Brief Summary of Minutes of Annual Meeting

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.