Brief Summary of Minutes of Annual Meeting

Minutes NC229 Meeting Chicago, IL, 12/02/2012. The meeting starts at 1:00 PM with the following list of speakers: Introduction and welcome by Dr. Jane Christopher-Hennings, Chair NC229 assisted by Dr. Fernando Osorio, Secretary, Vice-Chair NC229.,
Dr. Peter Johnson, Dr. Margo Holland, USDA, Updates from USDA: Drs Holland and Johnson described the structure of NIFA, overall NIFA fundings records , enumeration of the major challenge areas identified by USDA-NIFA, with emphasis on foundational program , listing of alternative funding sources other than research grants, and the titles of grants funded in FY2011.
Dr. David Benfield, Administrator NC229, addressed the significance of the task for the coming year, as it is the time in which the group should decide whether to write a renewal to continue by December 2013. Key dates to remember: Sept 30 2014 termination of old project, Sept 15, 2013: deadline for submission /justification of need for renewal.
Dr. Bob Rowland, Current PRRSV CAP2 Director, Thoughts on NC229 Directions: Review of the IPPRSV activities, and host genetics consortium. Recommendations: Pursue a Grand Challenge that reflects the talents of the group (PRRS, emerging diseases, respiratory diseases), Engage stakeholders (translational), Build larger collaborative groups Nutrition Host genomics Wildlife biology etc
Dr. Mike Murtaugh, University of Minnesota, Recommendations: Refocus on a more integrative perspective related to health, and away from a disease emphasis on direct effects of pathogen causing disease. Emphasize porcine respiratory health, since enteric diseases are covered by another NC multistate group, and since we do not have expertise in reproductive health. Build a team of rivals to turn weakness into strength.
Dr. William Laegreid, University of Wyoming, What are the most appropriate problems for NC-229 to address? What approaches are most appropriate for NC-229 to provide? Focus on disease control for PRRSv and other emerging diseases. Disease control is dependent on epidemiology: Surveillance/Measures of disease frequency; Diagnostic Test Evaluation/Validation: Cost-benefit analysis of disease interventions; Molecular epidemiology/Outbreak Investigation; Spatial epidemiology; Risk Analysis; Disease Modeling
Dr. Dan Rock, UIUC, sees three future roles of NC-229 : 1) A research role  given constraints, likely will see evolution of research strategies (research team makeup and project structure).Coordinated Agricultural Projects (CAP)  a role to play - but probably not the best research structure for high impact outcomes. 2) A scientific leadership role  a think tank function- for objective problem analysis, scientific debate and setting the national animal health research program strategies. 3) An educational role - organize scientific meetings/workshops for investigators, junior investigators, students and others.
Dr. Jane Christopher-Hennings announces release of Internet-based survey to be conducted between December 2012 and January 2013 to probe the attitude of the group towards renewal, theme and objectives. Meeting adjourns at 500 PM.

B. PROGRESS OF WORK AND PRINCIPAL ACCOMPLISHMENTS
Objective 1. Elucidate the mechanisms of host-pathogen(s) interactions.
1.1 (USDA/BARC) State-of-the-art genome wide association studies (GWAS) are ongoing to have identify genetic regions associated with resistance/susceptibility to primary PRRSV infection.. A 38-SNP (~ 1 Mb) region on swine chromosome 4 (SSC4) explained 14.6% and 9.1% of the genetic variance for VL and WG, respectively.
1.2 (USDA/BARC + Host Genetics Consortium-PHGC-) Evaluation of differences in gene expression of whole blood RNA from PRRS Host Genetics Consortium (PHGC) pigs revealed a range of responses to PRRS virus infection. PHGC pigs were allocated into four phenotypic groups according to their high/low serum viral level and weight gain. Functional analyses were performed to assess if immune related gene sets were enriched for genes differentially expressed across four phenotypic groups. Finally, a power analysis was performed to estimate sample size and sampling time-points for future experiments. The conclusion was that the best scenario for investigation of early response to PRRSV infection consists of sampling at 0, 4 and 7 DPI using about 30 pigs per phenotypic group. These experiments are still ongoing.
1.3 (KSU Rowland/Sang) characterizing the expression of 39 type I IFN genes in the PRRSV-infected fetus. The approach incorporates 454 sequencing.
1.4 (KSU (Wyatt/Rowland) are characterizing a newly discovered SCID pig as a model for understanding PRRSV immunity and pathogenesis.
1.5 (KSU, Sang) Analysis of type 1 and type 2 macrophages in PRRSV immunity.
1.6 (KSU Rowland + PHGC) reported a marker on SSC4 linked to increased weight gain and reduced virus load during PRRSV infection.
1.7 (UGA) Developed a PRRS-susceptible immortalized porcine stem cell line (iPSC). They are now characterizing PRSS persistence in iPSC cells, and plan to evaluate the host cell response to infection.
1.8 (OSU) This station is actively studying the immune modulatory responses of wt PRRSV(VR2332) in pigs at mucosal tissues (mainly lungs and lymphoid tissues). Natural killer (NK) cells, and ³´ T cells in the lungs and lymphoid tissues were significantly modulated favoring PRRSV persistence. The NK cell-mediated cytotoxicity was significantly reduced in infected pigs. In addition, increased population of immunosuppressive T-regulatory cells (Tregs) and associated cytokines were also increased in VR2332 infected pigs. In conclusion, although wild-type parental strain VR2332 is avirulent, still it dampens the most essential immune components at the site of its replication and in lymphoid tissues, resulting in weak and delayed anti-PRRSV immunity.
1.9 (OSU) Also involved in developing a inactivated PRRSV vaccine by nanotechnology based delivery strategy. They use nanoparticle-entrapped UV-killed PRRSV vaccine in pigs. We entrapped PLGA [poly (lactide-co-glycolides)] nanoparticles with killed PRRSV antigens (Nano-KAg) and detected its phagocytosis by pig alveolar macrophages. Single dose of Nano-KAg vaccine administered intranasally to pigs upregulated innate and PRRSV specific adaptive responses. In a virulent heterologous PRRSV challenge study, Nano-KAg vaccine significantly reduced the lung pathology and viremia, and the viral load in the lungs. Immunologically, enhanced innate and adaptive immune cell population and associated cytokines with decreased secretion of immunosuppressive mediators were observed at both mucosal sites and blood suggesting the feasibility of this approach for cross-protective immunity in pigs.
1.10 (UMD Zhang/Zhu) They have identified and characterized a PRRSV isolate that induces synthesis of type I interferons in cultured cells. The IFN induced by this strain is biologically active and presents normal antiviral effect. This atypical strain is closely related to the VR2332 reference strain.
1.11 (UMD) Ongoing experiments on the mechanism of PRRSV interference with IFN-activated JAK/STAT pathway. They report that nsp1² blocks STAT1/STAT2 nuclear translocation by interfering with their interaction with importin-±5.
1.12 (VA Tech Meng). Thery have discovered attenuation of PRRSV by molecular breeding of the virus envelope genes GP5 from 7 genetically divergent strains and GP5/M dimer of 6 divergent strains Using DNA shuffling for molecular breefing. . The GP5 envelope genes of 7 genetically divergent PRRSV strains and the GP5-M genes of 6 different PRRSV strains were molecularly bred by DNA shuffling, and the shuffled genes were cloned into the backbone of a DNA-launched PRRSV infectious clone. They developed two specific representative chimeric viruses, DS722 with shuffled GP5 genes and DS5M3 with shuffled GP5-M genes. An in vivo pathogenicity study revealed attenuation and also effective immunogenicity of these two strains.
1.13 (VA Tech Meng). DNA shuffling of the GP3 genes of PRRSV produces a chimeric virus with an improved cross-neutralizing ability against a heterologous PRRSV strain (led by XJ Meng). The GP3 genes of six different PRRSV strains were bred by traditional DNA shuffling in an attempt to improve its heterologous cross-neutralizing ability. Additionally, synthetic DNA shuffling of the GP3 gene was also performed. Four traditional-shuffled chimeras and four other synthetic-shuffled chimeras were successfully rescued. These chimeras displayed similar levels of replication, in vitro and in vivo, compared to the backbone parental virus, indicating that the GP3 shuffling did not impair the replication capability of the chimeras. One chimera GP3TS22 induced significantly higher levels of cross-neutralizing antibodies in pigs against a heterologous PRRSV strain.
1.14 (VA Tech LeRoith ) Pursuing the study of the ability of swine dendritic cells infected with PRRSV, PCV2, or both to induce Tregs in vitro. The induction of Tregs by co-infected DCs may be dependent on TGF-² and not IL-10.
1.15 (UMN, Murtaugh)Whole genome sequencing of virulent field viruses was performed to evaluate potential genetic changes characteristic of novel strains associated with seasonal PRRS.
1.16 (UMN, Murtaugh) Research was performed to analyze genetic variation in the population of PRRSV produced from permissive cells.
1.17 (UMN, Murtaugh)The role of ORF5a protein in immunity was investigated. Studies were initiated to investigate the neutralizing antibody response in sows from herds exposed to virulent PRRSV. Collaborative research was performed to determine the role of plasmacytoid dendritic cells in anti-PRRSV host response.
1.18 (SDSU, Fang) Studying the status of PKR activation with regards to phosphorylation of eIF2± in PRRSV-infected cells. They observe that PRRSV induced the phosphorylation of eIF2± during late infection. This may contribute to the observed cell death and virus release from infected cells. Additionally, they observed that PKR activation might contribute to PRRSV replication in an eIF2±-independent manner. These studies continue at SDSU.
1.19 (SDSU, Fang) This laboratory also centers on the role that PRRSV NSP2 plays on the anti-IFN effects of PRRSV infection. They focus on the mechanisms by which NSP2 Inhibits the Antiviral Function of Interferon-Stimulated Gene 15. They show results demonstrating that ISG15 and ISGylation play an important role in the response to PRRSV infection and that nsp2 is a key factor in counteracting the antiviral function of ISG15.
1.20 (UCONN ,Risatti): Using the yeast two-hybrid screening method they have identified a cadre of cellular proteins that interact with PRRSV NSP3. Identified proteins are involved in multiple cellular pathways including, metabolism of carbohydrates, and metabolism of lipids, chaperones, cell signaling, apoptosis, and innate immune response.
1.21 (UCONN, Garmendia) This lab centers their research on providing a mechanistic explanation to the diverse range of sensitivity to IFNbeta observed among different PRRSV isolates and between MARC-145 cells and porcine alveolar macrophages (PAM)
1.22 (Purdue + PHGC) Research at Purdue pursues the characterization of molecular markers important for immunological responses during PRRSV infections following the model of pigs that clear virus while still gaining weight. They report the following interesting observation: basal expression of CD69 as a factor leading to up regulation and activation of markers responsible for Th1 type immune response; however, high basal expression of transcription factors GATA3 and FOXP3 demonstrated activation of the markers responsible for Th2 type pathway during PRRSV infection.
1.23 (USDA NADC,Faaberg et al) They have compared and contrasted the pathogenesis in swine after challenge with novel Type 2 PRRSV field isolates.
1.24 (USDA NADC, Faaberg) NSP 2 isolated and characterized from purified PRRSV virions
1.25 (USDA NADC Faaberg, Lager et al) In vivo pathogenesis studies of Highly Pathogenic PRRSV (HP-PRRSV) of Asian origin .
1.26 (UIUC, Zuckermann) Defining the mechanism(s) by which porcine reproductive and respiratory virus (PRRSV) is able to inhibit the host interferon (IFN)-± response using ZMAC cells.. They report that infection of ZMAC cells with PRRSV does not inhibit the poly(I:C)-induced activation of NFºB, STAT-1 or IRF-3, nor does it inhibit IFN-± or IFN-ß gene transcription. They conclude that the mechanism by which PRRSV inhibits the secretion of IFN-± in PAMs shall involve events occurring at the post-transcriptional level.
1.27 ( UIUC, Yoo ) This lab has recently reviewed the overall strategies for modulation of type I IFN responses. At least three non structural proteins (Nsp1, Nsp2, and Nsp11) and a structural protein (N nucleocapsid protein) have been included in this manuscript.
1.28 (UIUC,Yoo) This lab has also focused on the role of GP4 on molecular pathogenesis of PRRSV. The GP4 was found to co-localize with CD163 in the lipid rafts on the plasma membrane, thus suggesting an important role of lipid rafts during entry of the virus.
1.29 (UIUC,Yoo) Studies on N protein have focused on immunomodulatory properties of the PRRSV N protein and the linkage between IL-10 production and development of PRRSV-induced Treg. Their results support an immunomodulatory function of the PRRSV N protein that may contribute to the immunopathogenesis of PRRSV.
1.30 (UNL) Using reverse genetics (alanine-scanning mutagenesis) this group has identified amino acid residues in NSP1 important for anti-IFN activity of porcine reproductive and respiratory syndrome virus non-structural protein 1. They were able to obtain a NSP1² mutant that presented an in vitro and in vivo distinct phenotype with relieved anti-IFN effect. However, the mutant reverted in vivo within the first week post-infection. The results indicate a strong selection pressure towards maintaining the IFN-inhibitory property of PRRSV for successful propagation in pigs.
1.31 (UNL) This group also studied the molecular determinants of anti-TNF± mediated by PRRSV NSP1. Using the same approach as in 1.30 ( see above). Two mutant viruses, with mutations at Nsp1± Gly90 orNsp1² residues7074, generated from infectious cDNA clones exhibited attenuated viral replication in vitro and TNF-± was found to be up-regulated in infected macrophages. In infected pigs,theNsp1² mutant virus was attenuated in growth. These studies provide insights into how PRRSV evades the effector mechanisms of innate immunity during infection.

Objective 2. Understand the ecology and epidemiology of PRRSV and emerging viral diseases of swine.

2.1 (MN) In the area of transmission and ecology of influenza in pigs, MN has documented the detection and isolation of influenza virus from air samples collected from pigs under different production conditions, highlighting the risk for influenza transmission in aerosols. Using the same air sampling methodology, they have determined transmission rates of influenza for immune (either vaccinate or with natural maternal immunity) and non-immune populations were assessed
2.2 (UGA) Role of re-assortment in influenza: At UGA is explored the potential for swine, human and avian influenza viruses to reassert on the TRIG backbone in primary swine epithelial cells, and on primary human epithelial cells.
2.3 (UGA) Research related to mechanisms of transmission in influenza. These involve UGA studies on potential for avian and swine origin influenza viruses to infect and transmit in cats and ferrets, as well as chicken-duck transmission and pathogenesis of H1 viruses from different species using the ferret model
2.4 (UGA) Research related to influenza Immunity and cross-protection  They have tested efficacy of aerosolized vaccines and the potential for hetero-subtypic immunity. They also explored the potential for a novel vaccine vector (PIV5) to serve as a vaccine against highly pathogenic avian influenza virus.
2.5(UGA) Research related to flu epidemiology. UGA Explored the potential for avian influenza viruses to infect felines and tested feral cats for exposure to AIVs to determine their potential as an alternate reservoir or vector. They also developed a novel surface enhanced Raman spectroscopic assay for detection of influenza virus. They are currently testing a handheld device for field use, as well as evaluating clinical specimens using the device.
2.6 (KSU Rowland, Dekkers) studies of PRRSV neutralizing antibody responses in large numbers of experimentally infected pigs. They identified subpopulation of pigs with high and broad titers of neutralizing antibody. This response has a host genetic component.
2.7 (KSU, Rowland) They have identified an immunodominant epitope, CP(169-180), linked to PCV2 immunopathogenesis.

Objective 3. Develop effective and efficient approaches for detection, prevention and control of PRRSV and emerging viral diseases of swine.

3.1 (UGA) the swine virology group at UGA reports studies on the host gene requirements for influenza virus replication, and how microRNA govern their expression. These studies have identified key miRNAs and multiple cellular targets for avian, human and swine influenza viruses.
3.2 (UGA) A research project at UGA is aimed at evaluating if genetic changes occur as PRRS replicates in iPSC cells ( continuous macrophage-like cell line) .
3.3(UMN) Air filtration systems have been demonstrated to reduce the risk of introduction of PRRSV contaminated aerosol into susceptible herds. A large retrospective study was designed to assess the incidence of PRRSV outbreaks in filtered when compared with non-filtered sow farms and the profitability of the investment based on real production data of these farms during the period October 2004 to June 2011.
3.4 (KSU Rowland, SDSU Fang, ISU Opriesnnig) these different labs are collaborating on the development of a Luminex platform for the detection of antibodies against PRRSV, PCV2 and SIV.
3.5 (KSU Gabler, Rowland) at KSU they are performing a study about the effect of PRRSV infection on feed digestibility.
3.6 (USDA BARC, SDSU + PHGC) A multiplex FMIA has been developed to simultaneously quantify porcine cytokines in serum using Luminex xMap" technology. Using such FMIA, they have evaluated the serum levels of different cytokines during the progress of PRRSV infection. They find that serum concentrations of IL-8, IFNa and CCL2 are significantly altered after PRRSV infection. These investigators are currently probing for genetic effects and correlations of cytokine profiles with serum viral levels and growth performance.
3.7 (USDA BARC,SDSU) These groups have used Fluorescent Microsphere Immunoassays (FMIAs) to test the cytokine levels in oral-pharyngeal fluid (OF) samples from PRRSV vaccinated or infected pigs that were either experimentally vaccinated for, or infected with, PRRSV. Their results suggest that OF can be used to evaluate the immune responses of pigs. The data showed that all pigs expressed the innate cytokines (IL-1b and IL-8) in OF whereas only vaccinated pigs mounted a T helper 1 (Th1) response (IL-12 and higher IFN³) and thus would be predicted to be able to control the PRRSV infection better than the infected pigs.
3.8 (UIUC) Zuckermanns lab has developed a porcine alveolar macrophage cell (ZMAC) that has been found to efficiently support the replication of many attenuated or virulent strains of PRRSV . These investigators have now compared the immunologic efficacy of one PRRSV MLV (Prime Pac) strain when propagated in either ZMAC or in MARC 145 cells. To test the immunologic efficacy to provide heterologous pretection they challenged all vaccinated and control pigs with PRRSV NADC 20 strain. Based on different immunologic parameters, they report equivalent levels of optimal protection in either case, with the addition of certain immunogenic advantage for the ZMAC-propagated vaccine group. The researchers propose that such advantage would be based on a distinct glycosylation pattern frequently observed on PRRSV GP2 gene occurring in the genome of ensuing vaccine strain when propagated in ZMAC cells but not when propagated in Marc 145 cells.
3.9 (VA Tech , Zhang )This lab reports a immunogenicity study of plant-made oral subunit vaccine against PRRSV. Corn calli were genetically engineered to produce PRRSV viral envelope-associated M protein. Both serum and intestine mucosal antigen-specific antibodies were induced by oral administration of the transgenic plant tissues to mice. In addition, serum and mucosal antibodies showed virus neutralization activity. The neutralization antibody titers after the final boost reached 6.7 in serum and 3.7 in fecal extracts, respectively. A PRRSV-specific IFN-³ response was also detected in splenocytes of vaccinated animals.
3.10 (SDSU) PRRSV Diagnostic news from SDSU: Lateral flow devices for the detection and quantitation of PRRSV in the field are in the process of being validated in collaboration with commercial companies.
3.11(SDSU) development of a fluorescent microsphere immunoassay for detection of antibodies against porcine reproductive and respiratory syndrome virus using oral fluid samples as an alternative to serum-based assays. The development, fine tuning and validation of the FMIA have been used in collaboration with USDA BARC in assessment in OF (see 3.7). This study provides a framework from which a more robust assay could be developed to profile the immune response to multiple PRRSV antigens in a single test.
3.12 (SDSU) Diagnostic advances: Swine influenza fluorescent microsphere immunoassays (FMIA) using xMAP® technology are being developed to measure the responses to vaccine constructs for vaccine potency evaluations in collaborations with commercial companies.
3.13 (SDSU, Hennings, Fang) These labs are experimenting the use of recombinant live PRRSV carrying immunomodulatory genes in their genome. They constructed a recombinant PRRSV (vP129/swIL1²) expressing swine IL-1² from the separate subgenomic mRNA inserted between the ORF1b and ORF2 genome region. The construct was tested in vitro (MARC 145 cells) and in vivo (nursery pig disease model). The vP129/swIL1² exhibited attenuated phenotype in infected pigs. The expression of various cytokines from peripheral blood mononuclear cells measured by fluorescent microsphere immunoassay showed that IL-1², IL4 and IFN³ expression levels were up-regulated in pigs infected with vP129/swIL1² at 7 and 14 days post-infection. However, no detectable level of IL-1² was found in serum samples from pigs infected with either vP129/swIL1² or parental virus. This study shows that a recombinant PRRSV can be used to study the role of different cytokines in disease progression and immune responses.
3.14 (Purdue) new diagnostic developments at Purdue: A relatively new method has been implemented allowing the detection of a wide variety of PRRSV strains by utilizing multiple primer sets and real time PCR technique. They have demonstrated that by using a single primer set in real time PCR, the PRRSV genome was detected across a wide diversity of the viral genome and produced comparable threshold cycle (CT) values to a similar assay available from Tetracore®.
3.15 (USDA NADC (Brockmeier et al) this team conducted animal study to evaluate modulation of innate immunity with G-CSF to prevent secondary bacterial infections and/or mortality induced by HP-PRRSV
3.16 (USDA NADC Nicholson et al) this team is working on the development of a diagnostic nucleotide array
3.17 (USDA NADC Faaberg and Spear) these researchers are working on the development of a PRRSV DIVA vaccine
3.18 (CNB-CSIC) Dr Enjuanes laboratory centers on rTGEV vectors co-expressing PRRSV GP5 and M proteins which they have shown to confer partial protection against PRRSV wt PRRSV infection. The work from this group during this period was focused on the improvement of rTGEV vectors stability and the generation of new antigenic structures that may confer protection against PRRSV: These included: I .Analysis of the stability and expression levels of rTGEVs expressing GP5-NH2 fragments and M protein. II. Purification of PRRSV GP2, GP3 and GP4 envelope proteins and successful generation of polyclonal antibodies, and III. Expression of other PRRSV envelope proteins. To analyze other correlates of protection, an rTGEV vector was constructed expressing PRRSV GP2a, GP3 and GP4. These minor structural proteins are exposed on virus surface assembled as a heterotrimer, and may play a role on protection against PRRSV. They developed a tricistronic rTGEV vector and rTGEV vector expressing GP4 alone respectively. This research is currently ongoing.
3.19 (UWM) Research at University of Wisconsin on genetic and antigenic diversity within PRRS virus was completed this year. They developed a novel analytical approach to identify a small number of representative viral genotypes from among the enormous diversity of viral sequences available in GenBank and PRRSVdb. The method ranks PRRSV sequences in terms of their importance among the diversity of sequences in nature. Viruses represented by the top ranking sequences are valuable targets for future study and can be eventually incorporated into a polyvalent vaccine.
3.20 ( UNL Osorio/Pattnaik, UIUC Zukermann/Laegreid) This collaborative project consisted of mapping, using pepscan technology, T-cell epitope candidates contained in NSP9 and NSP10, both of which are highly conserved. The peptides were probed for their ability to elicit a recall proliferative and interferon-gamma response in peripheral blood mononuclear cells obtained from pigs immunized against the type-II PRRSV strain FL-12. These studies led to the identification of four peptides, two from each NSP9 and NSP10 that appear to contain seemingly highly conserved T-cell epitopes. The identified epitopes may be important for the formulation of immunogens ( i.e peptide vaccines) to provide broad cross-protection against diverse PRRSV strains.

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