Brief Summary of Minutes of Annual Meeting

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.

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.