NC_OLD229: Porcine Reproductive and Respiratory Disease: Methods for the integrated control, prevention and elimination of PRRS in United States Swine

(Multistate Research Project)

Status: Inactive/Terminating

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SAES-422 Reports

Annual/Termination Reports:

[12/01/2004] [12/15/2006] [12/15/2006] [07/15/2008]

Date of Annual Report: 12/01/2004

Report Information

Annual Meeting Dates: 11/12/2004 - 11/13/2004
Period the Report Covers: 10/01/2003 - 09/01/2004

Participants

Gylling, Steven R. Gylling Data Management, Inc steve@gdmdata.com;
Faaberg, Kay University of Minnesota kay@mail.ahc.umn.edu;
Goldberg, Tony University of Illinois tlgoldbe@uiuc.edu;
Pattnaik, Asit University of Nebraska apattnaik2@unl.edu;
Schommer, Susan University of Missouri schommers@missouri.edu;
Johnson, Peter USDA:CSREES pjohnson@reeusda.gov;
Kehrli, Marcus USDA:ARS:NADC mkehrli@nadc.ars.usda.gov;
Kleiboeker, Steve University of Missouri kleiboekers@missouri.edu;
Laegreid, Will USDA:ARS:MARC laegreid@email.marc.usda.gov;
Lunney, Joan USDA:ARS:BARC jlunney@anri.barc.usda.gov;
McCaw, Monte North Carolina State University monte_mccaw@ncsu.edu;
Moore, Camille PRRS Integrated Program, Advisory Board camillemoore@videotron.ca;
Murtaugh, Michael University of Minnesota murta001@umn.edu;
Christopher-Hennings, J South Dakota State University jane.christopher-hennings@sdstate.edu;
Neumann, Eric National Pork Board eric.neumann@porkboard.org;
Osorio, Fernando University of Nebraska fosorio@unl.edu;
Rowland, RRR Kansas State University browland@vet.ksu.edu;
Yoo, Dongwon University of Guelph dyoo@uoguelph.ca;
Zimmerman, Jeff Iowa State University jjzimm@iastate.edu;
Zuckermann, Federico University of Illinois fazaaa@uiuc.edu;

Brief Summary of Minutes

NC-229 Business Meeting Minutes. Friday 11/12/04.

The meeting was brought to order at 1:12 pm by chair Jeff Zimmerman. An overview of the meeting agenda was presented. Financial support of the meeting was acknowledged (Boehringer Ingelheim Vetmedica, IDEXX Laboratories, Inc., Intervet, Inc., National Pork Board, Pig Improvement Company). Names of attendees were collected.

Old Business
The minutes from the last meeting were presented to the group and accepted without corrections.

New Business
The Annual Report for October 2003 to September 2004 (in progress) was presented for the purpose of soliciting corrections and/or additions. None were indicated at this time.

Peter Johnson (NC-229 USDA Representative) discussed the latest activities related to USDA. Dr. A. Palmisano was hired as a new administrative director and a new web site is under development. Investigators will be submitting grants this year by paper; next year, submission will be electronic.

Michael Murtaugh (Minnesota) presented the progress report for the PRRS Integrated Program Project, including a detailed analysis of expenditures. In summary, expenditures were $2.5M for Year One (ending Jan 15, 2005). A total of 17 proposals received $3.8M with $1.4M to 9 awards and 2 contracts for services.

Murtaugh suggested that we convene at six-month intervals, to discuss issues related to funding. The special issue of Veterinary Immunology and Immunopathology on PRRS virus immunology has been published.

Steve Gylling (President, Gylling Data Management, Inc., Brookings, South Dakota) gave a presentation on ARM Research Management Software. Handouts were provided and an overview of the software presented. Essentially, ARM software would provide NC-229 researchers web-based means to share data in a Good Laboratory Practice environment.

Eric Neumann (National Pork Board) presented the NPB report. NPB funding for the previous year consisted of $2M for 50 proposals. Eric identified two projects funded through Leverage and Discovery: 1) PRRSV protein expression, using Mike Murtaughs techniques will be performed by a private company; 2) a chamber for use in aerosol stability studies will be built at Iowa State University and subsequently made available for other projects.

NPB is interested in feedback from NC-229. Priorities for next year are similar, e.g., investment into vaccine research, regional eradication, outreach, and education. Funding for next year will include two calls for proposals. Following budget approval, the NPB may have another $2M in support of PRRS research. Proposal format for next year has been modified and includes a pre-proposal format. The principal message from the NPB proposal review committee is that researchers need to improve experimental design, focus on research that provides answers, and pay close attention to details in assembling their proposals. NPB will consider multi-year proposals, but Year One should be described in detail.

Eric Neumann handed out a draft annual report on PRRS and suggested bundling NPB, NRI, and NC-229 reports into a single PRRSV Research Report. This idea was well received. Target date is February 15, 2005. Eric Neumann described the PRRS website and communications, including a link to the PRRS initiative. He described several new features, including a message board. The areas of Education and Outreach are currently in the process of development.

Camile Moore (IPP Scientific Review Board) was asked to comment on the IPP. Overall, he was impressed by project selection and teamwork. He suggested that the next step is to build greater collaboration on projects; i.e. combine proposals into a collaborative network. He also suggested that the IPP needs to seek input from outside the current group of scientists.

Open Discussion
Host genetics related to resistance to PRRS was the first topic. The discussion was lively and participation was extensive among those present. The general consensus was that identification of host genes related to resistance to PRRS represented a long-term, difficult, and expensive project. A number of experimental approaches are possible, but it is not certain which would be most efficient in terms of arriving at a solution. Suggestions included: 1) additional input from geneticists, 2) additional input from the external advisory board, and/or 3) the formation of a committee on disease resistance.

This was followed by a discussion on the Annual Meeting of NC-229 / International Workshop on PRRS. Given the large amount of information being generated by NC-229 participants and IPP researchers, has it become necessary to appoint a Program Committee? In the context of active multi-station research programs, do we need to discuss station reports? The need for a scientifically strong program was expressed. The idea of extending the meeting was raised, but not received with enthusiasm. The addition of poster presentations was suggested as a method to accommodate the diversity and depth of current research.

The next issue discussed was financial issues related to funding. In particular, the question considered was whether PI salaries should be included on IPP grants. Although there are concerns regarding salaries, the general consensus was that reasonable requests are acceptable and excessive requests can be addressed during the proposal review process.

The final discussion concerned the planning of the next meeting of the PRRS Integrated Program Project in conjunction with the Annual Meeting of the American Association of Swine Veterinarians (March 2005, Toronto). The suggestion was made to expand the stakeholder meeting to include fundees of NC-229. This idea was well received.

The meeting was adjourned at 5:00 pm.

NC-229 reconvened on Saturday 11/13/04.
5TH ANNUAL MEETING OF NC-229 / INTERNATIONAL WORKSHOP ON PRRS

08:00 am Opening remarks. Jeff Zimmerman, Chair NC-229;
08:15 am Comments. David Benfield, Administrative Advisor NC-229;
08:30 am Comments. Michael Murtaugh, Director, PRRS Integrated Program Project. Overview of Integrated Program progress and forecast for Year Two;
08:45 am Comments. Eric Neumann, Director, Swine Health Information and Research, National Pork Board. NPBs perspective.;

09:00 am Creation of a Virtual Laboratory infrastructure. Moderator: Steve Kleiboeker;
Immunological reagents: What and how? Joan Lunney. USDA/ARS/BARC;
Protein reagents: A test case for common reagent use. M. Murtaugh. University of Minnesota;
A proposed listserv to facilitate communication. Steve Kleiboeker. University of Missouri;
NC-229 / NPB website Eric Neumann. National Pork Board;
ARM7 data management software Jeff Zimmerman. Iowa State University;

10:00 am Break

10:30 am The PRRS viral genome. Moderator: Dongwan Yoo;
Evolution and quasispecies of PRRSV in cells in vitro Susan Schommer. University of Missouri;
Evolution and quasispecies of PRRSV in pigs. K-J Yoon. Iowa State University;
Emergence of European-like PRRSV in North America Ying Fang. South Dakota State University;
Infectious clones and manipulation of PRRSV genome Dongwan Yoo. University of Guelph;

11:30 am Juxtaposition: Equine arteritis virus vs PRRSV - James MacLachlan

12:00 pm Lunch

01:00 pm Pathogenesis. Moderator: RRR Rowland;
Gene expression analysis of PRRSV pathogenesis Elisabetta Giuffra. Roslin Institute;
Engineering the PRRSV genome as an infectious bacterial artificial chromosome. Young-Min Lee. Chungbuk National University;
Heteroclite RNAs - potential role in pathogenesis. Kay Faaberg. University of Minnesota;
The PRRSV-macrophage molecular interface. Mark Rutherford. University of Minnesota;

02:00 pm The immune response. Moderator: Fernando Osorio;
PRRSV adaptive immunity M Murtaugh. University of Minnesota;
Effect of repeated PRRSV exposure on immune response Eileen Thacker. Iowa State University.;
Suppression of the humoral immune response to PRRSV M McCaw. North Carolina State Univ;
Mechanisms of immune subversion by PRRSV. F Zuckermann. University of Illinois;
Innate immune response to PRRS virus J Lunney. USDA / ARS / BARC / ANRI;
PRRSV humoral immunity F Osorio. University of Nebraska-Lincoln;

03:00 pm Break

03:30 pm Viral ecology, epidemiology, and eradication. Moderator: Locke Karriker;
PRRSV immunoepidemiology (?) on commercial farms Tony Goldberg. University of Illinois;
PRRSV transmission in nurseries Cate Dewey. University of Guelph;
Considerations for monitoring rate of transmission in populations C Muñoz-Zanzi. University of Minnesota;
PRRSV circulation in commercial nursery/finisher flows Locke Karriker. Iowa State University;

04:30 pm Defining the role of host genetics in disease control - Steve Bishop;

Accomplishments

Objective 1. Implement a virtual laboratory infrastructure through the development and open distribution of resources, materials, protocols and data among participating researchers. A workshop on diagnostics, reagents, immunology methods standardization and host genetics vis-à-vis PRRSV infection was held on March 5, 2004. The 5th Annual Meeting of the NC-229 hosted a demonstration of Agricultural Research Management (ARS version 7) software. This data management software facilitates web accessibility and sharing of databases under Good Laboratory Practice standards. Objective 2. Achieve biosecurity within herds by preventing the spread of virus within a herd and facilitating its elimination from endemically infected herds. 2.1 Cells of the immune system. Kansas State University (KSU) determined the effects of PRRSV infection on immunophenotypes of lymphoid cells. North Carolina State University (NCSU) is using a training grant to adapt and validate immune cell proliferation measurement by flow cytometry. The University of Guelph reported changes in host cell gene expression by PRRSV glycoproteins, GP4 and GP5. No apoptosis-related genes were regulated in cells expressing GP5, indicating that GP5 is not the protein responsible for apoptosis in PRRSV-infected cells. The University of Minnesota (UMn) evaluated gene expression patterns in macrophages infected with either virulent (VR-2332) vs vaccine strains of the virus using the Qiagen 70-meroligonucleotide array system. Replication in dendritic cells was studied by South Dakota State University (SDSU) reported that PRRSV underwent a productive replication in pig monocyte-derived dendritic cells (Mo-DC). University of Missouri (UMo), Ghent University, and the UMn examined the interaction of PRRSV with macrophage-matured DCs. 2.2 Cytokines. University of Illinois (UI) showed that IL-12 receptor (IL-12R) transcripts were only marginally induced in alloantigen-stimulated cultures of PBMC from PRRSV immune pigs, the IL-12R gene response increased on addition of recombinant porcine IL-18. One important defect during PRRSV infection is the apparent failure to initiate an appropriate IFN response. Institutions involved in the study of IFN responses included USDA:ARS:MARC, UMo, and UI. In MARC-145 cells, both IFN-alpha and -beta transcript abundance were unaffected by PRRSV infection. Further studies suggest that PRRSV infection directly interferes with type I IFN transcriptional activation early in its pathway, at the level of IFN-beta gene transcription. UMo extended these studies to include the IFN response in cultured macrophages (PAMs). PRRSV isolates were differentially sensitive to porcine recombinant IFN-alpha (rIFN-alpha) and varied in their ability to induce IFN-alpha. The relative number of IFN-alpha transcript copies did not correlate with IFN-alpha protein levels, suggesting a post-transcriptional mechanism of IFN suppression. Using ELIspots for the measurement of IFN-gamma-specific T cells, the UI, found that the T helper 1 response was variable and generally weak. UI studied the ability of DCs cells to produce IFN. Depending on the virus stock, PRRSV was at least 10-100 times less efficient than transmissible gastroenteritis virus (TGEV) or type-A CpG oligonucleotides at stimulating the secretion of IFN-alpha from DC. 2.3 Antibodies. The role of neutralizing antibody in the control of PRRSV remains under active study. University of Nebraska (UNL) previously demonstrated that transfer of antibodies highly enriched in neutralizing activity to PRRSV protected pregnant sows and conferred sterilizing immunity in sows and offspring. Augmentation of the neutralizing antibody response in a PRRSV-infected herd was investigated by NCSU. Antibody responses to nonstructural proteins was investigated at the UMn. Iowa State University (ISU) investigated auto-anti-idiotypic antibodies (Aab-2s) specific for antibodies against GP5 and M surface proteins. The early and late Aab-2s possessed different idiotype-binding specificities. 2.4. Persistent infection in pigs. SDSU and Ohio State University (OSU) examined the role of lymphoid and non-lymphoid tissue in acute and persistent infections of PRRSV for the detection and elimination of PRRSV infected pigs. 2.5. Viral genome. UNL used reverse genetics to identify viral genes involved in virulence. Using an infectious clone, they developed a series of chimeric constructs containing structural genes of a well studied PRRSV attenuated vaccine strain within the genomic context of a highly virulent PRRSV strain. The role of heteroclite RNAs in pathogenesis was studied at the UMn. It was found that there was a differential effect of sequential heteroclite transcript addition to transcripts of the infectious clone of full-length virus, in that varying amounts of added heteroclite transcripts could enhance or abolish viral replication. Studies were conducted on the evolution of virus at ISU. Three independent lines of in vivo replication were maintained for 2 years. Plaque-cloned viruses were obtained at each passage and sequenced for all major ORFs (1b, 2 to 7). All ORFs except 1b and 7 co-evolved, although at different rates. ISU examined the immunobiological significance of genetic variation among PRRSV. Correlation between the level of genetic divergence and cross-neutralization (both in vitro and in vivo) was studied. Isolates with less than overall 95% homology did not cross neutralize well. The UMo is using an infectious clone to evaluate in vitro quasispecies evolution. 2.6. Pathogenesis (virus factors). USDA:ARS:NADC looked at viral properties of pathogenesis. Preliminary data indicate the highest virus load in sera and lung tissue occurs with the most virulent wild-type virus. The UMn, SDSU, UMo, and Boehringer Ingelheim Vetmedica collaborated on studies to understand the in vivo growth properties of virulent field isolates and attenuated PRRSV isolates. Virulent PRRSV isolates were found to exhibit longer and more elevated levels of viremia that correlated with faster and more intense humoral immune responses. A microarray and semi-quantitative reverse-transcription polymerase chain reaction (sqRT-PCR) approaches were used by USDA:ARS:MARC to evaluate the gene response of cells to infection. Twenty-six apoptosis-related genes were examined during the first 24 h of infection and found to be unaltered, indicating that apoptotic induction was not occurring in PRRSV-infected cells. RNA silencing is being used at the UMn to study PRRSV-host cell interactions in cells infected in vitro. Conditions for siRNA transfection of primary porcine alveolar macrophages and MA-104 cells have been optimized and siRNA silencing of PRRSV replication demonstrated. 2.7. Pathogenesis (host factors). Research at KSU is directed at understanding the short and long-term consequences of congenital infection through assessment of virus replication and the induction of inflammatory/antiviral cytokines. UNL studied the response of pigs from either the NE Index Line (I) or Hampshire-Duroc cross pigs (HD) infected with PRRSV at 26 d of age. Pigs in each line responded differently to PRRSV challenge, indicating an underlying genetic variation. 2.8. Vaccines / Vaccination. USDA:ARS:BARC and UI used a novel cytokine adjuvant, IFN-alpha and molecular tests to evaluate immune factors that influence vaccine efficacy. Results showed that PRRSV vaccination, with or without IFN-alpha, stimulated low levels of protective IFN-gamma and only limited amounts of innate immune markers, interleukin-1 beta, IL-6 and IL-8 that should enhance immunity. At ISU, subunit preparations are being evaluated for their ability to induce neutralizing antibody and prevent disease due to PRRSV (subunit vaccine). Glycoproteins present in the envelope of the PRRSV will be modified by deglycosylation to prepare various subunit vaccine candidates. ISU is evaluating commercial killed and MLV vaccines to acclimate PRRSV negative gilts prior to breeding. ISU found that the degree of genetic homology of the ORF 5 between MLV PRRSV vaccines and the isolate that pigs are challenged with is not a good predictor of vaccine efficacy. Objective 3. Achieve biosecurity among herds by preventing viral spread between sites. 3.1. Virus diversity. Genetic variation among PRRSVs in pigs and farms was investigated at the UI. ORF5 from tonsils of naturally infected swine was amplified, cloned, and individual clones sequenced to characterize viral diversity in nine animals from two farms. All animals harbored multiple PRRSV variants at both the nucleic and the amino acid levels. Using viruses isolated from diagnostic samples submitted to the South Dakota ADRDL, UMn, SDSU, KSU, and OSU collaborated in an effort to understand the emergence of Type 1 PRRSV isolates in the U.S. Research interpreting genomic sequencing in the context of spatial analysis is being done to assess the regional epidemiology of PRRSV (UMn). Current results suggested that genomic variability correlated only with geographic and not temporal distance. 3.2. Transmission. PRRSV dose:response curves were produced by researchers at ISU. The ID50 for young pigs exposed orally and by the intranasal route to PRRS virus is approximately 5.0 TCID50/ml and 3.8 TCID50/ml, respectively. Work in progress at the UMn will provide quantitative estimates of PRRSV shedding by individual animals via the aerosol route for 25-and 120-kg pigs. ISU is also working to model virus transmission between herds under specific atmospheric conditions (temperature, relative humidity, sunlight, wind). 3.3. Herd immunity. The UI examined cellular immunity and protection against reproductive failure in sows on commercial swine farms during clinical outbreaks of PRRS. Evidence that a strong cellular immune response correlated with protection against clinical PRRS was found in 3/4 farms, but farms and animals within farms varied considerably in their immune response and the degree to which they were protected clinically. UMn is evaluating the effect of repeated immunization on persistence and transmission of a related virus in a population of pigs. A field study of PRRSV circulation in nursery and finisher phases of commercial herds indicated that persistent viremia and/or re-infection was found in 36 pigs post 20 weeks of age on the basis of PCR screening of serum (ISU). Objective 4. Improve diagnostic assays and create on-farm monitoring systems. 4.1 PCR-based assays. At the UMo, real-time (TaqMan) RT-PCR assays were developed for multiplex detection, differentiation, and quantification of NA and EU PRRSV field isolates. A multiplex fluorogenic PCR for PRRSV that detects NA and EU PRRSV in a differential manner was developed at ISU and used to monitor PRRS incidence. The efficiency of a SYBR green real-time PCR for detecting PRRSV in boar semen and serum was evaluated at KSU. The final stages of validation for a new NA/EU PRRSV TaqMan RT-PCR are in progress at the UMn. This test will replace two separate, NA and EU TaqMan, and will also provide increased sensitivity for EU PRRSV strains. A commercial, single-tube, real-time PCR assay was developed for the detection of U.S. and Euro/LV PRRSV isolates by UNL, SDSU, Pig Improvement Company (PIC), Syngen, Inc., Tetracore, Inc., and Boehringer Ingelheim Vetmedica. 4.2. Other work re diagnostics. NCSU characterized antibody and rtPCR responses following challenge with a high dose of homologous wild-type PRRSV in pigs initially immunized with multiple low doses of virus. Antibody levels to rORF 5-6 ectodomain chimera followed the SN antibody temporal response curve and to rORF7 followed the response curve observed with the commercial ELISAs. UMn, SDSU, PIC and Boehringer Ingelheim Vetmedica are developing and evaluating blocking ELISAs for the detection of antibodies against North American and EU-like PRRSV. This test appears to lack sensitivity in detecting antibodies produced against EU PRRSV. Virginia Polytechnic Institute and State University and ISU have collaborated on the development of a heteroduplex mobility assay (HMA) to identify PRRSV isolates with significant nucleotide sequence identities (>/=98%) with the modified live-attenuated vaccines. Objective 5. Develop and test PRRSV virus eradication protocols under various ecological settings. Research directed by the UMn is looking at the feasibility of controlling PRRS within a selected area (Rice County, MN). Semi-annual sampling of all sites will be conducted, seeking 95% confidence in detecting PRRSV pigs at various production stages (sows, nursery, finish) having at least 20% seroprevalence. ISU examined the question of how long herds that eliminated PRRSV remain free of infection. Information from 84 farms indicated that the estimate of probability of maintaining negative status for 2-years ranged 47 to 70%. Objective 6. Develop educational outreach tools for disseminating information through established outreach and extension networks to producers, veterinarians, educators, and researchers. A special issue (Dec 2004) of Veterinary Immunology and Immunopathology focused on PRRS was edited by KSU and the UMn, with contributions from participants from other Stations. In the past year, NC-229 participants have disseminated 71 refereed publications, 87 abstacts/proceedings, and 4 book chapters related to PRRSV. Objective 7. Create an information network to ensure rapid and efficient communication of PRRSV research.A national PRRS epidemiological registry and database was funded by The National Pork Board (NPB). Work-in-progress involves transferring the existing sequence database to a MySQL language and building a new web page for producers/veterinarians/ researchers to easily access the UMn data. New software tools are currently being created that will enable individuals to submit simple queries to the database. Communication activities were initiated in collaboration with the NPB (www.porkboard.org/prrs) for public dissemination of information. This website included an on-line catalog of a variety of PRRS-related resources, including The PRRS Compendium. Approximately 97 PRRSV researchers and scientists from the U.S., the U.K., Italy, France, Korea, and Mexico were in attendance at the annual meeting.

Publications

Refereed publications 1. Bassaganya-Riera J, Thacker B, Yu S, Strait E, Wannemuehler M, Thacker E. 2004. Impact of immunizations with porcine reproductive and respiratory syndrome virus on lymphocyte recall responses of CD8+ T cells. Viral Immunol 17:25-37. 2. Bastos RG, Dellagostini OA, Barletta RG, Doster AR, Nelson,E Zuckermann F, Osorio FA. 2004. Immune response of pigs inoculated with Mycobacterium bovis BCG expressing a truncated form of GP5 and M protein of porcine reproductive and respiratory syndrome virus. Vaccine 22:467-474; 3. Batista L, Dee SA, Rossow KD, Polson DD, Xiao Z, Olin M, Molitor TW, Murtaugh MP, Pijoan C. 2004. Detection of porcine reproductive and respiratory syndrome virus in pigs with low positive or negative ELISA s/p ratios. Vet Rec 154:25-26.; 4. Batista L, Dee SA, Rossow KD, Xiao Z, Olin M, Molitor TW, Murtaugh MP, Pijoan C. Virological and immunological responses to porcine reproductive and respiratory syndrome virus (PRRSV) in a large population of breeding age female swine. Can J Vet Res (In press).; 5. Batista L, Pijoan C, Dee S, Olin M, Molitor T, Xiao Z, Murtaugh M. Virological and immunological features of homologous and heterologous protection to porcine reproductive and respiratory syndrome virus (PRRSV) in gilts. Can J Vet Res (In press).; 6. Batista L, Pijoan C, Ruiz A, Utrera V, Dee SA. 2004. Assessment of the transmission of Mycoplasma hyopnuemoniae by personnel. J Swine Health Prod 12:75-77.; 7. Batista L, Pijoan C, Lwamba H, Johnson CR, Murtaugh MP. 2004. Genetic diversity and possible avenues of dissemination of PRRSV in two geographic regions of Mexico. J Swine Health Prod 12:170-175.; 8. Cancel-Tirado S, Evans R, Yoon K-J. 2004. Identification of PRRSV epitopes associated with antibody-dependent enhancement and neutralization of virus infection using monoclonal antibodies. Vet Immunol Immunopathol (in press).; 9. Cha S-H, Chang C-C, Yoon K-J. 2004. Instability of ORF5 RFLP pattern of PRRSV during sequential pig-to-pig passages. J Clin Microbiol (in press).; 10. Dawson HD, Royaee AR, Nishi S, Kuhar D, Schnitzlein WM, Zuckermann F, Urban JF, Lunney JK. 2004. Identification of key immune mediators regulating T helper 1 responses in swine. Vet Immunol Immunopathol 100:105-111.; 11. Dee SA, Boorman J, Moon RD, Fano E, Trincado C, Pijoan C. 2004. Transmission of porcine reproductive and respiratory syndrome virus under field conditions during a putative increase in the fly population. J Swine Health Prod 12:242-245.; 12. Dee SA, Deen J, Burns D, Douthit G, Pijoan C. 2004. An assessment of sanitation protocols for commercial transport vehicles contaminated with porcine reproductive and respiratory syndrome virus. Can J Vet Res 68:208-214; 13. Dee SA, Deen J, Otake S, Pijoan C. 2004. An assessment of transport vehicles as a source of porcine reproductive and respiratory syndrome virus transmission to susceptible pigs. Can J Vet Res 68:124-133.; 14. Dee SA, Deen J, Pijoan C. 2004. An evaluation of four intervention strategies to prevent mechanical transmission of porcine reproductive and respiratory syndrome virus. Can J Vet Res 68:19-26.; 15. Dee SA, Deen J, Pijoan C. Evaluation of disinfectant efficacy for sanitizing porcine reporductive and respiratory syndrome virus-contaminated transport vehicles. Can J Vet Res (in press).; 16. Dee SA, Jacobson L, Rossow K, Pijoan C. A laboratory model to evaluate the role of aerosols in the transport of porcine reproductive and respiratory syndrome virus Vet Rec (in press).; 17. Dee SA, Martinez BC, Clanton CJ. Survival and infectivity of porcine reproductive and respiratory syndrome virus in swine lagoon effluent. Vet Rec (in press).; 18. Dee SA, Torremorell M, Deen J, Thompson B, Pijoan C. An evaluation of the Thermo-Assisted Drying and Decontamination (TADD) system for the elimination of porcine reproductive and respiratory syndrome virus from contaminated livestock transport vehicles. Can J Vet Res (in press).; 19. Dee SA. 2004. Elimination of porcine reproductive and respiratory syndrome virus by test and removal on 30 farms. J Swine Health Prod 12:129-133.; 20. Dongwan Y, Wootton SK, Li G, Song C, Rowland RRR. 2003. Colocalization and interaction of the porcine arterivirus nucleocapsid protein with the small nucleolar RNA-associated protein fibrillarin. J Virol 77: 12173-12193.; 21. Fang Y, Kim DY, Ropp S, Steen P, Christopher-Hennings J, Nelson EA, Rowland RRR. 2004 Heterogeneity in Nsp2 of European-like porcine reproductive and respiratory syndrome viruses isolated in the United States. Virus Res 100:229-235.; 22. Fano E, Pijoan C, Dee SA. Evaluation of aerosol transmission of a mixed infection of Mycoplasma hyopneumoniae and porcine reproductive and respiratory syndrome virus. Vet Rec (in press).; 23. Ferrin NH, Fang Y, Johnson CR, Murtaugh MP, Polson DD, Torremorell M, Gramer ML, Nelson EA. 2004. Validation of a blocking enzyme-linked immunosorbent assay for the detection of antibodies against porcine reproductive and respiratory syndrome virus. Clin Diagn Lab Immunol 11:503-514.; 24. Gerrits RJ, Lunney JK, Johnson LA, Pursel VG, Rohrer GA, Dobrinsky JR. 2004. A vision for artificial insemination and genomics to improve the global swine population. Theriogenology. (in press); 25. Goldberg TL, Lowe JF, Milburn SM, Firkins LD. 2003. Quasispecies variation of porcine reproductive and respiratory syndrome virus during natural infection. Virology 317:197-207.; 26. Hermann JR, Honeyman MS, Zimmerman JJ, Thacker BJ, Holden PJ, Chang CC. 2003. Effect of dietary Echinacea purpurea on viremia and performance in porcine reproductive and respiratory syndrome virus-infected nursery pigs. J An Sci 81:2139-2144.; 27. Horter DC, Yoon K-J, Zimmerman JJ. 2004. A review of porcine tonsils in immunity and disease. Animal Health Research Reviews 4:143-155.; 28. Hyland K, Foss DL, Johnson CR, Murtaugh MP. 2004. Oral immunization to induce local and distant mucosal immunity in swine. Vet Immunol Immunopathol 102:331-340.; 29. Jiang Z, Zhou E-M, Ameri-Hahabadi M, Zimmerman JJ, Platt KB. 2003. Identification and characterization of auto-anti-idiotypic antibodies specific for antibodies against porcine reproductive and respiratory syndrome virus envelope glycoprotein (GP5). Vet Immunol Immunopathol 92:125-135.; 30. Johnson W, Roof M, Vaughn E, Christopher-Hennings J, Johnson CR, Murtaugh MP. 2004. Pathogenic and immunological responses to porcine reproductive and respiratory syndrome virus (PRRSV) are related to viral load in acute infection. Vet Immunol Immunopathol 102:235-250.; 31. Kleiboeker SB, Schommer SK, Lee S-M, Watkins S, Chittick W, Polson D. Simultaneous detection of North American and European porcine reproductive and respiratory syndrome virus using real-time quantitative RT-PCR. J Vet Diagn Invest (in press).; 32. Lee C, Bachand A, Murtaugh MP, Yoo D. 2004. Differential cellular gene expression regulated by the porcine reproductive and respiratory syndrome virus GP4 and GP5 glycoproteins. Vet Immunol Immunopath 102:189-198 (Published on-line Oct 18, 2004).; 33. Lee C, Bachand A, Murtaugh MP, Yoo D. Yoo. 2004. Differential host cell gene expression regulated by the porcine reproductive and respiratory syndrome virus GP4 and GP5 glycoproteins. Vet Immunol Immunopathol 102:179-188.; 34. Lee C, Calvert JG, Welch SK, Yoo D. A DNA-launched reverse genetic system for porcine reproductive and respiratory syndrome virus reveals that homodimerization of the nucleocapsid protein is essential for virus infectivity. Virology (in press); 35. Lee C, Rogan D, Erickson L, Zhang J, Yoo D. 2004. Characterization of the porcine reproductive and respiratory syndrome virus glycoprotein 5 (GP5) in stably expressing cells. Virus Res 104: 33-38.; 36. Lee S-M, Schommer SK, Kleiboeker SB. Porcine Reproductive and Respiratory Syndrome Virus Field Isolates Differ in in vitro Interferon Phenotypes. Vet Immunol Immunopathol (in press).; 37. Lemke CD, Haynes JS, Spaete R, Adolphson D, Vorwald A, Lager K, Butler JE. 2004. Lymphoid hyperplasia resulting in immune dysregulation is caused by PRRSV infection in neonatal pigs. J Immunol 172:1916-1925.; 38. Lopez OJ, Osorio FA. The role of antibodies in PRRSV protective immunity, a short survey. Vet Immunol Immunopathol (in press); 39. Lowe JF, Husmann R, Firkins LD, Zuckermann FA, Goldberg TL. Correlation of cellular immunity to porcine reproductive and respiratory syndrome virus and clinical disease during outbreaks of PRRS in commercial swine herds. J Am Vet Med Assoc (in press).; 40. Martens GW, Lunney JK, Baker JE, Smith DM. 2003. Rapid assignment of swine leukocyte antigen (SLA) haplotypes in pedigreed herds using a polymerase chain reaction based assay. Immunogenetics 55:395-401.; 41. Meier WA, Husmann RJ, Schnitzlein WM, Osorio FA, Lunney JK, Zuckermann FA. 2004. Cytokines and synthetic double-stranded RNA augment the T helper 1 immune response of swine to porcine reproductive and respiratory syndrome virus. Vet Immunol Immunopathol 102:299-314. ; 42. Miller LC, Fox JM. 2004. Apoptosis and porcine reproductive and respiratory syndrome virus. Vet Immunol Immunopathol (Published on-line October 18, 2004); 43. Miller LC, Laegreid WW, Bono JL, Chitko-McKown CG, Fox JM. 2004. 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Impact Statements

PRRS continues to be the number one viral disease problem for swine herds and producers in the United States and Europe. Economic losses from this viral disease approach $250 or higher per bred gilt or sow. Standardization of immunological procedures will provide investigators with similar protocols to compare studies on the immune response of this disease.
The development of molecular tools such as infectious clones and microarrays will be instrumental in delineating the pathogenesis of PRRSV. Key pathogenetic mechanisms as to how the PRRSV virus modulates the immune response, evades host immune responses and promotes persistent infections need to be determined to devise appropriate methods for prevention or control of this disease.
The development of commercial diagnostic assays to detect acute and persistently infected pigs is crucial to providing a means to identify pigs with the potential to shed and transmit the virus. These assays will also provide appropriate tools for surveillance necessay in programs to evaluate eradication protocols in commercial herds.

Date of Annual Report: 12/15/2006

Report Information

Annual Meeting Dates: 12/02/2005 - 12/03/2005
Period the Report Covers: 10/01/2004 - 09/01/2005

Participants

Benfield, David Ohio State University benfield.2@osu.edu
Christopher-Hennings, J South Dakota State University jane.christopher- hennings@sdstate.edu
Jodzio, John University of Minnesota jodz0001@umn.edu
Johnson, Peter USDA:CSREES pjohnson@reeusda.gov
Lager, Kelly USDA:ARS:NADC klager@nadc.ars.usda.gov
Laegreid, Will USDA:ARS:MARC laegreid@email.marc.usda.gov
Lunney, Joan USDA:ARS:BARC jlunney@anri.barc.usda.gov
Murtaugh, Michael University of Minnesota murta001@umn.edu
Osorio, Fernando University of Nebraska fosorio@unl.edu
Rowland, RRR Kansas State University browland@vet.ksu.edu
Schommer, Susan University of Missouri schommers@missouri.edu
Sundberg, Paul National Pork Board paul.sundberg@porkboard.org
Yoo, Dongwon University of Guelph dyoo@uoguelph.ca
Zimmerman, Jeff Iowa State University jjzimm@iastate.edu
Zuckermann, Federico University of Illinois fazaaa@uiuc.edu

Brief Summary of Minutes

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Accomplishments

Objective 1. Implement a virtual laboratory infrastructure through the development and open distribution of resources, materials, protocols, and data among participating researchers. 1.1 Development of a PRRSV isolate reference panel and repository. Fourteen isolates composed the panel to date (one from NCSU; 7 from Boehringer Ingelheim Vetmedica, 6 from MO). An in vitro transcript to the ORF 2 gene through the end of the 3untranslated region of the U.S. prototype isolate VR-2332 is available. The transcript is in high concentration (>1011 copies/ul), adequate for use to determine the analytical accuracy and detection limit for real-time RT-PCR assays. 1.2 Development of a PRRSV Sequence Database. A web-accessible MySQL database was developed by MN with funding from the National Pork Board (NPB). The web-site (http://prrsv.ahc.umn.edu) contains 4100 PRRSV ORF5 nucleotide sequences generated at MN and associated history, sequence and RFLP pattern. Investigators can use this database to compare field isolates for genetic homo-or heterogeneities. 1.3 Data sharing and analysis. The project acquired through the PRRS CAP-1 USDA grant the ARM7 (Grylling Management, Brookings, SD) database software to facilitate data collection, data sharing, and data analysis in collaborative research conducted at multiple institutions. The software has been used to facilitate a large, collaborative project coordinated by KS and IA that provided standard research samples for experiments described in the other objectives in this report. 1.4 Recombinant PRRSV polypeptides from strain VR2332 encoding portions of nsp2, envelope glycoprotein 5 (GP5), and nucleocapsid (N) were produced with funds provided by the NPB and are available upon request with a materials transfer agreement to all PRRS investigators 1.5 Oligonucloetide microarray. PRRS-CAP 1 has collaborated with NPB and the national swine genome project (NRSP-8) to acquire a copy of the second-generation swine oligonucleotide array, that will facilitate functional genomic studies to better understand the virus-pig interaction and identify key swine targets of immunological and pathogenic significance. Objective 2. Achieve biosecurity within herds by preventing the spread of virus within a herd and facilitating its elimination from endemically infected herds. Research is focused on functional genomics of PRRSV resistance, mechanisms of protective immunity for PRRSV prevention, evaluation of immune modulators to stimulate/enhance antiviral immunity and agents that reduce virus replication in the pig. 2.1 Cells of the immune system. SD, IL, IA and KS are investigating various aspects of the cellular immune response to PRRSV. PRRSV modulates the functions of dendritic cells (the most important antigen presenting cells for cytotoxic T cell response) impairing normal antigen presentation and interferon production. These results guide future vaccine research to develop vaccines that enhance rather these innate immune functions. IA is determining the effects of PRRSV virulence and antigen-presenting cells (APC) on T-cell activation and antiviral cytokine production. Results suggest that: PRRSV suppresses T-cell functions in both virulence-dependent and virulence-independent fashions; suppresses porcine APC that interact with T-cells; and T-cell suppression is partly due to PRRSV-induced IL-10. KS is mapping T-cell epitopes on PRRSV proteins. Using mesenteric lymph nodes (MLN) as the source of lymphoid cells the following has been determined: 1) While proliferation is evident when cultured MLN lymphocytes are compared with freshly isolated, non-cultured cells, there is a background level of proliferating lymphocytes in culture that is not enhanced by polyclonal activation or antigenic stimulation by PRRSV; 2) There are activated T cells in the cultures as determined by co-expression of CD25 and MHC II on CD4+ T cells; 3) There is a background of interferon-gamma secreting cells in cultures of MLN lymphocytes; however, polyclonal activation and antigenic stimulation result in increased numbers of secreting cells compared to unstimulated cultures; 4) A residual percentage of adherent MLN node cells remain positive for PRRSV antigen, but there is no apparent association between the percentage of antigen-positive cells and results from the cultures; and 5) Control pigs have remained antibody negative throughout the study and MLN cells from these animals do not respond to viral antigens indicating that many of the above changes in MLN lymphocytes are induced by PRRSV infection.. 2.2 Cytokines. BARC (USDA ARS) provided immune gene expression analyses for several projects including: 1) Testing gene expression in samples from PRRSV infected boars in collaboration with SD; 2)Assessing in vitro test parameters (time in culture, use of crude or recombinant PRRSV antigen) that produces the maximum immune gene expression data in cooperation with IL and NC; and 3) Comparing immune gene expression of pigs infected or vaccinated with type 1 and type 2 PRRSV in collaboration with SD; and 4)Looking at how different phenotypes of pigs respond to PRRSV at NE and MARC (USDA ARS). Results from these studies may explain why PRRSV infection modulates the immune response. NADC showed that IFN-gamma was induced in pigs much earlier that previously reported and that that the immune pathways used against PRRSV, unlike other swine virus infections, more closely resembled immune responses to infections by intracellular bacteria and protozoa. KS determined that the permissiveness of porcine alveolar macrophages (PAMs) to PRRSV is dependent on new cellular mRNA synthesis and regulated by certain cytokines. 2.3 Antibodies. IA produced anti-idiotypic antibodies against the Gp5 of PRRSV that blocks PRRSV infection of MARC145 cells and PAMs and may have the potential to prevent infection in pigs. MN and IA are collaborating on a project to better understand the induction and maintenance of anti-PRRSV immunity and to gain insights into mechanisms of viral persistence. The most significant B-cell response is to the nsp2, GP5 and N viral antigens in that order. These B-cells are localized to the sternal lymph node (SLN) and spleen at 37 dpi, indicating that these tissues are the major sites of antibody production. Antigen-specific B-cell responses peaked at 37 dpi and declined by 98 dpi. However memory B-cell responses remained high up to 150 dpi. In contrast there was no significant difference in tissue distribution of memory B-cell responses. Both ASC and memory B-cell responses were extremely low in bone marrow, although it is regarded as the primary site of antibody production in vertebrates. Since no difference in IgG total B-cell responses was observed in a variety of lymphoid tissues from both infected and uninfected pigs in acute and persistent PRRSV infection, we conclude that there was no polyclonal B-cell activation in PRRSV infection. The protease activities encoded in the 5-end of ORF1a are the first viral proteins synthesized in cells infected with PRRSV. These proteins are expressed early in the viral life cycle, hence they are available from the earliest time of infection to the macrophage proteosome antigen processing pathway for presentation to the immune system. Since PRRSV induces cell lysis at 2-3 days after infection, releasing cellular contents and viral proteins into interstitial spaces, the hypothesis was that an early antibody response to nsps would be generated. In fact, swine mount an immediate response to nsp1;peak levels of anti-nsp1 antibody exceed anti-PRRSV N;and the levels of anti-nsp1 are maintained at the same level as anti-N antibodies for at least 120 dpi. The antibody response to nsp2 is greater than to any other PRRSV structural or non-structural protein. Responses to nsp4, by contrast, appeared after acute infection and were weaker that the response to nsp2. The antibodies appear to be cross-reactive. Protein refolding is essential for immunoreactivity to nsp1, but not nsp4. Refolding may restore non-linear antigenic epitopes that appear to be dominant on nsp1, since no immunoreactivity was observed in the absence of refolding. 2.4 Persistent infection in pigs. IA studies indicated that all genes of PRRSV co-evolve at different rates and that recombination readily occurs within a pig infected with at least two PRRS strains of virus. IA and KS created a sample bank of various tissues and blood for studies on acute and persistent PRRSV infections in a large population of pigs. Affectionately titled Big Pig this experiment has provided over 20,000 samples (serum and tissues) to 5 institutions to identify virological/immunological correlates of persistence and clearance, and to assist in development of a model for persistence at the population level. 2.5 Viral genome. Infectious cDNA clones of PRRSV allow us to manipulate the viral genome and create specifically defined mutant viruses that are used to study individual viral protein functions in vivo and serve as the backbone for developing the next generation of PRRS vaccines. Using an infectious cDNA clone developed for a North American genotype the University of Guelph and Pfizer, generated a knock-out of the nuclear localization signal (NLS) of the N protein. While NLS was required for PRRSV replication, the viral growth rate was reduced 100-fold, compared to wild-type (wt) PRRSV. This NLS-mutant virus did not localize in the nucleus of infected cells, replicated to lower titers in pigs, viremia was of shorter duration and neutralizing antibody titers were higher. When the small envelope (E) protein expression was suppressed in mutated infectious cDNA clone, virus replication was also suppressed suggesting that the E protein is essential for viral replication and may function as an ion-channel protein for PRRSV. Overall, the data suggest that the N protein functions as a virulence factor modulating the immune response of the host .and the E protein may be involved in the uncoating process during viral entry of the PRRSV into the host cell. In addition to the above studies on structural proteins, MN in collaboration with SD and Northern Michigan University (NMU) has constructed infectious clones to determine the effects of deletions on the nsp2 region of the replicase. Also MN and NMU have developed 15 recombinant full-length constructs with alterations to the N-glycosylation site of GP5. Studies are now in progress to determine if these constructs are infectious and can be stably passaged in MA-104 cell culture. Along similar lines KS is using a different infectious clone to construct recombinant viruses that express an nsp2-GFP fusion protein. MN, Guelph, SD and NMU collaborated on determining the role of PRRSV minor structural proteins (GP2, 2b, 3 and 4) in PRRS disease. To date the group has produced plasmids and eukaryotic expression clones to produce protein in vitro for immunization of rabbits to develop polyclonal antibodies to each of these proteins. MN and IA performed an in vivo study of PRRSV isolates naturally deficient in GP5 N-glycosylation. In a study funded by Pig Improvement Company, three isolates deficient in N-glycosylation at three key locations in GP5 were forwarded to IA for infection of swine. Serum samples were taken at various timepoints and forwarded to UMN for testing by peptide ELISA and ORF5 nucleotide sequence analysis. The results were mixed, but seemed to suggest that inoculation of pigs with a strain deficient in N-glycosylation in the hypervariable region induced a better protective response in pigs to protect against virus challenge with the MN184 strain. However, the studies were not conclusive as animal numbers were small, biosecurity was questioned, and the studies assumed only the N-glycosylation of GP5 had an effect on immunity. Additional studies by NE examined the influence of N-linked glycosylation of GP5 on virus infectivity, antigenicity and the ability to induce neutralizing antibodies. Three putative N-linked glycosylation sites (N34, N44, and N51) and major neutralization epitope both exist on the GP5 ectodomain. Using a panel of GP5 mutants containing single and multiple amino acid substitutions at these sites it was determined that mutations involving N44 residue were not infectious. Viruses with mutations at N34, N51, and N34/51 grew to lower titers than wt virus and exhibited reduced cytopathic effects in MARC 145 cells. In serum neutralization assays, the mutant viruses exhibited enhanced sensitivity to neutralization by wt PRRSV-specific antibodies and pigs inoculated with these viruses produced significantly higher levels of neutralizing antibodies against mutant and wt PRRSV. These results suggest that the loss of glycan residues in the ectodomain of GP5 enhances the sensitivity of these viruses to neutralization and the immunogenicity of the nearby neutralization epitope. Thus, these mutant viruses may have be significant candidates for PRRSV vaccines of enhanced protective efficacy. SD and KS are using infectious clones of European-like PRRSV to determine the usefulness of nsps as potential epidemiological tools. Phylogenetic analysis using ORF5 nucleotide sequences from 6 U.S. Type 1 isolates from geographically separated swine herds showed that 15/16 isolates formed a monomorphic clade of four subgroups. Comparative analysis with the genomic sequences of European prototypic strain, Lelystad virus (LV) and North American (VR-2332) revealed that each of the European-like viral genomes had higher nucleotide homology with the LV than the VR-2332 strain of PRRSV. Nsps1², 2, 6 and 12 were identified as the most variable nsp regions. Nsp2 showed similar genetic heterogeneity among isolates as GP5, which has been used most frequently for PRRSV genetic diversity and evolution studies. This study represents the largest type 1 PRRSV full-length genome sequence database available for future comparative studies of Type 1 and 2 PRRS viruses. Researchers at MO are using infectious clones to identify regions within nsp2 (targeted for deletions) and short genomic region between ORF 4 and 5 (targeted for insertions) that can be stably manipulated. A series of constructs has been prepared representing increasing lengths targeted for deletion and a unique restriction endonuclease site was inserted into the ORF 4 and 5 regions. If rescue of viable recombinant (deletion mutant) viruses is successful, future constructs will be prepared in which heterologous sequences (such as the marker protein GFP) can be inserted into one or both regions being investigated. SD in collaboration with KS, MN and NE constructed a European-like Type 1 PRRSV full-length cDNA infectious clone (pSD 01-08) to further characterize this group of U.S. Type 1 PRRSV and provide an essential tool for the future construction of a new generation of genetically engineered PRRSV vaccines for both Type 1 and Type 2 PRRSV. This virus has low virulence in pigs and induces an early and robust neutralizing antibody response The full-length cDNA infectious clone derived from SD 01-08 P34 could be an ideal viral backbone for future recombinant PRRSV vaccine construction. 2.6. Pathogenesis (virus factors). NADC is investigating the dysregulation of immune responses induced by PRRSV. Antigen-specific antibody responses are augmented after PRRSV infection, followed by heightened polyclonal antibody activation especially in gnotobiotic pigs that have 50-fold higher antibody titers than sham-inoculated, colonized controls. Furthermore, secondary antibody responses to a thymus-dependent antigen were not altered by PRRSV infection. Hence PRRSV does not impair either the primary or secondary immune responses. However, the proportion of IgG to a thymus dependent and type 2 thymus-independent immunogen produced by immunized, infected piglets was highest 1-week after infection and progressively decreased over time, while that of uninfected controls did not. These results suggest that that PRRSV initially stimulates B-cell proliferation, but as infection proceeds, the continual activation effect on pathogen-recognition receptors such as Toll-like receptors generates much more IgG to the thymus dependent and independent immunogens. In short, antibodies from specific antibody-producing cells are overwhelmed by non-specific IgG, an observation consistent with previous reports that <1% of the total IgG in PRRSV-infected germ-free piglets was virus specific. Nucleo-cytoplasmic shuttling of the PRRSV N protein is being studied at KS in collaboration with Guelph. The 123 amino acid N protein of PRRSV localizes to the nucleus and nucleolus of infected cells. In the nucleolus, N appears to regulate rRNA processing, but must also leave the nucleus to be assembled into nucleocapsids in the cytoplasm. The purpose of this study was to determine the mechanism for export of N from the nucleus. The addition of inhibitors to the nuclear export shuttle protein, and RNA polymerase I and II, blocked nuclear export. The C-terminal 34 amino acid polypeptide covering amino acids 90-123, when tagged with EGFP, was retained in the cytoplasm and substitutes for the nuclear export signal (NES) sequence of equine infectious anemia virus Rev (ERev) protein. Replacement of two hydrophobic residues within an LXL-like motif failed to prevent nuclear export, suggesting that the C-terminal region of the PRRSV N possess a CRM1-dependent mechanism for N protein export from the nucleus. However, the NES deviates from classical NES sequences common to other viral proteins and inhibition of export by high concentrations of actinomycin D suggests that N protein export is dependent on de novo nucleolar rRNA synthesis. Activation of the innate immune response pathways plays a critical role in the control of virus infections. The antiviral properties of the unfolded protein response (UPR) are related to the detection of perturbations in ER function, such as the accumulation of misfolded or aggregated proteins. PRRSV replication is primarily in the ER-Golgi, resulting in disintegration of ER and formation of double membrane vesicles. The capacity of PRRSV to induce the unfolded protein response (UPR) in cells is being investigated at KS. One outcome of UPR activation is the altered splicing of XBP-1 mRNA, which codes for a transcription factor that is rapidly transported to the nucleus. To study this, MARC 145 cells were infected with low passage and cell-adapted PRRSV isolates SD23983 P4 and P136, respectively. Uninfected cultures served as negative controls and culture wells treated with the UPR activator, DTT, served as positive controls. At 2 days after infection and one hr after DTT treatment, total RNA was extracted and XBP-1 amplified by RT-PCR. Results showed that DTT treatment resulted in the accumulation of Pst-resistant XBP-1 cDNA; whereas PCR products from PRRSV-infected and control cultures retained sensitivity to Pst-1, supporting the concept that PRRSV blocks the induction of antiviral responses during replication. 2.7. Pathogenesis (host factors). KS identified Vimentin as a cellular receptor for PRRSV and produced a monoclonal antibody 7G10 that binds to cytoskeletal filaments Vimentin also bound to PRRSV N protein, and anti-vimentin Abs blocked PRRSV replication. Vimentin is expressed on the surface of the permissive MARC-145 cell line and the addition of simian vimentin to nonpermissive BHK-21 and CRFK, renders these cells permissive to PRRSV infection. These results suggest that that vimentin is part of a PRRSV receptor complex and function in PRRSV binding to cytoskeletal filaments that mediate virus transportation to the cytosol. Research at IA indicated that certain viral genes were found to account for differences in the replication of attenuated and wt PRRSV in porcine alveolar macrophages and MARC-145 cell lines. MN has determined that pathogenicity of PRRSV strains, coinfections with Mycoplasma hyopneumoniae and age (2 months vs 6 months) significantly impacted viral loads in experimentally infected animals. NE and USDA-BARC are collaborating to determine the impact of host genetics on gene expression and immune responses by comparing Duroc/Hampshire (HD) crossbred pigs (n=100) and NE Index (I) line pigs (n=100) infected with PRRSV. Comparisons were evaluated for resistance/susceptibility. Viremia (V), weight change (WT), and rectal temperature at 0, 4, 7, and 14 dpi with lung, bronchial lymph node (BLN), and blood tissue collected at necropsy 14 dpi. Results indicated that genetics influences the responses to PRRSV. Low (L) responding pigs had high WT, low V, and few lung lesions; high (H) responders had low WT, high V, and many lesions. Significant under-expression of immune genes in L pigs was detected in lung and BLN, particularly in the I line. Also, prior to infection low serum levels of the cytokine, interleukin-8, and low expression of interferon-gamma in cDNA and in serum correlated with resistance. KS and IA are evaluating the effect of PRRSV infection on growth in a relatively large population of experimentally infected pigs over a period of 200 days. Within two weeks after infection, approximately 15% of the pigs appeared to be smaller, had a rough appearance and a high degree of variability in the weights of PRRSV exposed pigs. For example, at 33 dpi weights ranges from 242-271 lbs (mean=258, n=10) for the control group versus 150-251 (mean 217, n=5) for the PRRSV group, indicating that PRRSV infections directly impact growth rates in the absence of a secondary infection, such as circovirus. KS is defining the pathogenesis of congenital infection. When gilts are infected with PRRSV at 90-days gestation and fetuses examined between 109 and 112 days, the infection rate is not 100%. Rather infected fetuses were clustered within uterine horns, suggesting that virus is transmitted fetus-to-fetus. At necropsy gross pathology was not a reliable indicator of infection and the thymus was the principal site of PRRSV replication in the fetus. Thus, fetal alterations may result from the effect of PRRSV on maternal tissues and diagnosticians should consider the different clinical presentation of congenital PRRSV when dealing with abortion or stillborn pigs. 2.8. Vaccines / Vaccination. IL measured virus neutralizing and non-neutralizing antibody titers and the frequencies of virus-specific interferon-secreting cells in circulating lymphocytes generated following exposure to either a commercial MLV vaccine, wt virus alone or wt virus followed by an injection of killed virus in gilts for 84-days. Exposure to the wt virus, irrespective of boosters with inactivated virus, elicited a faster and higher strain-specific neutralizing antibody response and a more rapid generation of interferon-secreting memory T cells. However, these animals averaged 2.45 fewer piglets/litter compared to sows exposed to the MLV. Thus, current immunological assays that measure responses to PRRSV fail to correlate with protection and the practice of controlled exposure of sows to virulent PRRSV should be used with caution as it reduces litter size. IA is investigating immune responses and protection by vaccine and various vaccine adjuvant candidates to virulent PRRSV. The study reported the influence of various vaccine adjuvants on humoral-mediated immune (HMI) and cell-mediated immune (CMI) responses to PRRS MLV vaccine (Ingelvac®, Boehringer Ingelheim Vetmedica, St. Joseph, MO) as well as on protection from virulent PRRSV MN-184 challenge. The study found that PRRS MLV vaccine alone successfully primed CD4-CD8+³´- T cells as demonstrated by a significant increase in percent IFN gamma + cells when live PRRSV was used as a recall antigen. Booster immunizations of mixed ORF5 peptides and co-administration of IL-12 with PRRS MLV vaccine significantly enhanced IFN gamma expression by some T cell subsets. All groups receiving MLV vaccine with or without adjuvants had reduced lung lesions after challenge. The group immunized with only ORF5 peptide/cholera toxin did not have significant T cell recall responses and was not protected from challenge. Expression of IFN gamma by several T cell subsets correlated with reduced lung lesions and viremia, whereas expression of CD25 did not correlate with either fewer lesions, viremia or IFN gamma production. PRRSV ELISA s/p ratio prior to challenge also correlated with reduced lung lesions and viremia. In conclusion, booster immunizations of the mixed ORF5 peptides and co-administration of IL-12 effectively enhanced the CMI response to PRRS MLV vaccine. However, neither adjuvant significantly contributed to reducing clinical effects when compared to PRRS MLV alone. IA conducted a study at understanding the role of PCV2-infection on vaccine efficacy. In healthy pigs, commercial respiratory (PRRSV, SIV, M. hyo) vaccines are generally considered to be effective. The objective of this study was to investigate the effect of acute PCV2-infection on the ability of a PRRSV vaccine to protect pigs against PRRSV-induced clinical disease and lesions. Overall, the results indicate that vaccine efficacy was reduced when administered at the time of PCV2-infection. The adverse effect of PCV2-infection on development of immunity to PRRSV and other respiratory vaccines may be a very important factor in controlling porcine respiratory disease complex and other diseases in growing pigs. SD is collaborating with the U of Rochester to determine the immunogenicity of a herpes virus-based construct containing the complete GP5 gene of PRRSV 23983 (HSV-Gp5) in pigs. Preliminary immunization studies with mice indicated that the HSV-Gp5 construct induced an inconsistent virus neutralization antibody response and a relatively poor T cell response in mice. This vector is being further refined for future immunity studies in pigs. NE and KS are currently evaluating pseudorabies and PRRSV (M and GP5) recombinants for expression and use as vaccines. Iinvestigators at the Eastern Virginia Medical School studied the potential of antisense phosphorodiamidate morpholino oligomers (PMOs) to suppress PPRSV replication in cell culture. One of six PMO compounds (PMO-1) showed promise as an antiviral and blocked PRRSV replication (reduction in virus yield by 4.5 logs) in a dose-dependent manner. Objective 3. Achieve biosecurity among herds by preventing viral spread between sites. 3.1 Virus Diversity. Researchers at SD and KS evaluated the pathogenic and immunological properties of U.S. Type 1 European-like PRRSV isolates recovered between 2001- 2003. Pigs were infected with Type 1 PRRSV strains to investigate the pathogenesis of each isolate and produce a pool of well-characterized serum samples for other researchers and diagnostic laboratories. Results indicate that: clinical signs and pathology were variable and mild in severity; all animals seroconverted with ELISA titers by14 dpi; and neutralizing antibody responses against homologous challenge isolates appeared at 21 dpi and reached peak titer of 1:128 by 56 dpi. However, sera failed to neutralize selected North American (Type 2) PRRSV isolates and demonstrated intermediate levels of neutralization against heterologous Type 1 strains and LV. Genetic analyses provides insights into the diagnostic, antigenic, molecular properties, emergence and strategies for control of U.S. Type 1 PRRSV. 3.2. Immunity and/or Cross Protection. When PRRSV infects a previously exposed sow herd by infection or vaccination, the virus will either be eliminated or continue to circulate. Veterinarians at MN and the Pipestone Veterinary Clinic (Pipestone, MN) are conducting field studies to obtain quantitative information on serological and virological responses following serum inoculation in a PRRS-positive herd. Results indicate that herd immunity induced by commercial MLV vaccine is not increased by exposure to wt virus at mid-gestation. Virus was detected in these herds at very low levels and low frequency by PCR., suggesting that PRRSV may be maintained in herds at or below the sensitivity of PCR assays. At IA researchers are examining the immunobiological significance of genetic variation among PRRS viruses to determine the correlation between genetic divergence and cross-neutralization (both in vitro and in vivo). The goal is to identify a single genetic marker on viral genes that predicts whether immune responses would be cross protective between viruses. IL conducted a study in collaboration with swine producers to investigate the relationships among immunity, reproductive performance, and viral genetic variation in swine herds infected with PRRS. Thirty PRRSV-naïve replacement gilts were exposed to PRRSV by intramuscular injection upon their introduction to the farm. Serial clinical samples (blood, serum and/or tonsil biopsies) were collected until 85 days of the first gestation. Tonsil biopsies were used for RT-PCR testing for viral RNA and genetic characterization of PRRSV (ORF 5 sequences). Ten weeks post-infection (2 weeks after exposed and non-study pigs were intermingled), two genetic clusters of ORF5 sequences were identified: one cluster was closely related to the exposure strain, and one was genetically divergent. Phylogenetic relationships among strains indicated that three study pigs were re-infected with a co-circulating, genetically divergent viral variant at the time of sampling. Cellular and humoral immune responses were examined in all pigs using ELISPOT and FFN tests. These results indicate that poor immunity to PRRSV may facilitate re-infection. Finally, there was a positive correlation (r = 0.63) between the number of pigs born alive and the intensity of the virus-specific IFN-gamma response, indicating that cellular immunity provides some protection from clinical disease even for pigs housed in an environment with multiple, co-circulating viral strains. 3.3. Transmission. Researchers at MO are using a non-invasive antemortem technique to obtain tonsillar crypt exudate from pigs inoculated with either MLV vaccine or a PRRSV field isolate. The purpose of the study is to determine the duration of time pigs harbor vaccine or field strains of virus in the tonsil as am indicator as to the length of time new animals should be isolated prior to introduction into a PRRSV positive herd. Studies are in progress at NC to determine whether Stomoxys calcitrans (stable fly) can transmit PRRSV infection to naïve pigs. This is important because Stomoxys calcitrans is capable of traveling long distances (12 to 20 miles) in a 24 hr period and could account for possible lateral spread of PRRSV between herds. NC and MN have shown that the prairie dog (Cynomys ludovicianus) is not a host for PRRSV. Investigators at IA and MN have ongoing research to: determine the quantity of virus excreted by PRRSV-infected pigs; estimate the influence of relative humidity and temperature on the half-life (T1/2) of aerosolized PRRSV; construct the infectious dose-response curve for pigs exposed to aerosolized PRRSV; develop a computational (predictive) model that will make it possible to predict spread of PRRSV via aerosols. Also research at MN suggested that the frequency and transmissibility of PRRSV in aerosols was related to isolate pathogenicity. Results also indicated that HEPA-based filtration systems provided the best protection against PRRSV entrance into facilities through the air, but alternative systems (95% DOP @ 0.3 micron filters) were cost-effective alternatives. Methods for preventing PRRSV transmission are being evaluated at MN. First, a series of five studies evaluated the ability of industry-based sanitation protocols (disinfection, thermo-assisted drying and decontamination) to sanitize PRRSV-contaminated transports. The results indicated that while drying was a superior method of sanitation, two commercially available disinfectants (Virkon and Syngergize) were also highly efficacious. 3.4. PRRS Risk Factors. MN investigators are determining the relation between geographical distance and genetic homology among PRRSV isolates from a single pork producing company. The findings indicated that, under the conditions of this study, the greater distance between farms, the less genetic homology between the PRRSV isolates In partership with the National Pork Board, IA researchers plan want to characterize the physical and environmental components that affect the rate of pig-to-pig PRRSV transmission within swine herds. At IL scientists determined that increasing cell-mediated immunity within infected herds has the potential to decrease clinical reproductive disease. Objective 4. Improve diagnostic assays and create on-farm monitoring systems. IA investigators identified anti-PRRSV IgG in meat juice and IgA in oral fluid. Studies are in progress to validate the commercial ELISA for meat juice samples and examine the dynamics of IgA antibody responses in infected pigs. Aattenuated viruses induce antibody responses less optimally than wt PRRSV. Furthermore, while ELISA and IFA test results were less affected by genetic variation (i.e., potential antigenic changes), serum-virus cross neutralization was severely affected. MN is evaluating various test protocols including frequency of sampling (daily, twice/week, weekly, every two weeks, etc), type of test (PCR in serum, blood swab or semen, ELISA), individual testing or pooling of samples. Preliminary results indicated that PCR testing of serum or blood swab were more efficient for early detection than antibody testing. Investigators at the MO are developing biosensors to detect PRRSV antigen in clinical samples. The biosensor utilizes Fluorescence Resonance Energy Transfer (FRET) to detect the PRRSV. MN is developing a high throughput TaqMan RT-PCR for simultaneous detection of Type 1 and 2 PRRSV isolates. Objective 5. Develop and test PRRSV virus eradication protocols under various ecological settings. MN initiated studies on the regional transmission of PRRSV using actual swine farms in Rice County MN. Six groups of PRRSV isolates were identified in this area, but no evidence of area spread was documented. In a second study commercial vaccine administered to swine significantly reduced transmission of field isolates compared to non-vaccinated herds. Objective 6. Develop educational outreach tools for disseminating information through established outreach and extension networks to producers, veterinarians, educators, and researchers. Eighty participants attended the American Association of Swine Veterinarians workshop held March 4-5 in Toronto with topics on antivirals, genetic resistance and a comparative immune protection evaluation study. PRRS-CAP, NPB and NC-229 sponsored an International PRRS Symposium in St. Louis December 2005. The symposium had 225 registrants and 77 posters from the U.S., Mexico, Canada, Europe and Asia. Objective 7. Create an information network to ensure rapid and efficient communication of PRRSV research. Three communication initiatives were started: PRRS CAP 1 website (prrs.org); quarterly newsletter to provide information about NC-229/PRRS-CAP 1 activities; and monthly conference calls among PRRS CAP 1 funded researchers.

Publications

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Impact Statements

The PRRSV database has opened the door to new research opportunities in epidemiology, diagnostics, genetics and allows practitioners assess to real time data from PRRSV experiments. The integration of ARM-7 into a shared data collection and analysis network provides an interactive method for data sharing among researchers that should increase the efficiency and timeliness of research projects.
Using the reverse genetics system, mutant PRRS viruses can now be constructed and mutations may be introduced to specific sites of the virus to alter respective protein function. This approach will identify virulence factors of PRRS virus and useful vaccine candidates (mutants) can now be generated in the laboratory.
Studies in progress on PRRSV (Type 1 and Type 2) continue to demonstrate that genetic diversity affects cross-neutralization of viruses, susceptibility of PRRSV to cell-mediated immunity, and our ability to stop circulation of virus in swine populations. Work in progress will provide important information on transmission by arthropods, aerosols, and the duration of persistent infection. This information is critical to the development of strategies for protecting herds and eliminating infections.
Efficient, cost-effective diagnostics are paramount for producer support of elimination and control projects. NC-229 researchers are investigating novel approaches for diagnostic assays that detect both viral structural and nonstructural proteins. These new assays will enhance the ability to detect persistently infected pigs, one of the main impediments to successful eradication of this disease.
The Rice County (Minnesota) project is an early experiment with regional eradication of PRRSV from commercial swine hereds. Interesting, despite concerns over area spread, initial results indicate minimal area spread between independent farms. This is good news to veterinary practitioners and producers as the mechanism of area spread is unknown and a major impediment to elimination of PRRSV in herds.
NC-229 sponsors an annual International PRRS Symposium and discussion among PRRSV research scientists throughout the world. This symposium is a new means to openly present and discuss PRRS research progress. The participation and input of non-NC229 entities is especially valuable to the PRRS community. The participation of researchers from all over the world makes NC-229 unique. The quarterly newsletter provides an up to date PRRS progress report to researchers, practitioners and stakeholders

Date of Annual Report: 12/15/2006

Report Information

Annual Meeting Dates: 12/01/2006 - 12/02/2006
Period the Report Covers: 10/01/2005 - 09/01/2006

Participants

Please see attached participant roster

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Brief Summary of Minutes

Please see attached minutes

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Accomplishments

Objective 1. Implement a virtual laboratory infrastructure through the development and open distribution of resources, materials, protocols, and data among participating researchers. 1.1 PRRSV Sequence Database. A web-accessible MySQL database was developed by UMN with funding from the National Pork Board (NPB) and SDSU. The web-site (http://prrsv.ahc.umn.edu) contains 5400 PRRSV ORF5 nucleotide sequences generated at MN and associated history, sequence and RFLP pattern. Up to 25 isolates can be multi-sequence aligned for further phylogenetic analysis using tools available at the same site, including ClustalX, Jalview and PFAAT. The effort was aimed at producing an easy to use database for nucleotide sequence mining. 1.2 Data sharing and analysis. The multi-institutional "Big Pig" project (KSU, ISU, SDSU, BARC, IDEXX) supplied thousands of biological samples at no charge to several other laboratories. It continues to distribute samples to researchers. All labs have participated in the movement toward a virtual laboratory through involvement in the development of ARM-7 database. 1.3 The PRRS virus isolate bank that was established will continue to be maintained at UMO. 1.4 Recombinant PRRSV polypeptides from strain VR2332 encoding portions of nonstructural protein 2 (nsp2), envelope glycoprotein 5 (GP5), and nucleocapsid (N) were produced at UMN with funds from NPB and are available to all PRRS investigators. Recombinant proteins encoding the nsp1, 7, 8, 12, and functional domains of nsp 2, 4, 9, 10, and 11 were in vitro expressed in E. coli by SDSU researchers. A full panel of monoclonal antibodies (mAbs) against PRRSV nsps is being developed at SDSU to study protein structure-function and design antiviral intervention strategies. A panel of mAbs against nsp1, 2, 3, 4, 7 and 8 were produced and screened by indirect immunofluorescence against and Western blotting or immunoprecipitation; other mAbs are in progress. These are basic reagents for study of the fundamental biology of PRRSV nsps and good candidates for future development of diagnostic assays. 1.5 Oligonucloetide microarray. PRRS-CAP 1 has collaborated with NPB and the national swine genome project (NRSP-8) to acquire a copy of the swine oligonucleotide array http://www.pigoligoarray.org/, that will facilitate functional genomic studies to better understand the virus-pig interaction and identify targets of immunological and pathogenic significance. Objective 2. Achieve biosecurity within herds by preventing the spread of virus within a herd and facilitating its elimination from endemically infected herds. Research is focused on functional genomics of PRRSV resistance, mechanisms of protective immunity for PRRSV prevention, evaluation of immunomodulators to stimulate/enhance antiviral immunity and agents that reduce virus replication in the pig. 2.1 Cells of the immune system. Flow cytometric and fluorescent antibody analysis of PRRSV infection of MARC-145 cells were used by SDSU scientists to clarify viral dynamics and the mechanism of viral spread. The roles of viral permissiveness and the cytoskeleton in PRRSV infection and transmission were examined in conjunction with antiviral and cytotoxic drugs. PRRSV infection and cell-to-cell transmission were efficiently suppressed by interferon-gamma (IFNg), and the more potent antiviral agent AK-2, indicating that an intact cytoskeleton is critical for PRRSV infection and that viral permissiveness is an efficient drug target. KSU is mapping T-cell epitopes on PRRSV proteins. Using mesenteric lymph nodes (MLN) they determined that activated T cells co-express CD25 and MHC II on CD4+ T cells. Polyclonal activation and antigenic stimulation increased IFNg secreting cells. Fluorescent cell sorting-based assays have been adapted for the identification of T cell epitopes in PRRSV proteins. A peptide epitope of PRRSV GP5 stimulates IFNg secretion and CD4+ T cell proliferation. This information is critical for vaccines that assure stimulation of cell mediated immunity. Plasmacytoid dendritic cells (PDC) are the most potent source of IFNa and thus are primarily responsible for the initial anti-viral protective response. UIUC scientists evaluated the behavior of pig PDC exposed to PRRSV both in vitro and in vivo. When freshly purified PDC were incubated with PRRSV, the resultant IFNa response was meager, much less intense than that stimulated by transmissible gastroenteritis virus (TGEV). That PRRSV affected PDC function was affirmed since it repressed the vigorous IFNa responsiveness to TGEV of PBMC. A similar impact of PRRSV on PDC was observed in vivo and may be unique since it was not found when swine were infected with Pseudorabies virus. 2.2 Cytokines. BARC provided immune gene expression analyses for several projects including: 1) Testing immune gene expression in samples from PRRSV infected boars with SDSU; 2) With UIUC and NCSU, assessing in vitro test parameters (time in culture, PRRSV antigen) that produce the maximum immune gene expression data; 3) Comparing immune gene expression of pigs infected or vaccinated with type 1 and type 2 PRRSV with SDSU; and 4) Assessing the genetic basis of response by testing how different genetic lines of pigs respond to PRRSV with UNL. Results from these studies may explain why and how PRRSV infection modulates the immune response. An early warning biomarker of infection would improve diagnosis and facilitate better PRRSV preventive strategies. UMN scientists hypothesized that PRRSV infection should produce a serum protein profile characteristic of early infection. They obtained a serum profile of low mw proteins in sera from PRRSV-infected and non-infected pigs by mass spectrometry. Comparative analysis revealed a protein in PRRS sera within 1 dpi, with sensitivity = 0.92 and specificity = 0.94 at 7dpi. When sera from pigs infected with non-PRRSV pathogens were included specificity = 0.83. The protein was identified as the alpha1S subunit of porcine haptoglobin (a1Hp) as confirmed by immunoblotting. The results suggest that the a1Hp, a well known acute phase protein, is a potential protein biomarker for acute PRRS, thus providing new insights into biochemical processing of Hp and its role in PRRSV pathogenesis. 2.3 Antibodies. In previous studies NADC and Univ. IA scientists observed in gnotobiotic isolator piglets infected with PRRS virus (PRRSV) the development of lymphoid hyperplasia, hypergammaglobulinemia and autoimmunity. Preliminary characterization of the expanded B cell population in these animals reveals that gnotobiotic isolator piglets infected with PRRSV do not diversify the VDJ repertoire of their circulating B cells or that of B cells in infected tissues. This may indicate that B cells with hydrophobic HCDR3s in PRRSV-infected piglets are targeted for T cell-independent proliferation without repertoire diversification. It has been hypothesized from gnotobiotic pig studies that polyclonal humoral responses are responsible for viral pathogenesis and establishment of persistence in PRRSV infection. UMN and ISU scientists quantified actively secreting IgG total plasma cell responses in conventional pigs by ELISPOT assay. No change in IgG total blood plasma cell responses among PRRSV infected animals, KLH immunized and uninfected animals was found, indicating that there is no non-specific polyclonal B-cell activation in conventional pigs in PRRSV infection. There is a slight elevation in serum Ig levels but that might be common in viral infections. Altogether PRRSV infection did not induce any pathological, non-specific polyclonal B-cell activation or hypergammaglobulinemia in conventionally raised pigs. 2.4 Persistent infection in pigs. The "Big Pig" project, a multi-disciplinary, multi-institutional (KSU, ISU, SDSU, BARC, IDEXX Labs) project, monitored PRRSV infection responses of 165 pigs for as long as 203 days. BARC scientists compared RNA prepared from lung and tracheobronchial lymph nodes (TBLN). Infected pigs showed up regulation of innate and IFNG stimulated, T helper 1 (Th1) genes in the first 84 days after infection followed by overall down regulation. However, there was no pattern of TBLN cytokine expression that was associated, or might help predict, which pigs will clear virus and distinguish them from those that remain persistently infected. UMN, KSU and ISU scientists investigated the antibody response to PRRSV GP5 and M. Little is known about the role of the GP5-M heterodimeric structure in anti-viral immunity. They hypothesized that antibodies directed to conformational epitopes on the ectodomains of GP5 and M proteins may be involved in virus neutralization. Pigs infected with PRRSV developed anti-GP5-M antibody response that was long-lasting, but neutralizing antibody titers were low at 193 dpi. There was no apparent correlation between anti-GP5-M antibody response and the serum total neutralization titers, nor to resistance to challenge. Pigs immunized with GP5-M developed high antibody response and had partial protection against challenge, but no neutralizing activity was detected. This suggests that antibodies to GP5-M may play a role in protection against PRRSV that is not dependent on viral neutralization. 2.5 Viral genome. Guelph scientists worked to understand the pathogenic role of viral proteins during infection and their interactions with host cell proteins. They identified a specific signal responsible for nuclear translocation of the viral capsid protein, and by mutating the signal sequence, the translocation was blocked. This was the basis for constructing a genetically modified PRRSV, using an infectious cDNA clone, of which the viral capsid protein is no longer translocated in the nucleus. This newly constructed PRRSV induced higher neutralizing antibodies in pigs than wild-type PRRSV, and the clinical outcome tends to be somewhat milder. This study reveals one of the pathogenic mechanisms that PRRSV plays during infection, and shows how this process can be blocked by genetic modification of the virus. SDSU in collaboration with KSU, UMN and UNL constructed a European-like Type 1 PRRSV full-length cDNA infectious clone (pSD 01-08) to further characterize this group of U.S. Type 1 PRRSV. This virus has low virulence in pigs and induces an early and robust neutralizing antibody response. SDSU, KSU, BARC and Boehringer Ingelheim Vetmedica Inc. (BIVI) scientists characterized the immune response to this cDNA infectious clone. In vivo studies showed similar observations form animals challenged with cloned viruses as those with the parental virus or vaccine. The full-length cDNA infectious clone derived from SD 01-08 P34 could be an ideal viral backbone for future recombinant PRRSV vaccine construction. SDSU and KSU are using infectious clones of European-like PRRSV to determine the usefulness of nsps as potential epidemiological tools. Phylogenetic analysis using ORF5 nucleotide sequences from 6 U.S. Type 1 isolates from geographically separated swine herds showed that 15/16 isolates formed a monomorphic clade of four subgroups. Comparative analysis with the genomic sequences of Lelystad virus (LV) and North American (VR-2332) revealed that each of the European-like viral genomes had higher nucleotide homology with the LV than the VR-2332. Nsps1², 2, 6 and 12 were identified as the most variable nsp regions. Nsp2 showed similar genetic heterogeneity among isolates as GP5, which has been used most frequently for PRRSV genetic diversity and evolution studies. At UMN numerous infectious clones have been developed for the study of mechanisms of PRRSV pathogenesis. In collaboration with BIVI, recombinant constructs were generated, characterized and forwarded to BIVI for in vivo testing. The UMN laboratory is also studying the effects of deletions made to nsp2 region of the replicase; 2 deletion mutants represent a 111 and a 200 aa deletion in nsp2. Additional constructs were generated in collaboration with Northern MI Univ. (NMI) to study the effects of N-glycosylation of GP5; 11 successfully produced intact virus. Several had a mutation at N-51, the N-glycan site critical for viral growth and are being analyzed further. 2.6. Pathogenesis (virus factors). In a Multistate initiative, UMN, Guelph, SDSU and NMI scientists are further characterizing GP2, 3 and 4 of 3 North American PRRSV strains. They both have successfully generated plasmids that express only the ORF in question, and have begun to study its ability to produce protein in rabbit reticulocyte lysates. Eukaryotic expression is underway at UMN; proteins produced were sent to SDSU for immunization of rabbits. UNL scientists verified that certain PRRSV nsps and two structural genes, GP5 and GP2, are heavily involved in virulence. Since GP5 does actually interact with the cell receptor, attention switched to GP2, now shown to be associated with virulence. PRRSV evades the immune system by means of a glycan-shielding mechanism; the deglycosylation of the PRRSV GP5 enhances PRRSV protective antibody response. These concepts, will be used to differentiate vaccinated pigs from infected pigs (DIVA). This is a major target for PRRSV vaccinology. A reverse genetics experimental system for PRRSV has been established at UNL and is being used by labs worldwide. Efforts at VA Tech have been aimed at developing novel antivirals against PRRSV by suppressing PRRSV replication using a novel class of antisense compounds, testing their ability to suppress PRRSV replication in cell culture. Of six tested, one (5UP1) was found to be highly effective at reducing PRRSV replication, generating up to 4.5log reduction in infectious viral titer, as confirmed by immunofluorescence assay. Production of PRRSV negative-sense RNA was reduced if 5UP1 was added to cells at up to 6hpi. These results indicate that 5UP1 has potential as an anti-PRRSV agent. 2.7. Pathogenesis (host factors). Understanding the effects of PRRSV infection on expression of host genes will provide targets for pharmacologic and genetic interventions to control infection. Serial analysis of gene expression (SAGE) has the ability to quantify expression of all genes within a population of cells. MARC and NADC scientists have initiated the development of SAGE libraries from PRRSV-infected PAMs. These libraries will be available to researchers; a feature of SAGE is that rare gene transcripts may be detected simply by sequencing more clones from the libraries. UIUC scientists asked whether cell-mediated immunity (CMI) against PRRSV virus is correlated with protection against reproductive failure in sows during clinical outbreaks of PRRS in 4 commercial breeding herds. A negative association between the intensity of the CMI response and the number of pigs born dead per litter was detected on 1 farm. Evidence that a strong CMI response was correlated with protection against clinical PRRS was detected in 3 of 4 farms. However, farms and sows within farms varied considerably in their immune responsiveness and in the degree to which they were protected clinically. A UIUC longitudinal prospective cohort study was designed to compare the reproductive performance of randomly assigned pigs inoculated with a farm strain of PRRSV to that of naturally infected pigs on an operational US swine farm. Cohort-1 consisted of pigs exposed to an attenuated farm strain of PRRSV and maintained in isolation. Cohort-2 were contact exposed by cohabitation with cohort-1 pigs. Cohort-3 were pigs co-mingled with pigs of cohorts 1 and 2 when all were moved into a grow-finish facility at 8 wks age (delayed contact exposure). Viral RNA could not be detected in tonsil of any pig pre-breeding. At 17 wks age (half way between entry into grower finisher and pre-breeding), the ELISPOT responses were 96 ± 48, 97 ± 54, and 83 ± 49 respectively. The total number of piglets weaned averaged 9.9 ± 2.8, 8.9 ± 3.6 and 9.7 ± 3.3 in cohorts 1, 2, and 3 respectively. 2.8. Host Genetic resistance With UNL collaborators BARC scientists performed immune gene and protein expression analyses to study the genetics of PRRS resistance/susceptibility. Principal component analyses ranked 200 PRRSV infected pigs from two genetic sources for phenotypic response to PRRSV. Low PRRSV burden pigs had high weight gain, low viremia, and few lung lesions. Resistant, low PRRS burden pigs had a quicker immune response to PRRSV, low expression of IFNg in cDNA and in serum, and low serum antibodies. High pre-infection serum levels of the innate cytokine, IL-8, were also significantly associated with PRRS virus-resistance, possibly implicating activation of the innate immune system as a step to prevent viral expansion. 2.9. Vaccines / Vaccination. Scientists at VA Tech developed a unique procedure to infect pigs with infectious cDNA clone-based RNA transcripts. Direct inoculation of RNA transcripts from PRRSV infectious clones into the lymph nodes and tonsils of live pigs produces active PRRSV infection in pigs. This unique system should help scientists to directly test the effects of genetic manipulations on virus pathogenesis and replication in vivo without having to propagate the virus in cell cultures. Development of Edible Vaccines against PRRSV has been pursued by VA Tech and ISU scientists. Preliminary work has been undertaken to create lines of transgenic maize suitable for use as an edible vaccine for PRRSV. A codon optimized ORF 5 sequence for protein production in maize was designed from cDNA of U.S. isolate ATCC VR 2385. Maize callus was transformed and recombinant GP5 identified in extracts by Western Blot. The long-term goal to help eliminate the PRRS virus from swine herds by developing transgenic plants expressing PRRSV immunogens. Vaccines that can differentiate infected from vaccinated animals (DIVA) are a new development in PRRS vaccine design. Using reverse genetics and a PRRSV infectious cDNA clone, KSU scientists constructed a viable PRRS virus that contained a 132 amino acid deletion in nsp2, in a region that is relatively conserved and immunogenic. The deleted nsp2 peptide was recognized by sera from pigs infected with wild-type, but not recombinant viruses. The results from this study can be directly applied to the development of tagged MLV vaccines that can 1) identify vaccinated pigs, 2) distinguish vaccinated from naturally infected pigs, and 3) detect the loss of immune protection following vaccination. ISU scientists attempted to apply a DIVA concept to PRRS serology using a vaccine-virus specific antigen. The effect of PCV2 infection on the efficacy of MLV PRRSV vaccination was assessed by VA Tech and ISU researchers. PCV2-infected, PRRSV-vaccinated, and PRRSV-challenged pigs had significantly more-severe macroscopic lung lesions pigs that were not exposed to PCV2 prior to PRRSV vaccination. Non-vaccinated PRRSV-infected pigs had a higher incidence of PRRSV antigen in lungs than did all other groups except the group infected with PCV2 prior to PRRSV. The non-vaccinated PRRSV-challenged group and the group challenged with PCV2 prior to PRRSV had lower average daily weight gain than control and vaccinated groups. This work suggests that PCV2 infection has an adverse effect on the development of protective immunity induced by PRRSV vaccine. Objective 3. Achieve biosecurity among herds by preventing viral spread between sites. 3.1 Virus Diversity. UMN scientists developed a High Throughput TaqMan RT-PCR for Simultaneous Detection of Type 1 and 2 PRRSV. The new format allows diagnosis of several hundred isolates per day and can also be run as a strain differentiation assay. With the emergence of new strains of PRRSV and demand for increased sensitivity, they are continually updating the test to meet the demands of veterinarians and producers. European-like Type 1 PRRSV, known as North American (NA) Type 1 PRRSV, appeared in US swine herds in 1999. Their diversity and evolution were studied by SDSU and KSU scientists by constructing phylogenetic trees using nsp2 and ORF5 of 20 US Type 1 isolates. All but two isolates possessed a unique 51 nt deletion in nsp2, suggesting a clonal origin. Viruses could be placed into distinct groups; however, the forces driving genetic separation are complex. Incongruity between the nsp2 and ORF5 trees, identified recombination in one isolate. ISU researchers assessed recombination events among field isolates and its impact on PRRSV molecular diagnostics and studied phenotypic and genetic difference between wild-type and attenuated PRRSV isolates. They assessed the effects of genetic variation on cross neutralization among PRRS viruses. They are examining the immunobiological significance of genetic variation among PRRS viruses to determine the correlation between genetic divergence and cross-neutralization (both in vitro and in vivo). The goal is to identify a single genetic marker on viral genes that predicts whether immune responses would be cross protective between viruses. The N-terminal of PRRSV nsp2 encodes a putative cysteine protease (CP) responsible for nsp2/3 cleavage and predicted to function as a co-factor with the nsp4 serine protease to process the other nsp cleavage products. Using a reverse genetic system, SDSU scientists deleted immunogenic epitopes on nsp2 of a Type 1 PRRSV. All epitope deletion mutants were viable except for a mutant containing a deleted ES2 epitope located in the CP domain. Antibody responses to the CP domain were evaluated and results suggest the PRRSV nsp2 CP not only plays a key role in virus replication but may also be involved in modulation of host immunity. 3.2. Immunity and/or Cross Protection Compared to other viruses that infect the respiratory system, PRRSV appears to induce only modest levels of IFNa. For this study by ARS NADC and PIADC scientists, pigs were injected with a non-replicating adenovirus vector expressing porcine IFNa (AdIFNa). Pigs were inoculated with AdIFNa, or adenovirus that does not express IFNa (Adnull), and challenged with PRRSV 1 day later. IFNa levels in all AdIFNa inoculated pigs were elevated the day of challenge (1 dpi) but were undetectable by 3dpi in unchallenged pigs. AdIFNa pigs had lower clinical responses, at 10dpi, delayed viremia and antibody response, and higher serum IFNa levels as a result of PRRSV infection, as compared to other pigs. Thus IFNa can have protective effects if present during the time of infection with PRRSV. 3.3. Transmission. Understanding the dynamics of PRRSV infection within the host and at the herd level is essential for designing monitoring protocols. UMN scientists developed a Monte Carlo model that simulates the introduction and transmission of PRRSV into a negative herd (boar studs), including the changes in infection, shedding, and serology status in each boar over time. They compared different testing protocols based on frequency of sampling, type of test, individual testing or pooling. PRRSV makes a small membrane protein, termed E protein, which when knocked-out in an infectious cDNA clone was replication-defective. Studies by Guelph researchers showed that E protein is an ion-channel protein, which is the key function for virus uncoating and subsequent multiplication in infected cells. Since the blocking of E protein function prevents PRRSV infection, a specific drug that binds to the E protein will be explored as a specific antiviral drug for PRRSV. A similar category of antiviral drugs have been developed for influenza virus in humans. 3.4. PRRS Risk Factors. Before large-scale PRRS eradication programs can begin, a clear understanding of how PRRSV is transmitted between farms and how to prevent virus entry to naïve farms is critical. UMN investigators found that the greater the distance between farms, the less genetic homology between the PRRSV isolates. In partnership with NPB, ISU researchers have started to characterize the physical and environmental components that affect the rate of pig-to-pig PRRSV transmission within swine herds. Objective 4. Improve diagnostic assays and create on-farm monitoring systems. Monitoring of boar studs for PRRS is critical to minimize the risk of transmission to sows via contaminated semen. However, current protocols for monitoring PRRSV in boar studs are diverse, sometimes very costly, and their effectiveness has not been examined quantitatively. Studies conducted by SDSU and UMN evaluated 29 naïve boars inoculated with PRRSV. Results indicate that PCR using blood swabs or serum undiluted or at 1:3 or 1:5 dilutions have similar sensitivities and are useful in detecting early infection in adult boars. This study supports field observations suggesting that an intensive monitoring protocol (testing a large number of boars by PCR and at a high frequency) needs to be in place in order to detect a PRRSV introduction as early as possible. ISU researchers investigated the use of oral fluids as a diagnostic specimen. Tests have been completed indicating the utility of ropes for stimulation of saliva production and viral load monitoring. Investigators at UMO continue to work on nanosensor technology for a fast, penside test for PRRSV. Two methods being pursued include an optical based nanobiosensor and capillary electrophoresis on a microchip. Developing sensitive biosensors will help to quickly detect PRRSV antigen in clinical samples. The cysteine protease domain (CP) of PRRSV nsp2 was evaluated by SDSU scientists as a potential new antigen for sensitive, specific and differential diagnostic ELISA tests since antibody to CP can detected as early as 14 dpi. The CP-based ELISA was determined with 93% agreement with the IDEXX ELISA. To differentiate Type 1 and 2 PRRSV, an epitope-based ELISA using the conserved ES2 epitope of Type 1 PRRSV was developed and showed good specificity (94.4%) and sensitivity (94.5%) for Type 1 PRRSV. Objective 5. Develop and test PRRSV virus eradication protocols under various ecological settings. NCSU scientists experimentally determined that stable flies do internalize infectious PRRSV by consuming viremic blood meals, however, they were unable to infect naïve pigs while obtaining their next meal by natural intra-dermal feeding. PRRSV was detectable from dissected fly gut following blood meals spiked with PRRSV and was detectable by qcRTPCR up to 96 hr post-feeding (hpf); with a linear decay from 12 to 96 hpf. However, 3 attempts failed to transmit PRRSV infection to naïve pigs; with 30 to 60 bite sites observed per pig. Similar methods were used to estimate that the non-biting house fly only consumed 1 - 5 PRRS virions/meal. Aerosol transmission studies of PRRSV by UMN and ISU scientists demonstrated a significant association between PRRSV isolate pathogenicity and shedding/transmission via aerosols. Isolate pathogenicity did not influence the concentration of PRRSV in aerosols. Pig age and co-infection with Mycoplasma hyopneumoniae (MHYO) did not impact aerosol shedding. Utilizing a model of a swine production region to evaluate routes of transmission of PRRSV, UMN and ISU scientists evaluated seasonal risk factors and tested the ability of various biosecurity protocols to reduce the risk of PRRSV entry into naïve populations. Preliminary observations suggest that contaminated fomites, insects and aerosols are important risk for spread of PRRSV between farms and that farms equipped with an air filtration system may reduce risk. Objective 6. Develop educational outreach tools for disseminating information through established outreach and extension networks to producers, veterinarians, educators, and researchers. NC229 leaders serve as CoChairs for the International PRRS Symposium in Chicago IL. The 2006 symposium had 190 registrants and 70 posters. An international Scientific Advisory Board has developed the 2007 program with 77 posters, 153 registrants to date including attendees from the U.S., Mexico, Canada, Europe and Asia. The full 2007 program and 2006 abstract book are available (http://www.prrssymposium.org/). The North American PRRS Eradication Task Force (NAPPRSETF) was created by an NCSU scientist and is a working group of the American Association of Swine Veterinarians (AASV),. The NAPPRSETFs mission is to facilitate communication of PRRS control research results and needs between producers, veterinarians, and researchers with the ultimate goal of building producer confidence and demand for PRRSV Eradication. Each NAPPRSETF Member is tasked to form Producer Veterinary Area Task Forces to identify problems specific to their areas and communicate the need for, or propose, research projects designed to address their regions specific problems. Objective 7. Create an information network to ensure rapid and efficient communication of PRRSV research. Four communication initiatives were continued: the PRRS CAP 1 website (prrs.org) was expanded; quarterly newsletter provides information about NC-229/PRRS-CAP 1 activities via email; past issues are posted at the website; conference calls among CAP1 researchers expanded plans for CAP2 renewal; and many NC229 members served on the CAP2 writing team. ISU and KSU have participated in the movement toward an open information network through our involvement in the development of the ARM-7 database using the "BigPig" data as the original dataset to expand the database for animal disease data.

Publications

Please see attached publications report

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Impact Statements

Work in progress through NC229 researchers will provide important information on transmission by arthropods, aerosols, and the duration of persistent infection. This information is critical to the development of strategies for protecting herds and eliminating infections.
Using the reverse genetics system, mutant PRRS viruses are now being constructed. This infectious cDNA clone approach has helped to identify virulence factors, compare properties of different viral strains, and assess how the virus replicates. These clones have enabled NC229 researchers to engineer new vaccine candidates, by stably mutating the virus to provide more effective immunity after vaccination.
NC229 researchers have developed a unique procedure to infect pigs with these infectious cDNA clone-based RNA transcripts, thus bypassing the traditional cell cultures. This will have major implications for PRRSV research and will facilitate PRRSV structural and functional studies and vaccine developments in the future.
Studies in progress on PRRSV (Type 1 and Type 2) continue to demonstrate that genetic diversity affects cross-neutralization of viruses, susceptibility of PRRSV to cell-mediated immunity, and our ability to stop circulation of virus in swine populations. Reverse genetics studies using a Type 1 PRRSV infectious clone suggest the PRRSV nsp2 cysteine protease not only plays a key role in virus replication but may also be involved in the modulation of host immunity. Infectious clones from Type I PRRSV may provide an important backbone for type 2 PRRSV vaccine constructs.
Monoclonal antibodies (mAbs) against the PRRSV non-structural proteins (nsps) have been produced and will provide basic key reagents for study of the fundamental biology of the PRRSV nsps. Many PRRSV proteins have been expressed using recombinant techniques. These recombinant proteins and mAbs produced are good candidates for future development of diagnostic assays.
Dendritic cells (DCs) play a role in anti-viral immunity by stimulating early innate immunity. The adaptive response to PRRSV is ineffective, suggesting an aberrant activation of DCs. Results with 2 different DCs indicated that both responded to PRRSV with an induction of IFNb mRNA, but the magnitude and duration of the response differed between cell types. Without IFNa DC response to PRRSV was limited to IFNb transcription, which may be inadequate in triggering the adaptive immune response.
Genes encoding the innate cytokine interleukin-8 (IL8) and the anti-viral protein interferon-gamma (IFNG) may help determine whether pigs will effectively resist PRRS virus infection based on a study of 400 pigs by NC229 scientists. These data outline targets for future studies to determine if specific immune gene alleles are associated with PRRS virus resistance or susceptibility.
NC-229 researchers affirmed experimentally that stable flies do internalize infectious PRRSV by consuming viremic blood meals, however, they were unable to infect naïve pigs while obtaining their next meal by natural intra-dermal feeding. PRRSv was detectable by qcRTPCR up to 96 hr post-feeding of PRRSV-spiked heparinized blood, a linear decay in pooled gut content PRRSV was observed. However, 3 attempts to transmit PRRSV infection to naïve pigs failed; pigs remained negative for 35 days.
Efficient, cost-effective diagnostics are paramount for producer support of elimination and control projects. NC-229 researchers are investigating novel approaches for diagnostic assays that detect both viral structural and nonstructural proteins. These new assays will enhance the ability to detect persistently infected pigs, one of the main impediments to successful eradication of this disease.
Monitoring of boar studs for PRRS is critical to minimize the risk of transmission to sows via contaminated semen. Studies conducted by NC-229 researchers affirmed that protocols based on PCR of sera will detect a PRRSV introduction earlier than PCR on semen. This study supports field observations suggesting that an intensive monitoring protocol (testing a large number of boars by PCR and at a high frequency) needs to be in place in order to detect a PRRSV introduction as early as possible.
Studies conducted by evaluate surveillance methods for PRRSV detection in boars. Sensitivity estimates for pooled serum and blood swabs ranged from 71-100%. Sensitivity for raw and extended semen samples was low (14 to 29%). Results indicate that using blood swabs or serum undiluted or at 1:3 or 1:5 dilutions have similar sensitivities and are useful in detecting early infection in adult boars.
The North American PRRS Eradication Task Force (NAPPRSETF), organized by an NC-229 researcher, is an AASV working group whose mission is to facilitate communication of PRRS control research results and needs between producers, veterinarians, and researchers with the ultimate goal of building producer confidence and demand for PRRSV Eradication. Members are committed to developing or validating PRRS control production management strategies or tools for swine producers.
The PRRSV database has expanded research opportunities in epidemiology, diagnostics, and genetics. Its 5400 PRRSV isolate based sequences provide an essential tool for veterinarians and epidemiologists. It enables practitioners access to real time data from PRRSV studies.
NC-229 sponsors an annual International PRRS Symposium for scientists worldwide. This symposium is a means to openly present and discuss PRRS research progress with the participation and input of non-NC229 researchers. The quarterly PRRS CAP supported newsletter provides an up to date PRRS progress report to researchers, practitioners and stakeholders.

Date of Annual Report: 07/15/2008

Report Information

Annual Meeting Dates: 05/21/2008 - 05/22/2008
Period the Report Covers: 10/01/2007 - 09/01/2008

Participants

NC229 Representatives:

;Lunney, Joan K. - USDA, ARS, BARC, APDL, (joan.lunney@ars.usda.gov);
Rowland, Raymond R.R. - Kansas State University (KSU) (browland@vet.ksu.edu);
Zimmerman, Jeff - Iowa State University (ISU) (jjzimm@iastate.edu);
Schommer, Susan - University of Missouri (UMO) (schommers@missouri.edu);
Nelson, Eric A. - South Dakota State University (SDSU) (eric_nelson@sdstate.edu);
Zuckermann, Federico A. - University of Illinois at Urbana-Champaign (UIUC) (fazaaa@uiuc.edu);
Faaberg, Kay - NADC (kay.faaberg@ars.usda.gov);
Murtaugh, Michael P. - University of Minnesota (UMN) (murta001@umn.edu);
Meng, X.J. - Virginia Polytechnic Institute and State University (VA Tech) (xjmeng@vt.edu);
McCaw, Monte B. - North Carolina State University (NCSU) (monte_mccaw@ncsu.edu);
Osorio, Fernando A. - University of Nebraska-Lincoln (UNL) (fosorio@unl.edu);
Pogranichniy, Roman - Purdue (rmp@purdue.edu);
Guillermo R. Risatti - University of Connecticut (UCONN) (guillermo.risatti@uconn.edu);
Benfield, David - Ohio State University (benfield.2@osu.edu);
Johnson, Peter - USDA CSREES (pjohnson@reeusda.gov)

Other NC229 Scientists:

;Wysocki, Michal - BARC;
Christopher-Hennings, Jane - SDSU;
Johnson, Rodger - Univ NE;
Smith, Doug, Ho, Sam - Univ. MI;
Munoz-Zanzi, C., Rovira, Albert - UMN;
Steibel, JP, Ernst, Cathy - MSU;
Wyatt, Carol - K-State;
Hesse, Dick - K-State;
Sang, Yongming - K-State;
Chang, KC - K-State;
Blecha, Frank - K-State;
Calvert, Jay - Pfizer Animal Health;
Fang, Ying - SDSU;
Cafruny, William - USD;
Richt, Juergen - ARS;
Roof, Mike - BIV;
Yoo, Dongwan - UIUC;
Erdman, Matthew - ISU;
Halbur, Patrick - ISU;
Harris, D.L. (Hank) - ISU;
Karriker, Locke - ISU;
Opriessnig, Tanja - ISU;
Platt, Kenneth - ISU;
Roth, JA - ISU;
Yoon, Kyoung-Jin - ISU;
Wang, Xiuqing - SDSU;
Kerhli, Marcus Jr. - NADC;
Lager, Kelly - NADC;
Brockmeier, Susan - NADC;
Miller, Laura - NADC;
Loving, Crystal - NADC;
Neill, John - NADC;
John Butler - University of Iowa;
Dee, Scott - UMN;
Joo, Han Soo - UMN;
Molitor, Tom - UMN;
Morrison, Robert - UMN;
Deen, John - UMN;
Munoz-Zanzi, Claudia - UMN;
Rossow, Kurt - UMN;
Rovira, Albert - UMN;
Rutherford, Mark - UMN;
Davies, Peter - UMN;
Zhang, Chenming - VA Tech;
LeRoith, Tanya - VA Tech;
Boyle, Stephen M. - V A Tech;
Zhang, Yanjin - University of Maryland at College Park;
Pattnaik, Asit - UNL;
Doster, Allan - UNL;
Johnson, Rodger - UNL;
Garmendia, Antonio - UConn;

Brief Summary of Minutes

NC229 Meeting Beltsville, MD Wednesday-Thursday, 5/21/2008 05/22/2008.

1. Attendees:

Joan Lunney, USDA-BARC, Chair

X.J. Meng, Virginia Polytechnic Institute and State University, Secretary

Bob Rowland, Kansas State University, Immediate Past Chair

David Benfield, Ohio State University, NC-229 Administrative Advisor

Ralph Tripp, University of Georgia

Mark Tompkins, University of Georgia

Ying Fang, South Dakota State University

Alberto Rovira, University of Minnesota

Susan Schommer, University of Missouri

Roman Pogranichniy, Purdue University

Mike Murtaugh, University of Minnesota

Frederick Leung, University of Hong Kong

Lisa Becton, National Pork Board

Fernando Osorio, University of Nebreska-Lincoln

Guillermo Risatti, University of Connecticut

Antonia Garmendia, University of Connecticut

Yanjin Zhang, University of Maryland

Xiao-ping Zhu, University of Maryland

Jeff Zimmerman, Iowa State University - (via telephone)

Richard Hesse, Kansas State University - (via telephone)

Jane Christopher-Hennings, South Dakota State University - (via telephone)

Eric A. Nelson, South Dakota State University - (via telephone)

2. Meeting opened by Chair, Joan Lunney; Welcomed everyone and introduction of participants around the table.

3. NPB updates: Lisa Becton (replacing Pam Zaabel at the NPB): PCV2 RFP out already, there will be a PRRSV RFP in late June. NPB may match the funds for "pig high fever disease" from USDA.

4. Comments on Pig High Fever Disease:

Bob Rowland: BSL-3 facility available at KSU to put PHFD virus into pigs. Can we build our own infectious clone of the PHFD virus and work in labs here in the United States?

X.J. Meng: The problem will be how can we import the samples to the United States?

Mike Murtaugh: perform the research in China on site, will the USDA be receptive to allow the research on pig high fever disease to be done in China?

Fred Leung: Maybe easier to get samples from Hong Kong. Suggest direct dialogue with China at the USDA level.

Joan Lunney: NC projects, sharing resources and ideas and collaborative efforts.

5. Update on PRRS CAP-2: Bob Rowland, PRRS CAP-2 RFA is out, and the deadline is August 20, 2008. Coordinating with Lisa Becton from NPB; inform Bob Rowland if you plan to submit a proposal for CAP-2.

6. Updates on PRRSV host genetics consortium. Joan Lunney, the Consortium database that stores extensive phenotypic and genotypic information will be available for PRRSV CAP-2 initiatives.

7. Updates on U.S. Veterinary Immune Reagents Network. Joan Lunney, adding additional reagents to new species in including swine. Vetimm.org website for Swine Toolkit. Priority for new reagent development will be reagents that are not commercially available.

8. Updates on 2008 Intl PRRS Symposium. X.J. Meng, the Symposium website is up and running. Four keynote speakers are invited and all confirmed. The website is now open for registration and Abstract submission.

9. New member Lecture. Ralph Tripp University of Georgia, Center for Disease Intervention, Athens, GA.

Summarized plans for use of the Animal Health Research Center BSL3 facility to study host dynamics of infection; plans for translational disease intervention strategies; pathogen biosensing; viral interfering RNAi; Bench to bedside/barnside. There is a GMP vaccine facility at BSL1/2/3 as needed; current work on swine influenza, PRRS, PCV2; West Nile; and human viruses: RSV, SARS, flu, others.

10. NC-229 Annual Reports: Each station representative presented their annual report (10 min each). Written annual reports are due to XJ Meng.

11. Dave Benfield: Farm Bill CREATE-21 update, the new Farm Bill established the Natl Inst for Food and Agriculture (NIFA). NIFA will replace CSREES. A distinguished scientist, nominated by the President and confirmed by the Senate, will be appointed for a 6-year term as the director of NIFA.

12. Discussion on NC-229 renewal and re-write: break out into groups, discussed and identified important work and objectives to be included in the NC-229 renewal. Identified leaders for each objective, and assembled writing teams for the NC-229 renewal. The new NC229 title will be "Detection and control of PRRSV and emerging viral diseases of swine" with Objectives: 1) Elucidating the mechanisms of host-pathogen(s) interactions; 2) Understanding ecology and epidemiology of PRRSV and emerging viral diseases of swine; and 3) Develop effective and efficient approaches for detection, prevention and control of PRRSV and emerging viral diseases of swine.

13. Peter Johnson - CSREES, new grant RFAs will come from AFRI, not NRI with possibly more money; Congratulations on PRRS CAP-2; 6 weeks until final paperwork completed.

Accomplishments

Objective 1. Implement a virtual laboratory infrastructure through the development and open distribution of resources, materials, protocols, and data among participating researchers. 1.1. In collaboration with J Lunney, USDA-BARC, R Rowland at Kansas State University worked to establish a PRRS Host Genetics Consortium. Rowland distributed samples from the Big Pig Project to other investigators. 1.2. PRRSV Database (prrsvdb.org) Faaberg, UMN, now NADC, initiative; Continued development of the database, now including SDSU (Christopher-Hennings), ISU (Yoon), and University of Hong Kong (Leung) sequences. Over 9500 unique sequences have been deposited in the database to date. 1.3. Miller, Neill NADC initiative: 5 SAGE libraries with tag counts of in vitro PRRSV-infected porcine alveolar macrophages at 0, 6, 12, 16, and 24 h p.i. have been submitted to GenBank GEO (accession number GSE10346), available July 2008. Porcine macrophage SAGE modified Identitag database has been created with Dr Greg Harhay, USDA, ARS, USMARC, 2007. 1.4. Miller NADC initiative: MARC-145 cells provided to: Dr. Dick Hesse, Diagnostic Medicine/Pathobiology, College of Veterinary Medicine, Kansas State University; Dr. Guolong (Glenn) Zhang, Department of Animal Science, Oklahoma State University; Dr. Fernando Osario, Department of Veterinary and Biomedical Sciences, University of Nebraska-Lincoln; Dr William Laegreid, Department of Veterinary Pathobiology, University of Illinois. 1.5. The Lunney Lab at USDA-BARC served as reference immune gene expression lab for multiple PRRSV studies. J Lunney participated in coordinating testing and analyses of Big Pig samples of virus persistence and SLA effects on anti-viral responses, and obtained NPB funding for PRRS Host Genetics Consortium (PHGC): A proposal to develop a consortium to study the role of host genetics and resistance to PRRSV. J Lunney at USDA-BARC used PHGC funds to 1) establish barcoding software for organized sample collection, storage and cataloguing; and 2) with Iowa State Univ. scientists develop relational database for recording PRRS phenotypic and genotypic records. 1.6. Dee at UMN, two mobile nurseries for transmission and biosecurity research acquired with PRRS CAP and matching funds for use in controlled studies of intervention strategies that prevent PRRSV infection of nurseries have been used by UMN and ISU for studying the value of filtration in preventing aerial virus and bacterial transmission in herd-dense regions. 1.7. 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. Primary participants include Fang, Christopher-Hennings, Nelson (SDSU), Rowland (KSU), Murtaugh (UMN), Faaberg (UMN & NADC), and Yoo (UIL). Objective 2. Achieve biosecurity within herds by preventing the spread of virus within a herd and facilitating its elimination from endemically infected herds. Research is focused on functional genomics of PRRSV resistance, mechanisms of protective immunity for PRRSV prevention, evaluation of immunomodulators to stimulate/enhance antiviral immunity and agents that reduce virus replication in the pig. 2.1. In collaboration with Jay Calvert from Pfizer Inc, Kansas State University researchers identified a deletion in nsp2 of the P129 infectious clone that attenuated the virus and expressed a GFP tag. 2.2 Iowa State University researchers focus on interpreting PRRS sequence information using BEAST (Bayesian Evolutionary Analysis Sampling Trees), provided new estimates of R0 for PRRSV in different operations, conducted survival analysis of PRRSV transmission within herds, and overviewed PRRSV routes of transmission and patterns of circulation. 2.3 NADC researchers conducted nsp2 deletion mutant studies. Select VR-2332 nsp2 deletion mutants were chosen for an in vivo study. Young swine (4 pigs/group; 5 control swine) were inoculated intramuscularly with one of 4 nsp2 deletion mutants (?727-813, ?543-726, ?324-523, ?324-726) and full-length recombinant virus (VR-V7). Serum samples were collected on various days post-inoculation. Samples were analyzed by HerdChek ELISA, RT-PCR, interferon g ELISA, and nucleotide sequence analysis. Lymph node weight compared to body weight was recorded for each animal and used as a clinical measurement of viral pathogenesis. Results showed that all deletion mutants grew less robustly than full-length recombinant virus, yet all but the large deletion virus (?324-726) recovered to parental virus levels by study end. Swine receiving mutants ?727-813 and ?324-726 had a significant decrease in lymph node involvement compared to rVR-V7. Three of the 4 deletion mutants had significant reductions in serum IFN-g levels; only the D543-726 nsp2 mutant mimicked VR-V7 in inducing a host serum IFN-g response. Sequencing results suggested that all nsp2 deletions were stable. The data suggest that selected nsp2 deletion mutants may indicate regions responsible for inducing IFN-g and domains that cause lymph node enlargement. This study must be repeated with additional swine to verify the results. In addition, MN184 and VR-2332 viruses were inoculated into four-week old swine and examined in parallel for viral growth kinetics and various host responses, including changes in gene expression and antigen recall. The results of this study have not been completed. 2.4 Researchers at Purdue University studied viral receptors for PRRSV and blocking mRNA of these receptors with RNAi in vitro and blocking PRRSV virus replication in vitro as well. RNA interference (RNAi) has been used increasingly for reverse genetics in invertebrate and mammalian cells. The results showed the potential of anti-sense antiviral oligos with pegylated liposome formulation known as stealth for control of PRRSV infection in vitro. 2.5 Identifying host gene expression changes that are involved in regulating responses to PRRSV infection and vaccination. With NCSU collaborators BARC scientists tested the effect of PRRSV infection or vaccination on pigs using RNA prepared from tracheobronchial lymph nodes (TBLN), the cranial and distal part of the lung, and tonsils. Pigs were either infected with Minnesota strain (MNW2B) of the PRRSV or vaccinated with ATP or non-treated controls and samples collected between 3 and 6 days post treatment do that the early innate immune response could be evaluated. RNAs were prepared and hybridization to the new swine long oligo array [pigoligoarray.org] assessed. Analyses are underway at BARC with statistical collaboration from MSU. Tests of heterologous PRRS vaccination and challenge are also underway at NCSU and samples will be analyzed by both BARC and NCSU labs. 2.6 Researchers at the University of Connecticut completed a project on Mapping PRRSV Genetic Determinants of Macrophage Host-Range and Immune Modulation to identify PRRSV genetic determinants associated with macrophage host range and with modulation of pro- and anti-inflammatory cytokine expression. Mapping of these pathobiologically relevant PRRSV genes/determinants will permit rational design of differential vaccines of unprecedented safety, efficacy, and utility. Eight chimeras (Ch1-Ch8) represented the entire SP genome in the genetic background of NVSL 97-7895. Chimeric viruses Ch2v and Ch5v, containing regions encoding for SP nsp2 protein and SP ORF1B, respectively, were largely defective for replication relative to NVSL 97-7895, showing limited growth titers in both MARC-145 cells and swine pulmonary alveolar macrophage (PAMs) primary cell cultures. Five chimeric viruses (Ch1v, Ch4v, Ch6v-Ch8v) were able to replicate but demonstrated a small-plaque phenotype relative to parental virus in MARC-145 cells, consistent with a restriction in virus attachment and/or spread. Only Ch3v, a virus containing genes for nsp3 to nsp9 proteins from SP, showed plaque phenotypes resembling parental viruses. Notably, while other chimeric viruses induced a pattern of PAM gene expression (among 58 genes) similar to that of virulent NVSL 97-7895, Ch3v induced a pattern more similar to that induced by attenuated SP. These data suggest that, in the region encompassing genes for nsp3 to nsp9, PRRSV contains genetic determinants affecting host gene expression, and they indicate a potential role for these proteins in regulating macrophage inflammatory response during PRRSV infection. 2.7. The project Assessment of Virulence of PRRSV Isolates Based Both on their Sensitivity to IFNb and Ability to Induce Type I IFN Responses was conducted by scientists at the University of Connecticut. The study is aimed at determining mechanisms as to how PRRSV subverts the hosts innate immune responses and establishes long lasting infections. In order to accomplish this goal we have initiated efforts to phenotype PRRSV isolates in terms of their sensitivity to IFNb and ability to induce IFNb both in vitro and in vivo. Virus isolates with opposing phenotypes will be tested comparatively in cell culture in attempts to identify potential steps in the IFN type I pathway that may be blocked. Differences in pathogenicity will be ultimately investigated in swine. Initial tests conducted in our laboratory indicate that there are significant differences in sensitivity to IFNb among different PRRSV isolates. The results also indicated that there are differences among PRRSV isolates in their individual capacities to induce IFNb in vitro. Both characteristics will be used to formulate their phenotype for further studies. 2.8. Co-infections may affect the response of pigs to PRRSV infection, including immune response and transmission characteristics. Porcine circovirus type 2 (PCV2) may modulate the response to PRRSV since it disrupts lymph node structure and causes lymphodepletion. To better understand PCV2 in the U.S. swine herd, researchers at the University of Minnesota developed ELISA and PCR tests and characterized these tests to specifically differentiate and quantitatively assess the status of PCV-1 and PCV-2 infection in serum samples collected from finishing pigs in 2006 as part of the NAHMS swine health survey. A quantitative Tetra-Nucleotide Discrimination (TND) Assay, based on detection of a highly conserved sequence that specifically varies between PCV1 and PCV2, was developed to measure simultaneously the amount of PCV-1 and PCV-2 in a test serum sample. The test was validated on serial dilutions of known positive control samples, by comparison to other tests that independently measured PCV1 or PCV2, by analysis of spiked test samples, and by comparison to virus isolation. The PCR assay was applied to a random subset of 800 samples in the NAHMS survey. Seventy nine percent of serum samples were positive or suspect positive for PCV2 virus, whereas only 2.5% were positive for PCV1 virus. For the ELISA, amino-terminal deleted capsid proteins expressed in bacteria were sensitive and specific antigens. To establish baseline serological data for exposure to PCV, more than 2,600 serum samples from 97 farms throughout the U.S. were evaluated for serological response to PCV1 and PCV2. Eighty percent of samples were positive for PCV2, 4% were suspect positive, and 16% were negative. With five exceptions, more than 50% of animals sampled on farms were seropositive; on only one farm were all samples test-negative. PCV1 antibodies were detected in 23% of serum samples and an additional 9% were suspect positives. Only 7 farms had more than 50% of samples test-positive. The findings to date indicate that PCV1 is only rarely present in finishing pigs. PCV2 is widespread in individual pigs, indicative of a viremic, active infection. Interestingly, in 81% of cases the serological and virological status of a sample were concordant for PCV2, either both positive and negative, and were discordant 19% of the time. While not a specific finding of this study, the data also suggest that the practice of serum inoculation might result in extensive dissemination of PCV2 within a herd. 2.9. Projects Mapping Genes of PRRSV involved in virulence, and Attenuated vaccines X PRRSV with marker capability were conducted by scientists at the University Nebreska The major conclusions on these two projects include the following new information on PRRSV biology: (a).Virulence of PRRSV. We know now that certain PRRSV non-structural proteins and two structural genes (ORF 5 and ORF2) are involved in virulence. This invites considering our novel information about GP2 in light of the previous reports stating that the major envelope of PRRSV (GP5) would not be the gGP that interacts with the cell receptor; this may then attract the attention of researchers to GP2 that we know now, through our research, is importantly associated to virulence. Along this line, fine mapping of virulence will likely involve site directed mutagenesis and reverse genetics, using a pair of homologous wt /attenuated strains (JA142/ATP strain). The attenuated strain in this pair is the most commonly used vaccine in the US market. (b).Immunopathogenesis and vaccinology of PRRSV. We have found that PRRSV evades the immune system by means of a glycan-shielding mechanism and that the de-glycosylation of the PRRSV GP5 enhances significantly the ability of the PRRSV strain to induce protective antibody response. These two concepts, added to the notion that PRRSV may have several serologic markers (immunodominant B-cell epitopes) that could be used for DIVA differentiation, make together a significant contribution to PRRSV vaccinology. A paper in press describes proof of the concept of slected epitopes serving as markers in a DIVA vaccine. 2.10 Research tools to study PRRSV pathogenesis and immunity issues. Scientists at the University of Nebreska have been able to successfully establish a reverse genetics experimental system for PRRSV that serves as national and international reference. Several laboratories worldwide have requested and are successfully using our IC system. It is now known that the infectious clone is fully functional, being the PRRSV infectious clone system with best recorded evidence of stability and reproducible pathogenesis in vivo. 2.11 Examining PRRS strain diversity by researchers form Univ of NE. By means of cross neutralization of PRRSV strains, using strain-specific neutralization sera, it may be possible to set the basis to cluster or subgroup the wide universe of strains of PRRSV in subtypes. These subtypes may have a direct correlation with cross protection. If true, this concept may help to define the minimal number of valences or specificities that should be present in a PRRSV vaccine for this product to be broadly protective. 2.12 Markers for vaccines and diagnostic tests: One of the key steps 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, researchers at SDSU (Fang, Christopher-Hennings, Nelson) in collaboration with J. Lunney (USDA-BARC) 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, but induced a higher level of neutralizing antibody response compared to parental virus. To compliment the 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 a high level of antibody response to GFP by 14 days post-infection. This recombinant marker virus, in conjunction with the diagnostic tests, enables serological differentiation of vaccinated animals from wild-type virus infected animals. This rationally designed marker virus will provide a basis for further development of PRRSV marker vaccines to assist with the control of PRRSV. 2.13. Purification of the major envelop protein GP5 of porcine reproductive and respiratory syndrome virus (PRRSV) from native virions. This project lead by Dr. C. Zhang at Virignia Tech in collaboration with XJ Meng is to develop an improved vaccine against PRRSV. Towards this end, they have developed a process for purification of GP5 protein from native virions. Cation exchange chromatography (CEX) was used for partial fractionation of GP5, although the N protein was a major impurity in CEX elution fractions. Pure GP5 protein was eluted from the HIC resin in the second hydrophobic interaction chromatography (HIC) elution stage by Triton X-100 displacement; however the protein is present as a homodimeric/tetrameric aggregate. Currently we are studying the Gp5 and N interaction under native conditions. 2.14. Peptide-conjugated morpholino oligomers inhibit porcine reproductive and respiratory syndrome virus replication. This project is lead by Dr. Y.J. Zhang of University of Maryland, in collaboration with Virginia Tech (Meng) and Iowa State University (Halbur, Opriessnig). The inhibition of PRRSV replication by peptide-conjugated antisense phosphorodiamidate morpholino oligomers (PPMO) was characterized. Four PPMOs were found to be highly effective at inhibiting PRRSV replication in cell culture in a dose-dependant and sequence-specific manner as evidenced by reduction in virus titer, viral RNA and virus-induced CPE. The inhibitory effect of these PPMO in pigs are being tested. Objective 3. Achieve biosecurity among herds by preventing viral spread between sites. 3.1. Iowa State University scientists conducted studies to estimate the k rate (inactivation rate) of PRRSV by ultraviolet. 3.2. Researchers at Kansas State University (F Blecha, and R Rowland) are studying the role of Tol-like receptor (TLR) activation in the regulation of virus replication. Activation of TLR 3 reduced replication. KC Chang (KSU) in collaboration with Dongwan Yoo (UIIC) studied factors that influence stimulation with LPS or phorbol ester decreased CD163 expression, while treatment with IL-10 increased expression. Rowland in collaboration with Ying Fang (SDSU) showed that viruses of European origin that are found in the U.S. are a diverse group, but originated from a single virus introduction. These data demonstrate that European viruses are well-established in U.S. swine herds and are present at a relatively higher frequency than previously reported. Carol Wyatt (KSU) identified a T cell epitope in GP5 that appears to be relatively conserved among isolates. Long-term infected pigs consistently responded to this epitope. Studies conducted by a collaboration between Lunney (BARC) and Rowland were directed at identify cytokine alterations that are important for viral persistence and growth performance during infection. 3.3. Analyzed respiratory immune responses of pigs with persistent PRRSV infections. The "Big Pig" project, a multi-disciplinary, multi-institutional (Kansas State, Iowa State, SD State, BARC; and IDEXX Laboratories) project, monitored PRRSV infection responses of 165 pigs for as long as 203 days. BARC scientists compared RNA prepared from tracheobronchial lymph nodes (TBLN). There was no pattern of TBLN cytokine gene expression that was associated, or might help predict, which pigs will clear virus and distinguish them from those that remain persistently infected. However, tests of sera collected early in infection (14-84 dpi) indicated the pigs that cleared their infection had higher levels of serum cytokine interleukin-8 (IL-8) at 14 dpi and faster interferon-gamma (IFNg) responses 28 dpi versus 42 dpi for persistently infected pigs. This data indicates that non-persistently infected pigs may stimulate viral regulatory pathways more effectively and thus stimulate protective anti-PRRSV immunity. 3.4. Assess boar mucosal reproductive tissue for immune genes associated with responses to PRRSV infections. Tests are planned to assess local reproductive tissue for immune genes that influence virus shedding and persistence in breeding boars by SDSU, U MN, and BARC researchers. Cytokines act as immunomodulators that can influence viral infectivity but there is no information on cytokine levels in boar seminal plasma. Therefore, we measured cytokine levels in PRRSV-infected and non-infected boars by ELISA. Both infected and non-infected boars had high levels of IL-12, though the significance of this in boar seminal plasma is unknown. IL-12 promotes inflammatory and cytotoxic T lymphocyte responses which may stimulate the uterine inflammatory response in sows after insemination and may protect against transmission of viral infection in utero. 3.5. Using a production region model, a 2-year study was initiated by scientists at the University of Minnesota in November 2007 to evaluate aerosol transmission and biosecurity. This model involves a 300 head grow-finish source population experimentally inoculated with PRRSV and Mycoplasma hyopneumoniae. As of May 2008, 6 replicates have been completed. Airborne transmission of PRRSV from the source population to a population of pigs housed in the non-filtered facility has occurred in 3/6 replicates, while airborne spread of Mycoplasma has been observed in 4/6. No transport or transmission of either agent to pig populations housed in the filtered facility has been observed. Through the use of an on-site weather station, real-time meteorological data are been collected which will allow documentation of environmental conditions present during periods when virus was present and not present in air. In addition, an evaluation of alternative aerosol biosecurity strategies is underway for inclusion in year 2 of the study. 3.6. European-like Type 1 PRRSV isolates appeared in U.S. swine herds in 1999. Their diversity and evolution were studied by researchers from South Dakota State University and Kansas State University (Fang, Christopher-Hennings, Nelson, Rowland) over a five-year period by constructing phylogenetic trees using nsp2 and ORF5 sequences of 20 NA Type 1 isolates. All but two of the isolates possessed the same 51-nt deletion in nsp2, suggesting a clonal origin. Parsimony and distance analysis showed that viruses could be placed into two distinct sub-clades, which were similar for both nsp2 and ORF5. An incongruity between the two trees identified one isolate, 04-41, as the product of recombination. Recombination analysis using SimPlot identified a break point located downstream of the nsp2/3 junction. Results from this study suggest that Type 1 PRRSV in the U.S. is well-established and rapidly evolving. However, the forces driving genetic diversity and separation are complex and remain to be elucidated. 3.7. Development of a unique in vivo transfection strategy using PRRSV infectious cDNA clone to study PRRSV evolution. Scientists at Virginia Tech showed that direct in vivo transfection with RNA transcripts from an infectious clone initiated PRRSV infection in pigs. Quasispecies evolution of PRRSV during acute infection resulting from an infectious cDNA clone is examined. Quasispecies populations were identified in each pig as early as 7 dpi. The sequences from the 4 pigs at dpi 7 had 0.8% nucleotide and 1.5% amino acid sequence variation with each other, whereas the 2 positive pigs at 14 dpi had 1.7% nucleotide and 3.5% amino acid sequence variation with each other. Objective 4. Improve diagnostic assays and create on-farm monitoring systems. 4.1. Development and improvement of diagnostic assays. A new, simple, on-site diagnostic test to detect PRRSV acute infection was developed with funding from the NPB by scientists from the University of Minneasota. The accuracy of this test is currently being evaluated. Saliva samples were collected on days 0, 2, 6, 14, 17 pi. Saliva sample tips were suspended in 300 µl of 10 mM Tris HCl, pH 7.5, allowed to stand for 10 minutes at room temperature then liquid eluted in a microfuge tube funnel apparatus. Saliva and serum samples were used at 1/100 and 1/50 dilutions, respectively, for determination of total immunoglobulin (IgA) and PRRSV specific antibody. To determine total IgA levels, plates were coated with goat anti-pig IgA-affinity purified antibody (1 ¼g/well) in carbonate buffer at pH 9.6 overnight at 4°C. PRRSV nucleocapsid-specific IgA and IgG for saliva were determined on plates coated with nucleocapsid protein (200 ng/well) under the same conditions. Ten ul of saliva sample was applied in 100 ul total volume, followed by goat anti-pig IgA-HRP conjugate and goat anti-pig IgG-HRP conjugate respectively at 1/100,000 dilution for 2 hours. Color was developed with TMB for 20 minutes. PRRSV nucleocapsid-specific serum antibodies for IgG and IgM were determined at 2, 7, and 17 days. Serum was diluted 1/50. Goat anti-pig IgG- and IgM-HRP conjugates were used at 1/100,000 dilution for 2 hours, followed by color development. Saliva dilution effects were controlled by normalizing to total protein with the following formula: (Measured [IgA]) x ([Total protein]/average [Total protein]of all samples) x 100 fold-dilution x 4 (assuming 100 ul of swab volume plus 300 ul of Tris buffer). 4.2. Improvement of on-farm monitoring systems. The feasibility of pooling serum samples for detection of PRRS virus antibodies by ELISA in surveillance protocols for negative sow farms was evaluated by researchers at the University of Minneasota. Pooling serum samples resulted in a decrease in sensitivity and an increase in specificity, compared to testing individual samples. However, an approach that can increase the herd sensitivity of a surveillance protocol for breeding herds, while maintaining high herd specificity and low testing costs was described. This can be achieved by sampling a larger number of animals and running the samples in pools. 4.3. Iowa State University scientists study on the use of oral fluids for the detection of PRRSV, anti-PRRSV antibody, and other pathogens. 4.4. To further characterize the humoral immune response of pigs to PRRSV, the kinetics of antibody response to the PRRSV non-structural proteins (nsp) was determined by ELISA in experimentally infected pigs by researchers from SDSU (Fang, Christopher-Hennings, Nelson) and UMN (Murtaugh). The nsp1, nsp2 and nsp7 induced higher antibody response than the other nsps, with nsp7 being the most consistent. Using nsp7 recombinant protein as antigen, we further validated a dual nsp7 ELISA for the simultaneous detection and differentiation of serum antibodies against type 1 and type 2 PRRSV. This assay is convenient with respect to antigen production and is highly sensitive and specific. Thus, it is considered to be a potential tool for routine diagnostics, epidemiological surveys, and outbreak investigations. Objective 5. Develop and test PRRSV virus eradication protocols under various ecological settings. 5.1. Regional projects in Rice County and Stevens County Minnesota, started 3 years ago by scientists from the University of Minnesota, are making steady progress in reducing the number of PRRS-positive sites. Communication among producers and veterinarians is critical to success. Objective 6. Develop educational outreach tools for disseminating information through established outreach and extension networks to producers, veterinarians, educators, and researchers. 6.1. NC-229 leaders serve as chair, co-chair, scientific program chair and proceeding editor for 2007 as well as 2008 International PRRS Symposium (IPRRSS), Chicago IL for approximately 200 registrants (http://www.prrssymposium.org/). Organized plan with CRWAD for AVMA continuing education credits for 2008 IPRRSS. Develop plans for PRRS CAP2 genetics objective with NC1037 swine genome researchers during May 14-15, 2008 meeting. Organized a PRRS diagnostic workshop (Chicago, IL, Dec. 5th, 2008). 6.2. Collaboration is underway with the AASV by University of Minnesota scientists to develop an online sample size calculator for negative boar studs that have a routine PRRSV surveillance program. The calculator has been evaluated and approved by the Boar Stud Committee of the AASV and will be offered freely through the AASV website. 6.3. NC-229 members made many scientific presentations on PRRS. For examples, Seminar presentation by UMN scientists, 3 Jan 2008, Structural analysis of PRRSV by mass spectrometry at the Department of Population Health and Pathobiology, North Carolina State University, Raleigh NC; 5 March 2008, invited seminar, PRRS immunology: knowledge is power. Points of Pride Day, at the College of Veterinary Medicine, Univ of Minnesota; 9 April 2008, PRRS Immunology at the 2nd Asian PRRSpective, Macao. Objective 7. Create an information network to ensure rapid and efficient communication of PRRSV research. 7.1. NC-229 members contribute and maintain the PRRSV genomic database, organized and participated in the International PRRS Symposium. 7.2. NC-229 members served on writing team for PRRS CAP2 renewal which is directed by R Rowland (Kansas State University), helped develop plans for PRRS Symposium. NC-229 members wrote the renewal proposal for the NC-229 with expanded objectives.

Publications

Please refer to the attached file for Publications.

Publications Attachment:

Attachment

Impact Statements

Research advances over the last year continue to expand our understanding of PRRSV pathobiology and provide new ideas for countering and/or eliminating the infection. Extensive work has been done regarding the emergence of genetic and antigenic variation during replication in pigs and its role in persistence. Continued assessment and research in diagnostic technology contributes to the improvement and refinement of our ability to detect and diagnose PRRSV infection.
On-going work on immunity and vaccine development holds great promise. In particular, the most challenging task to eradicate PRRSV from pigs is to induce strong protective immunity. Numerous NC229 researchers have identified novel approaches to address this important task.
The appearance of Type 1 PRRS viruses is a relatively recent phenomenon. Phylogenetic studies of Type 1 isolates show that this re-emerging group are genetically diverse and successfully compete with endemic Type 2 isolates. These data show that European PRRSV is a significant and largely un-recognized problem. Vaccines and diagnostics need to be developed that target the Type 1 group of viruses.
Studies of TLR receptor function during PRRSV infection are still in their infancy. Treatments that activate TLR3 provide a means to block PRRSV infection of macrophages. Immune modulators that target TLR function need to be developed.
Vaccines primarily target humoral immunity. T cells provide a critical role in memory responses following vaccination. The identification of a conserved T cell epitope that stimulates a memory response to PRRSV is a target for vaccines that can provide long term protective immunity.
Nsp2 is a multi-functional protein that is critical for virus replication. The insertion of nucleotides into Nsp2 is a novel approach for virus attenuation and marker expression. The impact is the identification of a new means to develop modified live vaccines that contain immunological markers.
Identified novel changes in gene expression in porcine alveolar macrophages (PAM) infected with PRRSV using Serial Analysis of Gene Expression (SAGE) libraries. Demonstrated suppression of the host innate immune response which may explain how PRRSV successfully evades a protective immune response. This comprehensive evaluation of gene expression in PRRSV-infected PAM identified potential proteins and pathways affected by PRRSV infection and new targets for the control of PRRSV infection.
A potential target site on PRRSV viral genome that effectively blocked virus replication in vitro was identified. The protective effect of RNAi and other compounds against PRRSV virus infection in pigs are being evaluated. The circovirus type2 group1 pathogenesis study clearly demonstrated that a new virus was emerging in the population and is a much more virulent strain. Virulence depends on the presence of co-factors in the infected pig population.
An essential component of PRRS control is preventing and quickly identifying infection of boar studs. An NPB funded study in boars and boar semen optimized testing strategies for detection of PRRSV by PCR. High levels of interleukin-12 (IL-12) were identified in seminal plasma from infected and non-infected boars. IL-12 promotes inflammatory and cytotoxic responses and may stimulate uterine inflammatory response in sows after insemination and protect against transmission of viral infection.
Identifying PRRSV genetic determinants associated with macrophage host range and modulation of pro- and anti-inflammatory cytokine expression, provides critical information for engineering of PRRS live attenuated vaccines and antivirals. This will permit rational design of differential vaccines of unprecedented safety and efficacy. Identifying PRRSV isolates with different phenotypic and pathotypic characteristics enables comparison of genotypic differences and mapping of virulence determinants.
Successful completion of the biosecurity projects will provide a clear understanding of PRRSV transmission and cost-effective interventions to prevent its spread between farms. The conventional PRRSV surveillance protocols for sow farms based on ELISA on individual samples can be improved by using pooled-sample testing.
A cDNA infectious clone that was produced through funding received from USDANRICGP and from NPB was provided to different laboratories. Successful applications of this cDNA infectious clone in those labs are now being reported in the literature.
Marker vaccines are needed for differentiation of vaccinated from naturally infected animals. Using an infectious clone of Type 1 PRRSV, a recombinant green fluorescent protein (GFP) tagged PRRSV was constructed containing deletion of an immunogenic epitope, ES4, in the nsp2 region. This marker virus, in conjunction with developed companion diagnostic tests, enables serological differentiation of vaccinated from wild-type virus infected.
Understanding the PRRSV virus GP5 and N and M protein interactions could aid in the development of improved vaccines. The peptide-conjugated antisense phosphorodiamidate morpholino oligomers are effective against PRRSV in vitro and could be useful as anti-PRRSV agents. The in vivo transfection strategy will help delineate structural and functional relationship of PRRSV genes.
NC-229 sponsored and organized annual International PRRS Symposiums for scientists worldwide. The symposium is a means to openly present and discuss PRRS research progress with the participation and input of non-NC229 researchers worldwide.