Brief Summary of Minutes of Annual Meeting

NC229 Meeting Chicago, IL, 12/02/2010 Brief Summary of Minutes of Annual Meeting. (Held on Friday, Dec. 2, at 1:00 PM -3:00 PM) 1:00 XJ Meng, opening remarks 1:05 Update of NC229, Dr. David Benfield, Administrative Advisor ; 2012 appears to be flat for NC229; 2013 budget unknown due to election For NC229 funding , current funding expires Sept.. 30th, 2014, so a new project would need to be written in 2013 for 2014. In 2012, there is a mid-year review. The original NC229 project was written for PRRSV research primarily (with a push for the PRRSV CAP programs) and then expanded to PRRSV + emerging viral diseases of swine. With the CAP program ending, for the next written proposal, may want to consider emphasis on other viruses since some stations do not get any funding from their station directors (eg. to leverage other opportunities for funding). 1:15 Election of new NC-229 officer (Dr. Fernando Osorio was elected Secretary for NC229. Dr. Jane Christopher-Hennings is the Chair starting 2012, replacing Dr. XJ Meng who was chair for last 2 years) 1:20 Update from USDA-NIFA (Drs. Peter Johnson/Margo Holland): Dr. Margo Holland: FY 2012 funding is flat. AFRI Indirect costs will increase to 30% for 2012 funding. No less than 40% for applied research No less than 2% for equipment grants 60% is for basic research (with 30% focused on multi-disciplinary teams) 2012 Foundation Programs to be released in early 2012 (need letter of intent) Areas of interest include Animal Health and Production; Agricultural systems (may want to put emphasis on nanotechnology) Grants generally $500,000 total 2011 Animal Health Foundation program had > 200 letters of intent with 160 proposals received; 12 proposals recommended for funding (8.3% success rate) with 4 awards for PRRSV research (3 standard research proposals and 1 seed grant) representing vaccine, health surveillance and host-pathogen interactions. PRRSV CAP has been $1.2 million/year and is considered a success story of a multi-disciplinary group. $ 3 million has been leveraged (eg. NPB, University, Fed. Gov. private companies) NIFA Fellowships: Pre-docs $75,000 (for 2 years) Post-docs $130,000 (for 2 years) Dr. Peter Johnson: NEW funding on Ecology and evolution of disease (NIFA, NSF, NIH) Due 1st Wed in Dec (this year Dec. 7th). Need to appeal to all agencies, http://nsf.gov/pubs/2011/nsf11580/nsf11580.htm

Objective 1. Elucidate the mechanisms of host-pathogen(s) interactions. 1.1 (NADC, Faaberg/Brockmeier/Loving/Miller) Compared pathogenesis in swine after challenge with 3 new Type 2 PRRSV field isolates in vivo and in vitro. Differences seen in viral growth kinetics and pathogenesis. 1.2 (NADC, Faaberg/Brockmeier/Loving/Miller) Compared growth and disease in swine after vaccination with nsp2 deletion mutant viruses and challenge with novel Type 2 isolate. 1.3 (NADC, Kehrli/Miller/Faaberg) Adenovirus expression of a region of PRRSV nsp 2. 1.4 (NADC Faaberg/Lager/Miller/Brockmeier/Kerhli/Nicholson) Compared high and low dose challenge of US swine with Chinese and Vietnamese HP-PRRSV. 1.5 (NADC, Faaberg)Development of infectious clone of a new vaccine strain. 1.6 (NADC, Faaberg) Examined the interaction of nsp2 with host genes. 1.7 (NADC, Miller/Kehrli) Developed PRRSV infected tissue culture assay to screen for polyclonal B-cell activation. 1.8 (NADC, Miller/Kehrli/Faaberg)Assessed PRRSV strains that have a reduced capacity for the induction of polyclonal B-cell activation for potential vaccine candidates. 1.9 (NADC, Miller; G Rohrer USMARC) Sample collection and genomic DNA purification for SNP chip analysis to examine genotype in host susceptibility to PCV2. 1.10 (NADC, Miller) Compared the transcript expression of tracheobronchial lymph nodes of pigs infected with PRV/PRRSV/SIV/PCV-2 in vivo. 1.11 (NADC, Miller) Determine the transcriptomic immune response to contemporary US and Asian PRRSV in vivo. 1.12 (NADC, Faaberg/Miller; B. Guo, Visiting Scientist) Developed type 1 IFN bioassay for examination of PRRSV strain specific pathology. 1.13 (NADC, Faaberg) Developing chimeric vaccine to Asian HP-PRRSV. 1.14 (UNL) Understanding the role of the nsp1 in PRRSV--Using nsp1², a proteolytically processed functional product of nsp1 as bait, we have identified the cellular poly (C)-binding proteins 1 and 2 (PCBP1 and PCBP2) as 2 of its interaction partners. Interactions of PCBP1 and 2 with nsp1² was confirmed. In MARC cells, the cytoplasmic PCBP1 and 2 partially co-localize to the viral replication-transcription complexes. Recombinant purified PCBP1 and 2 bound to the viral 5 UTR. SiRNA-mediated silencing of PCBP1 and 2 in cells resulted in significantly reduced replication and transcription without effects on initial polyprotein synthesis. ID of cellular factors involved in PRRSV lifecycle give a better understanding of virus biology and has potential for development of anti-viral therapeutics. 1.15 (UMN) discovered a novel PRRSV protein expressed in infected cells that may have a role in cellular pathogenesis. 1.16 (UMN) characterized the molecular pathogenesis of PCV2 infection by transcriptome profiling. Discovered a pronounced gene expression induction profile characteristic of a classical IFN response to viral infection. 1.17 (PURDUE) Host immune responses to PRRSV infection may be correlated with genetic control. The PRRS Host Genetics Consortium (PHGC) studies are aimed at identifying genes and pathways associated with pigs that clear PRRS virus while continuing to gain weight. Analyses of data from each PHGC trial [viral load from 0-21 days post infection (dpi) and weight gain from 0-42 dpi] were used to statistical identify 4 groups of pigs: those with the best phenotype, low virus and high growth (LvHg), high virus and high growth (HvHg), high virus and low growth (HvLg), and, the worst, low virus and low growth (LvLg). Real time PCR was used with primers corresponding to markers important for immune system activation. Markers included: transcription factors TBX21 (T-bet), GATA3 (presumed Th1 and Th2 regulators, respectively), FOXP3, cytokines IL10, IFNg, CD163, the PRRSV receptor, and CD69, the early marker for T cell activation and proliferation. LvHg animals at 4-10 DPI exhibited increased ratio of TBX21/GATA3. LvHg animals had a robust increase in expression of IL10 and IFNG within 4-14 DPI, with high basal expression of CD69 relative to other groups at 0 DPI. In HvHg, TBX21/GATA3 was the highest during PRRSV infection, up regulation and expression of TBX21 and GATA3 was minimal, along with other markers. HvLg animals had high base line expression of GATA3 and FOXP3 transcription factors and delayed increase of TBX21/GATA3. In LvLg animals a high basal expression of GATA3 and FOXP3 was detected with delayed increased of TBX21/GATA3. In LvLg animals higher baseline expression for CD163 and cytokine IL10 markers was detected. The HvLg and LvLg animals exhibited up regulation of the markers responsible for Th2 immunity during infection. Our data correlated basal expression of CD69 as a factor leading to up regulation and activation of markers responsible for Th1 immune response. Basal expression of GATA3 and FOXP3 demonstrated activation of the markers responsible for Th2 pathway during infection. 1.18 (KSU) Sang, Blecha,Rowland characterized the expression 39 type I IFN genes and related receptors in the PRRSV-infected fetus. 1.19 (KSU) Hesse, Rowland performed an analysis of cross-protection between diverse PRRSV strains. 1.20 (KSU) Rowland identified a decoy epitope CP (169-180) in PCV2 capsid protein produced to immunization with capsid monomer. Immunization with whole capsid offered protection. This explains why production of large quantities of virus during infection are non-protective. 1.21 (SHVRI) report viable chimeric viruses in which the envelope protein genes from ORF2a to ORF5 of vSHE (type 1) were swapped into the genetic backbone of vAPRRS (type 2). Found that the envelope proteins of type 1 were fully functional in type 2 PRRSV and the rescued chimeric progeny viruses showed robust genetic stability and similar replication properties to the parental strains in vitro. For PRRSV N protein, we found that type 1 N protein was functional in the type 2 PRRSV backbone. All cysteines in the N protein are non-essential for type 1 and 2 PRRSV viability. NEM treatment prevents disulfide-linked N dimerization in cells but not in extracellular virions. 1.22 (UCONN; Garmendia): Aims are to determine the sensitivity to and induction of IFNb by PRRSV isolates, to identify mechanisms of evasion of host's innate immune responses by PRRSV and to determine correlations with virulence. Field viruses were previously tested in their sensitivity to IFNb in vitro. Significant differences in sensitivity to IFNb among different PRRSV isolates and between MARC-145 cells and porcine alveolar macrophages (PAM) were demonstrated. The induction of IFNb by PRRSV was tested in PAMs using field isolates and a series of chimeric viruses derived from infectious clones of FL12 (virulent isolate) and a vaccine virus (attenuated), provided by Dr. Osorio, UNL. The induction tests demonstrated that PRRSV isolates do induce IFNb in PAMs but such induction is variable. The IFNb induced was bioactive as shown by a dose-dependent reduction of VSV infectivity in cell culture. Mx and TNF were measured in PRRSV-infected PAMs. Mx is expressed at significant levels that generally correlate with the corresponding levels of IFNb produced. TNF production is detectable but levels are not significant and do not follow a pattern related to IFNb. 1.23 (ISU) Studies on enhanced pneumonia and disease in pigs vaccinated with an inactivated human-like (´-cluster) H1N2 vaccine and challenged with 2009 pH1N1 influenza virus. 1.24 (ISU) Studies on cytokine and chemokine mRNA expression profiles in tracheobronchial LN from pigs singularly infected or coinfected with PCV2 and Mycoplasma hyopneumoniae. 1.25 (ISU) Studies on genetic and phenotypic characterization of a 2006 US PRRSV isolate associated with high morbidity and mortality in the field. 1.26 (ISU) Studies on the establishment of a DNA-launched infectious clone for a highly pneumovirulent type 2 PRRSV: Identification and in vitro and in vivo characterization of a large spontaneous deletion in the nsp2 region. 1.27 (OSU) To elucidate both cellular and innate cytokine response in growing pigs at very early stages of PRRSV infection in a commercial pig herd premises, 7 wk old pigs were infected. 1 in a pen of 25 was PRRSV infected and responses were assessed 2 DPI. All the infected and a few of the contact neighbor pigs were viremic. A majority of viremic pigs had more than 50% reduction in NK cell-cytotoxicity. At 2 DPI 1 fold increase in innate IFNa production was detected in plasma. Enhanced secretion of IL4, IL10 and IL12 (but not IFNg) in a majority of infected pigs was seen. A reduced frequency of myeloid cells, CD8+ and CD4+CD8+ T cells and increased T-regulatory cell population was detected in all the viremic pigs. Our results suggest that PRRSV modulate the innate and adaptive immune mediators from 2 DPI, resulting in subversion of host innate immunity from early stages post-infection. 1.28 (VA) We identified a large spontaneous deletion of 435-bp in the nsp2 gene of a highly pneumovirulent PRRSV, VR2385 . We established a DNA-launched infectious clone (passage 14) containing the 435-bp nsp2 deletion (pIR-VR2385-CA) and another DNA-launched infectious clone, pIR-VR2385-R, in which we restored the deleted 435-bp nsp2 sequence back to the pIR-VR2385-CA backbone. The growth characteristics of the 2 rescued viruses were compared, and results showed that the VR2385-CA virus with the nsp2 deletion replicated more efficiently in vitro than the VR2385-R virus with the restored nsp2 sequence but, VR2385-CA virus had a significantly reduced serum viral RNA load in vivo. In pigs, the nsp2 deletion had no effect on virulence. The spontaneous nsp2 deletion has a role for enhanced virus replication in vitro but has no effect on pathogenicity. 1.29 (VA) To determine whether cellular microRNAs (miRNAs) play a role in host response to PRRSV infection, we performed a global profiling of both cellular miRNA and mRNA in MARC cells infected with type 1 (SD01-08) or type 2 (VR2385) PRRSV. Results showed that the expressions of approximately 240 miRNAs were significantly altered with infection by PRRSV type 1 or 2 (114 for type 1, and 82 for type 2), and at least 15 specific miRNAs were shared by both types. Approximately 4,500 genes showed differential expression with infection by either virus type (p<0.05). We conducted a global human/bovine/porcine miRNA and porcine gene expression microarray analyses using a pool of lung homogenates of 10 SPF pigs at 14 DPI. Compared to the negative control, PRRSV infection resulted in significant changes of the expression level (>2-fold) of 17 miRNAs (p<0.05) and 3,713 mRNAs (p<0.01) including genes involved in host innate and adaptive immune responses. We found 270 unique miRNAs that were differentially up- (151 miRNAs) or down-regulated (119 miRNAs) in response to PRRSV infection. Deep sequencing data showed 1,892 novel putative porcine miRNAs that do not align to any known Sus scrofa miRNAs. We have correlated inverse regulation between miRNAs and putative target genes to build a miRNA-gene network. 1.30 (SDSU: Z Sun, Y Li, Y Fang) investigated ISG15 and PRRSV nsp2 OTU domain mediated deisgylation function. IFN-stimulated gene 15 (ISG15) is an ubiquitin-like protein which is stimulated by type I IFN ±/² or induced by viral or bacterial infection. Over-expression of ISG15 in cells significantly reduced the PRRSV titer and was confirmed using small interfering RNAs against ISG15-conjugating enzymes. IFN-induced antiviral activity is significantly alleviated by inhibiting ISG15 conjugation. In vitro deISGylation assay showed the N-terminal OTU domain of nsp2 has deconjugating activity towards ISGylated products. A 19 AA deletion plus a single AA mutation partially relieved the nsp2 de-ISGylation function. This showed that ISG15 conjugation has an important role in PRRSV infection and modifying certain regions of nsp2 could reduce the deISGylation ability of the virus. 1.31 (SDSU, KSU Lawson,Li, Patton, Langenhorst, Sun, Jiang, Hennings, Nelson, Knudsen, Fang, Chang). Constructed a recombinant PRRSV that encodes swine IL-1b as a separate subgenomic mRNA inserted between ORFlb and ORF2. MARC cells infected with recombinant virus secreted ILlb and had a similar growth rate to parental virus. No clinical signs were observed in the recombinant virus-infected nursery pigs and IL-lb, IL4 and IFNg were up-regulated in PBMCs. 1.32 (SDSU, LUMC, NL: Li, Tas, Sun, Snijder, Fang). MAbl and polyclonal antibodies were generated against PRRSV ORF1a-encoded nsps. Using these antibodies, we identified and characterized these ORFla-encoded nsps in infected MARC cells. This study confirmed the existence of proteolytic processing products of PRRSV ORFla encoded polyprotein in virus infected cells and provides a basis for both applied and basic research on the role of PRRSV nsps in viral replication and pathogenesis. 1.33 (BARC) The PRRS Host Genetics Conso rtium (PHGC) has expanded efforts to determine the role of host genetics in resistance to PRRS and in effects on pig health and related growth effects. The PHGC is a multi-year project that is funded by a US consortium representing the US National Pork Board (NPB), USDA ARS and NIFA, universities and private companies; it represents the first-of-its-kind approach to food animal infectious disease research. The project has used a Nursery Pig Model to assess pig resistance/susceptibility to primary PRRSV infection. Ten sets of 200 crossbred pigs from high health farms were donated by commercial sources and transported KSU. Pigs were infected with PRRSV and followed for 42 DPI. Blood was collected at 0,4,7,10,14,21,28,35 and 42 DPIi and weekly weights recorded. All 10 trials have been completed; each trial has affirmed that all pigs become PRRSV infected but some pigs clear virus from serum quicker with variable weight effects. Data is being stored in the PHGC database at ISU www.animalgenome.org/lunney. 1.34 (BARC) Genome wide association studies have identified genomic regions that determine PRRS resistance/susceptibility. DNA from PHGC pigs has been genotyped with the PorcineSNP60 Genotyping BeadChip (> 60K single nt polymorphisms or SNPs). (Data stored at ISU www.animalgenome.org/lunney. Multivariate analyses of viral load and weight data have identified PHGC pigs in different virus/weight categories, so that ongoing serum cytokine and gene expression studies can compare data from PRRS resistant/maximal growth pigs to susceptible/reduced growth pigs. PRRS CAP, NRSP8 Swine Genome Coordinator and Genome Alberta have supported SNP chip analyses and PRRS CAP the state-of-the-art genome wide association studies (GWAS) to identify genetic determinants of PRRS resistance/susceptibility. Based on evaluation of phenotypic traits, viral load [VL, area under the curve 0-21 dpi], and 0-42dpi weight gain (WG), response to PRRSV challenge was shown to be moderately heritable at 0.30 each. Regions on swine chromosomes 4 (SSC4) and SSCX appear to be associated with VL, and on SSC1, 2, 3, 4, 7, and 17 with WG. Regions on SSC4 for VL and WG are almost perfectly negatively correlated. Investigations revealed that the BB genotype for this region of SSC4 is the desirable genotype and has a low frequency (0.02), suggesting that genetic progress can be made by selective breeding. Functional gene and protein transcriptomic analyses are ongoing to ID gene networks and resistance associated biomarkers that differ in high versus low VL pigs. 1.35 (BARC) Identifying host gene expression changes that are involved in regulating responses to PRRSV infection. Grants from USDA NIFA and Genome Alberta are supporting whole blood gene expression analyses using microarrays and RNAseq (next generation transciptomics), respectively. The goal is to assess differential expression of individual genes and to discover networks and pathways enriched for those genes, in pigs showing different responses to PRRSV infection. RNA from 3 pigs per group was hybridized to the 20K 70-mer oligonucleotide Pigoligoarray following a blocked reference design with time 0 of each individual animal as a reference sample. Expression levels for 491 genes showed significant viral level-growth interaction for all time-points. Differentially expressed (DE) genes at early time-points (4, 7, 14 dpi) were evaluated by enrichment analysis with Ingenuity Pathways Analysis software www.ingenuity.com; for comparisons of viral level, 308 genes were DE with 16 significant gene networks (p d0.0001); for growth level, 367 genes were DE with 17 significant gene networks. Confirmatory qPCR work will explore these DE genes and their roles in PRRS control. The more significant biological functions identified (FDRd 5%) were those related to cell death, cellular function, maintenance and compromise, and inflammatory disease. For the growth comparison at 4 dpi in Lv animals, the antigen presentation pathway was over-represented (FDRd5%). 1.36 (UMD) found that PRRSV inhibits IFN downstream signaling and continued the studies to identify the interference mechanism. We found that NSP1² of PRRSV VR2385 interferes with IFN signaling pathway. VR2385 is a virulent strain whereas NSP1² of Ingelvac PRRS MLV does not affect IFN signaling. NSP1² of VR2385 blocks nuclear translocation of the IFN-induced STAT1/STAT2/p48 heterotrimer. IFN-induced phosphorylation of both STAT1 and STAT2 and their heterodimer formation were not affected. The NSP1² of VR2385 interferes with the interaction of STAT1 and importin. NSP1² may be the viral protein that interferes with the activation and signaling of type IFN. 1.37 (UMD) During studies of IFN signaling, we noticed 1 lab mutant induces synthesis of type I IFNs in cultured cells and has no effect on IFN downstream signaling. This mutant was plaque purified and named A2MC2. A2MC2-infected MARC cells resulted in strong inhibition of replication of an IFN-sensitive virus, Newcastle disease virus (NDV). Analysis of A2MC2-infected MARC cells showed that the transcripts of IFN-stimulated genes were elevated. Inhibition of A2MC2 replication abolished its capability to induce IFNs. A2MC2 does not affect IFN downstream signaling and induces IFN in PAMS. Infection of PAMs leads to little cytopathic effect. 1.38 (UIUC D Yoo) found that the suppression of NF-kB activation and sumoylation of PRRSV Nsp1± is mediated by protein inhibitor of activated STAT1 (PAIS1). We examined the role of PRRSV Nsp11, and endoribonuclease, on virus replication and IFN regulation. 1.39 (UIUC, Zuckermann) found that the influence of PRSV on the IFNa response of macrophages to infection with PRRSV is strain-dependent. Macrophages appeared non-responsive in the presence of either of 2 wild-type PRRSV, copious amounts of this cytokine was released by ZMAC cells exposed to a 3rd individual. This unconventional isolate was unique since it induced type I gene transcription and stimulated the phosphorylation of IRF3 to a greater extent than ZMAC cells infected with either conventional virus. Since the phosphorylation of a 2nd transcriptional factor, NFºß, also involved in the initiation of IFNa/ß gene transcription was comparatively unaffected in any of the virus-infected cells, the conventional viruses may be blocking IFNa production by interfering with a step(s) in the pathway leading to IRF3 activation. Objective 2. Understand the ecology and epidemiology of PRRSV and emerging viral diseases of swine. 2.1 (UGA) Studied ability of swine, human and avian influenza viruses to reassort on the TRIG backbone in swine and primary swine and human epithelial cells with the goal to elucidate the potential for reassortment. We also explored the evolution of AIVs in poultry and waterfowl. 2.2 (UGA) Explored the potential for avian and swine origin influenza viruses to infect and transmit in mice and ferrets. 2.3 (UGA) Explored receptor specificity of avian, human and swine influenza viruses. 2.4 (UGA) Explored the potential for avian influenza viruses to infect felines and tested for exposure of feral cats to AIVs to test their potential as an alternate reservoir/vector. 2.5 (UMN, Guelph, Hong Kong). PRRSV diversity based on sequencing/RFLP typing was described for type 2 PRRSV. 2.6 (UMN). Influenza Transmission dynamics and transmission parameters were evaluated in pigs with passive immunity induced by influenza vaccination. Homologous passive immunity decreased transmission but did not prevent it. Transmission was similar in pigs without passive immunity and in pigs with heterologous immunity. Influenza ecology studies showed that influenza infections in closed grow-finish populations were prolonged (70 DPI). Oral fluids were a sensitive method to detect influenza infections in populations. Weaned pigs are a source of virus introduction for grow-finish populations. Methods to study aerosol transmission of influenza virus in pigs were validated to detect influenza virus from aerosols generated from infected pigs. Detection of infectious aerosols may be related to the number of pigs shedding virus in a population. Positive aerosols could be detected when just a few pigs were known to be shedding. Detection of infectious aerosols has been shown in the field, inside pig barns and exhaust fans. 2.7 (UMN, ISU, SDSU) showed long-distance airborne spread of PRRSV and identified climactic conditions which support it. Multiple air filtration interventions were validated to prevent airborne spread of PRRSV and M hyopneumoniae. 2.8 (UMN, Pipestone) PRRSV shedding was decreased in pigs that had been vaccinated with a MLV vaccine compared to non-vaccinates. Decrease shedding was seen in oral fluids at 36 DPI and in air samples collected in exhaust fans. PRRSV was detected in air samples from non-vaccinated pigs for up to 70 days and 45 days for vaccinates. 2.9 (KSU, BARC, others) participated in the PHGC. Results include a genome wide association study of the first 600 pigs. The results show the ID of genomic markers that are linked to virus load and weight gain. To date, 2000 pigs are in the study. 2.10 (SHVRI) Viral RNA synthesis regulatory elements identification. The 5' untranslated region (UTR) of the genomic RNA is believed to be vital for the replication of PRRSV yet its functional mechanism remains largely unknown. Using a full-length cDNA clone we found SL1 was essential for infectivity of PRRSV. SL2 was a key regulatory structural element for PRRSV replication, particularly sg mRNA synthesis. 2.11 (ISU) Studies on the systematic review of factors that influence the persistence of influenza in environmental matrices. 2.12 (ISU) PCV2: Studies on commercially produced spray dried porcineplasma contains high levels of PCV2 DNA but did not transmit PCV2 when fed to naïve pigs. Establishment and maintenance of a PCV2-free breeding herd on a site that experienced a natural outbreak of PCV2-associated reproductive disease. Studies on high prevalence of PCV viremia in newborn piglets in 5 clinically normal swine breeding herds in North America. PRRSV influences infection dynamics of PCV2 subtypes PCV2a and PCV2b by prolonging PCV2 viremia and shedding. Shedding and infection dynamics of PCV2 after experimental infection and after natural exposure. 2.13 (ISU) Median infectious dose (ID50) of PRRSV isolate MN-184 via aerosol exposure. 2.14 (OSU) To potentiate the effect of killed PRRSV vaccine, poly(lactide-co-glycolide) (PLGA)- nanoparticles were prepared to encapsulate killed-PPRSV antigens. In nanoparticle-killed-PRRSV vaccinated pigs a reduction in viremia with complete viral clearance by day 15 was seen. In the lungs of nanoparticle- PRRSV vaccinated MN184 challenged pigs a significant reduction in PRRSV antigen load and a reduction in inflammatory cells infiltration was observed. Immunologically, increased frequency of immune cells which initiate Th1response associated with production of IFN±, IL12, IFN³ and IL6 cytokines was detected. Enhanced titers of PRRSV specific total and virus neutralizing antibodies were detected in nanoparticle-killed-PRRSV vaccinates. 2.15 (UW) developed novel analytical approaches to identify a small number of representative viral genotypes from among the diversity of viral sequences available in GenBank and PRRSVdb. We adapted techniques from network theory to rank PRRSV sequences in terms of their importance and applied these methods to a highly-curated PRRSV database that combines high-quality, non-recombinant sequences from both GenBank and PRRSVdb. Viruses represented by the top ranking sequences are valuable targets for future study and can be eventually incorporated into a polyvalent vaccine. Objective 3. Develop effective and efficient approaches for detection, prevention and control of PRRSV and emerging viral diseases of swine. 3.1 (NADC, Faaberg/Nicholson) Analyzed efficacy of using a diagnostic microarray to detect different PRRSV isolates. 3.2 (NADC, Lager/Miller; E. Zanella, Visiting Scientist) Validation of a real-time PCR for PRV. 3.3 (UGA) Studied prophylactic and therapeutic application of PRRSV-specific swine mAbs. 3.4 (UGA) Explored aerosol vaccination for SIV vaccines in mice and ferrets. 3.5 (UGA) Established primary normal swine bronchoepithelial cell cultures to measure innate responses to swine respiratory viruses. 3.6 (UGA) Tested a variety of PIV5-based live-attenuated vaccines against influenza virus. 3.7 (UGA) Developed new method for rapid detection of influenza virus: 3.8 (UGA) Developed assay for simultaneous serological detection of PRRSV and PCV2. 3.9 (UNL) previously identified immunodominant B-cell linear epitopes in the proteins of a type 2 PRRSV strain FL12 (infectious cDNA clone-derived PRRSV pathogenic strain) with the epitope number 201 (EP-201) of the M protein as a marker candidate (conserved epitope). A triple mutant (TM) carrying 3 AA substitutions in the epitope 201 of PRRSV FL12 was generated. The TM was no longer recognized by anti-201 mAb, was stable in vivo, did not elicit antibodies to peptide 201 and can be used as a DIVA marker vaccine strain (with current development of a companion ELISA). We are pursuing the development of DIVA vaccines using PRRSV mutants devoid of the only dispensable structural gene of PRRSV (ORF5a) and by developing mutants devoid of antigenic reactivity in conserved epitopes of structural genes. 3.10 (UMN, ISU) Development of methods for viral and serological monitoring of PCV2 in oral fluids using an experimental model. Prevalence of PCV2 in US finishing herds was characterized. MN participated with IA, IN, KA, NC, Canada and WV to compare PCV2 serological detection methods for diagnostics. 3.11 (UMN) PCR for detection of cytomegalovirus and lymphotrophic herpesvirus. 3.12 (UMN, OH) Novel vaccine development against PRRSV. 3.13 (UMN, ISU) Multiplex methods for serological detection of PCV2 and PRRSV. 3.14 (UMN, IA, SD, Guelph, Newport Labs) identified new PRRSV RFLP types in D-lab submissions. 3.15 (UMN) discovered a novel PRRSV protein, ORF5a, that is immunogenic and induces antibody responses in pigs. 3.16 (KSU, ISU, SDSU) Rowland, Zimmerman, Fang, Opriessnig developed a multiplex Luminex assay for the detection of antibodies against PRRSV, PCV2 and SIV and initiated a multi-lab validation of Luminex assays for serum and oral fluids. 3.17 (KSU Wyatt) characterized a T cell epitope in the PCV2 capsid protein. 3.18 (SHVRI) DIVA vaccine development used JX143 (parental virus HP-PRRSV). After serial psg., 88 extra AA deletions were found in the Nsp2 region and was genetically stable up to the 100th psg. (JXM100). An Nsp2-88aa epitope-based ELISA was developed. 9 pigs were divided into 3 groups and inoculated with parental JX143 (A), JXM100 (B) or were mock-infected (C). Grp. A were immunized with JXM100 virus from which antibodies could not be detected against the corresponding 88 AA deleted epitope until JX143 challenge. This demonstrated that the recombinant marker virus with the diagnostic test, enables serological differentiation between marker virus-infected and wild-type infected pigs. 3.19 (ISU, USDA, AASV, NPB) produced an instructional video and poster on collection and processing of swine oral fluids for disease monitoring and was distributed to AASV members by NPB. 3.20 (ISU) Multiple publications on PCV2 vaccines. 3.21 (ISU) Disinfection protocols reduce the amount of PCV2-contaminated livestock transport vehicles. 3.22 (ISU) Prolonged detection of PCV2 and anti-PCV2 antibody in oral fluids following experimental inoculation. 3.23 (ISU) Comparison of RNA extraction and PCR methods for the detection of PRRSV in oral fluid. 3.24 (ISU) Inhibition of PRRSV infection in piglets by a peptide-conjugated morpholino oligomer. 3.25 (ISU) Kinetics of UV254 inactivation of selected viral pathogens in a static system. 3.26 (ISU) Multiplex method for the simultaneous serological detection of PRRSV and PCV2. 3.27 (ISU) Terminology for classifying swine herds by PRRSV status. 3.28 (SDSU: Langenhorst, Lawson, Sun, Li, Hennings, Nelson, Fang) developed an FMIA for detection of PRRSV antibodies in oral fluid and serum. Recombinant nucleocapsid and nsp7 from genotypes 1 and 2 were used as antigens. 3.29 (SDSU: Li, Chen, Sun, Fang). NSP1b is a strong IFN antagonist. Site-specific mutations were introduced into nsp1b and these had reduced ability to inhibit IFNb activation. Recombinant viruses with these mutations were produced for vaccine development. 3.30 (BARC) A multiplex FMIA was developed to simultaneously quantify porcine cytokines in serum or oral fluids (eg. IL1b, IL6, IL8, IFNa, TNF-a, IL-10, IL-12, IFNg, IL4, CCL2). The assay will be of value in immunity, vaccine, challenge studies, determining genetic resistance to PRRSV and responses to other swine pathogens. 3.31 (UIUC, Laegreid; UNL, Osorio, Pattnaik) confirmed that N-glycan moieties in GP5 of type 2 PRRSV are important for the virus to escape neutralizing antibodies and that the N-glycan in GP3 is important in protecting the virus from antibody neutralization.

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