Source: SOUTH DAKOTA STATE UNIVERSITY submitted to
PORCINE REPRODUCTIVE AND RESPIRATORY DISEASE: METHODS FOR THE INTEGRATED CONTROL, PREVENTION & ELIMINATION OF PRRS IN U. S. SWINE HERDS
 
PROJECT DIRECTOR: Christopher-Hennings, J. Nelson, E. A. Fang, Y.
 
PERFORMING ORGANIZATION
VETERINARY SCIENCE
SOUTH DAKOTA STATE UNIVERSITY
BROOKINGS,SD 57007
 
NON TECHNICAL SUMMARY: PRRS is the most economically significant diease facing the swine industry today. There is a significant lack of effective protocols for the control and/or prevention of this disease in herds. This project is a multi-state, multi-investigator approach to provide effective control and prevention procedures for the elimination of this disease.
 
OBJECTIVES: 1. Implementation of a virtual laboratory and infrastructure through the development and open distribution of resources, materials, protocols and data among participating researchers and states. 2. Achieve biosecurity within herds to prevent the spread of virus within a herd and faciitate its elimination from endemically infected herds. 3. Achieve biosecurity among herds by preventing viral spread between herds. 4. Improve diagnostic assays and create on-farm monitoring systems. 5. Develop and test PRRS virus eradication protocols under various ecological settings. 6. Develop educational outreach tools for disseminating information through established outreach and extension networks to producers, veterinarians, educators, and researchers. 7. Create an information network to ensure rapid and efficient communication of PRRS virus research.
 
APPROACH: 1. Protocols, methods, procedures and standards will be established to enable investigtors to directly compare research results. Shared materials (infectious clones, recombinant proteins nucleic acids, challenge strains, etc.) will be made available. 2. Establishing a resource popoulation of pigs for analysis of genetic resistance to PRRSV, investigating mechanisms of protective immunity for PRRS prevention, investigating mechanisms of immune stimulation that might increase anti-PRRS immunity and prevent persistence and evaluating novel antisense RNA reagents that target viral replication would be methods to achieve the 2nd objective. 3. To achieve biosecurity among herds, molecular epidemiology studies and environment stability and transmission of PRRSV would be investigated. 4. To improve diagnostic assays and create on-farm monitoring systems, exploring novel diagnostic or detection strategies such as gene chips and biosensors and contructing recombinant PRRSV virus strains that stably integrate marker tags for use in future vaccines would be investigated. 5. PRRSV eradication protocols for regional eradication of PRRSV will be established and studied through cooperating production sites. 6 & 7. The NC229 and NPB will work cooperatively to create educational and instructional materials for outreach with publications and web-based communication networks.
 
CRIS NUMBER: 0201549 SUBFILE: CRIS
PROJECT NUMBER: SD00194-R SPONSOR AGENCY: NIFA
PROJECT TYPE: HATCH PROJECT STATUS: NEW MULTI-STATE PROJECT NUMBER: NC-229
START DATE: Oct 1, 2004 TERMINATION DATE: Sep 30, 2009

GRANT PROGRAM: (N/A)
GRANT PROGRAM AREA: (N/A)

CLASSIFICATION
Knowledge Area (KA)Subject (S)Science (F)Objective (G)Percent
311351010404.240%
311351010904.210%
311351011014.240%
311351011604.210%

CLASSIFICATION HEADINGS
KA311 - Animal Diseases
S3510 - Swine, live animal
F1160 - Pathology
F1090 - Immunology
F1101 - Virology
F1040 - Molecular biology
G4.2 - Reduce Number and Severity of Pest and Disease Outbreaks


RESEARCH EFFORT CATEGORIES
BASIC 40%
APPLIED 50%
DEVELOPMENTAL 10%

KEYWORDS: swine; disease control; eradication; prrs virus; respiratory diseases; reproductive diseases; integrated control (diseases); disease prevention; united states; molecular biology; virus diseases (animals); biosecurity; disease transmission; herd health; disease diagnosis; information dissemination; extension; producers; veterinarians; teachers; scientists

PROGRESS: Jan 1, 2008 TO Dec 31, 2008
OUTPUTS: Porcine reproductive and respiratory syndrome virus (PRRSV) continues to be a major problem in the pork industry worldwide. A key step in future vaccine development is to include markers for diagnostic differentiation of vaccinated animals from those naturally infected with wild-type virus. Using a cDNA infectious clone of Type 1 PRRSV, we constructed a recombinant green fluorescent protein (GFP) tagged PRRSV containing deletion of an immunogenic epitope, ES4, in the nsp2 region. In a nursery pig disease model, the recombinant virus was attenuated with a lower level of viremia. To complement marker identification, we developed GFP and ES4 epitope-based ELISAs. Pigs immunized with the recombinant virus lacked antibodies directed against the corresponding deleted epitope, while generating strong antibody response to GFP. Our results demonstrated that this recombinant marker virus, in conjunction with the diagnostic tests, enables serological differentiation between marker virus-infected animals and those infected with wild-type virus. This rationally designed marker virus will provide a basis for further development of PRRSV marker vaccines to control of PRRS. To further characterize the humoral immune response of pigs to PRRSV, direct enzyme-linked immunosorbent assays (ELISA) were used to study the kinetics of antibody responses to PRRSV nonstructural proteins. The highest immunoreactivities were against nsp1, nsp2 and nsp7. Using the recombinant nsp7 as an antigen, we validated a dual-ELISA for the simultaneous detection and differentiation of serum antibodies against Type 1 and Type 2 PRRSV. Receiver operating characteristic analysis based on 1,334 known positive and 1,357 known negative samples showed good specificity (98.3% to Type 1 and 99.3% to Type 2) and sensitivity (97.4% for Type 1 and 99.8% for Type 2). To differentiate Type 1 and Type 2 PRRSV, 470 sera originating from experimentally challenged pigs were tested and positive sera were correctly differentiated in 469 of 470 samples. The nsp7 dual-ELISA possessed 97.6% agreement with the IDEXX HerdChek PRRS 2XR ELISA. In further testing of IDEXX suspected false positive samples, the nsp7 dual-ELISA resolved 98% of the samples as negative. In addition, new approaches to reduce the risk of PRRSV transmission through boar semen were evaluated. The use of 1- and 2-layer density gradients to purify sperm in semen samples reduced the levels of PRRSV present in samples as detected by PCR. A cysteine protease inhibitor, chymostatin, was shown to inhibit PRRSV replication as indicated through in vitro virus inhibition assays. Results of the studies above have been reported at scientific conferences and submitted for publication in refereed journals. Key reagents, infectious clones and other technologies have been shared with other research groups in the U.S. and around the world. Additionally, numerous monoclonal and polyclonal antibody reagents, virus isolates, well-characterized panels of pig sera and protocols have been developed and distributed among researchers, diagnostic laboratories and private companies. PARTICIPANTS: Principal investigators from SDSU included Drs. Jane Christopher-Hennings, Ying Fang and Eric Nelson. Collaborators at SDSU included Dr. Xiuqing Wang, Department of Microbiology and Drs. David Knudsen and Russel Daly, Department of Veterinary Science. Dr. Joan Lunney, USDA-ARS, Beltsville, MD participated in the immunological studies. Dr. Gary Althouse, Director/Head of Theriorgenology at Pennsylvania State University participated as a collaborator in the reproductive studies. Dr. Micheal Murtaugh, University of Minnesota, Dr. Raymond Rowland, Kansas State University and Drs. Jeffery Zimmerman and Ramon Molina, Iowa State University participated in the PRRSV serology studies. Dr. Terje Dokland, University of Alabama-Birmingham was a collaborator for the electron microscopy studies. Dr. Kay Faaberg at the National Animal Veterinary Services Laboratory in Ames, Iowa was a collaborator in some molecular studies. Post-docs trained at SDSU included Zhenhai Chen and Steven Lawson. SDSU graduate students trained through these studies included Travis Clement, Lindsey Reister, Xiaoxin Zhou, Haixia Liu, Xiaofei Gao, Jingjing Bao and Elizabeth Brown. TARGET AUDIENCES: Target audiences include veterinary researchers and practitioners, pork producers, veterinary vaccine and biologics companies, and national and international scientific groups. PROJECT MODIFICATIONS: Not relevant to this project.

IMPACT: 2008-01-01 TO 2008-12-31 A new serological assay for the detection of antibodies against PRRSV was developed. This nsp7 dual-ELISA can be used as a differential test for PRRSV serology with a high level of sensitivity and specificity. This ELISA offers an additional tool for routine or follow-up diagnostics, as well as having substantial value in epidemiological surveys and outbreak investigations. Results of reverse genetics studies suggested that the ES4 epitope in the nsp2 region of the PRRSV genome is nonessential for PRRSV replication but may play an important role in viral attenuation and pathogenesis in vivo. A Type 1 PRRSV infectious clone containing both positive and negative markers for differentiation of vaccine from wild-type virus was developed. This marker virus may provide a good backbone for future development of improved PRRSV vaccines. Companion diagnostic serology assays were also developed for the differentiation between vaccinated and wild-type virus infected animals. Potential mechanical and antiviral methods were also evaluated to help insure a PRRSV-free semen supply. Density gradient purification of sperm cells may provide an additional level of security in protecting high-value animals from exposure to PRRSV via artificial insemination. Numerous research and diagnostic reagents were developed for use by the larger PRRSV research community.

PUBLICATION INFORMATION: 2008-01-01 TO 2008-12-31
Fang, Y., J. Christopher-Hennings, E. Brown, H. Liu, Z. Chen, S. Lawson, R. Breen, T. Clement, X. Gao, J. Bao, D. Knudsen, R. Daly and E.A. Nelson. 2008. Development of genetic markers in the non-structural protein 2 region of a US type 1 porcine reproductive and respiratory syndrome virus: implications for future recombinant marker vaccine development. J. Gen. Virol. 89:3086-3096.

PROJECT CONTACT INFORMATION
NAME: Christopher-Hennings, J.
PHONE: 605-688-5171
FAX: 605-688-6003