IMPROVED METHODS FOR THE DIAGNOSIS OF PORCINE REPRODUCTIVE AND RESPIRATORY SYNDROME AND OTHER IMPORTANT VIRAL DISEASES OF SWINE

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: HATCH
• Reporting Frequency: Annual
• Accession No.: 0225301
• Project Start Date: Mar 15, 2011
• Project End Date: Sep 30, 2016
• Program Code: [(N/A)]- (N/A)

Recipient Organization
SOUTH DAKOTA STATE UNIVERSITY
PO BOX 2275A
BROOKINGS,SD 57007

Performing Department
Veterinary & Biomedical Sciences

Non Technical Summary
Infectious diseases continue to present substantial economic and animal welfare concerns to the swine industry. Quick and accurate diagnosis is one of the primary means to safeguard livestock species to prevent further disease transmission resulting in morbidity, mortality and economic consequences. Various diagnostic assays for porcine reproductive and respiratory syndrome virus (PRRSV); porcine circovirus type 2 (PCV2); swine influenza virus (SIV) and rotaviruses are currently available but substantial improvements in the quality, speed and affordability of these assays are needed. Areas of particular importance include assays that allow for the rapid differentiation of virus types or strains, such as the various H and N subtypes of influenza or the groups and types of rotaviruses, since immunity can be greatly impacted by antigenic differences. New diagnostic tests are also needed for the differentiation of infected from vaccinated animals (DIVA technologies). These tests are very important in disease elimination programs and will be linked with specifically engineered marker vaccines. We plan to develop an array of new veterinary diagnostic tests, using enzyme-linked immunosorbent assay (ELISA), multiplex fluorescent microsphere immunoassay (FMIA) and immunochromatographic strip or lateral flow technologies. New assays will be optimized and validated according to recognized quality assurance standards. This study will specifically focus on improved diagnostic methods for some of the most important viral diseases of swine. PRRSV alone is estimated to cost the US swine industry over $600 million annually. Some of these diseases also impact other species, including humans, so diagnostic technologies developed here will likely have applications beyond the swine industry. Funding and cooperative efforts for this project will be leveraged through competitive grant proposals submitted to the National Pork Board, USDA NIFA program and other appropriate agencies. Ongoing cooperative studies with collaborators at other Land Grant Universities and the private sector will provide additional funding leverage. Technologies and reagents developed during the course of this study will be readily available to the livestock industry and existing outreach programs will be utilized to assist with broad dissemination and application of new knowledge.

Animal Health Component

25%

Research Effort Categories

Basic

25%

Applied

25%

Developmental

50%

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
311	3510	1090	40%
311	3510	1101	30%
311	4030	1040	10%
311	4030	1101	20%

Knowledge Area
311 - Animal Diseases;

Subject Of Investigation
3510 - Swine, live animal; 4030 - Viruses;

Field Of Science
1040 - Molecular biology; 1090 - Immunology; 1101 - Virology;

Keywords

swine

viral diseases

diagnostics

immunoassay

fluorescent immunomicrosphere

prrsv

porcine circovirus

swine influenza

rotavirus

Goals / Objectives
The overall goal of this project is to improve the quality, speed and affordability of diagnostic assays for the accurate diagnosis of major viral diseases of swine, including porcine reproductive and respiratory syndrome virus (PRRSV), swine influenza virus (SIV), porcine circovirus type 2 (PCV2), rotaviruses and others. Development and validation of these assays will provide critical information and model approaches for additional assays targeting other existing and new emerging diseases. Current, specific objectives include: 1) Development and characterization of improved reagents for application to assay development. New diagnostic reagents including expressed proteins for specific antibody detection and monoclonal antibodies for specific antigen detection will be developed and fully characterized. 2) Development of improved enzyme-linked immunosorbent assays (ELISA) for detection of antibodies and antigens associated with the targeted infectious agents. ELISA-based assays are currently available for some diseases targeted in this proposal. However, improvement is needed in many areas and a broader array of assays is needed for the detection of both antibodies and viral antigens, and for the differentiation of infected from vaccinated animals (DIVA technologies). 3) Multiplex fluorescent microsphere immunoassays (FMIA) will be developed to detect multiple antibody and/or antigen targets at the same time in a single sample, such as serum, oral fluids or fecal samples. FMIA or Luminex-based assays offer the ability to identify and subtype some pathogens at the same time; such as determining influenza H and N types or rotavirus groups and types. They also have the potential to provide DIVA capability. 4) Reagents and assays developed in Objectives 1-3 will be used to initiate studies on the development of field-based or pen-side diagnostic assays utilizing immunochromatographic strips or lateral flow technologies to allow rapid preliminary diagnosis of select viral diseases on the farm. At the completion of this project, we expect to have developed a broad array of new diagnostic reagents for application in new diagnostic assays, as well as for use in classical viral diagnostic methods such as immunohistochemistry (IHC) and fluorescent antibody (FA) techniques. New or improved ELISA and FMIA tests will be developed, including assays for detection and differentiation of rotaviruses, PRRSV, SIV and PCV2 antigens and/or antibodies as appropriate. Development of new rapid field-based or pen-side diagnostics will be initiated. The various diagnostic reagents and assays developed will be made available to the livestock industry as each individual technology is optimized and validated through the course of this project. All data and new technologies will be presented at scientific conferences focused toward swine practitioners, producers and veterinary diagnosticians and submitted for publication in refereed journals. Outreach programs sponsored by the National Pork Board, the American Association of Swine Practitioners and universities will assist with further dissemination and application of new knowledge to better serve the industry and public.

Project Methods
Our laboratory has extensive experience in the development and validation of new veterinary diagnostic assays and technologies. This background will be utilized to achieve our overall goal of providing improved, rapid and affordable assays for the accurate diagnosis of major viral diseases of swine. We have previously developed a number of monoclonal antibodies and expressed protein reagents for diagnostic assay development and will expand our reagent panels in Objective 1 of the current study. We also have access to an extensive array of diagnostic samples and specimens from previous controlled research studies and diagnostic lab submissions for the optimization and validation of new diagnostic assays. Studies associated with Objective 1 to develop and characterize improved reagents for application in assay development will begin immediately and continue throughout the course of this project. Expressed proteins representing key antigenic epitopes of selected pathogens will be produced using standard methods in our laboratory (Wu et al. 2001 Virology 287:183-191; Wu et al. 2004 Virus Research 114:177-181; Brown et al. 2009 Clinical and Vaccine Immunology 16:628-635). Additional monoclonal antibodies for the detection and differentiation of specific antigen targets will be developed using routine methods in our laboratory (Nelson et al. 1993 J. Clinical Microbiology 31:3184-3189). Development of ELISA and FMIA systems will be initiated immediately and expanded as external competitive funding is obtained. Approaches to ELISA development will vary based on specific antigen and antibody targets and desired assay sensitivity and specificity and may include indirect and competitive ELISA formats. General methods for the development, optimization and validation of these assays have been thoroughly described in our previous publications (Ferrin et al. 2004 Clinical Diagnostic Laboratory Immunology 11:503-514; Brown et al. 2009 Clinical and Vaccine Immunology 16:628-635). Multiplex FMIA technologies will also be developed using procedures that are standard in our laboratory (Lawson et al. 2010 Vaccine 28:5383-5391). Initial FMIA test development priorities include antibody and antigen detection assays for PRRSV and PCV2. Assays for rotavirus antigen detection and differentiation will be initiated after appropriate reagents are developed in Objective 1. Serologic assays using FMIA technologies will be developed to simultaneously detect antibodies to 6 different swine influenza virus (SIV) subtypes including H1 alpha, H1 beta, H1 gamma, H1 delta, the novel, pandemic 2009 H1N1, H3 (cluster 4) and the SIV nucleoprotein (NP). Archived samples of known status and control materials available in our laboratory will be used for optimization and validation of each individual assay prior to grouping tests together in appropriate multiplex formats. Assay specificity, sensitivity and cut-off points will be determined using receiver-operator characteristic (ROC) analysis.

Progress 03/15/11 to 09/30/16

Outputs
Target Audience:Veterinary researchers and diagnosticians, veterinary practitioners, pork producers, veterinary biologics and diagnostics companies. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?This work has resulted in expanded collaborations with private industry and other academic institutions and has provided training opportunities for graduate and undergraduate students. Additional funding for this work was provided by the National Pork Board, the Swine Health Information Center, USDA-NIFA, the South Dakota Agricultural Experiment Station and the Animal Disease Research and Diagnostic Laboratory. How have the results been disseminated to communities of interest?Results of this work have been broadly disseminated through publications in appropriate scientific journals and presentations at scientific and stakeholder meetings. Technologies developed during the course of this project have been implemented in various veterinary diagnostic laboratories to serve the livestock industry. Products developed here have been licensed for commercial distribution. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? The overall goal of this project was to improve the quality, speed and affordability of diagnostic assays for the accurate diagnosis of major viral diseases of swine. Due to the emergence of porcine epidemic diarrhea virus (PEDV) in the US in the spring of 2013, the identification of porcine deltacoronavirus (PDCoV) in the US in early 2014, and Senecavirus A (SVA) outbreaks of late 2015, our effort over the past year was focused on these diseases. Substantial progress has been made in the area of diagnostic development related to PEDV, PDCoV, and SVA as reflected in the following accomplishments. Multiple monoclonal antibodies (mAbs) recently developed against the spike glycoprotein of PEDV were characterized and epitope specificity of each mAb was evaluated by peptide ELISAs, Western blotting and virus neutralization activity. Spike-specific monoclonal antibodies were used to characterize immune-dominant regions of PEDV spike protein. By using this approach we identified a 170 aa domain within the S1 region of the spike that appears to be immune-dominant. These reagents will be valuable for ongoing and future research studies. The PEDV fluorescent focus neutralization (FFN) assay developed previously by our group was used to assess PEDV neutralizing antibodies in sow serum, milk, and colostrum samples and in piglet serum samples following selected feedback protocols. Sow serum samples from two farm sites within different production systems (A, B) were tested. At least 24 sows per site were screened for neutralizing antibodies at 0, 3, 6, 7, and 24 weeks post feedback. These functional antibodies were detected in sow serum samples at both sites 3, 6, 7, and 24 weeks post feedback and in milk and colostrum samples by 7 weeks post feedback. At 6 weeks post feedback, neutralizing antibodies were detected in 27 of 30 Site A piglets (90%), compared to 15 of 29 Site B piglets (52%). Piglets at both sites presented neutralizing antibodies, and sentinel pigs were successfully introduced into both systems without re-infection with PEDV by 24 weeks post feedback. Additional optimization and validation of serological assays for PDCoV was completed using over 2000 samples of known status. Diagnostic sensitivity and specificity of an indirect ELISA assay were greater than 96% and the assay was licensed for commercial development. Since July 2015, approximately 200 cases of SVA associated with vesicular disease and/or neonatal mortality have been reported in the US. Currently, however, there are minimal diagnostic reagents and serological assays available for herd surveillance and confirmation of disease. To address these needs, we developed reagents for immunohistochemistry (IHC), and fluorescent antibody (FA) staining, along with serological assays including an indirect ELISA, fluorescent microsphere immunoassay (FMIA) and a fluorescent focus neutralization (FFN) assay. Serological reagent development began with the cloning, expression and purification of SVA capsid proteins including VP1, VP2 and VP3. Recombinant VP1, VP2 and VP3 were used to immunize mice and rabbits for monoclonal and polyclonal antibody production. ELISA and FMIA assays were optimized using samples of known serostatus from uninfected pigs (n = 612) and from SVA-infected animals (n=171). Initial testing via FMIA showed reactivity of SVA positive samples with VP2 was approximately 10-fold greater than VP1 and VP3, providing the rational for further development of a VP2 indirect ELISA and FMIA. Both tests were validated and ROC analysis showed diagnostic sensitivities and specificities of 96.9, 92.0 and 96.2 and 91.9 respectively. The ELISA and FMIA assays were compared to an indirect immunofluorescence assay (IFA) and kappa values were determined as 0.923 and 0.925, respectively. ELISA and FMIA results show the detection of SVA antibodies as soon as one to two weeks post infection. Although the sensitivity of both assays was adequate, the specificity was lower than desired (57/612 false positives). Monoclonal antibodies against VP1, VP2 & VP3 are being used to develop a blocking ELISA to increase specificity. These new diagnostic reagents and assays should aid in improved surveillance and control strategies for SVA. A model was developed to evaluate the transboundary risk of PEDV contaminated swine feed ingredients during a simulated transport event. The model considered a 37 day time-frame to simulate cargo transport from Beijing, China to Des Moines, IA and evaluated 11 different feed ingredients imported from China. Feed ingredients were inoculated with cell culture adapted PEDV and tested by PCR, virus titration and/or swine bioassay at the end of selected transport segments. An environmental chamber was used to duplicate historical temperature and relative humidity data corresponding with transport from December 23, 2012 to January 28, 2013. At the conclusion of all simulated transport segments, viable PEDV was still detected in certain ingredients, including soybean meal (organic and conventional), Vitamin D, lysine hydrochloride and choline chloride.

Publications

• Type: Journal Articles Status: Published Year Published: 2016 Citation: Dee, S., C. Neill, A. Singrey, T. Clement, R. Cochrane, C. Jones, G. Patterson, J. Christopher-Hennings, E. Nelson. 2016. Modeling the transboundary risk of feed ingredients contaminated with porcine epidemic diarrhea virus. BMC Vet. Res. 12:51.
• Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Dee, S., C. Neill, A. Singrey, T. Clement, R. Cochrane, C. Jones, G. Patterson, G. Spronk, J. Christopher-Hennings, E. Nelson. Modeling the transboundary risk of feed ingredients contaminated with porcine epidemic diarrhea virus. American Assoc. of Swine Veterinarians. Feb. 27-Mar 1, 2016. New Orleans, LA. P. 421.
• Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Setness, B., J. Bjustrom-Kraft, K. Woodard, L. Gim�nez-Lirola, Y. Sun, P. Lasley, Q. Chen, J. Zhang, E. Nelson, D. Baum, P. Gauger, J. Zimmerman, R. Main. Do sows and gilts previously infected with PEDV respond differently to PEDV vaccination? Feb. 27-Mar 1, 2016. New Orleans, LA. P.277.
• Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Dee, S., C. Neill, A. Singrey, T. Clement, A. Singrey, J. Christopher-Hennings, E. Nelson. An evaluation of porcine epidemic diarrhea virus survival in individual feed ingredients in the presence or absence of a liquid antimicrobial. American Assoc. of Swine Veterinarians. Feb. 27-Mar 1, 2016. New Orleans, LA. P.54-56.
• Type: Journal Articles Status: Published Year Published: 2016 Citation: Wang, X., H. Zhang, A.M. Abel, E. Nelson. 2016. .Protein kinase R (PKR) plays a pro-viral role in porcine reproductive and respiratory syndrome virus (PRRSV) replication by modulating viral gene transcription. Archives of Virology. 161:327-333. 10.1007/s00705-015-2671-0.
• Type: Journal Articles Status: Published Year Published: 2016 Citation: Diel, D.G., S. Lawson, F. Okda, A. Singrey, T. Clement, M.H.V. Fernandes, J. Christopher-Hennings, E.A. Nelson. 2016. Porcine epidemic diarrhea virus: an overview of current virological and serological diagnostic methods. Virus Research. doi.org/10.1016/j.virusres. 2016.05.013.
• Type: Journal Articles Status: Published Year Published: 2016 Citation: Song, Y., P. Singh, E. Nelson, S. Ramamoorthy. 2016. A computationally-designed serological assay for the porcine epidemic diarrhea virus. J. Clin. Micro. doi:10.1128/JCM.00460-16.
• Type: Journal Articles Status: Published Year Published: 2016 Citation: Vitosh-Sillman, S., J. Loy, B. Brodersen, C. Kelling, A. Doster, C. Topliff, E. Nelson, J. Bai, L. Peddireddi, B. Hause, R. Hesse. 2016. Experimental infection of conventional nursing pigs and their dams with porcine deltacoronavirus. J. Vet. Diagn. Invest. 28:468-497.
• Type: Journal Articles Status: Published Year Published: 2016 Citation: Okda, F., S. Lawson, X. Liu, A. Singrey, T. Clement, J. Nelson, J. Christopher-Hennings, E.A. Nelson. 2016. Development of monoclonal antibodies and serological assays including indirect ELISA and fluorescent microsphere immunoassays for diagnosis of porcine deltacoronavirus. BMC Vet. Res. 12:95. doi: 10.1186/s12917-016-0716-6.
• Type: Journal Articles Status: Published Year Published: 2016 Citation: Clement, T., A. Singrey, S. Lawson, F. Okda, J. Nelson, D. Diel, E.A. Nelson, J. Christopher-Hennings. 2016. Quantitation of porcine epidemic diarrhea virus neutralizing antibodies in sow serum, colostrum, milk and piglet serum after feedback. J. Swine Health and Production 24:147-153.
• Type: Journal Articles Status: Published Year Published: 2016 Citation: Joshi, L., K. Mohr, T. Clement, K. Hain, B. Myers, J. Yaros, E. Nelson, J. Christopher-Hennings, D. Gava, R. Schaefer, L. Caron, S. Dee, D. Diel. 2016. Detection of the Newly Emerging Senecavirus A in Pigs, Mice and Houseflies. J Clin Micro. 54:1536-1545. doi: 10.1128/JCM.03390-15.

Progress 10/01/14 to 09/30/15

Outputs
Target Audience:Veterinary researchers and diagnosticians, veterinary practitioners, pork producers, veterinary biologics and diagnostics companies. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?This work has provided training and professional development opportunities for graduate and undergraduate students as they participated in all aspects of the project. How have the results been disseminated to communities of interest?Results have been disseminated through refereed and popular press publications, scientific and stakeholder meetings, and regular formal and informal discussions with swine producers and practitioners. Additional, broad dissemination has been made possible by the National Pork Board's outreach programs. What do you plan to do during the next reporting period to accomplish the goals?Serological assays and diagnostic reagents for Seneca Valley Virus (Senecavirus A) will be developed and evaluated in response to the recent outbreaks in North America. We will continue to optimize and fully validate high throughput serological assays for PEDV and PDCoV. Studies designed to identify fluorescent microsphere immunoassay (FMIA) antigen targets that may correlate with functional neutralizing antibody responses will continue. Work will continue on validation of multiplex FMIA techniques for simultaneous detection of antibodies against multiple pathogens in a single sample. Additional monoclonal antibody-based reagents will be developed and evaluated for improved diagnosis of emerging diseases of swine

Impacts
What was accomplished under these goals? The overall goal of this project is to improve the quality, speed and affordability of diagnostic assays for the accurate diagnosis of major viral diseases of swine. Due to the introduction of porcine epidemic diarrhea virus (PEDV) into the US in the spring of 2013 and the identification of porcine deltacoronavirus (PDCoV) in the US in early 2014, much of our effort over the past year has continued to focus on these diseases. Substantial progress has been made in the area of diagnostic development related to PEDV and PDCoV, and is reflected in the following accomplishments. Multiple monoclonal antibodies (mAbs) against the spike glycoprotein of PEDV were developed. These antibodies are currently being characterized and epitope specificity of each mAb is under investigation. These reagents will be valuable for ongoing and future research studies. Spike-specific monoclonal antibodies have been used to characterize immune-dominant regions of PEDV spike protein. By using this approach we identified a 170 aa domain within the S1 region of the spike that appears to be immune-dominant. The PEDV fluorescent focus neutralization (FFN) assay for detection of functional neutralizing antibodies in serum, milk and colostrum was further validated with over 15,000 diagnostic samples tested to date. Detailed protocols were shared with other diagnostic laboratories and inter-laboratory comparisons completed to allow wider utilization of this assay. Further optimization and validation of serological assays for PDCoV was completed using over 1000 samples of known status. Diagnostic sensitivity and specificity of an ELISA assay were greater than 96%. This work has resulted in expanded collaborations with private industry and other academic institutions and has provided training opportunities for graduate and undergraduate students.

Publications

• Type: Journal Articles Status: Published Year Published: 2015 Citation: Collin, E.A., S. Anbalagan, F. Okda, R. Batman, E.A. Nelson, B.M. Hause. 2015. An inactivated vaccine made from a U.S. field isolate of porcine epidemic disease virus is immunogenic in pigs. BMC Veterinary Research. 11:62.
• Type: Journal Articles Status: Published Year Published: 2015 Citation: Dee, S., C. Neill, T. Clement, A. Singrey, J. Hennings, E. Nelson. 2015. An evaluation of porcine epidemic diarrhea virus survival in individual feed ingredients in the presence or absence of a liquid antimicrobial. Porcine Health Management. 1:9 doi:10.1186/s40813-015-0003-0.
• Type: Journal Articles Status: Published Year Published: 2015 Citation: Ran, Z., H. Shen, Y. Lang, E. Kolb, N. Turan, L. Zhu, J. Ma, B. Bawa, Q. Liu, H. Liu, M. Quast, G. Sexton, F. Krammer, B. Hause, J. Christopher-Hennings, E. Nelson, J. Richt, F. Li, and W. Ma. 2015. Domestic pigs are susceptible to infection with influenza B viruses. Journal of Virology 89:4818-4826.
• Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: Drebes, D., L. Greiner, E. Nelson, M. Murtaugh, D. King, and J. Connor. The effect of translactational antibodies on preweaning mortality in a porcine epidemic diarrhea virus exposed herd. 2015 American Association of Swine Veterinarians, Proceedings.
• Type: Journal Articles Status: Published Year Published: 2015 Citation: Okda, F., X. Liu, A. Singrey, T. Clement, J. Nelson, J. Christopher-Hennings, E.A. Nelson, S. Lawson. 2015. Development of an indirect ELISA, blocking ELISA, fluorescent microsphere immunoassay and fluorescent focus neutralization assay for serologic evaluation of exposure to North American strains of Porcine Epidemic Diarrhea Virus. BMC Veterinary Research 11:180.
• Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: Setness, B., J. Bjustrom-Kraft, K. Woodard, L. Gim�nez-Lirola, Y. Sun, P. Lasley, Q. Chen, J. Zhang, E. Nelson, D. Baum, P. Gauger, J. Zimmerman, R. Main. Do sows and gilts previously infected with PEDV respond to PEDV vaccination? 2015 American Association of Swine Veterinarians, Proceedings.
• Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: Vitosh-Sillman, S., C. Kelling, B. Brodersen, J. Loy, A. Doster, C. Topliff, E. Nelson, J. Bai, E. Schirtzinger, E. Poulsen, B. Meadors, D. Hesse. Histopathological and immunohistochemical characterization of pigs experimentally infected with porcine deltacoronavirus. 2015 American Association of Swine Veterinarians, Proceedings.
• Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: Caya, I., M. Bertrand, A. Broes, S. Lawson, E.A. Nelson, J. Christopher-Hennings. Development of an indirect enzymelinked immunosorbent assay (ELISA) for detection of antibodies against porcine epidemic diarrhea virus (PEDV). AAVLD, Kansas City, MO, Oct. 2014. p.191.
• Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: Lawson, S. F. Okda, X. Liu, A. Singrey, T. Clement, J. Christopher-Hennings, E.A. Nelson. Development of a fluorescent microsphere immunoassay (FMIA) and a blocking ELISA for detection of antibodies against porcine epidemic diarrhea virus (PEDV). AAVLD, Kansas City, MO, Oct. 2014. p.193.
• Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: Singrey, A., S. Lawson, F. Okda, X. Liu, T. Clement, J. Nelson, J. Christopher-Hennings, E.A. Nelson. Development and diagnostic application of monoclonal antibodies against porcine epidemic diarrhea virus (PEDV). AAVLD, Kansas City, MO, Oct. 2014. p.192.
• Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: Dee, S., T. Clement, A. Singrey, J. Christopher-Hennings, E. Nelson. An evaluation of a liquid antimicrobial (Sal CURB�) for reducing the risk of viral proxies for foreign animal diseases in contaminated feed. North American PRRS Symposium, Chicago, IL, Dec. 2014. p.118.
• Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: Dee, S., T. Clement, J. Christopher-Hennings, E. Nelson. Assessing the viability of porcine epidemic diarrhea virus in dry feed at 4 degrees C. North American PRRS Symposium, Chicago, IL, Dec. 2014. p.119.
• Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: Singrey, A., S. Lawson, F. Okda, K. Hain, T. Clement, J. Christopher-Hennings, E.A. Nelson. Development of monoclonal antibodies and other reagents for detection of porcine deltacoronavirus (PDCoV). North American PRRS Symposium, Chicago, IL, Dec. 2014. p.53.
• Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: Okda, F., S. Lawson, A. Singrey, X. Liu, J. Nelson, J. Christopher-Hennings, E.A. Nelson. Development of serological assays for detection of antibodies against porcine deltacoronavirus (PDCoV). North American PRRS Symposium, Chicago, IL, Dec. 2014. p.51.
• Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: Dee, S., T. Clement, A. Schelkopf, J. Nerem, D. Knudsen, J. Christopher-Hennings, E. Nelson. An evaluation of contaminated complete feed as a vehicle for porcine epidemic diarrhea virus infection of na�ve pigs following consumption via natural feeding behavior: Proof of concept. North American PRRS Symposium, Chicago, IL, Dec. 2014. p.116.
• Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: Dee, S., C. Neill, T. Clement, J. Christopher-Hennings, E. Nelson. An evaluation of a liquid antimicrobial (Sal CURB�) for reducing the risk of porcine epidemic diarrhea virus infection of na�ve pigs during consumption of contaminated feed. North American PRRS Symposium, Chicago, IL, Dec. 2014. p.117.
• Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: Lawson, S., F. Okda, X. Liu, A. Singrey, T. Clement, J. Christopher-Hennings, E.A. Nelson. Development of an indirect ELISA, a fluorescent microsphere immunoassay (FMIA) and a blocking ELISA for detection of antibodies against porcine epidemic diarrhea virus (PEDV). North American PRRS Symposium, Chicago, IL, Dec. 2014. p.48.
• Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: Vitosh-Sillman, S., C. Kelling, B. Brodersen, J. Loy, A. Doster, C. Topliff, E. Nelson, J. Bai, E. Schirtzinger, E. Poulsen, B. Meadors, D. Hesse. Porcine DeltacoronavirusKochs Postulates Fulfilled. North American PRRS Symposium, Chicago, IL, Dec. 2014. p.27.
• Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: Singrey, A., S. Lawson, F. Okda, K. Hain, T. Clement, J. Christopher-Hennings, E.A. Nelson. Development of monoclonal antibodies and other reagents for detection of porcine deltacoronavirus (PDCoV). 95th Conference of Research Workers in Animal Diseases, Chicago, IL, Dec. 2014. 083P.

Progress 10/01/13 to 09/30/14

Outputs
Target Audience:Veterinary researchers and diagnosticians, veterinary practitioners, pork producers, veterinary biologics and diagnostics companies. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?This work has provided training and professional development opportunities for graduate and undergraduate students as they participated in all aspects of the project. How have the results been disseminated to communities of interest?Results have been disseminated through refereed and popular press publications, scientific and stakeholder meetings, and regular formal and informal discussions with swine producers and practitioners. Additional, broad dissemination has been made possible by the National Pork Board's outreach programs. What do you plan to do during the next reporting period to accomplish the goals?We will continue to optimize and fully validate high throughput serological assays for PEDV and PDCoV. Studies designed to identify FMIA antigen targets that may correlate with functional neutralizing antibody responses will continue. Additional efforts will focus on validation of multiplex FMIA techniques for simultaneous detection of antibodies against multiple pathogens in a single sample.

Impacts
What was accomplished under these goals? The overall goal of this project is to improve the quality, speed and affordability of diagnostic assays for the accurate diagnosis of major viral diseases of swine. Due to the introduction of porcine epidemic diarrhea virus (PEDV) into the US in the spring of 2013 and the identification of porcine deltacoronavirus (PDCoV) in the US in early 2014, much of our effort over the past year has focused on these diseases. Substantial progress has been made in the area of diagnostic development related to PEDV and PDCoV, and is reflected in the following accomplishments. Three separate serological assays for the detection of antibodies against PEDV were developed including an indirect ELISA, a fluorescent microsphere immunoassay (FMIA) and a blocking ELISA (bELISA). All three tests use E. coli expressed, full length PEDV nucleoprotein (NP) as the detection antigen. Results of ROC analysis using >1400 samples for FMIA showed estimated sensitivity and specificity of 98.2% and 99.2%, respectively. The indirect ELISA and bELISA showed a sensitivity and specificity of 97.9% and 97.6% or 98.2% and 98.9%, respectively. These high throughput assays should be of value in controlling the spread of PEDV. Additionally, a fluorescent focus neutralization (FFN) assay was developed and validated to monitor neutralizing antibody responses in serum, milk and colostrum samples. Several serological assays for porcine deltacoronavirus (PDCoV) also were developed and optimized, including indirect ELISA, FMIA, indirect fluorescent antibody (IFA) and virus neutralization assays. A refolded version of E. coli expressed fulllength NP was used as an ELISA and FMIA antigen. The tests were optimized using expected seronegative and seropositive serum samples (n=375). Preliminary ROC analysis indicated diagnostic sensitivities and specificities of approximately 95% and 94%, respectively as compared to a validated IFA test. Both FMIA and ELISA showed seroconversion between 8-14 DPI. In addition, neutralizing antibody titers in serum are being quantified using a fluorescent focus neutralization assay (FFN). Since no specific antibody-based reagents were available to assist in diagnosis of PDCoV, we developed reagents for detection of PDCoV antigen in diagnostic tests, such as virus isolation, immunohistochemistry and fluorescent antibody techniques. The monoclonal antibodies, hyperimmune serum and related reagents produced in this project should prove of substantial value in the detection of PDCoV antigen. They have been shared with multiple laboratories and are currently being utilized in PDCoV IHC, virus isolation and FFN assays.

Publications

• Type: Journal Articles Status: Published Year Published: 2014 Citation: Dee S, T. Clement, A. Schelkopf, J. Nerem, D. Knudsen, J. Hennings, E. Nelson. 2014. An evaluation of contaminated complete feed as a vehicle for porcine epidemic diarrhea virus infection of na�ve pigs following consumption via natural feeding behavior: Proof of concept. BMC Veterinary Research, 10:176.
• Type: Journal Articles Status: Published Year Published: 2014 Citation: Dee, S., C. Neill, T. Clement, J. Christopher-Hennings, E. Nelson. 2014. An evaluation of a liquid antimicrobial (Sal CURB�) for reducing the risk of porcine epidemic diarrhea virus infection of na�ve pigs during consumption of contaminated feed. BMC Veterinary Research, 10:220.
• Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: S. Lawson, F. Okda, X. Liu, T. Clement, A. Singrey, J. Christopher-Hennings, E.A. Nelson. Development of an indirect ELISA for detection of antibodies against porcine epidemic diarrhea virus (PEDV). North American PRRS Symposium, December 7-8, 2013, Chicago, IL.
• Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: A. Singrey, S. Lawson, F. Okda, T. Clement, C. Welbon, J. Christopher-Hennings, E.A. Nelson. Development of monoclonal antibodies and other reagents for detection of porcine epidemic diarrhea virus (PEDV). North American PRRS Symposium, December 7-8, 2013, Chicago, IL.
• Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: S. Lawson, F. Okda, X. Liu, T. Clement, A. Singrey, J. Christopher-Hennings, E.A. Nelson. Development of an indirect ELISA for detection of antibodies against porcine epidemic diarrhea virus. Conference of Research Workers in Animal Diseases. December 8-10, 2013. Chicago, IL.
• Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: Lawson, S., F. Okda, X. Liu, A. Singrey, T. Clement, J. Christopher-Hennings, E.A. Nelson. Development of indirect and blocking ELISAs for detection of antibodies against porcine epidemic diarrhea virus (PEDV). 23rd Congress of the International Pig Veterinary Society, June 8-11, 2014, Cancun, Mexico. Abstract #1937.
• Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: Singrey, A., S. Lawson, F. Okda, X. Liu, T. Clement, J. Nelson, J. Christopher-Hennings, E.A. Nelson. Development and diagnostic application of monoclonal antibodies to porcine epidemic diarrhea virus (PEDV). 23rd Congress of the International Pig Veterinary Society, June 8-11, 2014, Cancun, Mexico. Abstract #1938.
• Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: Nelson, J., F. Okda, R. Parmar, A. Singrey, S. Lawson, X. Liu, J. Christopher-Hennings, E.A. Nelson. Environmental stability of a cell culture adapted U.S. isolate of porcine epidemic diarrhea virus (PEDV). 23rd Congress of the International Pig Veterinary Society, June 8-11, 2014, Cancun, Mexico. Abstract #1940.

Progress 01/01/13 to 09/30/13

Outputs
Target Audience: Veterinary researchers and diagnosticians, veterinary practitioners, pork producers, veterinary biologics and diagnostics companies. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Extensive training and professional development opportunities have been provided for two graduate students. How have the results been disseminated to communities of interest? Results have been published in refereed journals and presented at appropriate scientific conferences. They have also been presented in some popular press and news articles as well as through extensive direct communications with veterinary proactitioners and swine producers. What do you plan to do during the next reporting period to accomplish the goals? During the next year we plan to continue optimization and validation of selected diagnostic assays with a strong focus on porcine epidemic diarrhea virus (PEDV) due to the great economic impact of this new disease issue in the US. Initially we will focus on the further characterization of new monoclonal antibody-based diagnostic reagents developed in our laboratory and improved serological assays including ELISA and virus neutralization assays that we hope will contribute to the control of this disease. We will also use tools and methods developed in our laboratory to assist the swine industry in development and implementation of scientifically-based control measures including assesment of vaccination and oral feedback protocols.

Impacts
What was accomplished under these goals? The overall goal of this project is to improve the quality, speed and affordability of diagnostic assays for the accurate diagnosis of major viral diseases of swine, including porcine reproductive and respiratory syndrome virus (PRRSV), swine influenza virus (SIV), porcine circovirus type 2 (PCV2), rotaviruses and others. Due to the introduction of porcine epidemic diarrhea virus (PEDV) into the US in the spring of 2013 much of our effort over the past year has focused on this disease. Substantial progress has been made on several specific objectives, especially in the area of diagnostic development related to PEDV, and is reflected in the following accomplishments. PEDV was isolated from intestinal contents of several diagnostic cases submitted to SDSU. The PEDV-CO isolate was also received from NVSL and further adapted to cell culture. Consistent high-titer virus stocks approaching 7 logs/ml are now routinely being produced and are being used in studies involving PEDV environmental stability and the effectiveness of sanitation efforts. Availability of high-titer cell culture adapted PEDV is particularly valuable for virus stability and disinfectant studies as simple virus re-isolation can be used to assess presence of viable PEDV, rather than relying on time-consuming and expensive swine bioassay systems. A sensitive real-time PCR assay for PEDV was developed, validated and is now routinely used in diagnostic applications at the SD-ADRDL. It was licensed to Tetracore, Inc., Rockville, MD and is currently in production for commercial availability. This assay is a PEDV/TGE multiplex quantitative, real-time PCR which was validated using current US PEDV isolates and field samples and includes an internal control. Another area of focus was to develop high quality, readily available reagents for detection of PEDV antigen in research and diagnostic applications. Rabbits and mice were immunized with selected recombinant PEDV proteins including a full length nucleoprotein (NP) or portions of the spike (S) protein containing putative neutralizing epitopes. Rabbit antisera is being used in numerous diagnostic applications and mouse hybridoma clones producing monoclonal antibodies against the PEDV nucleoprotein and spike protein were produced for additional diagnostic applications. Cell culture adapted PEDV was used to develop an indirect fluorescent antibody (IFA) test for PEDV serology. This test was brought on-line at the SD-ADRDL in September and is regularly used by diagnostic clients. A similar test is offered by the Iowa State University-VDL and inter-laboratory comparisons between the laboratories have demonstrated excellent agreement. A serological ELISA using expressed and purified PEDV nucleoprotein (NP) was developed, optimized and is in the final stages of validation. It has demonstrated good correlation with IFA results from known PEDV seropositive and naïve populations. Final receiver operating characteristic (ROC) analysis including over 1000 samples is in progress to determine optimum cut-off values and diagnostic sensitivity and specificity. While the IFA and NP-ELISA tests should be very valuable in determining previous exposure to PEDV, they may provide limited information regarding protection from future challenge. However, virus neutralization assays would be expected to provide a reasonable indication of protection since they measure antibodies that actually neutralize the virus. Traditional CPE-based VN assays present some technical challenges when dealing with PEDV due to the strict trypsin requirements of PEDV replication. To minimize this concern, we developed a fluorescent focus neutralization assay that allows accurate endpoint determination and rapid turnaround time in under 24 h. Modifications of this assay minimize influence of test serum samples in trypsin inactivation and allow consistent PEDV infection. This work has resulted in expanded collaborations with private industry and other academic institutions. Additionally, SDSU participated in collaborative studies led by Iowa State University researchers to more fully validate the application of a commercially available ELISA for the detection of PRRSV antibodies in oral fluids. Results of this work were widely presented to swine producers and veterinary practitioners and the assay is now routinely offered at many veterinary diagnostic laboratories.

Publications

• Type: Journal Articles Status: Published Year Published: 2013 Citation: Christopher-Hennings, Jane, Karla Araujo, Carlos Souza, Ying Fang, Steven Lawson, Eric A. Nelson, Travis Clement, Mike Dunn, Joan K. Lunney Opportunities for bead-based multiplex assays in veterinary diagnostic laboratories. 2013. J. Vet. Diagn. Invest. 25:671-691.
• Type: Journal Articles Status: Published Year Published: 2013 Citation: Olsen C., Wang C., Christopher-Hennings J., Doolittle K., Harmon, K., Abate, S., Kittawornrat A., Lizano S., Main R., Nelson, E., Otterson T., Panyasing Y., Rademacher C, Rauh R, Shah R, Zimmerman J. 2013, Probability of detecting Porcine reproductive and respiratory syndrome virus infection using pen-based swine oral fluid specimens as a function of within-pen prevalence. J. Vet. Diagn. Invest. 25, 328-325.
• Type: Conference Papers and Presentations Status: Published Year Published: 2013 Citation: Kittawornrat A, Wang C, Anderson G, Ballagi A, Broes A, Carman S, Doolittle K, Galeota J, Johnson J, Lizano S, Nelson E, Patnayak D, Pogranichniy R, Rice A, Scherba G, Zimmerman J. Ring test evaluation for the detection of PRRSV antibody in oral fluid specimens using a commercial PRRSV serum antibody ELISA. Proceedings of the 44th annual meeting of the American Association of Swine Veterinarians. March 3, 2013, San Diego, California, p.61.
• Type: Conference Papers and Presentations Status: Published Year Published: 2013 Citation: Olsen C, Wang C, Christopher-Hennings J, Doolittle K, Abate S, Harmon K, Kittawornrat K, Lizano A, Main R, Nelson E, Otterson T, Panyasing Y, Rademacher C, Rauh R, Shah R, Zimmerman J. March 2013. Probability of detecting PRRSV infection using pen-based swine oral fluid specimens as a function of within-pen prevalence. Proceedings of the American Association of Swine Veterinarians. San Diego, California, p. 109-110.

Progress 01/01/12 to 12/31/12

Outputs
OUTPUTS: The overall goal of this project is to improve the quality, speed and affordability of diagnostic assays for the accurate diagnosis of major viral diseases of swine, including porcine reproductive and respiratory syndrome virus (PRRSV), swine influenza virus (SIV), porcine circovirus type 2 (PCV2), rotaviruses and others. Over the past year, substantial progress has been made on several of the specific objectives and is reflected in the following tangible outputs. First, full optimization and validation of a fluorescent microsphere immunoassay (FMIA) for detection of antibodies against PRRSV using oral fluid samples or serum samples was completed with additional funding support from the National Pork Board. Results and applications of this work were presented at multiple national and international conferences and published in Clinical and Vaccine Immunology. This work has led to a broad multi-institutional collaboration for further development of FMIA for the simultaneous serological monitoring of multiple swine pathogens. Second, preliminary data obtained, in part, from this project allowed the submission and successful funding of a competitive grant from the National Pork Board to sequence and characterize genes encoding selected capsid proteins from field strains of porcine rotavirus. This first step of the study is led by our collaborators at Kansas State University and represents an initial step toward the development of improved diagnostic reagents and assays for porcine rotavirus, as well as the development of improved vaccine candidates. Third, preliminary data attributable to this project resulted in other internal funding to develop FMIA-based assays for the simultaneous detection of multiple swine influenza subtype antibodies. This work is ongoing with additional industry collaborations. Forth, studies associated with this project and others have led to collaborative grants funded by the National Pork Board for development of shared reagent and reference standard resources for the development and validation of improved diagnostic assays. Also, additional expressed viral protein antigens and monoclonal antibodies have been developed by our group for application in new enzyme-linked immunoassay (ELISA) and FMIA-based diagnostic technologies, as well as new emerging technologies. This work has resulted in expanded collaborations with private industry and other academic institutions. Additionally, SDSU participated in collaborative studies led by Iowa State University researchers to more fully validate the application of a commercially available ELISA for the detection of PRRSV antibodies in oral fluids. Results of this work were widely presented to swine producers and veterinary practitioners and the assay is now routinely offered at many veterinary diagnostic laboratories. PARTICIPANTS: Molecular Virology Research Laboratory: Ying Fang, Associate Professor (co-PI); Steven Lawson, Post-doc Research Associate; Zhi Sun, Post-doc Research Associate; Robert Langenhorst, Graduate Research Assistant; Russell Ransburgh, Graduate Research Assistant; Yanhua Li, Graduate Research Assistant. Molecular Diagnostic Laboratory: Jane Christopher-Hennings, Professor (co-PI); Travis Clement, Research Associate. Serology Laboratory: Eric Nelson, Professor (PI); Aaron Singrey, Senior Microbiologist; Xiaodong Liu, Microbiologist. Primary collaborators at Iowa State University: Jeffery Zimmerman, Professor; Apisit Kittawornrat, PhD Student; Chris Olsen, PhD Student. Primary collaborators at Kansas State University: Richard Hesse, Professor; Jianfa Bai, Assistant Professor; Raymond Rowland, Professor. Training and education opportunities were provided for multiple graduate and undergraduate students. TARGET AUDIENCES: Veterinary researchers and diagnosticians, veterinary practitioners, pork producers, veterinary biologics and diagnostics companies. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
For effective disease diagnosis and surveillance, rapid and sensitive assays are needed to detect antigens associated with major pathogens and to detect antibodies developed in response to infection. Porcine reproductive and respiratory syndrome virus (PRRSV) continues to be the most important and costly viral disease of swine internationally, which makes this pathogen one of our primary focuses. The multiplexed fluorescent microsphere immunoassay (FMIA) developed for detection of PRRSV-specific antibodies in oral fluid and serum samples has been validated and is being considered for commercial development. The FMIA for detection of antibodies against PRRSV in both serum and oral fluids has laid the groundwork and provided a model for development of additional FMIA-based technologies for the rapid detection of antigens and/or antibodies associated with major swine pathogens in a multiplex format. Cell culture-based diagnostic assays previously developed in our laboratory have been modified for application in the rapid screening of candidate anti-viral compounds. Several compounds are being further evaluated for potential use as anti-virals against PRRSV and other swine pathogens. Our efforts to produce new expressed viral protein antigens and monoclonal antibodies are providing essential reagents for work on new immunoassays including multiplex FMIA techniques for swine influenza, porcine circovirus type 2 and other agents. Development and validation of these assays will provide critical information and model approaches for additional assays targeting other existing and new emerging diseases. This work represents the ongoing collaboration of three co-PIs at SDSU, their research groups and external collaborators. The expanding collaborations involving multiple academic institutions and private industry will help speed the optimization and validation of these diagnostic assays and make them available to veterinary diagnostic laboratories, practitioners and pork producers.

Publications

• Kittawornrat, A., C. Wang, G. Anderson, A. Ballagi, A. Broes, S. Carman, K. Doolittle, J. Galeota, J. Johnson, S. Lizano, E. Nelson, D. Patnayak, R. Pogranichniy, A. Rice, G. Scherba, J. Zimmerman. 2012. Ring Test Evaluation for the Detection of PRRSV Antibody in Oral Fluid Specimens using a Commercial PRRSV Serum Antibody ELISA. Proceedings of the American Association of Veterinary Laboratory Diagnosticians 55th Annual Conference, Greensboro, NC. p.50.
• Kittawornrat, A., C. Wang, A. Ballagi, S. Lizano, J. Johnson, K. Doolittle, E. Nelson, A. Broes, J. Zimmerman. 2012. Ring test evaluation of the PRRS oral fluid IgG ELISA. 4th European Symposium of Porcine Health Management, April 25-27, 2012, Bruges, Belgium.
• Kittawornrat A., C. Wang, G. Anderson, A. Ballagi, A. Broes, S. Carman, K. Doolittle, J. Galeota, J. Johnson, S. Lizano, E. Nelson, D. Patnayak, R. Pogranichniy, A. Rice, G. Scherba, J. Zimmerman. 2012. Ring test evaluation of the detection of PRRSV antibodies in oral fluid specimens using a commercial PRRSV serum antibody ELISA. Proceedings, 22nd International Pig Veterinary Society Congress. Jeju, Korea, p. 996.
• Langenhorst, R., S. Lawson, A. Kittawornrat, J. Zimmerman, Z. Sun, Y. Li, J. Christopher-Hennings, E. Nelson and Y. Fang. 2012. Development of a fluorescent microsphere immunoassay for detection of antibodies against PRRSV using oral fluid samples as an alternative to serum-based assays. Clinical and Vaccine Immunology 19:180-189.
• Anderson, T., W. Laegreid, F. Cerutti, F. Osorio, E. Nelson, J. Christopher-Hennings, T. Goldberg. 2012. Ranking viruses: Measures of positional importance within networks define core viruses for rational polyvalent vaccine development. Bioinformatics 28:1624-1632.
• Kittawornrat, A., C. Wang, G. Anderson, A. Ballagi, A. Broes, S. Carman, K. Doolittle, J.Galeota, J. Johnson, S. Lizano, E. Nelson, D. Patnayak, R. Pogranichniy, A. Rice, G. Scherba, J. Zimmerman. 2012. Ring test evaluation for the detection of PRRSV antibodies in oral fluid specimens using a commercial PRRSV serum antibody ELISA. 2012. J. Vet. Diagn. Invest. 24(6), 1057-1063.
• Langenhorst R., S. Lawson, A. Kittawornrat, J. Zimmerman, Z. Sun, Y. Li, J. Christopher-Hennings, E. Nelson, Y. Fang. 2012. Development of a fluorescence microsphere immunoassay for detection of antibodies against porcine reproductive and respiratory syndrome virus using oral fluid samples as an alternative to serum-based assays. American Society for Virology, Madison, WI, P25-8.
• Langenhorst R., S. Lawson, A. Kittawornrat, J. Zimmerman, Z. Sun, Y. Li, J. Christopher-Hennings, E. Nelson, Y. Fang. 2012. Simultaneous detection of antibodies against PRRSV nsp7 and nucleocapsid protein in swine oral fluid and sera using a fluorescence microsphere immunoassay. Nebraska Virology Center Annual Retreat, Nebraska City, NE.
• Langenhorst R., S. Lawson, A. Kittawornrat, J. Zimmerman, Z. Sun, Y. Li, J. Christopher-Hennings, E. Nelson, Y. Fang. 2012. Development of a fluorescent microsphere immunoassay for detection of antibodies against PRRSV using oral fluid samples as an alternative to serum-based assays. 2012 International PRRS symposium. Kansas City, KS.
• Olsen C., L. Karriker, C. Wang, B. Binjawadagi, J. Christopher-Hennings, K. Doolittle, K. Harmon, S. Jones, A. Kittawornrat, A. Kurtz, E. Kurtz, S. Lizano, J. Coetzee, R. Main, A. Meiszberg, E. Nelson, T. Otterson, Y. Panyasing, C. Rademacher, R. Rauh, G. Renukaradhya, R. Shah, J. Zimmerman. 2012. Swine Oral Fluid Diagnostics Update. Swine Disease Conference for Swine Practitioners. Ames, IA.
• Christopher-Hennings, J., G. Erickson, R. Hesse, S. Oliveira, E. Nelson. 2012. Diagnostic tests, test performance and considerations for interpretation. In: Diseases of Swine (10th edition). Wiley-Blackwell, Inc., p 77-93.
• Olsen C., C. Wang, J. Christopher-Hennings, K. Doolittle, K. Harmon, S. Abate, A. Kittawornrat, S. Lizano, R. Main, E. Nelson, T. Otterson, Y. Panyasing, C. Rademacher, R. Rauh, R. Shah, J. Zimmerman. 2012. Oral fluids: Detection of PRRSV as a function of within pen prevalence. Leman Swine Conference. St. Paul, Minnesota.
• Kittawornrat, A., C. Wang, G. Anderson, A. Ballagi, A. Broes, S. Carman, K. Doolittle, J. Galeota, J. Johnson, S. Lizano, E. Nelson, D. Patnayak, R. Pogranichniy, A. Rice, G. Scherba, J. Zimmerman. Ring test evaluation for the detection of PRRSV antibody in oral fluid specimens using a commercial PRRSV serum antibody ELISA. 93rd Annual Meeting of the Conference of Research Workers in Animal Diseases, December 2-4, 2012. Chicago, IL. Abstract 163.
• Kittawornrat, A., C. Wang, G. Anderson, A. Ballagi, A. Broes, S. Carman, K. Doolittle, J. Galeota, J. Johnson, S. Lizano, E. Nelson, D. Patnayak, R. Pogranichniy, A. Rice, G. Scherba, J. Zimmerman. 2012. Ring test evaluation for the detection of PRRSV antibody in oral fluid specimens using a commercial PRRSV serum antibody ELISA. International PRRS Symposium, November 29-30, 2012. Kansas city, MO. Poster number 22.
• Christopher-Hennings, J., S. Lawson, J. Lunney, K. Araujo, T. Clement, E. Nelson, Y. Fang, S. Wong, K. Kulas. 2012. Development of a swine-specific multiplexed cytokine assay for use in the boar--a potential biomedical model of viral infection in semen. Graduate Women in Science Conference, Madison, WI.

Progress 03/15/11 to 12/31/11

Outputs
OUTPUTS: This is a new project with the overall goal of improving the quality, speed and affordability of diagnostic assays for the accurate diagnosis of major viral diseases of swine, including porcine reproductive and respiratory syndrome virus (PRRSV), swine influenza virus (SIV), porcine circovirus type 2 (PCV2), rotaviruses and others. Over the past nine months, substantial progress has been made on several of the specific objectives and is reflected in the following tangible outputs. First, preliminary data allowed the submission and successful funding of a competitive grant obtained from the National Pork Board. This grant funding allowed the development of a fluorescent microsphere immunoassay for detection of antibodies against PRRSV using oral fluid samples as an alternative to serum-based assays. Results of this work were presented at the XII International Nidovirus Symposium, the 2011 International PRRS Symposium, several invited presentations and accepted for publication in Clinical and Vaccine Immunology. Second, preliminary data attributable to this project resulted in submission of a competitive grant proposal to develop FMIA-based assays for the simultaneous detection of multiple swine influenza subtype antibodies. This work is ongoing through internal funding sources and has led to additional collaborations with researchers at other institutions and industry. Third, multiple expressed viral protein antigens and monoclonal antibodies have been developed for application in new enzyme-linked immunoassay (ELISA) and FMIA-based diagnostic technologies, as well as new emerging technologies. This work has resulted in expanded collaborations with private industry and other academic institutions. Additionally, SDSU participated in collaborative studies lead by Iowa State University researchers to validate the application of a commercially available ELISA for the detection of PRRSV antibodies in oral fluids. This technology is now available to swine producers and veterinary practitioners through selected veterinary diagnostic laboratories. PARTICIPANTS: Molecular Virology Research Laboratory: Ying Fang, Associate Professor (co-PI); Steven Lawson, Post-doc Research Associate; Robert Langenhorst, Graduate Research Assistant; Zhi Sun, Graduate Research Assistant; Yanhua Li, Graduate Research Assistant; Molecular Diagnostic Laboratory: Jane Christopher-Hennings, Professor (co-PI); Michael Dunn, Microbiologist/MS Student; Serology Laboratory: Eric Nelson, Professor (PI); Xiaodong Liu, Microbiologist. Primary collaborators at Iowa State University: Jeffery Zimmerman, Professor; Apisit Kittawornrat, PhD Student. Training and education opportunities were provided for multiple graduate and undergraduate students. TARGET AUDIENCES: Veterinary researchers and diagnosticians, veterinary practitioners, pork producers, veterinary biologics and diagnostics companies. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
For effective disease diagnosis and surveillance, rapid and sensitive assays are needed to detect antigens associated with major pathogens and to detect antibodies developed in response to infection. Porcine reproductive and respiratory syndrome virus (PRRSV) continues to be the most important and costly viral disease of swine internationally, which makes this pathogen our initial focus. We developed a multiplexed fluorescent microsphere immunoassay (FMIA) for detection of PRRSV-specific antibodies in oral fluid and serum samples. Recombinant nucleocapsid protein and nonstructural protein 7 from both PRRSV genotypes (type I and type II) were used as antigens covalently coupled to Luminex fluorescent microspheres. Based on an evaluation of 488 oral fluid samples with known serostatus, the oral fluid-based FMIAs achieved over 92% sensitivity and 91% specificity. For serum samples (n=1,639), the FMIAs demonstrated over 98% sensitivity and 95% specificity. Kinetics of the antibody response in pigs was also investigated. Development of the FMIA for detection of antibodies against PRRSV in both serum and oral fluids has laid the groundwork and provided a model for development of additional FMIA-based technologies for the rapid detection of antigens and/or antibodies associated with major swine pathogens in a multiplex format. Our efforts to produce new expressed viral protein antigens and monoclonal antibodies are providing essential reagents for work on new immunoassays including multiplex FMIA techniques for swine influenza and other agents. Development and validation of these assays will provide critical information and model approaches for additional assays targeting other existing and new emerging diseases. This work represents the ongoing collaboration of 3 co-PIs at SDSU, their research groups and external collaborators. The expanding collaborations involving multiple academic institutions and private industry will help speed the optimization and validation of these diagnostic assays and make them available to veterinary diagnostic laboratories, practitioners and pork producers.

Publications

• Langenhorst R, Lawson S, Kittawornrat A., Zimmerman J, Sun Z, Li Y, Christopher-Hennings J, Nelson E, Fang Y. 2011. Simultaneous detection of antibodies against PRRSV nsp7 and nucleocapsid protein in swine oral fluid and sera using a fluorescence microsphere immunoassay. Proc. of The XIIth International Nidovirus Symposium. 072W.
• Fang, Y. 2011. PRRS control and diagnostics: from basic to applied science. Proc. of The XIIth International Nidovirus Symposium. SP13.
• Langenhorst R, Lawson S, Kittawornrat A., Zimmerman J, Sun Z, Li Y, Christopher-Hennings J, Nelson E, Fang Y. 2011. Simultaneous detection of antibodies against PRRSV nsp7 and nucleocapsid protein in swine oral fluid and sera using a fluorescence microsphere immunoassay. Proc. of the 2011 International PRRS Symposium. p. 40.