submitted to KSVDL/ISUVDL. Diagnostic sensitivity of individual APPV assays was ~90% (NS3) and 97% (NS5B), respectively. When these assays are combined into a multiplex format, the diagnostic sensitivity of the multiplex assay increased to 100% with a limit of detection of less than 10 copies of APPV target sequences, which corresponds to a Ct of 37. No cross reactivity was observed for any of the assays with other common swine pathogens indicating high assay specificity. Retrospective screening of 1214 clinical samples, submitted to KSVDL over a period of 2 years, revealed ~15% prevalence of APPV in the US swine herds. APPV has been detected in samples from all age groups pigs, suggesting the possibility of APPV persistence in infected pigs. Of all the specimens tested, oral fluids appear to have higher viral loads (as indicated by low Ct) suggesting oral fluids as a better diagnostic specimen for APPV detection. Phylogenetic analysis of 35 NS5B partial sequences from APPV positive clinical samples, obtained from different states, revealed considerable sequence diversity (85.8% to 100% nucleotide identity) among APPV strains within the US. In addition, two full genome sequences obtained in this study exhibited significant sequence diversity (~12%) compared to the first isolate reported from the US in 2014. Notably, our qRT-PCR assay detected APPV in clinical samples submitted from Canada, which is the first report of APPV in this region. In summary, the triplex qRT-PCR assay developed in this study offers rapid and reliable detection of APPV in the US swine herds. Sequence variation among APPV filed strains reported in this study provides a basis for our understanding of genetic diversity and molecular epidemiology of APPV, currently being circulated within the US swine herds.
Assessment of Slaughter Surveillance Based on Oral Fluids Samples
Project #: 16-175
Principal Investigator: Daniel Linhares
Co-Investigators: Jeff Zimmerman & Marcelo Almeida
Institution: Iowa State University
Development of practical, affordable, and effective monitoring and surveillance systems (MOSS) for tracking pathogens in swine populations over time and space is crucial to the future of the industry. Although serum is the traditional surveillance sample, oral fluid specimens are increasingly recognized as a bona fide alternative.
The objective of this study was to determine whether MOSS can be done using oral fluid samples collected in an U. S. abattoir. Porcine reproductive and respiratory syndrome virus (PRRSV) and Senecavirus A (SVA) were used to represent endemic and emerging pathogens, respectively.
A total of 36 lots of pigs (300-450 pigs per lot) were included in the study. On-farm oral fluid (n = 10) and serum (n =10) samples collected within two days of shipment to the abattoir were used to establish the reference PRRSV and SVA status of the study groups. At the abattoir, environmental samples were collected immediately before (n= 32) and after (n = 32) the pigs were placed in lairage. Three veterinary diagnostic laboratories (VDLs) tested the sera and oral fluids for anti-PRRSV antibody (ELISA), PRRSV RNA (rRT-PCR), and SVA RNA (rRT-PCR). Environmental samples (n = 64) were tested for PRRSV RNA and SVA RNA at one VDL.
Oral fluids (n = 3 per lot) were successfully collected from 32 lots (89%) at the lairage. All oral fluids (collected at the farm and abattoir) tested positive for PRRSV antibody at all VDLs. PRRSV positivity frequency on serum samples ranged from 92.4% to 94.6% among VDLs, with an overall agreement of 98% among the laboratories. PRRSV RNA was detected on 2%, 18%, and 18% of sera, farm oral fluids and abattoir oral fluids, respectively. Between-VDLs agreement for rRT-PCR on sera, and oral fluids was 98% and 81%, respectively. For SVA testing, all oral fluids, all farm samples tested negative at all VDLs. However, 70% of oral fluids collected at the abattoir tested positive in at least one VDL. Results demonstrate the need to further investigate the source of SVA RNA.
In summary, anti-PRRSV antibodies, and PRRSV and SVA RNA were successfully detected in abattoir oral fluids from pigs. There was a perfect agreement of PRRSV and SVA ELISA results between locations. There is opportunity to improve the between locations agreement of PRRS and SVA PCR testing. Abattoir surveillance based on oral fluids is an alternative to current practices with tests available or endemic and exotic diseases (including African Swine fever virus, Erysipelothrix rhusiopathiae, Influenza A virus, PCV2, PEDV, Classical swine fever virus, Foot-and-mouth disease virus, and PDCoV), but further studies are needed to better understand how to further improve agreement between abattoir and farm group results.
Detection and Differentiation of PCV3 from PCV2a, PCV2b and the Highly Prevalent PCV2d Mutant Strains
Project #: 16-257
Principal Investigator: Jianfa Bai
Institution: Kansas State University
The newly identified porcine circovirus 3 (PCV3) is causing problems in swine similar to that caused by porcine circovirus 2 (PCV2). Yet the PCV3 genome shares little similarity to the PCV2 genome. One objective of this study was to develop a molecular diagnostic assay that can detect and differentiate the majority of the field strains of PCV3 and PCV2. As PCV3 is a new virus, there was limited number of genome sequences available. The other objective was to sequence about 50 PCV3 genomes to study how fast the PCV3 genome is changing, and to use the new sequence information to guide the development, or modification of the detection assay developed in this study. Polymerase-chain reaction (PCR) that is the most used detection technology was used in this study. Analyzing all available genome sequences in the PCR assay design may be the most important first step to ensure the diagnostic coverage of the assay. In this study we have analyzed 1907 available PCV2 full- or near-full genomes, and designed two sets of tests that in combination can detect 98.9% of PCV2 strains including PCV2a, 2b, 2c, 2d and 2e genotypes. This is a significant improvement to several current PCV2 detection assays. The PCV3 assay was designed based on the limited 32 genome sequences available at the time of design. The assay was designed to cover all 32 sequences (100% coverage). However, when more sequences become available, both from the public database and from our home-sequenced ones (n=89), the original design had mismatches to a few strains. To overcome this potential issue, a second set of test was designed and in combination with the first design, they covers all 89 sequences with 100% coverage. An internal control is included in the assay to reduce the false-negative rate. Phylogenetic analysis of the 89 PCV3 full genomes indicated that the largest genetic mutation rate for PCV3 is currently 3.2%. Out of 51 PCV3 genomes we sequenced, 37 were unique genomes, and most of them was grouped into different clusters together with published PCV3 genomes of different locations. As most of our home-sequenced samples were collected from the state of Kansas, our data indicated that the mutations in PCV3 strains do not show an geographic distribution pattern, and they rather mutated randomly in the genome. The 3.2% mutation in the PCV3 genome in just two years indicated that the virus is changing, and continued monitoring the evolution of the virus may be necessary to monitor the emerging strains or genotypes of the virus and to modify molecular detection assays accordingly in order to keep assays up to date. Jianfa Bai, PhD, Director of Molecular Research and Development, Kansas State Veterinary Diagnostic Laboratory, Kansas State University. 785-532-4332; email@example.com.
Describing the Cull Sow and Cull Hog Market Networks in the US: A Pilot Project
Project #: –
Principal Investigator: Benjamin Blair and James Lowe
Institution: Integrated Food Animal Medicine Systems, Department of Veterinary Clinical Medicine, College of Veterinary Medicine, University of Illinois at Urbana-Champaign
What is the range of locations of sows that enter a slaughter plant? How many stops along the way do they make? How long do they remain the slaughter channel? Currently there is little data to investigate such questions allowing the industry and regulators to make informed decisions about how to respond to an animal disease outbreak. This project set out to collect data from a harvest plant to see if such information could lead to answers to those questions allowing the industry and animal health officials to better make decisions to prevent and control animal health emergencies.
In this study, data was captured from a single cull harvest plant, over a period of one week during the spring of 2017. We collected Premise ID tags of the culls as they moved through the plant and grouped them by shipping lot. This allowed for the final point of collection to be identified for the purposes of this study. The premise IDs were then cross referenced against a database containing origin information for each unique premise ID to identify the cull’s proposed farm of origin.
In total, we collected premise data on 90.4% of the culls that moved through the harvest plant that week. The animals originated from a total of 297 unique source farms. Sows originated from farms in 21 states and Canada. To determine whether movements to plants derive locally or nationally the distances between origin farms and plant were calculated. We defined the local region for this plant as the radius needed to meet the plant’s capacity at an industry standard 50% cull rate per year. USDA census surveys where used to calculate the breeding inventory of this area at a county level, and determined a 250km radius sufficient to provide the culls to meet capacity. With this in mind, 23.5% of culls originate from farms in the described local region and 43% of final collection points also reside within 250km of the plant. This depicts nature of the cull movements in the market network as national.
The data above presents information on how the cull network begins and ends however little is known about how culls move through collection points. To learn more about how these culls move after leaving the farm and before arriving at the plant, a simple distribution of the distance between the farm of origin and the final collection point was graphed. We also screened the data for statistical outliers and found that culls originating from distances greater that 240km from the terminal collection point were classified as outliers in the network. The majority of culls (86%) originate less than 240km from the final collection point. This interaction is deemed to be a primary interaction meaning that it is very likely the culls moved direct from the farm of origin to the final collection point. 14% of the culls travel a distance greater than 240km to the terminal collection point. Of these 14%, 17.7% or 2.5% of all culls traveled 5 times as far to the last point of collection from the farm than they did from collection point to plant. We hypothesize that 2.5% to 14% of culls moved between multiple collection points prior to arrival at the harvest plant.
We believe to be the first data set collected that allows for this level of detail in describing cull movement from farm until harvest. Although or study has limitations in both the size of dataset and limited timeframe, we believe it provides a unique insight into animal movements and serves as a platform for further work such as this, using larger sets of data to be completed. A better understanding of how culls move throughout the network may provide more detail about disease transmission in the cull market in the US.
Identifying 90.4% of culls over a short time period demonstrates that tracking culls through harvest plants is a realistic method to capture the complexity of the cull network. Although only 2.5%-14% of culls are believed to have moved between multiple collection points prior to harvest. We believe that this is significant and suggest, as was suspected for PEDV, that culls could be an efficient means of transferring diseases across large geographical regions. Being able to understand the way not only sows but diseases move through the slaughter chain holds great value in making the correct decisions to effectively control and prevent disease outbreaks, and why further work must be completed to effectively and efficiently track culls sows through harvest plans to prepare for such an event.
SHIC – Final Research Grant Report
Project #: 15-195
Principal Investigator: Pablo Pineyro, DVM PhD (firstname.lastname@example.org) and Luis Gimenez-Lirola, PhD (email@example.com)
Institution: Iowa State University
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
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
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
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:
- 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.
- 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
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
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
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.
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.
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.
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.
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.
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.
Published: Longitudinal Study of Senecavirus A Shedding in Sows and Piglets on a Single United States Farm During an Outbreak of Vesicular Disease
Project #: 15-199
Principal Investigator: Tousignant, Steve, et al.
The study illustrates the variation of SVA shedding patterns in different sample types over a 9 week period in sows and piglets, and suggests the potential for viral spread between piglets at weaning.