NC_OLD229: Detection and Control of Porcine Reproductive and Respiratory Syndrome Virus and Emerging Viral Diseases of Swine

(Multistate Research Project)

Status: Inactive/Terminating

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SAES-422 Reports

Annual/Termination Reports:

[12/29/2009] [12/03/2010] [12/02/2011] [12/02/2012] [12/08/2013]

Date of Annual Report: 12/29/2009

Report Information

Annual Meeting Dates: 12/04/2009 - 12/04/2009
Period the Report Covers: 06/01/2008 - 11/01/2009

Participants

Chair: Lunney, Joan K. ( joan.lunney@ars.usda.gov ) USDA, ARS, BARC, APDL;
Secretary: Meng, X.J. ( xjmeng@vt.edu )Virginia Polytechnic Institute and State University (VA Tech);
Rowland, Raymond R.R. (browland@vet.k-state.edu) Kansas State University (KSU);
Tompkins, S. Mark (smt@uga.edu) University of Georgia (UGA);
Enjuanes, Luis (L.Enjuanes@cnb.csic.es) Centro Nacional de Biotecnología (CNB), CSIC;
Zimmerman, Jeff (jjzimm@iastate.edu) Iowa State University (ISU);
Schommer, Susan (schommers@missouri.edu ) University of Missouri (UMO);
Christopher-Hennings, Jane (jane.hennings@sdstate.edu) SDSU;
Goldberg, Tony (tgoldberg@vetmed.wisc.edu ) University of Wisconsin-Madison;
Zuckermann, Federico A. (fazaaa@uiuc.edu) University of Illinois at Urbana-Champaign (UIUC);
Faaberg, Kay (kay.faaberg@ars.usda.gov) NADC;
Gourapura, Renukaradhya J.(gourapura.1@osu.edu ) The Ohio State University (OSU);
Murtaugh, Michael P (murta001@umn.edu ) University of Minnesota (UMN);
Osorio, Fernando A. (fosorio@unl.edu) University of Nebraska-Lincoln (UNL);
Zhang, Yanjin (zhangyj@umd.edu ) University of Maryland (UMD);
Pogranichniy, Roman (rmp@purdue.edu) Purdue (IN);
Guillermo R. Risatti (guillermo.risatti@uconn.edu ) University of Connecticut (UCONN);
Benfield, David (benfield.2@osu.edu) Ohio State University (OSU);
Johnson, Peter (pjohnson@reeusda.gov )USDA, CSREES;

Other NC229 Scientists;
Garmendia, Antonio - UConn;
Tripp, Ralph - UGA;
Baker, RB - ISU;
Halbur, Patrick - ISU;
Holtkamp, Derald J - ISU;
Harris, DL (Hank) - ISU;
Johnson, John K - ISU;
Karriker, Locke - ISU;
Main, Rodger G - ISU;
McKean, JD - ISU;
Opriessnig, Tanja - ISU;
Platt, Kenneth - ISU;
Strait, Erin - ISU;
Ramamoorthy, Sheila - ISU;
Ramirez, Alejandro - ISU;
Roth, JA - ISU;
Yoon, Kyoung-Jin - ISU;
Yoo, Dongwan - UIUC;
Laegried, Will - UIUC;
Wyatt, Carol - KSU;
Hesse, Dick - KSU;
Sang, Yongming - K-State;
Chang, KC - KSU;
Blecha, Frank - KSU;
Zhu, Xiaoping -UMD;
Davies, Peter - UMN;
Dee, Scott - UMN;
Deen, John - UMN;
Joo, Han Soo - UMN;
Molitor, Tom - UMN;
Morrison, Robert - UMN;
Rossow, Kurt - UMN;
Rovira, Albert - UMN;
Kerhli, Marcus Jr. - NADC;
Lager, Kelly - NADC;
Brockmeier, Susan - NADC;
Miller, Laura - NADC;
Loving, Crystal - NADC;
Neill, John - NADC;
John Butler - University of Iowa;
Saif, Linda J - OSU;
Fang, Ying - SDSU;
Wang, Xiuqing - SDSU;
Nelson, Eric - SDSU;
Wysocki, Michal - BARC;
Chen, Hongbo - BARC;
Smith, Doug - Univ. MI;
Ho, Sam - Univ. MI;
Steibel, JP; Michigan State Univ. (MSU);
Ernst, Cathy - Michigan State Univ. (MSU);
LeRoith, Tanya - VA Tech;
Roberts, P.C. - VA Tech;
Elankumaran, S. - VA Tech;
Mullarky, I.K. - VA Tech;
Pattnaik, Asit - UNL;
Ciobanu, Daniel C. - UNL;
Johnson, Rodger - UNL;

Brief Summary of Minutes

NC229 Meeting Chicago, IL Friday, 12/04/2009 - 12/04/2009.

1. Meeting opened by Chair Joan Lunney; Welcome everyone.

2. Brief remarks by David Benfield, NC229 advisor.

3. Nominations of new officers and voting by station reps: Jane Christopher-Hennings (SDSU) was nominated and approved by station reps as the new NC229 Secretary, and X.J. Meng (Virginia Tech) rotated into the new NC229 chair.

4. Discussion of progress and plans by objective. The NC229 attendees split into groups according to the 3 new objectives, for informal discussions led by the respective objective coordinators and co-leaders. Objective 1: Elucidate the mechanisms of host-pathogen(s) interactions is led by Mike Murtaugh (co-leaders: Roman Pogranichniy, Ying Fang, Aradhya Gourapura)
Objective 2: Understand the ecology and epidemiology of PRRSV and emerging viral diseases of swine is led by Scott Dee (co-leaders: Fred Leung, Jeff Zimmerman) Objective 3: Develop effective and efficient approaches for detection, prevention and control of PRRSV and emerging viral diseases of swine is led by Mark Tompkins (co-leaders: Luis Enjuanes, Antonio Garmendia).

5. Reports from each Objective team: Mike Murtaugh summarized and provided a brief on objective 1 (Elucidate the mechanisms of host-pathogen(s) interactions); Jeff Zimmerman summarized the discussion and progress on objective 2 (Understand the ecology and epidemiology of PRRSV and emerging viral diseases of swine); Mark Tompkins summarized the discussion and progress reports on objective 3 (Develop effective and efficient approaches for detection, prevention and control of PRRSV and emerging viral diseases of swine). Plans for sharing samples and protocols were presented by each group.;
6. Joan Lunney, closing remarks. The NC229 plans its next meeting for Friday December 3, 2010, prior to next year's PRRS Symposium. The leadership of NC229 was turned over to its new Chair X.J. Meng.

Accomplishments

Objective 1. Elucidate the mechanisms of host-pathogen(s) interactions. 1.1. Investigators (Risatti&339;s group) at UCONN studied the transcriptional activation in macrophages upon PRRSV infections. A sustained transcriptional activation was observed for interleukin-1± (IL-1±), IL-6, TNF-±, IFN-², IRF-7, PKR and Mx1, consistent with activation of an antiviral state within PAMs upon infection with virulent FL12v. At 24 hour post-infection (hpi), which corresponds to the peak logarithmic phase of FL12v infectious virus assembly and release, transcription of IL-1±, IL-10, IL-15, IRF-7, VCAM, and Mx-1 genes was significantly different in PAMs infected with cP5U.NSP12 or cPNSP3.8 relative to PAMs infected with FL12v. Live attenuated vaccine (LAV) SP infection induced a pattern of transcriptional activation at 24 hpi similar to that of FL12v. IL-1±, IL-1², IL-10, IL-15, TNF-±, MCP-2, IRF-7, and Mx1 mRNAs accumulation was significantly different in cells infected with the recombinant viruses or LAV SP relative to cells infected with FL12v. The data on induction obtained thus far, shows that PRRSV isolates do induce IFNb in PAM but at variable levels. 1.2. ISU scientists (Zimmerman) conducted studies that led to new estimates of PRRSV persistency for up to 175 days post-inoculation. They provided new information on virus evolution indicating that persistent PRRSV infection does not depend on mutations in ORFs 1b, 5, or 7. The ISU group also showed that among a variety of antibody assays and ELISPOT, the SVN antibody response was the best predictor of both level and duration of viremia. Antibody responses (IDEXX ELISA, N ELISA, and M 3' ELISA) predicted prior exposure to PRRSV, but provided little information regarding the ontogeny of the protective immune response. ELISPOT was a poor prognosticator of PRRSV infection status. 1.3. The UIUC station scientists (Zuckermann) analyzed the expression of CD163 on PAMs and macrophages derived from CD14 positive blood monocytes (MDMs), in correlation with PRRSV replication. By flow cytometric analysis, they showed that the levels of CD163 expression correlated well with the overall level of PRRSV replication. They further examined the effects of modulators of macrophage function, including 12-O-tetradecanoylphorbol-13-acetate (TPA), lipopolysaccharide (LPS), and interleukin (IL)-10 on the expression of CD163 and PRRSV replication. Pre-treatment of PAMs or MDMs with TPA or LPS resulted in decreased expression of CD163 and reduction in PRRSV replication. On the contrary, the incubation of CD14 positive monocytes with IL-10 during differentiation into MDMs resulted in up-regulated expression of CD163 with a corresponding increase in PRRSV infection. By utilizing a yeast two-hybrid screening, they identified that the inhibitor of MyoD family-a (I-mfa) domain-containing protein (HIC) is a cellular partner for PRRS virus (PRRSV) N protein. 1.4. Purdue scientists (Pogranichniy, IN) designed stealth RNAi antisense from specific PRRSV cellular receptor CD163 and co-receptor Siglec-1 (sialoadhesin, Sn, CD169) and 5-UTR region of viral genome and demonstrated significant inhibition of PRRSV infection and spread in MARC-145 cell culture line. PRRSV infection in MARC-145 cultured cell in the absence of endogenous Sn expression resulted in enhanced cellular expression of PTEN which is indicative of negative regulation of the Akt pathway leading to cell arrest and apoptosis. However, in the presence of endogenous Sn, the activation of the Akt pathway was demonstrated by the up regulation of tissue metalloproteinase -9 (MMP-9) mRNA leading to cell growth and survival. 1.5. Scientists at KSU (Sang, Blecha and Rowland) performed a study which identified 39 type I IFN genes. Recombinant IFN proteins expressed from these genes including some novel IFNs, showed a wide range of activities in controlling PRRSV replication. Some are quite effective while others have no activity against PRRSV infections. In addition, Hesse and Rowland are performing an analysis of cross-protection between diverse PRRSV strains. 1.6. Scientists at UMD (Zhang) continued their efforts on developing anti-PRRSV peptide-conjugated phosphorodiamidate morpholino oligomers (PPMOs). Compared with mock-infected controls, the expression of CXCL10, IFN-², and CCL2 in PRRSV-infected PAMs were 2157-, 1740-, and 108-fold higher, respectively. The expression of double-stranded RNA-activated protein kinase R (PKR), interferon regulatory factor 1 (IRF-1), signal transducers and activators of transcription 1 (STAT-1), tumor necrosis factor-± (TNF-±), toll-like receptor 3 (TLR-3), and nuclear factor-kB p65 (NF-kB) in infected PAM s were 7.5-, 6.4-, 6.4-, 8.3-, 2.8-, and 1.7-fold higher, respectively. 1.7. Scientists at the UMN (Murtaugh) demonstrated that pig age influences the pathogenesis of PRRSV infection. Clinical signs were markedly more severe and prolonged in young piglets than in finishers or sows. Viremia was prolonged in weaned pigs for both attenuated and virulent PRRSV. Viremia was reduced in magnitude and duration in finisher pigs and sows. Many sows did not show evidence of viremia following infection with attenuated PRRSV. They also showed that, based upon whole genome sequencing on field isolates, recombination occurs in the field. 1.8. UMN scientists also showed that IFN ³ secreting peripheral blood mononuclear cells were more abundant in sows but not specifically increased by PRRSV infection in any age group, and IL-10 levels in blood were not correlated with PRRSV infection status. These findings show that animal age, perhaps due to increased innate immune resistance, strongly influences the outcome of acute PRRSV infection, whereas an antibody response is triggered at a low threshold of infection that is independent of age. Prolonged infection was not due to IL-10-mediated immunosuppression, and PRRSV did not elicit a specific IFN ³ response, especially in non-adult animals. Equivalent antibody responses were elicited in response to virulent and attenuated viruses, indicating that the antigenic mass necessary for an immune response is produced at a low level of infection, and is not predicted by viremic status. Thus, viral replication was occurring in lung or lymphoid tissues even though viremia was not always observed. 1.9. OSU scientists (Gourapura, Saif) studied natural killer (NK) cell-mediated innate immune cytoxicity in PRRSV and porcine respiratory coronavirus (PRCV) infected pigs. The PRRSV/PRCV dual virus-infected pigs had significantly suppressed innate immune responses, as evidenced by reduced IFN-± level in lung and blood. In addition, they identified a significant reduction in systemic NK cell-mediated cytotoxicity in PRRSV alone infected pigs. Further, upon co-infection with PRCV, there was a synergistic suppression of NK cell-mediated cytotoxicity. Co-infection by PRRSV and PRCV led to enhanced PRRSV replication in lung and a trend towards increased serum Th1 (IFN³ and IL-12), but decreased Th2 (IL-4) cytokine responses, thus clinically exacerbating PRRSV pneumonia. These findings imply that a prior innate immune suppression by immunomodulating respiratory viruses (like that induced by PRRSV) may be a contributing factor to more severe pneumonia due to PRCV infection. 1.10. Scientists at SDSU (Fang) showed that the nonstructural protein 2 (nsp2) of PRRSV has a role in viral replication and may modulate host immunity. Each of the six identified immunodominant nsp2 B-cell epitopes (ES2 through ES7) was deleted from a Type I PRRSV cDNA infectious clone. Deletion of ES3, ES4, or ES7 allowed the generation of viable virus. The ?ES3 mutant showed increased cytolytic activity and more vigorous growth kinetics, while ?ES4 and ?ES7 mutants displayed decreased cytolytic activity and slower growth kinetics in vitro. In a nursery pig model, ?ES4 and ?ES7 mutants exhibited attenuated phenotypes and the ?ES3 mutant produced higher peak viral loads. IL-1² and TNF-± expression levels were down-regulated in cells stimulated (or infected) with the ?ES3 mutant. 1.11. At SDSU, scientists determined that the PRRSV nsp1 protein can antagonize beta interferon (IFN-²) responses. In PRRSV infected cells, we detected the presence of nsp1± and nsp1² and the cleavage sites between nsp1±/nsp1² and nsp1²/nsp2 were identified. Both nsp1± and nsp1² dramatically inhibited IFN-² expression and nsp1² inhibited nuclear translocation of STAT1 in the JAK-STAT signaling pathway. These results demonstrated that nsp1² inhibits both interferon synthesis and signaling, while nsp1± alone strongly inhibits the synthesis of interferon. 1.12. Scientists at USDA-BARC (Lunney) developed PRRS Host Genetics Consortium (PHGC) 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 NPB, USDA, universities and private companies; it represents the first-of-its-kind approach to food animal infectious disease research. The project uses a Nursery Pig Model to assess pig resistance/ susceptibility to primary PRRSV infection. Crossbred pigs were infected with PRRSV and followed for 42 days post infection (dpi). Blood samples were collected at 0,4,7,10,14,21,28,35 and 42 dpi and weekly weights recorded. DNA from all PHGC pigs has been prepared and is being genotyped with the PorcineSNP60 Genotyping BeadChip (containing over 60K single nucleotide polymorphisms or SNPs). Results from the first 5 trials of 200 pigs each have affirmed that all pigs become PRRSV infected; some pigs clear virus from serum quicker and weight effects are variable. 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 PRRS susceptible/reduced growth pigs. Overall, the PHGC project will enable researchers to verify important genotypes and phenotypes that predict resistance/susceptibility to PRRSV infection. 1.13. BARC scientists also identified host gene expression changes that are involved in regulating responses to PRRSV infection and vaccination. With samples collected McCaw at NC State Univ. BARC scientists are testing the effect of PRRSV infection or vaccination on pigs using RNA prepared from tracheobronchial lymph nodes (TBLN), the cranial and distal part of the lung, and tonsils. Pigs were either infected with Minnesota (MNW2B) or NC Powell strains of PRRSV or vaccinated with ATP or were non-treated controls. Mucosal tissue samples were collected from pigs between 3 and 6 days post treatment so that the early innate immune response could be evaluated. RNAs were prepared and hybridization to the swine long oligo array [www.pigoligoarray.org; Steibel et al. 2009] could be assessed. Analyses are underway at BARC with statistical assessment of gene expression patterns performed in collaboration with MSU scientists. Tests of the effect of samples collected after homologous or heterologous PRRS vaccination and challenge are also underway. 1.14. Scientists at VA Tech (Meng) identified and characterized a porcine monocytic cell line supporting PRRSV replication and progeny virion production by using an improved DNA-launched PRRSV reverse genetics system. We developed an improved DNA-launched (plasmid DNA transfection-based) reverse genetics system with reduced cost and labor for PRRSV by introduction of ribozyme elements at both termini of the viral genomic cDNA that were placed under the control of a eukaryotic hybrid promoter. The rescue efficacy of PRRSV with this system was approximately 10-50-fold higher than the in vitro-transcribed RNA-based system and the traditional DNA-launched system without the engineered ribozyme elements, as determined by reporter GFP level in transfected cells and the peak titer of the recovery virus. By using this new reverse genetics system, they identified and characterized a porcine monocytic cell line, 3D4/31, capable of supporting PRRSV replication, progeny virion production, and attachment on the cell surface. 1.15. The VA Tech group (Meng) described the molecular cloning, gene structure, tissue distribution and PRRSV binding characteristics of Porcine DC-SIGN. They cloned and characterized the cDNA and gene encoding porcine DC-SIGN (pDC-SIGN). The full-length pDC-SIGN cDNA encodes a type II transmembrane protein of 240 amino acids. Phylogenetic analysis revealed that pDC-SIGN, together with bovine, canis and equine DC-SIGN, are more closely related to mouse SIGNR7 and SIGNR8 than to human DC-SIGN. pDC-SIGN has the same gene structure as bovine, canis DC-SIGN and mouse SIGNR8 with eight exons. pDC-SIGN mRNA expression was detected in pig spleen, thymus, lymph node, lung, bone marrow and muscles. pDC-SIGN protein was found to express on the surface of monocyte-derived macrophages and dendritic cells, alveolar macrophages, lymph node sinusoidal macrophage-like, dendritic-like and endothelial cells but not of monocytes, peripheral blood lymphocytes or lymph node lymphocytes. A BHK cell line stably expressing pDC-SIGN binds to human ICAM-3 and ICAM-2 immunoadhesins in a calcium-dependent manner, and enhances the transmission of PRRSV to target cells in trans. 1.16. Scientists at UNL (Osorio, Pattnaik) studied the effects on innate and acquired immune responses. The group identified certain pathogenic mechanisms (such as decoy epitope deploying or glycan shielding) that would suggest that PRRSV employs diverse strategies to subvert and/or evade the hosts immune response, thus securing an abundant and unrestricted viral replication during the acute phase of infection and a long persistence in the host. They recently confirmed this weak IFN induction phenotype exhibited by PRRSV in monocyte-derived swine macrophages. They have recently screened all non-structural proteins (NSPs) of PRRSV identifying at least four (NSP1, NSP2, NSP4 and NSP11) having inhibitory activity towards IFN production. Of these, the strongest inhibitor of IFN production is NSP1², affecting primordially dsRNA signaling pathways. 1.17. UNL station scientists (Osorio, Pattnaik) also studied the role of viral genes in virulence and determinants. They initiated reverse genetics experiments to determine the molecular basis of attenuation of virulence in PRRSV. They have used the infectious clone for the development of chimeras between PRRSV strains of different degrees of virulence, and shown that a NSP-coding area of the PRRSV genome is a major cluster of virulence. Also ORF5 and ORF2 contain structural determinants of virulence. Likewise, site-specific mutagenesis of GP5 (product of ORF5) indicated that PRRSV evades the pigs immune system by means of a glycan-shielding mechanism. They have also studied epitopes in different PRRSV proteins that could be deleted or modified to be used as serologic differential markers. Objective 2. Understand the ecology and epidemiology of PRRSV and emerging viral diseases of swine. 2.1. ISU scientists (Zimmerman) performed studies that led to new estimates of R0 for PRRSV in different operations. They performed survival analysis of PRRSV transmission within herds and provided an overview of PRRSV routes of transmission and patterns of circulation. In addition, they continued work on PRRSV aerosols transmission, including methods of inactivating airborne viruses, and their research shows that PRRSV is difficult to transmit to susceptible pigs via consumption of meat from PRRSV-infected animals. 2.2. Scientists (Rowland and Hesse) at KSU in collaboration with Lunney (BARC) and others participated in the PRRS Host Genetics Consortium. The results for the year include the infection and sample collection of 400 pigs at K-State. The results reveal the appearance of stratified subpopulations which possessed wide variations in weight, virus load and growth performance. 2.3. At the UMN, diagnostic tests for PCV2 and differential PCR for subtypes 2a and 2b were refined, and ultrastructural studies were conducted to detect coinfecting agents in clinical samples. The tools were applied to epidemiologic studies of PCV2, including the ecology of PCV2 in boar studs, and associations of PCV2 subtypes with clinical disease. 2.4. Scientists at NADC continued to develop and provide materials and reagents to investigators. (Miller) Porcine modified Identitag (16bp tags) annotated database has been created with Dr Greg Harhay, USDA, ARS, USMARC, 2009; (Miller) MARC-145 cells were provided to Dr. Moiz Kitabwalla, Lipid Sciences; Dr S. Mark Tompkins, UGA; (Faaberg)- MARC-145 cells provided to Dr. Margo Brinton, Georgia State University; MARC-145, MA-104 and CL2621 cells provided to Dr. Jens Kuhn, Integrated Research Facility Frederick; (d) Faaberg group - Infectious clone pVR-V7 provided to Dr. Frederick Leung, Hong Kong University; Dr. Sergey Parinov, The National University of Singapore; Dr. István Kiss, National Veterinary Institute. 2.5. NADC scientists (Faaberg)- showed that nsp2 has been shown to be immunogenic, contains hypervariable segments, encodes a protease responsible for replicase cleavage and harbors B-cell epitopes. They studied the nature of the PL2 protease, when nsp2 was individually expressed in CHO cells and not associated with virus. They found that the PL2 cysteine protease domain possesses both trans- and cis-cleavage activities, and cleaved only at or near the G|G at nsp2 amino acids 1196|1197|1198. They also analyzed nsp2 when expressed from the viral genome in MARC-145 cells, and found that nsp2 was now found as at least 6 isomers, all containing the N-termini, but differing in size. They also discovered that heat shock 70kDa protein 5 (HSPA5) was bound to nsp2. 2.6. Scientists at NADC (Faaberg) also conducted in vivo study of two new isolates along with strains MN184 and SDSU-73. Serum and lavage samples are now being analyzed by virus isolation, qRT-PCR, IFN-gamma, ELISA, and lyphadenopathy. Also, previous results suggested that one section of strain VR-2332 nsp2, when deleted, resulted in virus that did not cause lymphadenopathy when infected into young swine. The NADC scientists are now examining that polypeptide when expressed in adenovirus. The viruses have been made, but no in vivo studies have been done. 2.7. Research at UWI (Goldberg) has begun to characterize genetic and antigenic diversity within PRRS virus, in an effort to identify a small number of representative viral genotypes for further testing, and that can be eventually incorporated into a polyvalent vaccine. Because of the large number of PRRSV sequences available (over 10,000 in the literature), the computational aspect of this work is challenging. The project has just begun, and data are currently being compiled and edited. Objective 3. Develop effective and efficient approaches for detection, prevention and control of PRRSV and emerging viral diseases of swine. 3.1. Investigators at the UGA (Tompkins, Tripp) intended to develop novel vaccine strategies for prevention of PRRSV, however all PRRSV efforts were set aside because of the influenza pandemic caused by H1N1 virus. The novel pandH1N1 virus was first identified in April 2009. UGA received the virus (A/CA/04/09) and initiated pathogenesis studies in swine and ferrets. The ferret serves as the primary model of human influenza virus infection. It is susceptible to infection with human influenza viruses without adaptation, sheds virus, and presents similar symptoms. Remarkably, the pandH1N1 replicated to very high titers in ferrets (>1e8 TCID50/ml of nasal wash), even when inoculated with very low 100 TCID50) doses of virus but failed to demonstrate significant clinical disease. Virus primarily replicated in the upper respiratory tract, but in some cases was detected in the lungs of infected ferrets. A/CA/04/09 infected pigs and caused clinical symptoms including loss of appetite, coughing, nasal discharge, and reduced activity. 3.2. ISU scientists (Zimmermann) conducted extensive studies on the use of oral fluids for the detection of PRRSV, anti-PRRSV antibody, and other pathogens - a new highly cost-effective approach for surveillance PRRSV and other pathogens in commercial settings. 3.3. Scientists at UIUC (Zuckermann) recently developed a porcine alveolar macrophage cell line. This cell line, named ZMAC, was found to efficiently support the replication of a number PRRS virus isolates, often achieving high titers (>107 TCID50/ml). Given the apparent high permissiveness of ZMAC cells to PRRS virus, they tested the proficiency of this cell line to isolate field PRRS virus from clinical samples. The ZMAC line proved highly efficient (>90%) at isolating PRRS virus within 72 hours after exposing ZMAC cells to pig serum samples known to be positive to PRRSV by real-time PCR, from which attempts to isolate PRRS virus in MARC-145, and even primary alveolar macrophages, had failed. The ZMAC cell line may prove useful for PRRSV vaccine development. 3.4. Scientists at UIUC engineered the viral genome to transcribe an additional subgenomic RNA initiating between non-structural and structural genes. Two unique restriction sites and a copy of the transcription regulatory sequence for ORF6 (TRS6) were inserted between ORFs 1b and 2a, yielding a general purpose expression vector. The enhanced green fluorescent protein (GFP) gene was cloned between the unique sites such that the inserted gene was transcribed from TRS2 which was located upstream within ORF1b, while the copy of TRS6 drives ORF2a/b transcription. Cells infected with P129-GFP produce virus progeny and showed fluorescence and the inserted gene was phenotypically stable for at least 37 serial in vitro passages. Subsequently, a capsid protein gene was cloned from PCV2 and inserted into the PRRSV infectious clone vector, generating virus "P129-PCV". Pigs immunized with either P129-GFP or P129-PCV2 produced antibodies specific for GFP or PCV2 capsid respectively. 3.5. Scientists at KSU (Rowland, Hesse) along with several others are incorporating Luminex for the detection of IgM and IgG antibodies to PRRSV and other pathogens. The results provide the opportunity to develop assays for the purpose of profiling multiple agents within a herd. 3.6. UMD scientists continued to develop anti-PRRSV PPMOs. PPMOs are single-stranded DNA analogs containing a modified backbone and cell-penetrating peptide. They examined PPMO-mediated inhibition of PRRSV replication in PAMs and found that (a) PAMs uptake PPMO efficiently. The uptake efficiency of PAMs of 72-hour pre-incubation was higher than PAMs of 24-h pre-incubation; (b) Treatment of PAMs with PPMO 5UP2 resulted in protection from PRRSV-induced cell death for at least seven days, and produced no elevation in the activity of the caspase 3, 7, 8 and 9; (c) 5UP2 treatment of PRRSV-infected PAMs also prevented the vigorous induction of interferon-² and chemokines observed in infected and mock-treated PAMs 3.7. Scientists at the UMN (Dee) completed the year 3 of the evaluation of air filtration as a means to reduce the risk of aerosol transmission of PRRSV at the SDEC production region model. A standardized means to validate the efficacy of biosecurity interventions to reduce the risk of airborne spread of PRRSV was developed. Assessment of air filtration to reduce the risk of airborne spread of PRRSV in large sow units in swine dense regions was initiated. Efficacy of regional control of PRRS was evaluated in Stevens County, Minnesota. Communication and implementation of best practices has reduced PRRS incidence to one infected herd in 89 existing sites. 3.8. Scientists at the OSU (Gourapura) evaluated the mucosal immune responses in the respiratory tract of pigs using innate immune cell specific agents as candidate adjuvants administered by the intranasal (IN) route. They initially determined the adjuvanticity of nine different bacterial preparations belongs to Mycobacterium tuberculosis, Streptoccocus pyogenes, and Vibrio cholera, administered intranasally (IN) to pigs. Based on the general mucosal immune responses elicited by the individual candidate adjuvants, we chose M. tuberculosis whole cell lysate (M. tb WCL) for further studies. Analyses are underway to explore the extended adjuvanticity of M. tb WCL, used with PRRSV modified live virus vaccine administered IN to PRRSV sero-negative pigs. Also they will perform challenge studies in mucosally immunized pigs using homologous (VR2332) and heterologous (MN184) PRRSV strains. 3.9. At SDSU station (Christopher-Hennings), a long-term objective is to provide a PRRSV-free semen supply for artificial insemination so mechanical and anti-viral methods were evaluated to reduce risk of transmission. A unilayer density gradient centrifugation method to purify PRRSV contaminated semen allowed for some risk reduction by eliminating PRRSV from 71% of semen samples tested. The antiviral chymostatin inhibited PRRSV infection in-vitro. However, further testing is needed to determine an effective animal dose and evaluate effects on sperm quality parameters. 3.10. Scientists at SDSU (Christopher-Hennings, Lawson) developed a multiplex assay to simultaneously quantify 9 porcine cytokines in serum using Luminex xMap" technology, and the assay was optimized to detect innate (IL-1b, IL-6, IL-8, IFN-a, TNF-a); regulatory (IL-10), T helper 1 (Th1) (IL-12, IFN-g) and Th2 (IL-4) cytokines. The assay will be of value in vaccine and challenge studies as well as for determining genetic resistance to PRRSV and immune responses to other swine pathogens. 3.11. Scientists at CSIC in Spain (Enjuanes) conducted animal experiments using the TGEV vector expressing PRRSV GP5 and M proteins, it was found that all animals present a high antibody response against TGEV, therefore, the vector infected target tissues as expected. Also, vaccinated animals showed a clear antibody response against the PRRSV antigens (i.e., GP5 and M proteins). After a challenge with a virulent PRRSV isolate, a fast recall response was observed, as vaccinated animals induced higher antibody titers against PRRSV antigens and earlier than control animals. Nevertheless, the immune response was not strong enough to provide full protection against PRRSV. That was likely due to the low levels of neutralizing antibodies produced before challenge. 3.12. Scientists at CSIC in Spain (Enjuanes) generated a set of rTGEV vectors expressing M protein and GP5 mutants with a modified glycosylation pattern. Just one of them was stable, a rTGEV expressing PRRSV M protein and a GP5 N46S mutant, lacking the glycosylation site overlapping neutralizing epitope. This rTGEV vector expressed high levels of GP5 and M PRRSV proteins in 74 % and 85% of the infected cells, respectively. A short in vivo immunization protocol was performed. One-week old piglets were inoculated with 1x108 pfu of the rTGEV by three routes: oral, nasal and intragastric. All animals present a high antibody response against TGEV, therefore, the vector infected target tissues as expected. Vaccinated animals showed a clear humoral response against PRRSV. A killed vaccine was also developed based on rTGEV expressing GP5-N46S mutant and M proteins. The protection conferred by this vaccine was tested. Vaccinated animals induced higher and faster antibody titers against PRRSV antigens than control animals. Neutralizing antibodies titers were also higher in the vaccinated animals when compared with non-vaccinated ones, suggesting that the elimination of glycosylation site close to the neutralizing epitope improves protective immune response against PRRSV. The presence of an immunodominant (decoy) epitope close to the neutralizing epitope in GP5 could be deleterious for a strong neutralizing immune response. Therefore, an rTGEV vector was constructed, expressing a GP5 protein lacking the non-neutralizing (decoy) epitope and the N46 glycosylation site. The virus was recovered with high titer and GP5 and M protein expression was stable in 65% and 90% of the infected cells, respectively.

Publications

Ando A, Uenishi H, Kawata H, Tanaka M, Shigenari A, Flori L, Chardon P, Lunney JK, Kulski JK, Inoko H. 2008. Microsatellite diversity and crossover regions within homozygous and heterozygous SLA haplotypes of different pig breeds. Immunogenetics. 60: 399-407. Beura, L. K., Sarkar, S. N., Kwon, B. J., Subramaniam, S., Jones, C., Pattnaik, A. K., and Osorio, F. A. (2010). Porcine Reproductive and Respiratory Syndrome Virus nonstructural protein nsp1b modulates host immune response by antagonizing IRF3 activation. J. Virology In press. Boyd P, Hudgens E, Loftus JP, Tompkins D, Wysocki M, Kakach L, LaBresh J, Baldwin CL, Lunney JK. 2009. Expressed gene sequence and bioactivity of the IFN³-response chemokine CXCL11 of swine and cattle. Vet. Immunol. Immunopathol. Submitted. Brockmeier, S. L., K. M. Lager, M. J. Grubman, D. E. Brough, D. Ettyreddy, R. E. Sacco, P. C. Gauger, C. L. Loving, A. C. Vorwald, M. E. Kehrli, Jr., and H. D. Lehmkuhl. 2009. Adenovirus-mediated expression of interferon-alpha delays viral replication and reduces disease signs in swine challenged with porcine reproductive and respiratory syndrome virus. Viral Immunol. 22:173-180. Brown, E., S. Lawson, C. Welbon, M. P. Murtaugh, E. A. Nelson, J. J. Zimmerman, R. R. R. Rowland, Y. Fang. 2009. Antibody response of nonstructural proteins: implication for diagnostic detection and differentiation of Type I and Type II porcine reproductive and respiratory syndrome virus. Clinical and Vaccine Immunology. 16(5):628-35. Butler, J.E., P. Weber, N. Wertz and K.M. Lager. 2008. Porcine reproductive and respiratory syndrome virus (PRRSV) subverts development of adaptive immunity by proliferation of germline-encoded B cells with hydrophobic HCDR3s. J. Immunol. 180: 2347-2356. Calzada-Nova, G., Schnitzlein, W., Husmann, R., Zuckermann, F.A. 2009. Characterization of the cytokine and maturation responses. of pure populations of porcine plasmacytoid dendritic cells to porcine viruses and toll-like receptor agonists. Vet. Imm. Immunopath. doi: 10.1016/j.vetimm.2009.10.26. Cano JP, Dee SA Murtaugh MM, and Morrison RB. Infection dynamics and clinical manifestations following experimental inoculation of gilts at 90 days of gestation with porcine reproductive and respiratory syndrome virus. . Can J Vet Res (Accepted for publication). Cano, J.P., S.A. Dee, M.P. Murtaugh, A. Rovira, and R.B. Morrison. 2008. Infection dynamics and clinical manifestations following experimental inoculation of gilts at 90 days of gestation with a low dose of porcine reproductive and respiratory syndrome virus. Can. J. Vet. Res. 73:303-307. Chang C-C, Yoon K-J, Zimmerman JJ. 2009. Persistent porcine reproductive and respiratory syndrome virus (PRRSV) infection in pigs does not require significant genetic change. J Swine Health Prod 17(6):318324. Chen Z, Zhou X, Lunney JK, Lawson S, Sun Z, Brown E, Christopher-Hennings J, Knudsen D, Nelson EA. Fang Y. 2009. Immunodominant epitopes in nsp2 of porcine reproductive and respiratory syndrome virus are dispensable for replication but play an important role in viral pathogenesis. J Gen Virology. Epub. 11/18/09. Chen, Z, S. Lawson, Z. Sun, X. Zhou, X. Guan, J. Christopher-Hennings, E. A. Nelson, Y. Fang Identification of two auto-cleavage products of nonstructural protein 1 (nsp1) in porcine reproductive and respiratory syndrome virus infected cells: nsp1 function as interferon antagonist. Virology (accepted). Chen, Z., X. Zhou, J. K. Lunney, S. Lawson, Z. Sun, E. Brown, J. Christopher-Hennings, D. Knudsen, E. Nelson, Y. Fang. Immunodominant epitopes in nsp2 of porcine reproductive and respiratory syndrome virus are dispensable for replication but play an important role in modulation of host immune response. J. Gen. Virology (accepted). Cruz, J.L.G., Zuñiga, S., Sanchez, C.M., Ceriani, J.E., Urniza, A., Plana-Duran, J. and Enjuanes L. Immunogenicity of a TGEV-based vector expressing porcine reproductive and respiratory syndrome virus GP5 and M proteins. Vaccine. Submitted. Das, P. B., Dinh, P. X., Ansari, I. H., de Lima, M., Osorio, F. A., and Pattnaik, A. K. (2010). The Minor Envelope Glycoproteins GP2a and GP4 of Porcine Reproductive and Respiratory Syndrome Virus Interact with the Receptor, CD163. J. Virology In press. Deendayal Patel, David A. Stein, and Yan-Jin Zhang: Morpholino Oligomer-Mediated Protection of Porcine Pulmonary Alveolar Macrophages from Arterivirus-Induced Cell Death. Antiviral Therapy 2009. In Press. de Lima, M. Ansari, I. H., Das, P. B., Ku, B., Martinez-Lobo, F. J., Pattnaik, A. K., and Osorio, F. A. (2009). GP3 is a Structural Component of the PRRSV Type II Virion. Virology, 390:31-36. de Abin, M.F, G. Spronk, M. Wagner, M. Fitzsimmons, J. Abrahante, and M.P. Murtaugh. 2009. Comparative infection efficiency of porcine reproductive and respiratory syndrome virus field isolates on MA104 cells and porcine alveolar macrophages. Can. J. Vet. Res. 73:200-204. Dee SA, Otake S, Oliviera S and Deen J. Evidence of long distance airborne spread of porcine reproductive and respiratory syndrome virus and Mycoplasma hyopnuemoniae. Vet Res 2009, 40(4)39. Dee SA, Pitkin AN and Deen J. Evaluation of alternative strategies to MERV 16-based air filtration systems for reduction of the risk of airborne spread of porcine reproductive and respiratory syndrome virus. Vet Microbiol doi:10.1016/j.vetmic.2009.03.019. Du, Y., Zuckermann, FA, and Yoo, D. 2009. Myristoylation of the small envelope protein of porcine reproductive and respiratory syndrome virus is non-essential for virus infectivity but negatively affects its growth. Virus Res. (submitted) Ellingson JS, Wang, Y., Layton, S., Ciacci-Zanella, J., Roof, M. B., and Faaberg, K. S. Vaccine efficacy of porcine reproductive and respiratory syndrome virus chimeras. Vaccine, submitted. Fang, Y., J. Christopher-Hennings, E. Brown, H. Liu, Z. Chen, S. Lawson, R. Breen, T. Clement, X. Gao, J. Bao, D. Knudsen, R. Daly and E.A. Nelson. 2008. Development of genetic markers in the non-structural protein 2 region of a US type 1 porcine reproductive and respiratory syndrome virus: implications for future recombinant marker vaccine development. J. Gen. Virol. 89:3086-3096. Gaudreault, N, RR Rowland, CR Wyatt. 2009. Factors affecting the permissiveness of porcine alveolar macrophages for porcine reproductive and respiratory syndrome virus. In press Archiv Virol. Gudmundsdottir I, Risatti GR. Infection of porcine alveolar macrophages with recombinant chimeric porcine reproductive and respiratory syndrome virus: effects on cellular gene transcription and virus growth. Virus Res. 2009 Oct;145(1):145-50. Lunney JK. 2008. Genetics of Infectious Disease Resistance in Animals. Proceedings American College of Veterinary Pathologists Meeting 2008. P. 240-242. Tuggle CK, Wang YF, Couture OP, Qu L, Uthe JJ, Kuhar D, Lunney JK, D. Nettleton D, J.C. Dekkers JC, Bearson SMD. 2008. Computational Integration of Structural and Functional Genomics Data across Species to Develop Information on Porcine Inflammatory Gene Regulatory Pathway. Dev Biol (Basel). 132: 105-13. Lunney JK, Ho C-S, Wysocki M, Smith DM. 2009. Molecular genetics of the swine major histocompatibility complex, the SLA complex. Dev Comp Immunol. 33: 362-74. Lunney JK, Fritz ER, Reecy JM, Kuhar D, Prucnal E, Molina R, Christopher-Hennings J, Zimmerman J, Rowland RRR. 2009. Interleukin-8, interleukin-1b and interferon-g levels are linked to PRRS virus clearance. Viral Immunology. In Revision. Han J, Rutherford, M. S., and K. S. Faaberg. 2009. Porcine reproductive and respiratory syndrome virus nsp2 cysteine protease domain possesses both trans- and cis-cleavage activities. J. Virol. 83:9449-9463. Han, J., M. S. Rutherford, and K. S. Faaberg. Proteolytic products of the porcine reproductive and respiratory syndrome virus nsp2 replicase protein. J. Virol., submitted. He D., C.C. Overend, R.J. Maganti, J. Ambrogio, , M.J. Grubman and A.E. Garmendia 2009. Marked differences between MARC-145 cells and alveolar macrophages in IFN ²-induced activation of antiviral state against PRRSV. Submitted to Vet Imm Immunopath Hermann JR, Brockmeier SL, Yoon KJ, Zimmerman JJ. 2008. Detection of respiratory pathogens in air samples from acutely infected pigs. Can J Vet Res 72:367-370. Hermann JR, Muñoz-Zanzi CA, Zimmerman JJ. 2009. A method to provide improved dose-response estimates for airborne pathogens in animals: An example using porcine reproductive and respiratory syndrome virus. Vet Microbiol 133:297-302. Hermann JR, Zimmerman JJ. 2008. Analytical sensitivity of air samplers based on uniform point source exposure to airborne porcine reproductive respiratory syndrome virus and swine influenza virus. Can J Vet Res 72:440-443. Ho C-S, Lunney JK, Franzo-Romain MH, Martens GW, Lee Y-J, Lee J-H, Wysocki M, Rowland RRR, Smith DM. 2009. Molecular characterization of swine leukocyte antigen (SLA) class I genes in outbred pig populations. Animal Genetics. 40: 468-78. Ho C-S, Lunney JK, Ando A, Rogel-Gaillard C, Lee J-H, Schook LB, Smith DM. 2009. Nomenclature for factors of the SLA system, update 2008. Tissue Antigens. 73: 307-315. Ho, C-S, Y-J Lee, JK Lunney, MH Franzo-Romain, GW Martens, J-H Lee, M Wysocki, RRR Rowland, DM Smith. 2009. Molecular characterization of swine leukocyte antigen (SLA) class I genes in outbred pig populations". Accepted for publication Anim Genetics. Holtkamp DJ, Yeske PE, Polson DD, Melody JL, Philips RC. 2009. A prospective cohort study evaluating duration of breeding herd PRRS virus-free status and its relationship with measured risk. Prev. Vet. Med. (submitted). Holtkamp DJ., Yu L, Polson DD, OConnor A. 2009. External and internal biosecurity factors associated with the occurrence of clinical PRRS breaks. Vet Res (submitted). Huang YW, Fang Y, Meng XJ. Identification and characterization of a porcine monocytic cell line supporting porcine reproductive and respiratory syndrome virus (PRRSV) replication and progeny virion production by using an improved DNA-launched PRRSV reverse genetics system. Virus Res. 2009 Oct;145(1):1-8. Huang YW, Dryman BA, Li W, Meng XJ. Porcine DC-SIGN: molecular cloning, gene structure, tissue distribution and PRRSV binding characteristics. Dev Comp Immunol. 2009 Apr;33(4):464-80. Jar, A.M., Osorio, F.A., Lopez, O.J. 2009 Mouse x pig chimeric antibodies expressed in baculovirus retain the same properties of their parent antibodies. Biotechnology Progress Mar-Apr;25(2):516-23. Jacobs AC, Hermann JR, Muñoz-Zanzi C, Prickett JR, Roof MB, Zimmerman JJ. 2010. Thermostability of porcine reproductive and respiratory syndrome virus in solution. J Vet Diagn Invest 22: (in press). Kim W-I, Kim J-J, Cha S-H, Yoon K-J. 2008. Different biological characteristics between wild-type PRRS viruses and vaccine viruses and identification of the corresponding genetic determinants. J Clin Microbiol 46:1758-1768. Kim W-I, Yoon K-J. 2008. Molecular assessment of the role of envelope-associated structural proteins in cross neutralization among different PRRS viruses. Virus Genes 37:380-391. Jung, K., G.J. Renukaradhya, K.P. Alekseev, Y. Fang, Y. Tang, and L.J. Saif (2009). Porcine reproductive and respiratory syndrome virus modifies innate immunity and alters disease outcome in pigs subsequently infected with porcine respiratory coronavirus: implications for respiratory viral co-infections. J. Gen. Virol. 90:2713-23. Kim, DY, TJ Kaiser, K Horlen, ML Keith, LP Taylor, R Jolie, JG Calvert, RR Rowland. 2008. Insertion and deletion in a non-essential region of the nonstructural protein 2 (nsp2) of porcine reproductive and respiratory syndrome (PRRS) virus: effects on virulence and immunogenicity. In press Virus Genes. Klinge, K.L., E.M. Vaughn, M.B. Roof, E.M. Bautista, and M.P. Murtaugh. 2009. Age-dependent resistance to Porcine reproductive and respiratory syndrome virus replication in swine. Virol. J. 6:177-187. Loving, C. L., S. L. Brockmeier, A. L. Vincent, K. M. Lager, and R. E. Sacco. 2008. Differences in clinical disease and immune response of pigs challenged with a high-dose versus low-dose inoculum of porcine reproductive and respiratory syndrome virus. Viral Immunol. 21:315-325. Lunney JK, Fritz ER, Reecy JM, Kuhar D, Prucnal E, Molina R, Christopher-Hennings J, Zimmerman J, Rowland RRR. 2010. Interleukin-8, interleukin 1² and interferon-³ levels are linked to PRRS virus clearance. Viral Immunol (in press). Metwally, S., F. Mohamed, T. Burrage, M. Prarat, K. Moran, A. Bracht, G. Mayr, M. Berninger, K. S. Faaberg, L. Koster, L.T. Thanh, V.L. Nguyen, M. Reising, S. Swenson, J. Lubroth and C. Carrillo. Pathogenicity and molecular characterization of emerging porcine reproductive and respiratory syndrome virus in Vietnam 2007. Transboundary and Emerging Diseases, submitted. Miller, L.C., Lager, K.M. and Kehrli Jr., M.E. 2009. Effect of porcine reproductive and respiratory syndrome virus infection of porcine alveolar macrophages on Toll-like receptors elicitation of type I interferon responses. Clinical and Vaccine Immunology 16:360-365. Molina, R.M., E.A. Nelson, J. Christopher-Hennings, R. Hesse, R.R. Rowland, J.J. Zimmerman, 2009. Evaluation of the risk of PRRSV transmission via ingestion of muscle from persistently-infected pigs. Transboundary and Emerging Diseases 56:1-8. Molina R, S.-H. Cha, W Chittick, S. Lawson, M.P. Murtaugh, E.A. Nelson, J Christopher-Hennings, K.-J. Yoon, R. Evans, R.R.R. Rowland and J.J. Zimmerman. 2008. Immune response against porcine reproductive and respiratory syndrome virus during acute and chronic infection. Veterinary Immunology and Immunopathology. Molina, R.M., W. Chittick, E.A. Nelson, J. Christopher-Hennings, R.R.R. Rowland and J.J. Zimmerman. 2008. Diagnostic performance of assays for the detection of anti-PRRSV antibodies in porcine muscle transudate (meat juice) samples. J. Vet. Diagn. Invest. 20:735-743. Mohammadi, H, S Sharif, RRR Rowland, D. Yoo. 2009. The lactate dehydrogenase-elevating virus capsid protein is a nuclear-cytoplasmic protein. Arch Virol. 154:1071-1080. Murtaugh, M.P., C.R. Johnson, Z. Xiao, R.W. Scamurra, and Y. Zhou. 2009. Species specialization in cytokine biology: is interleukin-4 central to the TH1-TH2 paradigm in swine. Develop. Comp. Immunol. 33:344-352. Opriessnig T, Patterson AR, Madson DM, PalM, Rothschild M, Kuhar D, Lunney JK, Juhan NM, Meng XJ, Halbur PG. 2009. Difference in severity of porcine circovirus type 2 (PCV2)-induced pathological lesions and disease between Landrace and Pietrain pigs. J. Animal Science. 87: 1582-90. Opriessnig T, Madson DM, Prickett JR, Kuhar D, Lunney JK, Elsener J, Halbur PG. 2008. Effect of porcine circovirus type 2 (PCV2) vaccination on porcine reproductive and respiratory syndrome virus (PRRSV) and PCV2 coinfection. Vet. Microbiol. 131: 103-14. Patton, J. B., R.R. Rowland, D. Yoo, and K.C. Chang. 2009. Modulation of CD163 receptor expression and replication of porcine reproductive and respiratory syndrome virus in porcine macrophages. Virus Res. 140:161-171. Pei, Y., D.C. Hodgins, J. Wu, S.K.W. Welch, J.G. Calvert, G. Li, Y. Du, C. Song, and Yoo, D.. 2009. Porcine reproductive and respiratory syndrome virus as a vector: Immunogenicity of green fluorescent protein and porcine circovirus type-2 capsid expressed from dedicated subgenomic RNAs. Virology 389:91-99. Pieters M, Dee SA, Fano E and Pijoan C. An assessment of the duration of Mycoplasma hyopneumoniae infection in an experimentally infected population of pigs. Vet Microbiol 2009;143:261-264. Pitkin AN, Deen J and Dee SA. Further assessment of fomites and personnel as vehicles for the mechanical transport and transmission of porcine reproductive and respiratory syndrome virus. Can J Vet Res (Accepted for publication). Pitkin AN, Deen J and Dee SA. Use of a production region model to assess the airborne spread of porcine reproductive and respiratory syndrome virus. Vet Microbiol 2009;136:1-7. Pitkin AN, Otake S, Deen J, Moon RD, Dee SA. Further assessment of houseflies (Musca domestica) as vectors for the mechanical transport and transmission of porcine reproductive and respiratory syndrome virus under field conditions. Can J Vet Res 2009;73:91-96. Prickett J, Kim W-I, Simmer R, Yoon K-J, Zimmerman JJ. 2008. Surveillance of commercial growing pigs for PRRSV and PCV2 infections using pen-based oral fluid sample: a pilot study. J Swine Health Prod 16(2):86-91. Prickett J, Simer R, Christopher-Hennings J, Yoon K-J, Evans RB, Zimmerman JJ. 2008. Detection of porcine reproductive and respiratory syndrome virus infection in porcine oral fluid samples: A longitudinal study under experimental conditions. J Vet Diagn Invest 20:156-163. Ran ZG, Chen XY, Guo X, Ge NX, Yoon K-J, Yang HC. 2008. Recovery of viable porcine reproductive and respiratory syndrome virus from an infectious clone containing partial deletion within Nsp2-encoding region. Arch Virol 153:899-907. Renukaradhya, G.J., K.P. Alekseev, K. Jung, Y. Tang, Y. Fang, and L.J. Saif (2009). Altered immune responses to porcine respiratory coronavirus in pigs previously infected with porcine reproductive and respiratory syndrome virus. Vet. Immunol. Immunopath. (In review). Rosenfeld, P., P.V. Turner, J.I. MacInnes, E. Nagy, and D. Yoo. 2009. Evaluation of porcine reproductive and respiratory syndrome virus replication in the laboratory rodents. Can. J. Vet. Res. 73: 313-318. Sang, Y, P Ruchala, R Lehrer, CR Ross, RRR Rowland, Frank Blecha. 2009. Antimicrobial Host Defense Peptides in an Arteriviral Infection: Differential Peptide Expression and Virus Inactivation. Viral Immun. Accepted for publication. Sang, Y, C Ross, RR Rowland, F Blecha. 2008. Toll-like receptor 3 (TLR3) activation decreases porcine arterivirus infection. Viral Immunol 21:303-313. Sang, Y, J Yang, C Ross, RRR Rowland, and F Blecha, 2008. Molecular identification and functional expression of porcine Toll-like receptor (TLR) 3 and TLR7. In press Vet Immunol Immunopath. Song, C., R. Lu, D. Bienzle, H.C. Liu, and D. Yoo. 2009. Interaction of porcine reproductive and respiratory syndrome virus nucleocapsid protein with the inhibitor of MyoD family-a domain containing protein. Biol. Chem. 390: 215-223. Spilman, M.S., C. Welbon, E.A. Nelson and T. Dokland. 2009. Cryo-electron tomography of porcine reproductive and respiratory syndrome virus (PRRSV): organization of the nucleocapsid. J. Gen. Virol. 90:527-535. Steibel JP, Wysocki M, Lunney JK, Ramos AM, Hu Z-L, Rothschild MF, Ernst CW. 2009. Validation of the Swine Protein-Annotated Oligonucleotide Microarray. Animal Genetics. 40: 883-893. Trible BR, Kerrigan, M., Faaberg, K. S., and R. R.R. Rowland. Identification of an immunodominant region the PCV2 capsid protein recognized by naturally infected and vaccinated pigs. Journal of General Virology, submitted. Vashisht, K., Erlandson, K.R., Firkins, L.D., Zuckermann, F.A., Goldberg, T.L. 2008. Evaluation of contact exposure as a method for acclimatizing growing pigs to porcine reproductive and respiratory syndrome virus. J Am Vet Med Assoc. 232:1530-5. Vashisht, K., Goldberg, T.L., Husmann, R.J., Schnitzlein, W., Zuckermann, F.A. 2008. Identification of immunodominant T-cell epitopes present in glycoprotein 5 of the North American genotype of porcine reproductive and respiratory syndrome virus. Vaccine. 26:4747-53. Vincent, A. L., Lager, K. M., Faaberg, K. S., Harland, M. L., Zanella, E., Ciacci-Zanella, J., Kehrli, Jr., M. E., Janke, B. H., Klimov, A. Susceptibility of Pigs to Pandemic 2009 A/H1N1 Influenza Virus. Plos Pathogens, submitted. Wenjun Ma, AL Vincent, KM Lager, BH Janke, SC Henry, RRR Rowland, RA Hesse, JA Richt. 2009. Identification and characterization of a highly virulent triple reassortant H1N1 swine influenza virus in the United States. Virus Genes, in press. Wu, J., J. Li, F. Tian, J. Shi, S. Ren, Z. Lan, X. Zhang, D. Yoo, and J. Wang. 2009. Porcine high fever disease: genetic variation and pathogenicity of porcine reproductive and respiratory syndrome virus in China. Arch. Virol. 154: 1579-1588. Xue Han, Sumin Fan, Deendayal Patel, and Yan-Jin Zhang: Enhanced Inhibitory Effect on PRRSV Replication by Combination of Two Morpholino oligomers. Antiviral Research 2009. 82:59-66. Yaeger M, Karriker L, Layman L, Halbur P, Huber G, Van Hulzen K. 2009. Survey of disease pressures in twenty-six niche herds in the Midwestern United States. J Swine Health Prod. 2009 17(5): 256-263. Yue F, Cui S, Zhang C, Yoon K-J. 2009. A multiplex PCR for rapid and simultaneous detection of porcine circovirus type 2, porcine parvovirus, porcine pseudorabies virus, and porcine reproductive and respiratory syndrome virus in clinical specimens. Virus Genes (in press) 2. List book chapters or monographs None 3. List abstracts or proceedings Beura L, Sarkar S , Kwon BJ, Subramaniam S, Jones C, Pattnaik AK, Osorio FA. Porcine reproductive and respiratory syndrome virus non structural protein 1 beta inhibits host innate immune response by antagonizing IRF3 activation. Proceedings of the 28th Annual Meeting American Society for Virology (25th ASV), Vancouver, BC, July 11-15, 2009 (Workshop 33-6). Butler, J.E. 2009 A comparative study of PRRSV, PCV-2 and SIV infections in germfree isolator piglets. Presented at 5th International Veterinary Vaccine Conference (5th IVVDC), Madison WI. July 23. Carley D, Ramamoorthy S, Opriessnig T, Wong C, Tobin G, Yoon KJ, Halbur PG, Messel R, Nara PL. 2009. Evaluation of the PRRSV antibody response following vaccination with a proprietary autogenous vaccine. Proceedings, Summer Scholar Program. Carley D, Ramamoorthy S, Opriessnig T, Wang C, Tobin G, Yoon KJ, Halbur PG, Messel R, Nara PL. August 2009. Evaluation of the PRRSV antibody response following vaccination with a proprietary autogenous vaccine. Research Day, College of Veterinary Medicine, Iowa State University. Ames, IA. Carmichael BA, Polson DD, Torremorell M, Holtkamp DJ. 2009. Pre-colostral stillborn piglet blood sampling procedure when a PRRSV positive sow herd is being monitored for time-to-negative interval. 40th AASV Annual Meeting. Dallas, Texas. Cha S-H, Dorman KS, Kim W-I, Yoon KJ. 2008. Viral recombination among field isolates of PRRSV type 2 implication for molecular epidemiology. PRRS Symposium. Chen Z, X Zhou, D Kuhar, S Lawson, J Lunney, Y Fang. 2008. Effect of PRRSV nsp2 epitope deletion mutants on the induction of cytokine response in porcine alveolar macrophages. 2008 CRWAD & 2008 PRRSV Symp. Chen Z, X Zhou, S Lawson, E Brown, R Breen J. Christopher-Hennings, E Nelson, Y Fang. 2008. Expression of foreign proteins in replicase gene regions of porcine reproductive and respiratory syndrome virus. CRWAD & 2008 PRRSV Symp. Chitko-McKown, C.G., Miller, L.C., Lager, K.M. and Kehrli Jr., M.E. Effects of PRRSV infection on TLR-dependent induction of NOS [abstract]. 2008 Conference of Research Workers in Animal Diseases (CRWAD), December 7-9, 2008, Chicago, IL. Chitko-McKown, C.G., Chapes, S.K., Miller, L.C., and Green, B.T. Characterization of the porcine monocyte-derived cell lines Cdelta2+ and Cdelta2-. 41st Annual Meeting of the Society for Leukocyte Biology, November 6-8, 2008, Denver, CO. Cutler T, Hoff S, Wang C, Warren K, Zhou F, Qin Q, Miller C, Ridpath J, Yoon K-J, Zimmerman J. 2009. UV254 Inactivation of Selected Viral Pathogens. 52st Annual Conference, American Association of Veterinary Laboratory Diagnosticians. San Diego, California, p. 124. Cutler T, Kittawornrat A, Hoff S, Wang C, Zimmerman J. 2009. Median infectious dose (ID50) of PRRSV isolate MN-184 for young pigs via aerosol exposure. 52st Annual Conference, American Association of Veterinary Laboratory Diagnosticians. San Diego, California, p. 110. Cruz, J. L. G., Zúñiga, S., Sánchez, C. M., Ceriani, J. E., Plana, J., and Enjuanes, L. 2008. Design of a recombinant TGEV vector to protect against porcine reproductive and respiratory syndrome. EuroPRRSnet Workshop. Combating PRRS in Europe. Cruz, J. L. G., Zúñiga, S., Sánchez, C. M., Urniza, A., Bru, T., Ceriani, J. E., Plana, J., and Enjuanes, L. 2008. Construction of a TGEV vector to protect against porcine reproductive and respiratory syndrome. 2008 PRRS Symposium. Cruz, J. L. G., Zúñiga, S., Sánchez, C. M., Ros, S., Juanola, S., Plana, J., and Enjuanes, L. 2009. Design of a TGEV vector to protect against porcine reproductive and respiratory syndrome. 2009 PRRS Symposium. Dee SA and Otake S. Investigation of alternative strategies for aerosol biosecurity for PRRSV. CRWAD, Chicago, Il, December 2008. Dee SA and Otake S. Use of a production region model to evaluate issues regarding the aerobiology of PRRSV and Mycoplasma hyopneumoniae. CRWAD, Chicago, Il, December 2008. Dee SA, AN Pitkin, Deen J. Alternative strategies for aerosol biosecurity for PRRSV. 2008 Pijoan Intl Symp on Swine Dis Erad St. Paul, September, 2008. Dee SA, Otake S, Deen J. Use of a production region model to evaluate the transmission and biosecurity of PRRS and Mycoplasma hyopneumoniae. AASV, Dallas, Tx, March 2009. Dee SA, Pitkin AN, Otake S and Deen J. Transmission of EP and PRRS. PIC Veterinary Conference, Stratford-upon-Avon, England, February 2009. Dion KR, Dau D, Delks AM, Hammer M, Holtkamp DJ. 2009. Feed medication protocol comparison in a PRRSv unstable nursery flow. 40th AASV Meeting. Dallas, Texas. Dixon PM, Yoon K-J. 2008. Estimating the mutation rate when not all mutations are detected. Proceedings, Iowa State University Fall Conference on Statistics in Biology. Dwivedi V, C. Manickam, R. Patterson, K. Dodson, and G. J. Renukaradhya. Steps towards development of a novel mucosal vaccine to PRRSV. Fourth International Scholar Research Exposition, The Ohio State University, November 19th, 2009. Dwivedi V, C. Manickam, R. Patterson, K. Dodson, and G. J. Renukaradhya. Development of a novel mucosal vaccine to protect against porcine reproductive and respiratory syndrome in pigs. 2009 PRRSV Symposium. Faaberg KS, J. Han, K. M. Lager, M. E. Kehrli, Jr., and M. S. Rutherford. 2008. PRRSV strain VR-2332 nsp2 deletion mutants attenuate clinical symptoms in swine. XIth International Symposium on Nidoviruses, P43, Oxford, Great Britain. Faaberg, K. S. 2008. State of the Art: Lessons learned through porcine reproductive and respiratory syndrome virus (PRRSV) recombinant technology. International Congress of Virology, 675, Istanbul, Turkey. Fang, Y. 2008. Structural and Function of PRRSV nonstructural proteins: where are we at? 2008 PRRSV Symp. Fritz E, Hu Z, Lunney J, Reecy J. 2008. The PHGC Database: management of large data sets. Intnl PRRS Symp. #324. 12/0. Guo, B., Faaberg, K. S., Lager, K., and M. E. Kehrli, Jr. 2009. Genetic stability of PRRSV VR-2332 nsp2 deletion mutants in swine. 28th Annual Meeting of the American Society for Virology, W33-12, Vancouver, Canada. Haley CA, Wagner B, Murtaugh MP. 2009. Estimating the sensitivity and specificity of a new ELISA test for porcine circovirus 2 exposure using a study pseudo gold standard and latent-class analysis. Proc AASV. pp 255-261. Harms PA, Holtkamp DJ, Quirk Z. 2009. Management of, and costs associated with, false positive when monitoring presumed PRRS-negative herds. 40th AASV Annual Meeting, Pre-Conference Seminar, Managing PRRS Introduction into High Risk Populations. Dallas, Texas. Holtkamp DJ, Melody JL, Burkgren TJ. 2008. Update On The AASV Production Animal Disease Risk Assessment Program (PADRAP) and the New Web-based Application. 20th IPVS Meetings. Durban, South Africa. June. p. 01.66. Holtkamp DJ, Melody JL, MacDougald D. 2008. A comparison of PRRSV risks for Canadian and U.S. breeding herds. 20th IPVS Meetings. Durban, South Africa. June. p. 01.77. Holtkamp DJ, Polson DD. 2008. PRRS herd classification: Can we speak the same language? Carlos Pijoan International Symposium: New Solutions to Old Problems. Pre-Conference Workshop 2006 Allen D. Leman Swine Conference. St. Paul, Minnesota. Hesse, R, R Rowland. 2008. Circovirus Vaccination Decisions: Herd Profiling and Next Generation Diagnostic Testing. 2008 Leman Swine Conference, St Paul, MN. Hiep Vu , Kwon BJ, Yoon KJ, Laegreid W, Pattnaik AK and Osorio FA. Analysis of the aberrant immune response induced by a PRRSV type 2 isolate naturally lacking glycan residues in two envelope glycoproteins. 2009 IPRRSS (#84) and 2009 CRWAD (poster # 64). Hicks, J., D. Yoo, and H.C. Liu. 2008. Identification of domains of PRRS virus GP5 and M proteins that interact with the host Snap-associated protein SNAPIN. 2008 IPRRSS. Hu J, Meng XJ, and Zhang C. 2008. Purification of native PRRSV virions from cell culture. 2008 PRRS Symposium. Huang YW, B. A. Dryman, X. J. Meng. 2008. Molecular cloning of porcine DC-SIGN and detection of its potential interaction with porcine reproductive and respiratory syndrome virus. 2008 International PRRS Symposium. 2008 PRRS Symposium. Huang YW, B. A. Dryman, X. J. Meng. 2008. Molecular cloning of porcine DC-SIGN and detection of its potential interaction with porcine reproductive and respiratory syndrome virus. 2008 International PRRS Symposium. 2008 CRWAD. Jung K, K. Alekseev, G.J. Renukaradhya, Y. Tang, Y. Fang, P. Lewis, X. Zhang, L.J. Saif. Altered pathogenesis of porcine respiratory coronavirus (PRCV) in the presence of PRRSV infection and their pathologic relationships: Potential effect of preexisting respiratory viral infections on SARS severity. XIV International Congress of Virology, ASV, Cornell University, Ithaca, NY, 11-15 August 2008. Jung K, K. Alekseev, G.J. Renukaradhya, Y. Tang, Y. Fang, P. Lewis, X. Zhang, L.J. Saif. Altered pathogenesis of porcine respiratory coronavirus (PRCV) subsequent to PRRSV infection: Model for effect of respiratory viral co-infections on SARS severity. 2008 CRWAD. Karriker L, Bowden J. 2009. PRRS virus vaccination strategies and efficacy. Proceedings of the George A. Young Swine Health and Management Conference. South Sioux City, Nebraska. Karriker L, Layman L, Yaeger M. 2008. Session 5531: Health challenges in niche production. Conference Notes CD of the 145th AVMA. New Orleans, Louisiana. Karriker L. 2008. Session 5543: PRRSV: management strategies. Current swine disease trends. Conference Notes CD of the 145th AVMA. New Orleans, Louisiana. Kim W-I, Bower L, Strait E, Harmon K, Yoon K-J. 2009. Simultaneous detection of multiple pathogens using high-throughput nanoliter real-time PCR. AAVLD. Kim W-I, Cho Y-I, Harmon K, Madson D, Opriessnig T, Yoon K-J. 2008. Comparison of three extraction methods for the detection of PCV2 and PRRSV in semen. AAVLD. Kim W-I, Sun D, Cho Y-I, Liu S, Cooper V, Yoon K-J. 2009. Identification of molecular markers for virulence of porcine reproductive and respiratory syndrome (PRRS) virus. AAVLD Kim W-I, Sun D, Cho Y-I, Liu S, Loynachan AT, Cooper VC, Yoon KJ. 2009. Genetic determinants associated with the virulence of PRRSV in pigs. AASV. Koziel JA, Yang X, Cutler T, Zhang S, Zimmerman J, Hoff S, Jenks WS, van Leeuwen JH, Laor Y, Ravid U, Armon R. 2008. Treatment of livestock odor and pathogens with ultraviolet photocatalysis. Proc AgEng 2008 International Conference on Agricultural Engineering and Industry Exhibition, (Abstr OP-575). Hersonissos, Greece. Koziel JA, Yang X, Zhang S, Cai L, Hoff SJ, Leeuwen HJ, Cutler T, Zimmerman J, Jenks WS, Laor Y, Ravid U, Armon R. October 2008. Treatment of livestock odor and pathogens with ultraviolet photocatalysis. Proc 3rd IWA Odour and VOCs Conference. Barcelona, Spain. Lawson S, Lunney JK, Fang Y, Nelson EA, Christopher-Hennings J. 2009. Development of a rapid, swine-specific microsphere assay to simultaneously detect multiple immune proteins (cytokines) affected by porcine reproductive and respiratory syndrome virus (PRRSV) infection. 2009 Intnl PRRS Symp. and CRWAD 12/09. Liu S, Kim W-I, Ramamoorthy S, Yoon K-J. 2009. Alternative assays and testing algorithm for confirmation of suspect false positives in a commercial ELISA for PRRSV. AAVLD. Lunney JK. 2008. Genetics of Infectious Disease Resistance in Animals: Pig and PRRS. Proc. Am Coll Vet Pathol. Meeting. 11/08 Lunney JK. 2009. PRRS Host Genetics Consortium: Current Progress and Potential for Canadian Involvement. Canadian Centre for Swine Improvement meeting, Quebec City, Canada 6/09 Lunney JK, Boyd P, LaBresh J, Kakach L, Wagner B, Tompkins D, Hudgens E, Baldwin C. 2009. Swine Toolkit progress for the US Veterinary Immune Reagent Network. 2009 Intnl PRRS Symp. and CRWAD 12/09. Lunney JK, Boyd P, Prucnal L, Zarlenga D, LaBresh J, Steffens C, Wagner B, Tompkins D, Hudgens T, C Baldwin C. 2008. Swine Toolkit progress for the US Veterinary Immune Reagent Network. Intnl PRRS Symp. #288 and CRWAD 95P. 12/08 Lunney JK, Reecy J, Rowland RRR. 2009. PRRS Host Genetics Consortium: Current Progress and Potential for Canadian Involvement. Canadian Swine Health Forum 2009: July 7-8, 2009, Saskatoon, SK, Canada. Lunney JK, Reecy J, Rowland RRR. 2009. Current Progress of US PRRS Host Genetics Consortium. Genomics for Animal Health: Outlook for the Future (EADGENE 2009) meeting, 10/09, Paris, France. Lunney JK, Rowland RRR, Chen Z, Zhou X, Lawson S, Sun Z, E. Brown E, J. Christopher-Hennings J, Nelson E, Fang Y. 2009. Genetic approaches to reveal immune response pathways and viral antigen targets for novel vaccine design. Intnl Vet Vaccines and Diagnostics Conference (IVVDC 2009), WI. 7/09. Lunney JK, Steibel JP, Reecy J, Rothschild M, Kerrigan M, Trible B, Rowland RRR. 2009. PRRS Host Genetics Consortium: Current Progress. 2009 Intnl PRRS Symp. and CRWAD 12/09. Lunney JK, Wysocki M, Steibel JP, Kuhar D, Ernst CW, McCaw M. 2009. Uncovering Genetic Components Involved In Regulating Early Immune Responses To Porcine Reproductive And Respiratory Syndrome (PRRS). PAG2009. PAG-XVII P640. 1/09. Loruzzo, A., Faaberg, K. S., Killian, M. L., Koster, L., Vincent, A. L. 2009. One step real-time RT-PCR for 2009 pandemic H1N1 matrix gene detection and quantitation in clinical samples. American Association of Swine Veterinarians 2010 Annual Meeting, March 6-9, 2010, Omaha, NE. Metwally S, C. Carrillo, F. Mohamed, K. Faaberg, M. McIntosh, L. Cox, L. Koster, S. Swenson, T. Burrage, T. Long, T. Beckham, E. Lautner, and J. Lubroth. 2008. Porcine High Fever Disease in Vietnam 2007; PRRS and Other Disease Agents. 51st Annual Meeting of the American Association of Veterinary Laboratory Diagnosticians, Greensboro, NC, USA. Miller, L.C., Harhay, G.P., Lager, K.M., Kehrli Jr., M.E., Laegreid, W.W. and Neill, J.D. In depth global analysis of gene expression levels in porcine alveolar macrophages following infection with porcine reproductive and respiratory syndrome virus [abstract]. ARK-Genomics Conference 2008: 3rd International Symposium on Animal Functional Genomics, April 7-9, 2008, Edinburgh, U.K. Paper No. ISAFG-P22.p. 38. Miller, L.C., Chitko-McKown, C.G., Lager, K.M. and Kehrli Jr., M.E. Differential roles of Toll-like receptors in the elicitation of type I interferon responses by alveolar macrophages [abstract]. 2008 International PRRS Symposium, December 5-6, 2008, Chicago, IL. Molina RM, Cha S-H, Rowland RRR, Christopher-Hennings J, Nelson E, Lunney J, Yoon K-J, Zimmerman JJ. 2008. Factores involucrados en la persistencia del virus de sindrome respiratorio porcino (PRRS). Memorias XXI Congreso Panamericano de Ciencias Veterinarias. Guadalajara, México, pp. 533- 534. Morrison RB, Davies PD and Dee SA. Regional and national eradication of PRRS. PIC Veterinary Conference, Stratford-upon-Avon, England, February 2009. Morrison, RB. Update on PRRS elimination in Stevens County, MN. Allen D Leman Swine Conference, preconference workshop. Pp. 67-74. Murtaugh M. 2009. Update on PRRSV immunology and viral genetics: from hopeless to hopeful. Proc AASV. pp 459-462. Osorio FA. Worldwide Research Efforts Towards a Broadly Protective and Effective Vaccine against PRRSV. Keynote presentation # 4 at the 2008 IPRRSS. Osorio FA. PRRSV immunology and vaccines Second Annual CVM Swine Health Initiatives Meeting UIUC, PRRSV protective immunity and immunization, Osorio FA presented at the XVIII Congreso Dia del Porcicultor, Navojoa, Sonora Mexico. 2008. Otake S, Deen J, Dee SA. New information aerosol transmission and biosecurity for Mycoplasma hyopneumoniae. 2008 Leman Swine Conference. Otake S, Deen J, Dee SA. Preliminary information from recent research on PRRSV and Mycoplasma hyopneumoniae transmission and biosecurity: Field application of air filters. 2008 Leman Swine Conference. Pires-Alves, M., Misra, A., Zuckermann, F.A., Laegreid, W. 2009. Comparison of two cell lines for the propagation of PRRSV. 2009 IPRRSS. Potter, ML, S Dritiz, R Hesse, R Rowland, J Nietfeld, R Oberst. 2008. Porcine Cirovirus Type 2 elimination study. 2008 AASV. Potter, ML, LM Tokach, SS Dritz, SC Henry, JM DeRouchey, MD Tokach, RD Goodband, JL Nelsen, RR Rowland, RD Hesse, RA Hesse. 2008. Genetic background influences pig growth rate responses to porcine circovirus type 2 (PCV2) vaccines. 2008 KSU Swine Day. Prickett J, Cutler S, Kinyon J, Naberhaus N, Stensland WR, Yoon K-J, Zimmerman J. 2008. PRRSV surveillance Stability of diagnostic targets in oral fluid: sample storage and critical techniques for testing. Proc 89th CRWAD, Abstr #38. Prickett J, Cutler S, Kinyon J, Naberhaus N, Stensland WR, Yoon K-J, Zimmerman J. 2008. PRRSV surveillance Stability of diagnostic targets in oral fluid: sample storage and critical techniques for testing. 2008 IPRRSS, p 26. Prickett J, Hoffmann P, Main R, Sornsen S, Johnson J, Zimmerman J. 2009. Cost-effective PRRS surveillance. AASV, pp. 467-469. Prickett J, Hoffmann P, Main R, Stensland W, Yoon K-J, Zimmerman J. 2008. Practical disease surveillance in growing pig populations. Proc 89th CRWAD, Abstr #39. Prickett J, Hoffmann P, Main R, Stensland W, Yoon K-J, Zimmerman J. 2008. Practical disease surveillance in growing pig populations. Proc 2008 IPRRSS, p 27. Prickett J, Zimmerman J. 2008. Practical disease surveillance in growing pig populations. Proceedings, Welfare and Epidemiology Conference: Across Species, Across Disciplines, and Across Borders. Ames, Iowa, p. 22. Prickett J, Zimmerman J. 2009. Practical disease surveillance in growing pig populations. J Appl Anim Welf Sci 12:156. Prickett JR, Cutler S, Kinyon J, Naberhaus N, Stensland W, Yoon KJ, Zimmerman J. 2008. PRRSV surveillance - Stability of diagnostic targets in oral fluid: sample storage and critical techniques for testing. Proc 51st Annual Conference, American Association of Veterinary Laboratory Diagnosticians. Greensboro, North Carolina, p. 140. Prickett JR, Cutler S, Kinyon J, Naberhaus N, Stensland W, Yoon K-J, Zimmerman J. 2008. PRRSV surveillance - stability of diagnostic targets in oral fluid: Sample storage and critical techniques for testing. Proc 47th North Central Conference of Veterinary Laboratory Diagnosticians. Madison, Wisconsin, pp. 8. Prickett JR, Cutler S, Kinyon J, Naberhaus N, Stensland WR, Yoon K-J, Zimmerman JJ. 2008. PRRSV surveillance stability of diagnostic targets in oral fluid: sample storage and critical techniques for testing. AAVLD. Prickett JR, Cutler S, Kinyon J, Naberhaus N, Stensland WR, Yoon K-J, Zimmerman JJ. 2008. PRRS surveillance Stability of diagnostic targets in oral fluid: Sample storage and critical techniques for testing. NCCVLD. Prickett JR, Hoffmann P, Main R, Sornsen S, Johnson J, Zimmerman J. 2008. Infectious disease surveillance in commercial swine populations. Proceedings 16th Annual Swine Disease Conference for Swine Practitioners, Iowa State University. Ames, Iowa, pp. 40-44. Prickett JR, Hoffmann P, Main R, Stensland W, Yoon K-J, Zimmerman J. June 2008. Practical disease surveillance in growing pig populations. Proc 47th North Central Conference of Veterinary Laboratory Diagnosticians. Madison, Wisconsin, pp. 10. Prickett JR, Hoffmann P, Stensland W, Yoon KJ, Zimmerman J. 2008. Practical disease surveillance in growing pig populations. Proc 51st Annual Conference, AAVLD, Greensboro, NC, p. 139. Renukaradhya GJ, Konstantin Alekseev, Kwonil Jung, and Linda J. Saif. Distorted immune responses in pigs to porcine respiratory coronavirus previously infected with porcine reproductive and respiratory syndrome virus: a respiratory viral co-infection model. 2009 CRWAD. Reister L, T Clement, E Nelson, J Christopher-Hennings. 2008. Potential mechanical and antiviral methods to insure PRRSV free semen. 2008 CRWAD & 2008 PRRSV Symp. Rovira A, Abrahante J, Murtaugh M. 2009. Detection of porcine reproductive and respiratory syndrome virus (PRRSV) by reverse transcriptase loop mediated isothermal amplification (RT-LAMP). Proc AASV. pp 109-110. Rowland RRR, Kerrigan M, Bujuru S, Trible B, Lunney JK. 2009. An infection model for the study of PRRS at the population level. 2009 IPRRSS. 12/09. Rowland, R, S Henry, S Dritz, R Hesse. 2008. Epidemiology of PCV2 and PCVAD. 2008 AASV. Rowland, R, R Hesse, K Horlen. 2008. Porcine circovirus vaccine trials: from the laboratory bench to the field. George Young Swine Conference, Sioux City. Rowland, R. 2008. PRRS vaccines. 2008 Leman Swine Conference, St Paul, MN. Rowland, R. 2008. Mapping host protective immunity in the PCV2 capsid protein. USDA NRI Prinicipal Investigators Meeting, Chicago. Sang, Y, P Ruchala, RI Lehrer, CR Ross, RRR Rowland, F Blecha. 2008. Antimicrobial host defense peptides in an arteriviral infection: differential expression and inactivation of PRRSV. 2008 IPRRSS. Sun D, Kim W, Cho Y, Cooper V, Cha S, Kim S, Choi E, Yoon K. 2009. Role of viral structural proteins in conferring protective immunity against PRRSV and its application to the development of vaccine candidates for broad cross-protection. 2009 AASV. Sun D, Kim W-I, Cho Y-I, Cha S-H, Kim S-H, Choi E-J, Yoon KJ. 2008. Cross-protection induced by chimeric mutant containing mixed structural genes of two different PRRS viruses. 2008 CRWAD. Sun D, Kim W-I, Cho Y-I, Yoon KJ. 2008. Attempt to achieve broader cross-protection among PRRS viruses by vaccination: use of chimeric mutant containing mixed structural genes of two different PRRS viruses. 2008 PRRS Symp. Trible, B, JG Calvert , RRR Rowland. 2008. Expression of enhanced green fluorescent protein (EGFP) in nonstructural protein 2 (nsp2) of PRRSV shows loss of fluorescence without affecting EGFP immunogenicity. Tuggle CK, Bearson SMD, Uthe JJ, Christian C, Couture O, Demirkale CY, Nettleton D, Lunney JK, Honavar V. 2009. Using transcriptomic data to develop tools for predicting shedding traits in growing pigs. CRWAD 12/09. Vincent, A. L., Lager, K. M., Faaberg, K. S., Harland, M. L., Zanella, E., Ciacci-Zanella, J., Kehrli, Jr., M. E., Janke, B. H., Klimov, A. Susceptibility of North American Swine to the Novel A/H1N1 Influenza A Virus. Center of Excellence Symposium, Minneapolis, MN. Vu HLX, M. Brito M, Kim WI, Yoon KJ, Laegreid W, Osorio FA. 2008. Sub-typing PRRSV isolates by means of measurement of cross neutralization reactions. CRWAD. Vu HLX, M. Brito M, Kim WI, Yoon KJ, Laegreid W, Osorio FA. 2008. Sub-typing PRRSV isolates by means of measurement of cross neutralization reactions. PRRS Symp. Waddell JM, Melody JL, Holtkamp DJ. 2009. Investigation of associations between risk factors, reported clinical PRRS breaks and reproductive performance in swine breeding herds. Proc. 40th AASV Annual Meeting. Dallas, Texas. Waddell JT, Polson DD, Holtkamp DJ. 2008. Assessment of changes in breeding herd PRRS site risk scores in a large production system over a three-year period. Proc 20th IPVS Meetings. Durban, South Africa. p. 01.135. Wong SJ, Lunney JK, Rowland RRR. 2009. Nucleocapsid protein-specific IgG and IgM responses in oral fluids during PRRSV infection. 2009 Intnl PRRS Symp. 12/09. Wong, SJ, R Hesse, R Rowland. Application of multiplex microsphere immunoassay techniques to the diagnosis of PRRSV and other infectious. 2008 International PRRS Symposium, Chicago. Wysocki M, SteibelJP, McCaw M, Kuhar D, Ernst CW, Lunney JK. 2008. Uncovering genetic components involved in early regulatory immune response during PRRSV infection. Intnl PRRS Symp. #285 and CRWAD #125. 12/08. Wu, J., Li, J., Tian, F., Shi, J., Ren, S., Lan, Z., Zhang, X., Niu, Z., Yoo, D., and Wang, J. 2008. Genetic variation and pathogenicity of porcine reproductive and respiratory syndrome virus in Shandong area of China. Intl PRRS Symposium, Chicago, IL. Yang X, Koziel JA, Cutler T, Zhang S, Zimmerman J, Hoff SJ, Jenks W, van Leeuwen J, Harmon J, Faulhaber C, Laor Y, Ravid U, Armon R. 2008. Treatment of livestock odor and pathogens with ultraviolet light. American Society of Agricultural and Biological Engineers (ASABE) Paper No. 085198. ASABE Annual International Meeting. Providence, Rhode Island. Yoon K-J. 2008. PRRS diagnosis as tool for evidence-based PRRS control. KASV. Zimmerman J, Prickett J, Johnson J. 2009. Oral fluid testing: Science-based applications. Carlos Pijoan International Symposium: New Approaches to Herd Diagnostics. 2009 Allen D. Leman Swine Conference. pp. 11-14. Zimmerman J, Prickett J, Johnson JH, Molina R. 2009. Evaluando fluidos orales: Base científica de su aplicación. XVIII Día del Porcicultor 2009. Asociación de Médicos Veterinarios Zootecnistas Especialistas en Ciencias Porcicolas del Sur de Sonora A.C. Navojoa, Sonora, Mexico (CD). Zimmerman J, Prickett J. 2008. Future epidemiologic / ecologic methods. 1st Annual Boehringer Ingelheim Swine Academy. Ames, Iowa, Vol 1, pp. 251-260. Zimmerman J, Prickett JR, Molina R, Hoffmann P, Main R, Sornsen S, Johnson J. 2008. Vigilancia epidemiológica de enfermedades infecciosasa en poblaciones porcinis comerciales utilizando fluidos orales. Memorias XXI Congreso Panamericano de Ciencias Veterinarias. Guadalajara, México, pp. 231-234. Zimmerman J. 2009. The Big Picture: Infectious disease, oral fluid testing, and swine health. 2nd Annual Boehringer Ingelheim Swine Academy. Ames, Iowa, pp. 181-200. Xiuqing Wang, Hanmo Zhang, Xueshui Guo. 2009. The interaction between PRRSV and type I interferon induction signaling pathways. 28th American Society for Virology. Yoo, D., Y. Sun, and N. Chen. 2008. One-step mutagenesis of the full-length infectious clone of PRRSV and generation of engineered viruses. Intl PRRS Symposium, Chicago, IL, Dec 6-7. Zuckermann, F.A. , Calzada-Nova, G., Schnitzlein, W., Husmann, R. 2009. Proficient isolation and titration of field PRRS virus from clinical samples using a porcine alveolar macrophage cell line. 2009 Intl PRRS Symposium, Chicago, IL, Dec 6-7. FUNDING SOURCES FOR PRRSV RESEARCH: Currently Funded Competitive Research Grants: Blecha, et al, USDA NRI, 2006-2009. Porcine antimicrobial peptides and Toll-like receptors in PRRS pathogenesis, $340,000. Christopher-Hennings J, Y Fang, J Lunney, EA Nelson. Development of a rapid, single tube, multiplex test to simultaneously detect immune parameters (cytokines) induced by PRRSV. National Pork Board, $101,107. Christopher-Hennings J, Y Fang, EA Nelson. Elimination of PRRSV from semen: On Farm Mechanical and Antiviral Methods. National Pork Board $94,558, 2007-2009. Enjuanes L. Induction of cross-protective immunity without exposure to live PRRSV (NPB #08-197). National Pork Board. 2008-2009. Enjuanes L. Mechanisms inducing protection against coronaviruses and arteriviruses. Fort Dodge, S.A. 2008-2009. Enjuanes L. Plant Production of Vaccines (PLAPROVA, EU 227056). European Communities. 2009-2011. Faaberg KS, Collins JE, Yoon K-J, Christopher-Hennings J, Crow JA, Leung FC. 09/01/07-08/30/08. Implementation of a PRRSV strain database. National Pork Board PRRS Initiative. $51,322. Faaberg, Guo, Miller: NPB, 11/01/09-11/01/10, $53,877, Molecular Identification of Type I Interferon Antagonistic Components of PRRSV Proteins. Faaberg: NRICGP, 10/1/06-6/30/10, $274,998, Biological Studies of Putative Nonstructural Protein 2 in Porcine Reproductive and Respiratory Syndrome Virus. Fang Y, JK Lunney, J Christopher-Hennings, E Nelson, A Young. The role of PRRSV non-structural proteins 1 and 2 in host immunity. USDA-NRI, $375,000, 1/08-12/2010. Fang Y, W Zhang, J Christopher-Hennings, EA Nelson, RB Baker. Development of an epitope-based vaccine against swine influenza A using a non-toxic enterotoxin as the carrier-adjuvant. National Pork Board. $49,993. Fang Y, Zimmerman J, Christopher-Hennings J, Nelson E, Murtaugh M, Lunney J. 01/01/10 to 12/31/11. Development of diagnostic assays for detecting PRRSV infection using oral fluid samples as an alternative to serum-based assays. National Pork Board - $99,989. Gourapura RJ. Evaluation of adjuvants at the mucosal area for the development of innovative mucosal vaccine against PRRS. National Pork Board. Nov. 2008 to May 1, 2010. Gourapura RJ. Study of mucosal immune responses in the respiratory tract of pigs infected with porcine reproductive and respiratory syndrome virus. OARDC Seed grant, The Ohio State University. March 2009 to February 2011. Gourapura RJ. Development of novel mucosal vaccines for the control of PRRSV outbreaks. National Pork Board. Dec. 2009 to May, 2011. Hesse and Rowland, 2009, Fort Dodge Animal Health, Heterotypic immunity as a platform for a new generation of modified live PRRS vaccines. $100,000 Holtkamp D, Ramirez A, O'Connor A, Zimmerman J. 01/01/09 to 12/31/09. Assessment of PRRS biosecurity in the field: Application of the American Assocation of Swine Veterinarians PRRS Risk Assessment Tool. USDA:CSREES National Research Initiative, Competitive Grants Program 230.1 Animal and Plant Biosecurity (subcontract: Kansas State University) - $24,300. Holtkamp D, Zimmerman J. 10/01/08 to 09/30/09. Quantifying risk factors for PRRSV introduction into swine herds through the use of the PRRS Risk Assessment Tool. National Pork Board - $89,875. Holtkamp D.J. A Cross-sectional Study Of PRRSV Positive Swine Breeding Herd Sites To Evaluate Associations Between Risk Factors And A Case Definition-based Number And Severity Of Clinical PRRS Episodes. Boehringer Ingelheim Vetmedica Inc. $115,000. Continuation of previous grant with same title. January 6, 2009. Kim W-I, Yoon K-J, Cooper VC. 11/01/07-10/30/08. Identification of protective epitopes toward developing a vaccine providing broad cross-protection among PRRS viruses. National Pork Board PRRS Initiative. $89,150. Kim W-I, Yoon K-J. 7/1/08-6/30/10. A new approach to PRRS vaccine that confers cross-protection against a broader range of PRRS viruses using chimeric mutant PRRS viruses. Iowa Healthy Livestock Initiative Research Grant. $39,980. Laegreid W., F. Osorio, T. Goldberg, J. Christopher-Hennings, E. Nelson. Immunological consequences of PRRSV Diversity. USDA-NRI, PRRSV CAP2. $947,885. Lunney J, J Christopher-Hennings, EA Nelson, Y Fang, JP Steibel, J Zimmerman. 01/01/10 to 12/31/11. Comparison of early immune responses of pigs which are genetically PRRS resistant/tolerant using a swine-specific immune protein (cytokine) multiplex assay. National Pork Board. $103,929. Lunney JK, J Dekkers, R Fernando, Z Jiang, H-C Liu, R Pogranichniy, JM Reecy, R Rekaya, M Rothschild, D Smith, JP Steibel, C Tuggle. PRRS CAP Host genetics: Characterization of host factors that contribute to PRRS disease resistance and susceptibility. USDA NIFA PRRS CAP2: Objective 3 Host Genetics. $560,000. 2009-2012. Lunney JK,C Ernst, V. Honavar, Z Jiang, R Pogranichniy, JP Steibel, C Tuggle. Identifying porcine genes and gene networks involved in effective response to PRRS virus using functional genomics and systems biology. USDA AFRI/NIFA Animal Genome, Genetics, and Breeding Program. $750,000. 2010-2012 Meng, X. J., Y. Fang, T. Opriessnig. Innovative approaches to develop a broadly protective and effective vaccine(s) against PRRSV. USDA PRRS CAP2, $100,000, 1/200912/2010. Miller, Harhay, Lager: NPB, 11/01/08-11/01/10, $139,152, Gene Expression in lymph nodes of PRRSV-infected pigs Murtaugh M (and MN stations). PRRS CAP 2, Minnesota Pork Board, National Pork Board, USDA, University of Minnesota Swine Disease Eradication Center, Minnesota Rapid Agricultural Response Fund. Murtaugh M, Gourapura RJ. Positive Prognosticators of Immune Protection and Prophylaxis against PRRSV in Swine Herds. PRRSV PRRS CAP 2. August 2009 to July 2013. Osorio FA: Immunologic Consequences of PRRSV Diversity, USDANRICGP CAP2 (Kansas State University subcontract), $74,368 August 2009-July 2010. Osorio FA: Development of a modified live vaccine against PRRSV with optimal DIVA marker potential, National Pork Board, Grant Period: 11/01/2008 - 12/31/2009 (extended at no cost) $125,700 Osorio FA: Porcine Reproductive and Respiratory Virus: role of viral genes in virulence/attenuation, USDA NRICGP Project No. No.2008-00903, Period: 09/01/2008 - 08/31/2011, $374,900. Pattnaik AK.; Molecular Structures of PRRSV that Contribute to PRRSV Protective Immunity. National Pork Board. $ 138,600; 12/01/2009-11/31/2010. Pattnaik AK.; Glycoproteins of Porcine Reproductive and Respiratory Syndrome Virus in Infection and Immunity?; States Department of Agriculture, AFRI (2009-01576), $371,230; 09/01/2009-08/31/2012. Pattnaik AK.: Role of All of PRRSV Glycoproteins in Protective Immune Response, 11/01/2008 - 10/31/2009 (extended at no cost) National Pork Board, $106,000. Rowland RRR, Lunney JK, Reecy J, Johnson, R, NPB, 2007-2008. PRRS host genetics consortium: A proposal to develop a consortium to study the role of host genetics and resistance to PRRSV. $300,000. Rowland RRR, Lunney JK, Reecy J, NPB, 2009-2010. PRRS host genetics consortium: A proposal to develop a consortium to study the role of host genetics and resistance to PRRSV. $247,000. Rowland et al., USDA NRI Coordinated Agricultural Program (CAP), 2008-2012. Integrated strategies to control and reduce the impact of PRRS virus control, $4.8 million. Wang, X. Interaction between PRRSV and interferon alpha/beta induction signaling pathways. USDA NRI 12/2008-11/2010 $ 100,000. Yoo D. Private industry, $90,000, Antiviral effects of tilmicosin on swine respiratory viruses. 2008-2009. Yoo D. USDA CSREES NRI, $375,000, Evasion strategies of PRRSV from the host defense. 2008-2011. Yoon K-J. 1/1/07-12/31/10. Development of surveillance program and vaccine for PRRS in Korea: Development of a differential test for PRRS vaccine virus and immunological study of viral factors for protective immunity. Korean Ministry of Agriculture and Forestry (c/o National Veterinary Research and Quarantine Service). $280,000. Zimmerman J, Dee SA, Davies PR, Holtkamp DJ, OConnor A. 08/01/09 to 07/31/10. Identifying ecologic and epidemiologic factors in the control of PRRS: A field-based approach. USDA:CSREES National Research Initiative, Competitive Grants Program 230.1 Animal and Plant Biosecurity (subcontract: Kansas State University) - $116,991. Zimmerman J, Hoff SJ. 07/01/09 to 06/30/10. Effect of temperature and humidity on ultraviolet (UV) inactivation of airborne PRRS virus. Innovative Swine Industry Enhancement Grant Program, Iowa Attorney Generals Office - $43,046. Zimmerman J. 05/01/09 to 04/31/10. PRRS CAP Graduate and Undergraduate Scholar Program Application - Iowa State University. USDA:CSREES National Research Initiative, Competitive Grants Program 230.1 Animal and Plant Biosecurity (subcontract: Kansas State University) - $23,999. Gold Sheet #97704. Zuckermann F. USDA NRI CPG, $369,064. In vivo analysis of PRRS virus immunopathogenesis. Tracking Number: GRANT00168948. 2007-2010.

Impact Statements

Research advances over the last year continue to expand our understanding of PRRSV epidemiology, pathobiology, virology, and provide new ideas for countering and/or eliminating the infection. Extensive work has been done regarding the emergence of genetic and antigenic variation during replication in pigs and its role in persistence. Continued assessment and research in diagnostic technology contributes to the improvement and refinement of our ability to detect and diagnose PRRSV infection. On-going work on new methods of surveillance promise to provide new, cost-effective methods of tracking infection and implementing area elimination/eradication programs. Accomplishments in these areas linked with research in viral ecology/epidemiology, will lead to the development of tools that will result in the eventual elimination and eradication of PRRSV from individual farms and regions.
The development of the porcine alveolar macrophage cell line ZMAC to isolate PRRS virus from field samples will aid better understand PRRSV as well as vaccine development.
The use of PRRS virus as vector for foreign gene expression is a first, which demonstrates the potential use of PRRSV as a vaccine vector for swine pathogens. The studies on the modulation of CD163 receptor expression and the replication of PRRSV in porcine macrophages data indicated that the expression of CD163 on macrophages in different microenvironments, in vivo, may determine the replication efficiency and subsequent pathogenicity of PRRS virus.
The interaction of PRRSV capsid with the cellular transcription factor implicates a possible regulation of host cell gene expression by the N protein during PRRSV infection
Genetic analysis of host response has revealed the diverse negative impacts of PRRSV on a population. Decreased performance demonstrated by lack of weight gain is a loss to the producers bottom line. New IFN genes that possess potent anti-PRRSV can be incorporated into vaccines and other antiviral therapies.
The development of Luminex system provides the means to 1) detect antibodies to multiple agents in a single small volume of sample, 2) increase sensitivity and specificity, 4) reduce the cost of testing, and 5) semi-quantitative output without need for serial dilution of a sample to an endpoint, and 5) test for agents in non-serum samples, such as oral fluids and meat juice.
The studies of PPMOs against PRRSV have demonstrated that PPMOs inhibited PRRSV replication and protected the cells from PRRSV-induced cell death. Administration of PPMO 5UP2 to piglets that were experimentally infected with a PRRSV strain resulted in lower viremia and less lung lesion. Specific antiviral PPMOs can complement other approaches for PRRS prevention and control, because there are highly conserved target sequences among PRRSV strains. Application of the antiviral PPMOs will yield significant economic benefits to the swine industry, especially for breeding farms.
The value of airborne transmission research results to stakeholders includes a comprehensive understanding of the airborne routes and significance for the spread of PRRSV between farms. The continued ability to demonstrate the efficacy of air filtration to reduce this risk, initially via the production region model and then under controlled field conditions provides producers and veterinarians with a tool to reduce this important risk factor.
Regional elimination of PRRS demonstrates to stakeholders that the disease can be eliminated from a region and provides tools and methods that can be implemented in other regions.
Immunity research informed stakeholders of significant age-dependent differences in the ability of pigs to resist PRRSV infection, providing important information on proper application of live vaccines in the field, and efficacy of serum inoculation in the control of PRRS disease in gestating sows and vertical transmission of PRRSV.
The identification and potential use of bacterial preparations as candidate adjuvants to augment anti-PRRSV mucosal immune responses in pigs will advance in the area of mucosal vaccine production and in understanding innate immune responses to PRRSV.
A multiplex assay to simultaneously quantify 9 porcine cytokines in serum using Luminex xMap" technology was developed and optimized to detect innate (IL-1b, IL-6, IL-8, IFN-a, TNF-a); regulatory (IL-10), T helper 1 (Th1) (IL-12, IFN-g) and Th2 (IL-4) cytokines. The assay will be of value in vaccine and challenge studies as well as for determining genetic resistance to PRRSV and immune responses to other swine pathogens.
The demonstration that nsp1² inhibits both interferon synthesis and signaling, while nsp1± alone strongly inhibits the synthesis of interferon provides important insights into the mechanisms of how nsp1 contributes to PRRSV pathogenesis and how this may impact future vaccine development strategies.
The PRRS Host Genetics Consortium (PHGC) has begun to determine the role of host genetics in resistance to PRRS and in effects on pig health and related growth effects. Using a Nursery Pig Model crossbred pigs from high health farms were infected with PRRSV and followed for 42 days. Results from the first 5 trials of 200 pigs each have affirmed that all pigs become PRRSV infected but pigs clear virus from serum at different rates; weight effects are variable. Overall, the PHGC project will enable researchers to verify important genotypes and phenotypes that predict resistance/susceptibility to PRRSV infection.
The establishment of an improved reverse genetic system and the identification of a porcine monocytic cell line supporting PRRSV replication will aid future studies of host-virus interaction of PRRSV. The identification and characterization porcine DC-SIGN and demonstration of its binding to PRRSV will help better understand the biological role(s) of DC-SIGN family in innate immunity during the evolutionary process.

Date of Annual Report: 12/03/2010

Report Information

Annual Meeting Dates: 12/03/2010 - 12/03/2010
Period the Report Covers: 10/01/2010 - 09/01/2011

Participants

Chair: Meng, X.J. - Virginia Polytechnic Institute and State University (VA Tech) xjmeng@vt.edu;
Secretary: Christopher-Hennings, Jane - SDSU jane.hennings@sdstate.edu;
Rowland, Raymond R.R. - Kansas State University (KSU) browland@vet.k-state.edu;
Benfield, David, - Ohio State University (OSU) benfield.2@osu.edu;
Enjuanes, Luis - Centro Nacional de Biotecnología (CNB), CSIC L.Enjuanes@cnb.csic.es;
Faaberg, Kay - (NADC) kay.faaberg@ars.usda.gov;
Garmendia, Antonio - UConn Antonio.garmendia@uconn.edu;
Goldberg, Tony - University of Wisconsin-Madison (UWI) tgoldberg@vetmed.wisc.edu;
Gourapura, Renukaradhya J. - The Ohio State University (OSU) gourapura.1@osu.edu;
Johnson, Peter - USDA, CSREES pjohnson@reeusda.gov;
Lunney, Joan - USDA-ARS, BARC, joan.lunney@ars.usda.gov;
Murtaugh, Michael P. - University of Minnesota (UMN) murta001@umn.edu;
Osorio, Fernando A. - University of Nebraska-Lincoln (UNL) fosorio@unl.edu;
Pogranichniy, Roman- (Purdue), IN rmp@purdue.edu;
Schommer, Susan - University of Missouri (UMO) schommers@missouri.edu;
Tompkins, S. Mark - University of Georgia (UGA) smt@uga.edu;
Yuan, Shishan - Shanghai Veterinary Res. Inst.(SHVRI), Chinese Academy of Agri. Sci., shishanyuan@shvri.ac.cn;
Zhang, Yanjin - University of Maryland (UMD) zhangyj@umd.edu;
Zimmerman, Jeff - Iowa State University (ISU) jjzimm@iastate.edu;
Zuckermann, Federico A. - University of Illinois at Urbana-Champaign (UIUC) fazaaa@uiuc.edu;

Other NC229 Scientists:;
Tripp, Ralph - UGA;
Baker, RB.- ISU;
Boddicker, N.- ISU;
Dekkers, Jack - ISU;
Halbur, Patrick - ISU;
Harris, DL. (Hank) - ISU;
Holtkamp, Derald J.- ISU;
Johnson, John K.- ISU;
Karriker, Locke - ISU;
Main, Rodger G.- ISU;
McKean, JD.- ISU;
Opriessnig, Tanj - ISU;
Platt, Kenneth - ISU;
Prickett, J.- ISU;
Ramamoorthy, Sheila- ISU;
Ramirez, Alejandro - ISU;
Reecy, J. - ISU;
Roth, JA.- ISU;
Rothschild M.- ISU;
Strait, Erin - ISU;
Wang, Chong - ISU;
Yoon, Kyoung-Jin - ISU;
Laegried, Will - UIUC;
Yoo, Dongwan - UIUC;
Blecha, Frank - KSU;
Chang, KC.- KSU;
Hesse, Dick - KSU;
Kerrigan M. - KSU;
Sang, Yongmin - KSU;
Trible, B. - KSU;
Wyatt, Carol - KSU;
Zhu, Xiaoping - UMD;
Davies, Peter - UMN;
Dee, Scott - UMN;
Deen, John - UMN;
Gramer, Marie - UMN;
Joo, Han Soo - UMN;
Molitor, Tom - UMN;
Morrison, Robert - UMN;
Rossow, Kurt - UMN;
Rovira, Albert - UMN;
Torremorell, Monteserrat - UMN;
Ciobanu, Daniel - UNL;
Johnson, Rodger - UNL;
Pattnaik, Asit - UNL;
Brockmeier, Susan - NADC;
Harhay, Greg - NADC;
Kerhli, Marcus Jr - NADC;
Lager, Kelly - NADC;
Loving, Crystal - NADC;
Miller, Laura - NADC;
Neill, John - NADC;
Butler, John - University of Iowa;
Saif, Linda J.- OSU;
Fang, Ying - SDSU;
Lawson, Steve - SDSU;
Nelson, Eric - SDSU;
Pohl, Stephen - SDSU;
Wang, Xiuqing - SDSU;
Chen, Hongbo - USDA-BARC;
Kuhar, D. - USDA-BARC;
Wysocki, Michal - USDA-BARC;
Steibel, JP. - Michigan State Univ. (MSU);
Ernst, Cathy - Michigan State Univ. (MSU);
Clark, A.- Purdue University;
Lazar V. - Purdue University;
Moore, B.- Purdue University;
Sina, R.- Purdue University;
LeRoith, Tanya - VA Tech;
Zhang, C. - VA Tech;
Ciobanu, Daniel C., UNL;
Johnson, Rodger, UNL;
Pattnaik, Asit, UNL ;
Calvert, Jay, Pfizer Animal Health;
Roof, Mike, Boehringer Ingelheim Animal Health;
McIntosh, Michael, PIADIC;
Leung, Frederick, Hong Kong University;
Carman, Susy, University of Guelph, Canada;
Anderson, Tavis, UWI

Brief Summary of Minutes

Dr. XJ Meng gave the opening remarks.
Dr. David Benfield gave a short history (started in 1999) and evolution of NC229 describing the uniqueness of this NC group being the first in a number of categories (eg. in submitting multi-investigator grants, formalizing an international meeting, promoting specialized editions of journals, to receive an excellence in multi-states award). It has continued to promote integrated approaches to research and may need to submit foundation or 501c3 organization grants to continue.
Dr. Peter Johnson and Dr. Margo Holland discussed funding opportunity changes in USDA (NIFA). Two handouts were received on categories of NIFA discretionary funding and areas funded in 2010 with the presidents budget and senate committee action for 2011, along with a brief description of NIFA (see http://www.nifa.usda.gov).
Dr. Bob Rowland discussed post-PRRS CAP funding for NC229 with some discussion.
Closing remarks were made by Dr. Jane Christopher-Hennings.

Minutes Attachment:

Attachment

Accomplishments

B. PROGRESS OF WORK AND PRINCIPAL ACCOMPLISHMENTS Objective 1. Elucidate the mechanisms of host-pathogen(s) interactions. 1.1 (KSU-Sang/ Blecha/Rowland) performed a study identifying 39 type I interferon (IFN) genes. Recombinant IFN proteins show a wide range of activities, including some novel IFNs that are effective in controlling PRRSV replication. An analysis of cross-protection between diverse PRRSV strains is also being performed (Hesse, Rowland). 1.2. (UMD-Zhang/Zhu) PRRSV interferes with IFN signaling. Transcripts of IFN-stimulated genes, ISG15 and ISG56, and the STAT2 protein in PRRSV infected MARC cells were significantly lower than in mock-infected cells after IFN± treatment. PRRSV blocks STAT1/STAT2 nuclear translocation. IFN-induced phosphorylation of STAT1 and STAT2, and their heterodimer formation in the PRRSV-infected cells were not affected. Most STAT1/STAT2/IRF9 heterotrimers remained in the cytoplasm of infected cells, indicating that the nuclear translocation of the heterotrimers was blocked suggesting PRRSV interferes with activation and signaling of type I IFN by blocking the STAT1/STAT2 nuclear translocation. Overexpression of nsp1² inhibited expression of ISG15 and ISG56 and blocked nuclear translocation of STAT1 suggesting that nsp1² might be responsible for IFN inhibition. PRRSV infection of primary porcine pulmonary alveolar macrophages (PAMs) also inhibited IFN± signaling. PRRS MLV activated expression of IFN-inducible genes including chemokines and antivirals in PAMs without the addition of external IFN, and had no detectable effect on IFN signaling. 1.3. (USDA-BARC), the PRRS Host Genetics Consortium (PHGC) was developed to determine the role of host genetics in PRRSV resistance. It is a multi-year project funded by NPB, USDA, universities and private companies. 8 sets of 200 nursery pigs were infected with PRRSV. Pigs became infected but some pigs clear virus from serum quicker with variable weight effects. DNA from all pigs is genotyped with PorcineSNP60 Genotyping BeadChip and data stored at www.animalgenome.org/lunney. Comparison of resistant/maximal growth pigs to susceptible/reduced growth pigs is performed. Support is from USDA, NRSP8 Swine Genome Coordinator and Genome Alberta. Initial results mapped viral load and weight gain during infection to multiple swine chromosomes. The effect of PRRSV infection or vaccination on pigs was tested. Pigs were given a low or high virulent PRRSV, vaccine or were controls. Tissues were collected for innate immune response evaluation by swine oligo array pigoligoarray.org with statistical assessment of gene expression patterns (MSU). Cellular immune response, chemokine signaling and apoptosis were significantly activated in infected tonsils vs. those from vaccinated and control pigs. 1.4. (SHVRI) studied the 3UTR of type 2 PRRSV using site-directed mutagenesis. At least 40 nt after the ORF7 stop codon were dispensable for the PRRSV viability. A chimeric PRRSV (type 2 with 3UTR from type 1) was viable and was similar to the parental strain. PRRSV expresses its genes via a set of nested subgenomic (sg) mRNAs. The utilization of TRS remains a puzzle, as many TRS-like sequences exist in viral genomes, yet only 6 or 7 sg mRNAs were transcribed in arterivirus infected cells. A PRRSV infectious cDNA clone pCPV expressing the capsid gene of PCV2 between PRRSV ORF1b and ORF2a was developed. The recombinant viruses contained a range of disparate deletions of the inserted PCV2 sequence, yet 2 stable recombinant viruses containing 41 and 275 nt of foreign sequences were generated. Further analysis of the sg RNA2 profile revealed that an array of novel sg RNA species was generated in infected cells. PRRSV can utilize foreign TRS-like sequences as transcriptional promoter and the insertion of foreign sequence provoked the generation of novel subgenomic RNAs utilizing cryptic TRS-like sequences that remain non-functional in native PRRSV. 1.5. (NADC-Faaberg/Brockmeier/Loving/Miller). Compared pathogenesis in pigs from challenge with 3 new Type 2 PRRSV. Comparison in growth and disease after vaccination with nsp2 deletion mutant viruses and novel Type 2 challenge. Faaberg/Lager compared virulence of a recombinant Chinese porcine high fever disease strain or VR-2332 in US swine. Kehrli/Miller/Faaberg provided adenovirus expression of a region of PRRSV nsp 2. Developed an infectious clone of Vietnamese porcine high fever disease strain and described cell culture phenotype (Faaberg, Guo). Developed nsp9 and ORF6 mutants of MN184, an infectious clone of a new vaccine strain and examination of the interaction of nsp2 with host genes (Faaberg). Developed an in vitro PRRSV infected tissue culture assay to screen for polyclonal B-cell activation (Miller/Kehrli). Study of PRRSV strains with a reduced induction of polyclonal B-cell activation for possible vaccine (Miller/Kehrli/Faaberg). Genomic DNA purification for SNP chip analysis to examine genotype in PCV2 host susceptibility (Miller/Rohrer-USMARC). Compared acute cytokine responses after infection with PRRSV, PRV, PCV2 and SIV (Miller). Transcript expression analysis of TBLN of PRRSV/PRV/SIV/PCV-2 in vivo (Miller). Conducted transcriptome and cytokine assays for PRV animal experiment (Miller/Lager/Zanella). Developed type 1 IFN bioassay to examine PRRSV strain specific pathology (Faaberg/Miller/Guo). 1.6. (UIUC) PRRSV nsp1± has a suppressive activity for IFN² production mediated through the RIG-I pathway. Nsp1± inhibited IºB phosphorylation and NF-ºB translocation to the nucleus, causing inhibition of NFºB stimulated gene expression. Nsp1 blocked dsRNA-induced IRF3 and IFN promoter activities through nsp1 degradation of CBP, leading to the block of IFN response. Nsp1 may form a new class of viral antagonists for IFN. Myristoylation of PRRSV E protein is non-essential for PRRSV infectivity, but promotes the growth of the virus. Enveloped viruses trigger secretion of IFNa by pDC but they remain quiescent when exposed to PRRSV, possibly due to virus-mediated suppression. An augmented phosphorylation of NFkB seen in activated pDC was not only unaffected by PRRSV but actually occurred in its presence. PRRSV may interact with a cell-surface protein(s) to impede signaling cascades involved in IFN-a production by stimulated pDC. 1.7. (UConn) studies on sensitivity to IFNb and ability to induce type I IFN responses by PRRSV show significant differences in sensitivity to IFNb among different PRRSV isolates and between MARC cells and PAMs. Field isolates and chimeric viruses were tested for induction of IFNb in PAMs. Although variable, PRRSV induces IFNb in PAMs. A flow cytometry assay, using anti-swine IFNb Abs produced in this laboratory, was developed. swIFNb was detectable in PAMs and chimeric and field strains of PRRSV induce IFNb. Extracellular or secreted swIFNb was detected by indirect ELISA in infected cells. Efforts are currently in identifying segments in the type I IFN pathway that may be blocked by the virus. Expression of TNF± and Mx was tested in PRRSV-infected PAMs. Results show that regardless of the IFNb levels detected in ELISA, TNF± expression appeared to be blocked early after infection, whereas, Mx expression was detected early, but appeared variable suggesting that blocking of type I IFNb activity may occur at the signaling phase. 1.8. (Purdue) How PRRSV controls host immune cell responses via Foxp3 expression and AKt pathway is being investigated. Goal: to determine putative gene sets and pathways that predict a pig's ability to clear infection and maintain weight gain. A 2nd study evaluates the host immune response to homologous and heterologous PRRSV challenge by validating the utility of gene sets and pathways for prediction of responsiveness to PRRSV infections in multiple populations. 1.9. (UMN) whole genome sequencing continued on field isolates indicating that recombination occurs in the field. Evolution of type 2 PRRSV was analyzed (UMN, Hong Kong Univ., Univ. of Guelph, Canada). Research on homologous immune protection in reproductive disease was completed. Virulent virus infection prior to breeding provides solid protection but not complete prevention of reproductive losses and fails to prevent transmission of PRRSV to piglets. At UMN/OSU, research was initiated on positive prognosticators of protection. Growing pigs were inoculated with a virulent field isolate in 2 studies. Unexpectedly, the virus did not cause clinical disease. 1.10. (UNL-Pattnaik/Osorio). Studied interactions of GP5, 2a, 3 and 4 and with the cellular receptor for PRRSV. Cloned each GP and CD163 receptor in expression vectors and examined their expression and interaction in transfected cells. A strong interaction with GP4 and 5 is seen, weak interactions with other minor envelope GPs and GP5. GP2a and 4 interacted, resulting in formation of multi-protein complex and interactions with CD163. The carboxy-terminal of CD163 is not required for interactions with GP2a or 4, but is for susceptibility to PRRSV infection in cells. GP4 with GP2a, serves as the viral attachment protein for mediating interactions with CD163 for virus entry. There is an anti-IFN effect of nsp1². 4 of 10 nsps had inhibitory effects on ²IFN promoter activation. The strongest inhibitory effect was by nsp1, then nsp 2, 11, and 4. Nsp1±, 1² and 11 had strong inhibitory activity, inhibiting dsRNA signaling. Nsp11 inhibited IRF3 and NFºB-dependent gene induction by dsRNA and Sendai virus. dsRNA-induced phosphorylation and nuclear translocation of IRF3 were strongly inhibited by nsp1. Nsp 3-8 and ORF5 are important for virulence. Single and triple mutants of ORF5 were generated. A change in AA position 64 seemed to contribute most of the virulence. There are certain key AA in ORF5 contributing to PRRSV virulence. 1.11. (OSU), Evaluation of the efficacy of MLV-PRRSV vaccine administered IN to pigs with choleratoxin and OK432 to enhance the anti-PRRSV specific immunity was performed. OK-432 is a killed Strep. pyogenes product, is a DC maturation agent and promotes the production of inflammatory cytokines. OK432 upregulated the frequency of NK cells, CTLs, Th/memory and Th cells. The choleratoxin upregulated the frequency of PRRSV specific CTLs and Th/memory cells while both adjuvants upregulated IFNg, IL-12, and IL-6 in lungs to MN184 challenge compared to mock or only MLV received and challenged pigs. This suggests that higher levels of Th1 and Th2 responses induced by choleratoxin and OK432 are beneficial. PLGA microspheres were prepared and then killed PRRSV Ags were entrapped. Engulfment of nanoparticles by PAMs was observed and entrapped PRRSV antigens were co-localized in the early endosome compartment of PAMs, indicating these PRRSV antigens are targeted to APCs. In vivo studies are in progress. 1.12. (ISU) Findings: PRRSV can persist in pigs without significant viral genetic change. It was demonstrated that IL-8, IL-1² and IFN³ levels are linked to PRRSV clearance. 1.13. (VA Tech) The effect of an IFN-stimulated response element (ISRE) mutant of PCV2 on PCV2-induced pathological lesions in a PRRSV co-infection model was evaluated. An ISRE-mutant PCV2 was used to infect pigs with either ISRE mutant or wt PCV2 singly or in combination with PRRSV. The ISRE mutation reduced viral replication. Lesions were more severe in pigs coinfected with ISRE-mutant PCV2 and PRRSV than in pigs coinfected with wtPCV2 and PRRSV. The hypothesis that current PRRS vaccines do not differ from pathogenic strains in the ability to induce Tregs was tested. PRRSV vaccine and parent strain are equally able to induce Tregs in pigs naturally infected with M. hyopneumoniae (LeRoith). Tregs induction in DC from pigs co-infected with PRRSV and PCV2 were studied (LeRoith). DCs were split into 4 groups: PRRSV alone, PCV2 alone, PCV2 plus PRRSV, and controls. The PCV2 infected group induced more Tregs than controls or singly infected groups. In the PCV2 negative pigs, the coinfected group induced significantly more Tregs than controls or individually-infected groups, and the coinfected and PCV2 infected groups had greater Treg induction. 1.14. (SDSU-Wang) Investigation of the molecular mechanism of PI3K/Akt activation mediated by PRRSV and possible link between PI3K/Akt pathway and IFN± during infection of Mo-DC. (Fang). Identification of 2 products of nsp1 in PRRSV infected cells function as IFN antagonists. Nsp1² significantly inhibited expression from an IFN-stimulated response element promoter after Sendai virus infection or IFN treatment and inhibited nuclear translocation of STAT1. Nsp1² inhibits IFN synthesis and signaling. Nsp1± inhibits IFN synthesis. (Fang), the cysteine protease domain of PRRSV nsp 2 possesses deubiquitinating and IFN antagonism functions. To determine if the nsp2 protein antagonist function can be ablated from the virus, point mutations in the OUT domain region were made. The mutations targeting a B-cell epitope in the OTU domain region generated viable recombinant viruses. Certain mutations lethal to virus replication impaired the ability of nsp2 to inhibit NFkB activation. Recombinant viruses didnt inhibit NFkB as effectively as wt virus. (Fang) Immunodominant epitopes in PRRSV nsp2 are dispensable for replication, but play an important role in modulation of the host immune response. Objective 2. Understand the ecology and epidemiology of PRRSV and emerging viral diseases of swine. 2.1. (VA Tech) found PCV2 ORF3 is dispensable for virus infection but evidence of reduced pathogenicity is limited in pigs infected by an ORF3-null PCV2 mutant. ORF3 of PCV2 reportedly induces apoptosis and is associated with PCV2 pathogenicity. An ORF3-null PCV2 mutant (muPCV2) was created and demonstrated that the dimerized plasmid DNA of muPCV2 clone is infectious in pigs. The pathogenicity of the muPCV2 and the wt PCV2 was compared and pigs inoculated with muPCV2 had delayed seroconversion and lower serum viral load, but no significant differences were seen in the mean scores of histologic or gross lesions or the amount of PCV2-specific antigen in tissues. 2.2. (KSU/BARC & others) participated in the PHGC. Infection and sample collection of approximately 1600 pigs revealed the appearance of stratified subpopulations which possessed wide variations in weight, virus load and growth performance. The 60K SNP chip analysis is started on the first 600 pigs (Dekker). 2.3. (UMN/ISU/SDSU), the role of aerobiological mechanisms in PRRSV transmission was investigated. Biosecurity protocols to reduce airborne spread were validated, specifically, 2 air filtration options (antimicrobial and electrostatic filters). 2.4. (UMN) SIV transmission in naïve and vaccinated pigs was studied. Studies on spread of pH1N1, calculation of the basic reproduction ratio (Ro) based on the outcome of transmission experiments, effect of homologous or heterologous vaccination on transmission and protection was investigated. Vaccination reduced transmission from 100% to 37.5% with heterologous and to 0% with homologous vaccine. Ro was from 3.8-4.6 in controls to ~1 in heterologously vaccinated pigs, 0 in homologously vaccinated pigs. 1155 nasal swabs: 13% SIV PCR+; 46 pens were positive since at least 1 animal was PCR+. Of 105 ropes, 38/105 (36%) were positive by PCR. Nasal swabs and oral fluids were strongly correlated. Most negative oral fluids were from vaccinated pigs. Predicted probability to detect SIV in a pen was 69% if 9% of pigs were infected, 99.67% if 18% were infected, 99.99% if the % of infected pigs was 27-100%. Disease ecology of SIV in breeding farms showed SIV was not detected in sows/gilts on the study farms, but was detected in neonatal pigs. Sows were predominantly viremic, shed PCV2 in colostrum, oral fluids. High viral loads seen in the presence of high levels of anti-PCV2 Abs in serum and colostrum; PCV2 was observed on skin and in serum of pre-suckling piglets (~30%). PCV2 infection is persistent in the presence of Abs; piglet infection in utero is common. 2.5. (SHVRI-Yuan, Shishan) A molecular survey was conducted of PPV4 in China from 2006-2010. PPV4 is present in swine herds, 2.09% (12/573) in clinical samples and 0.76% (1/132) from healthy animals. None were detected in samples prior to 2009. The Chinese and American PPV4 sequences are closely related. Viral genomes in head-to-tail configuration of various lengths of the non-coding region were detected confirming that PPV4 is a unique, recently discovered virus in pigs. PPV4 is most closely related to bovine parvovirus 2 (BPV2); shares limited ORF1 and 2 identity. PPV4 encodes an ORF3 resembling the Bocavirus genus, but shares minimal AA identity with Bocavirus genus ORF3 proteins. First evidence of infection by porcine bocavirus (PBoV) in Chinese swine; was more prevalent in weanling pigs with respiratory signs. Partial VP1/2 genes were highly conserved and only 5 frequent nt mutation positions exist in Chinese PBoV indicating it might be an emerging porcine respiratory virus. 2.6. (UGA) pH1N1 contained the TRIG cassette that predominates in SIV and has infected both swine and avian populations. Exploration of the potential for swine, human and avian influenza viruses to reassort on the TRIG backbone in swine and primary swine epithelial cells, and primary human epithelial cells to elucidate the potential for reassortment in these species. 2.7. (Purdue) Study of TTV infection in commercial wean-to-finish populations concurrently infected with PRRSV, PCV2, and SIV is being investigated. Of 600 oral fluid samples, 120 have been tested, 25/120 (21%) were positive for TTV-1, 97 (80%) were positive for TTV-2; 23/25 TTV-1 positive samples were positive for TTV-2 suggesting TTV infection was common in the 10 commercial wean-to-finish cohorts. 2.8. (UNL/UWI/SDSU) PRRSV CAP2 project: Immunologic Consequences of PRRSV Diversity, understanding what defines PRRSV strains as immunologically homologous or heterologous is critical to the development of vaccines. 1) Sequence a core set of PRRSV representing the breadth of gene variation 2) associate relevant immunologic phenotypes with PRRSV genomic variation. Immunologic cross-protection among a subset of viruses from the core set will be quantified in pigs. Isolates in the core set will be characterized for cross-neutralization in vitro. Serum is used to evaluate inter-isolate variation for in vivo immune responses. (UNL) Demonstration of potential T-cell epitopes present in nsp 9, 10 of type 2 PRRSV eliciting IFN³ responses. Characterization of the epitopes is important in modifying CMI. Nsp9 and 10 and were analyzed to determine their T-cell epitopes with proliferation assays and ELISPOT. In 4/78 nsp9 peptides and 2/54 nsp10 peptides were found to induce T-cell proliferation. Only 2 peptides of nsp9 and 2 peptides of nsp10 were detected using ELISPOT. Sequencing of 34 NA strains showed these epitopes were highly conserved, possible use for vaccines to provide cross-protection against PRRSV. 2.9. (ISU) Evaluation of duration of breeding herd PRRS virus-free status and its relationship with measured risk was performed. Surveyed disease pressures in 26 Midwest herds. Described reproductive failure associated with PPV and PCV2 coinfection. Evaluated factors that influence the environmental stability of SIV and PRRSV. Evaluated PRRSV transmission via ingestion of meat and exposure to aerosols from persistently-infected pigs. Described ecology of influenza virus in non-porcine species. Compared detection of endogenous retrovirus viremia in diseased vs healthy pigs by qtPCR. 2.10. (VA Tech) identified 4 distinct full-length genomic sequences of PTTV strains from a single pig. Results showed that these 4 prototype US strains of PTTV represent distinct genotypes or subtypes and a revised classification system for PPTV is proposed. Objective 3. Develop effective and efficient approaches for detection, prevention and control of PRRSV and emerging viral diseases of swine. 3.1. (KSU/others) are incorporating Luminex for the detection of PRRSV Abs and other pathogens for profiling multiple agents within a herd. 3.2. (SDSU/BARC) An FMIA to simultaneously quantify 8 porcine cytokines (IL-1², IL-8, IFN± TNF±, IL-10, IL-12, IFN³, IL-4) in serum using Luminex xMap" technology was developed. Levels were evaluated in pigs vaccinated with a MLV vaccine or killed virus vaccine with adjuvant and then challenged with a non-identical PRRSV. Studies are ongoing to measure cytokines in PHGC sera to identify biomarkers that may predict which pigs are resistant to PRRSV. US Veterinary Immune Reagent Network produced additional mAbs for FMIAs. FMIA assays to detect Ags and Abs to multiple swine pathogens simultaneously (eg. PRRSV, PCV2, SIV) is ongoing. 3.3. Studies on viruses are hampered by lack of in vitro systems for propagation and amplification. SHVRI (Yuan) have devised an alternative culturing system by recombining the PRRSV infectious cDNA into a baculovirus vector for generation of infectious virus particles by expression of the full-length cloned genome from the modified baculovirus vector. The recombinant baculovirus, AcAPRRS, was used to infect sf9 cells and IFA demonstrated the presence of nsp2 and N protein. EM showed PRRSV particles. Infectious particles were produced in MARC cells inoculated with AcAPRRS, and growth characteristics were similar to the parental strain. Infectious PRRSV was generated following AcAPRRS transduction of BHK-21 cells and Vero cells that are not sensitive to PRRSV. 3.4. (NADC-Faaberg), developed and tested the ability to detect and sequence Asian porcine high fever disease strain transcript RNA. Validation of a PRV rtPCR assay (Miller/Lager/Zanella). 3.5. (UIUC) Goal: to associate immunologic parameters (SN, T-cell responses) with specific genomic signatures in PRRSV. A novel method of sequencing library construction was developed which allows unbiased sequencing of full-length genomes using pyrosequencing. Full genome sequencing of 8 serologic groups was performed. 3.6. (UGA) the potential for prophylactic and therapeutic application of PRRSV-specific swine mAbs is studied along with its cost-effectiveness. A human virus-like particle vaccine was tested in swine for induction of protective Abs. Both IM and aerosol delivery were tested. While the H1N1-specific vaccine was immunogenic, it failed to induce a protective response. A PIV5-vectored vaccine is being tested for immunogenicity with SIV antigens. If efficacy is observed with this vector against SIV, PRRSV antigens will be tested with future PIV5 constructs. Cross-validating human and mouse Abs for reactivity to swine and ferret cells and cytokines using a multiplex assay is studied. Establishing primary normal swine bronchoepithelial cell cultures has been done to directly measure innate responses to swine respiratory virus infection. 3.7. (UMN) The sensitivity of PRRSv detection by PCR from serum and oral fluids embedded in FTA cards stored for 14 days was 101 TCID50/ml. Sensitivity and specificity was equal in fresh serum and serum on FTA cards, independent of storage time (overnight or 2 weeks) or temperature (4ºC or 25ºC). Regional PRRSV eradication studies in Stevens Co, MN consists of 87 farms. The approx. number of sows is 17,844, with 16,700 sows owned by 5 entities. Surveillance has decreased since most, and possibly all farms have eliminated PRRSV. A risk-based surveillance system was developed. PRRS appears under control in the Co. and has now expanded. 3.8. (UNL-Ciobanu/Osorio/Johnson), studies found that host genetic variation influences the incidence of PCVAD. Obj 1) Detect regions of the genome that affect PCVAD severity and harbor key modulators of the changes in gene expression following infection. 2) Identify genes, pathways and combination of allelic variants that influence PCVAD severity. Hamps x Duroc pigs experimentally infected with PRRSV gained less weight, had higher rectal temperature, viral loads, Ab titer, and incidence of lung lesions compared to NE Index Line. High pre-inoculation levels of IL8 and low post-inoculation levels of IFN³ were significantly associated with potential resistance to PRRSV. There was variation in magnitude and time of immune response after PCV2b challenge. Individuals displaying early or no immune response were less affected by inoculation. The lack of immune response was associated with reduced viremia, likely due to a mechanism that inhibits virus replication. There were moderate to high heritabilities for viremia and IgG in pigs naturally infected with PCV2. Viral load affected growth. In natural infection, viremia was highly genetically correlated with a PCVAD score. 3.9. (UMD-Zhang, Zhu), studies continued on developing anti-PRRSV PPMOs which are ssDNA analogs that exhibit highly specific binding to complementary RNA. A PPMO (5UP2) designed to complement sequence in the 5 region of the genome was effective in inhibiting PRRSV replication in cell culture. PPMO was given to 3 wk old piglets IN at 24 hrs before, and 2 and 24 hrs after PRRSV inoculation. Pigs given PPMO had significantly milder pneumonia, reduced viremia at 6 dpi; at 14 dpi, had a lower level of anti-PRRSV Abs than controls. All pigs had similar weight gain. 3.10. (CNB), animal studies using a TGEV vector expressing PRRSV M protein and GP5 mutant (altered glycosylation) showed all animals had high Abs against TGEV. After virulent PRRSV, there was a fast recall response; vaccinated animals had higher Abs, lung lesions in vaccinated animals were low. No full protection possibly due to low levels of neutralizing Abs prior to challenge. A rTGEV vector was constructed, expressing PRRSV GP2a, GP3 and GP4. This rTGEV vector was not stable as GP3 gene was lost. rTGEV vectors expressing PRRSV Ags were not fully stable. Loss of PRRSV Ag expression could be the cause for the modest results obtained in protection experiments using live rTGEV vectors compared to killed vaccines. Therefore, as PRRSV M protein is fully stable when cloned in rTGEV vectors, it has been the base for vectors co-expressing this protein and different small GP5 domains containing the neutralizing epitope (GP5-NH2). Eventually, this strategy would allow the elimination of GP5 domains providing T cell negative regulatory signals that may reduce the strength of the immune response to PRRSV antigenic domains involved in protection. The construction of rTGEV expressing GP5-NH2 and M proteins (rTGEV-GP5-NH2 -M) is planned. Abs to detect GP5-NH2 expression are not available, so an HA tag will be introduced. Recombinant TGEV viruses co-expressing GP5 fragments and M have been obtained. Abs recognizing PRRSV GP2, GP3 and GP4 are not available. Each gene was cloned and high levels of each protein have been generated and purified. Polyclonal Ab generation is in progress. 3.11. (UWI) Novel approaches are used to identify a small number of representative viral genotypes from among a diversity of sequences. Techniques from network theory were adapted to rank PRRSV sequences in their importance among sequences (>10,000 in the literature). and these methods were applied to a highly-curated PRRSV database that combines high-quality, non-recombinant sequences from GenBank and PRRSVdb. Viruses represented by the top ranking sequences are valuable for study and can be incorporated into a polyvalent vaccine. Dr. Tavis Anderson, has performed analysis with support of an exchange program with the Univ, of Torino, Italy. 3.12. (ISU) evaluated Ab responses of nsps for detection and differentiation of Type 1 and 2 PRRSV. Compared efficacy of PRRSV extraction and PCR for oral fluids showing PCR detection in oral fluids and serum over time is similar. Evaluated PRRSV stability and anti-PRRSV Ab in oral fluids, determined samples should be chilled or frozen. Described performance of a nucleoprotein ELISA for Ab detection and an ELISA in pigs using a commercial avian influenza epitope blocking ELISA. Developed a matrix-gene based multiplex PCR for detection and differentiation of p H1N1 and other influenza A viruses in NA. Showed that Mchip differentiates human H1N1, NA swine H1N1, and pH1N1. Developed PCR for simultaneous detection of PCV2, PPV, PRV, and PRRSV. Showed prolonged detection of PCV2 and anti-PCV2 Ab in oral fluids. Developed an SN for Nipah Virus using pseudotype particles. Compared efficacy of commercial PCV2 vaccines using a mixed PRRSV-PCV2-SIV infection. Compared efficacy of passive vs active vaccination against PCV2 and impact of passive Abs on vaccination. Compared PCV2 vaccines on challenge with PCV2, PRRSV and PPV. Presented terminology for classifying swine herds by PRRSV status. Reviewed veterinary vaccines for Henipaviruses in the OIE Manual. 3.13. (VA Tech) developed a SYBR green rtPCR and duplex nPCR assay for quantitation and differential detection of porcine TTV and 2 SYBR green-based PCR assays to quantify viral loads and differentiate 2 porcine TTV species (PTTV1 and PTTV2). A type-specific duplex nPCR was developed to simultaneously detect and distinguish between PTTV1a and 1b.

Publications

See attachment under Summary of Minutes for complete NC229 Annual Report

Impact Statements

See attachment under Summary of Minutes for complete NC229 Annual Report

Date of Annual Report: 12/02/2011

Report Information

Annual Meeting Dates: 12/02/2011 - 12/02/2011
Period the Report Covers: 06/01/2010 - 11/01/2011

Participants

Chair: Meng, X.J. - Virginia Polytechnic Institute and State University (VA Tech) - xjmeng@vt.edu;
Secretary: Christopher-Hennings, Jane - SDSU - jane.hennings@sdstate.edu;
Rowland, Raymond R.R. - Kansas State University (KSU)- browland@vet.k-state.edu;
Benfield, David - Ohio State University (OSU)- benfield.2@osu.edu;
Faaberg, Kay, - National Animal Disease Center (NADC)- kay.faaberg@ars.usda.gov;
Goldberg, Tony - University of Wisconsin-Madison - tgoldberg@vetmed.wisc.edu;
Gourapura, Renukaradhya J. - The Ohio State University (OSU)- gourapura.1@osu.edu;
Johnson, Peter - USDA, CSREES - pjohnson@reeusda.gov;
Lunney, Joan - USDA-ARS, BARC - joan.lunney@ars.usda.gov;
Murtaugh, Michael P - University of Minnesota (UMN)- murta001@umn.edu;
Osorio, Fernando A.- University of Nebraska-Lincoln (UNL) - fosorio@unl.edu;
Pogranichniy, Roman - (Purdue), IN - rmp@purdue.edu;
Risatti, Guillermo - University of Connecticut - guillermo.risatti@uconn.edu.;
Tompkins, S. Mark - University of Georgia (UGA)- smt@uga.edu;
Yuan, Shishan - Shanghai Veterinary Res. Inst. (SHVRI), SHVRI - shishanyuan@hotmail.com;
Zhang, Yanjin - University of Maryland - zhangyj@umd.edu;
Zimmerman, Jeff - Iowa State University (ISU)- jjzimm@iastate.edu;
Zuckermann, Federico - University of Illinois at Urbana-Champaign (UIUC) - fazaaa@illinois.edu;

Other NC229 Scientists: ;
Abrams, Sam - BARC;
Anderson, Tavis - UWI;
Araujo, Karla - BARC;
Araujo, Karla - BARC;
Arceo M - Purdue;
Baker, RB - ISU;
Blecha, Frank - KSU;
Boddicker, Nick - ISU;
Brockmeier, Susan - NADC;
Calvert, Jay - Pfizer Animal health;
Carman, Susy - University of Guelph, Canada;
Chang, KC - KSU;
Chen, Hongbo - USDA-BARC;
Choi, Igseo - BARC;
Ciobanu, Dan - UNL;
Clark, A. - Purdue University;
Davies, Peter -UMN;
Dee, Scott - UMN;
Dekkers, Jack - ISU;
Ernst, Cathy - MSU;
Fang, Ying - SDSU;
Garmendia, Antonio - UCONN;
Garrick, Dorian - ISU;
Gourapura, Aradhya - OSU;
Gramer, Marie - UMN;
Halbur, Patrick -ISU;
Haley, Charles - USDA-APHIS;
Harhay, Greg - NADC;
Harris, DL (Hank) - ISU;
Hause, Ben - Newport Labs, MN;
Hesse, Dick - KSU;
Holtkamp, Derald J - ISU;
Huang T - Purdue;
Huang, Tinghua - ISU;
Jiang, Zhihua - WSU;
Johnson, John K - ISU;
Joo, Han Soo - UMN;
Karriker, Locke - ISU;
Kerhli, Marcus Jr. - NADC;
Kerrigan M. - KSU;
Kittawornrat Apisit - ISU;
Kuhar, D. - USDA-USDA-BARC;
Laegried, Will - UIUC;
Lager, Kelly - NADC;
Lawson, Steve - SDSU;
Lazar V -Purdue ;
Leung, Frederick - Hong Kong University;
Loving, Crystal - NADC;
Lunney, Joan - BARC;
Main, Rodger G - ISU;
McCaw, Monte B. (deceased) - NCSU;
McKean, JD - ISU;
Moore, B - Purdue;
Morrison, Robert - UMN;
Nelson, Eric - SDSU;
Nerem, Joel Pipestone - Vet Clinic, MN;
Nicholson, Tracy - NADC;
Opriessnig, Tanja - ISU;
Pattnaik, Asit - UNL;
Prickett, J. - ISU;
Ramamoorthy, Sheila - UGA;
Ramirez, Alejandro - ISU;
Ramirez-Nieto, Gloria - Universidad Nacional de Colombia;
Raney NE - Purdue;
Raney, Nancy - MSU;
Reecy, Jim - ISU;
Rossow, Kurt - UMN;
Roth, JA - ISU;
Rothschild, Max - ISU;
Rovira, Albert - UMN;
Rowland, R.R.R. - KSU;
Sang, Yongming - KSU;
Schwartz, Kent J. - ISU;
Sina, R - Purdue;
Souza, Carlos - BARC;
Steibel, J.P. - MSU;
Stevenson, Greg W. - ISU;
Strait, Erin - ISU;
Srinivas, Jay - CVB-PEL/APHIS/USDA;
Torremorell, Montserrat - UMN;
Trible B. - KSU;
Tripp, Ralph - UGA;
Tuggle, Chris - ISU;
Waide, Emily - ISU;
Wang, Chong -ISU;
Wang, Xiuqing -SDSU;
Wyatt, Carol - KSU;
Wysocki, Michal -USDA-BARC;
Yoo, Dongwan -UIUC;
Yoon, Kyoung-Jin -ISU;
Zhang, C. - VA Tech;
Zhu, Xiaoping - UMD;
Zimmerman, Jeff - ISU;

Brief Summary of Minutes

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

Minutes Attachment:

Attachment

Accomplishments

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.

Publications

Abrahante JE, Martins K, Papas KK, Hering VJ, Schuurman H-J, Murtaugh MP. 2011. Microbiological safety of porcine islets: comparison with source pig. Xenotransplantation. 18:88-93. Baker SA, Mondaca E, Polson D and Dee SA. Evaluation of a needle-free injection device (AcuShot) for reduction of hematogenous transmission of PRRS virus. Swine Health Prod (accepted for publication). Baker SR, ONeil K, Gramer M and Dee SA. An estimate of seroprevalence of production-limiting diseases in wild swine. Vet Rec 2011;168:564 Beura LK, Dinh PX, Osorio FA, Pattnaik AK.(2011) Cellular Poly(C) Binding Proteins 1 and 2 Interact with Porcine Reproductive and Respiratory Syndrome Virus Non-Structural Protein 1{beta} and Support Viral Replication. J Virol. 2011 Oct 5. In press [Epub ahead of print] Binjawadagi B, V. Dwivedi, C. Manickam, J.B. Torrelles, and G.J. Renukaradhya (2011) Intranasal delivery of an adjuvanted modified live porcine reproductive and respiratory syndrome virus vaccine reduces the ROS production. Viral Immunol., In press. Bishop SC, Lunney JK, Pinard-van der Laan MH, Gay CG. 2011. Report from the Second International Symposium on Animal Genomics for Animal Health: Critical Needs, Challenges and Potential Solutions. Proc. Intnl. Symp. Animal Genomics for Animal Health (AGAH 2010). BMC Proceedings 5 Suppl 4:S1. Boddicker, N, EH Waide, RRR Rowland, JK Lunney, DJ Garrick1, JM Reecy, JCM Dekkers. Major QTL associated with host response to porcine reproductive and respiratory syndrome virus challenge. J Anim Sci. In Revision. Bradley, K.C., Jones, C.A., Tompkins, S.M., Tripp, R.A., Russell, R.J., Gramer, M.R., Heimburg-Molinaro, J., Smith, D.F., Cummings R.D., and D.A. Steinhauer. 2011 Comparison of the receptor binding properties of contemporary swine isolates and early human pandemic H1N1 isolates (novel 2009 H1N1). Virology. 413(2):169-82. PMID: 21353280 Brar MS, Shi M, Carman S, Ge L, Murtaugh MP, Leung FC-C. 2011. Porcine reproductive and respiratory syndrome virus in Ontario, Canada 1999-2010: diversity and restriction fragment length polymorphism. J Gen Virol. 92:1391-1397. PMID: 21346028. Calzada-Nova, G., Schnitzlein, W., Husmann, R., Zuckermann, F.A. 2011. North American porcine reproductive and respiratory viruses suppress the type I interferon response of activated porcine plasmacytoid dendritic cells. J. Virol. 85:2703-13. Chittick WA, Stensland WR, Prickett JR, Strait EL, Harmon K, Yoon K-J, Wang C, Zimmerman JJ. 2011. Comparison of RNA extraction and RT-PCR methods for the detection of porcine reproductive and respiratory syndrome virus (PRRSV) in porcine oral fluid specimens. J Vet Diagn Invest 23:248-253.C Cino-Ozuna, AG, S Henry, R Hesse, JC Nietfeld, J Bai, HM Scott, RRR Rowland.2011. Characterization of a new disease syndrome associated with porcine circovirus type 2 (PCV2) in previously vaccinated herds. J Clin Micro. In press. Corzo C, Mondaca E, Wayne S, Torremorell M, Dee S, Davies P, Morrison B (2010). Control and elimination of porcine respiratory syndrome virus. Virus Res, 154:185-192, doi:10.1016/j.virusres.2010.08.016 Cutler T, Wang C, Qin Q, Zhou F, Warren K, Yoon K-J, Hoff SJ, Zimmerman J. 2011. Kinetics of UV254 inactivation of selected viral pathogens in a static system. J Appl Microbiol 111:389-395. Cutler TD, Wang C, Hoff SJ, Kittawornrat A, Zimmerman JJ. 2011. Median infectious dose (ID50) of porcine reproductive and respiratory syndrome virus isolate MN-184 via aerosol exposure. Vet Microbiol 151:229-237. Cutler TD, Zimmerman JJ. 2011. Ultraviolet irradiation and the mechanisms underlying its inactivation of infectious agents. Anim Health Res Rev 12:15-23. Das PB, Vu HL, Dinh PX, Cooney JL, Kwon B, Osorio FA, Pattnaik AK.(2011)Glycosylation of minor envelope glycoproteins of porcine reproductive and respiratory syndrome virus in infectious virus recovery, receptor interaction, and immune response. Virology. 2011 Feb 20; 410(2):385-94. Epub 2010 Dec 30. Debin Tian, Haihong Zheng, Rong Zhang, Jinshan Zhuang, Shishan Yuan. 2011. Chimeric Porcine Reproductive and Respiratory Syndrome Viruses reveal full function of genotype 1 envelope proteins in the backbone of genotype 2. Virology. YVIRO-06057; No. of page: 8; 4C: 4. Dee S, Spronk G, Reicks D, Ruen P and Deen J. Further assessment of air filtration for preventing PRRSV infection in large breeding herds. Vet Rec 2010;167:976-977. Dee SA, Otake S and Deen J. An evaluation of ultraviolet light (UV 254) as a means to inactivate porcine reproductive and respiratory syndrome virus on common farm surfaces and materials. Vet Microbiol 2011;150:96-99 Dee SA, Otake S and Deen J. Use of a production region model to assess the efficacy of various air filtration systems for preventing the airborne transmission of porcine reproductive and respiratory syndrome virus and Mycoplasma hyopneumoniae. Results of a 2-year study. Virus Res 2010. 154:177-184. Dee SA, Otake S, Pitkin A and Deen J. A 4-year summary of air filtration system efficacy for preventing airborne spread of porcine reproductive and respiratory syndrome virus and Mycoplasma hyopneumoniae. Swine Health Prod 2011;19:292-294. Dlugolenski, D., Jones, L., Saavedra, G., Tompkins. S.M., Tripp R.A., and E. Mundt. 2011 Passage of low-pathogenic avian influenza (LPAI) viruses mediates rapid genetic adaptation of a wild-bird isolate in poultry. Arch Virol. 156(4):565-76. PMID: 21197555 Driskell, E.A., Jones, C.A., Berghaus, R.D., Stallknecht, D.E., Howerth, E.W., and S.M. Tompkins. 2011 Domestic cats are susceptible to infection with low pathogenic avian influenza viruses from shorebirds. In press, Veterinary Pathology. Driskell, E.A., Jones, C.A., Stallknecht, D.E., Howerth, E.W., and S.M. Tompkins. 2010 Avian influenza isolates from wild birds replicate and cause disease in a mouse model of infection. Virology. 399(2):280-9. PMID: 20123144 Driskell, J.D., Jones, C.A., Tompkins, S.M., and R.A. Tripp. 2011 One-Step Assay for Detecting Influenza Using Dynamic Light Scattering and Gold Nanoparticles. Analyst 136(15):3083-90. PMID: 21666913 Cover Article. Du, Y., D. Yoo, M. A. Paradis, and G.Scherba. 2011. Antiviral activity of tilmicosin for type 1 and 2 porcine reproductive and respiratory syndrome virus in cultured porcine alveolar macrophages. J. Antivirals Antiretrovirals 3:28-33. Dwivedi V, C. Manickam, R. Patterson, K. Dodson, and G.J. Renukaradhya (2011). Intranasal delivery of whole cell lysate of Mycobacterium tuberculosis induces protective immune responses to a modified live porcine reproductive and respiratory syndrome virus vaccine in pigs. Vaccine, 29(23):4067-76. Dwivedi V, Manickam C, Patterson R, Dodson K, Murtaugh M, Torrelles JB, Schlesinger LS, Renukaradhya GJ. 2011. Cross-protective immunity to porcine reproductive and respiratory syndrome virus by intranasal delivery of a live virus vaccine with a potent adjuvant. Vaccine 29:4058-4066.PMID: 21419162 Faaberg, K. S., Kehrli, Jr., M. E., Lager, K. M., Guo, B., and J. Han. 2010. In vivo growth of porcine reproductive and respiratory syndrome virus engineered nsp2 deletion mutants. Virus Res. 154:77-85. Fei Gao, Jiaqi Lu, Huochun Yao, Zuzhang Wei, Qian Yang, Shishan Yuan, Cis- Acting Structural Element in 5 UTR Is Essential for Replication of Porcine Reproductive and Respiratory Syndrome Virus. Virus Research, [J] 2011. 10.1016/j.virusres.2011.08.018. Feifei Tan, Zuzhang Wei, Yanhua Li, Rong Zhang, Jinshan Zhuang, Zhi Sun, Shishan Yuan. 2011. Identification of non-essential regions in nucleocapsid protein of Porcine and Respiratory Syndrome Virus for replication in cell culture. Virus Research. Volume 158, issues 1-2, June 2011, page 62-71. Gauger PC, Faaberg KS, Guo B, Kappes MA, Opriessnig T. 2011. Genetic and phenotypic characterization of a 2006 United States porcine reproductive and respiratory virus isolate associated with high morbidity and mortality in the field. Virus Res [Epub ahead of print] Gauger PC, Lager KM, Vincent AL, Opriessnig T, Cheung AK, Butler JE, Kehrli ME, Jr (2011) Leukogram abnormalities in gnotobiotic pigs infected with porcine circovirus type 2. Veterinary Microbiology 154:185-190 Gauger PC, Lager KM, Vincent AL, Opriessnig T, Kehrli ME, Jr, Cheung AK (2011) Postweaning multisystemic wasting syndrome produced in gnotobiotic pigs following exposure to various amounts of porcine circovirus type 2a or type 2b. Veterinary Microbiology 153:229-239 Gauger PC, Vincent AL, Loving CL, Lager KM, Janke BH, Kehrli Jr. M.E, Roth JA. 2011. Enhanced pneumonia and disease in pigs vaccinated with an inactivated human-like (´-cluster) H1N2 vaccine and challenged with pandemic 2009 H1N1 influenza virus. Vaccine 24:2712-2719. Gordy, J.T., Jones,C.A., Rue, J., Crawford, P.C., Levy, J.K., Stallknecht, D.E., Tripp, R.A., and S.M. Tompkins. 2011 Surveillance of Feral Cats for Influenza A Virus. In Press, Influenza and Other Respiratory Pathogens. Gorres JP, Lager KM, Kong W-P, Royals M, Todd JP, Vincent AL, Wei C-J, Loving CL, Zanella EL, Janke B, Marcus E. Kehrli J, Nabel GJ, Rao SS (2011) DNA Vaccination Elicits Protective Immune Responses against Pandemic and Classic Swine Influenza Viruses in Pigs. Clinical and Vaccine Immunology 18:1987-1995 Guo, B., Vorwald, A.C., Alt, D.P., Lager, K.M., Bayles, D.O. and Faaberg, K.S. 2011. Large scale parallel pyrosequencing technology: PRRSV strain VR-2332 nsp2 deletion mutant stability in swine. Virus Res. 161:162-169 Haley C, Wagner B, Puvanendiran S, Abrahante J, Murtaugh MP. 2011. Diagnostic performance measures of ELISA and quantitative PCR tests for porcine circovirus type 2 exposure using Bayesian latent class analysis. Prev Vet Med. 101:79-88. PMID: 21632130 Hamir AN, Greenlee JJ, Stanton T, Smith JD, Doucette S, Kunkle RA, Stasko JA, Richt JA, Kehrli ME, Jr (2011) Experimental inoculation of raccoons (Procyon lotor) with Spiroplasma mirum and transmissible mink encephalopathy (TME) Canadian Journal of Veterinary Research 75:18-24 Hamir AN, Kehrli ME, Jr, Kunkle RA, Greenlee JJ, Nicholson EM, Richt JA, Miller JM, Cutlip RC (2011) Experimental interspecies transmission studies of the transmissible spongiform encephalopathies to cattle: comparison to bovine spongiform encephalopathy in cattle. J Vet Diagn Invest 23:407-420 He D, C. Overend, J. Ambrogio, R.J. Maganti, M.J Grubman, A.E Garmendia 2011. Marked differences between MARC-145 cells and swine alveolar macrophages in IFN²-induced activation of antiviral state against PRRSV. Vet Immunol Immunopathol 139(1):57-60. Hiep, L. X. Vu., Byungjoon, K., Kyoung-Jin Y., LAegreid, W., Pattnaik, A.K., Osorio, F.A. 2011. Immune Evasion of Porcine Reproductive and Respiratory Syndrome Virus through Glycan Shielding Involves both Glycoprotein 5 as Well as Glycoprotein 3. J. Virol. 85:5555-64. Holtkamp DJ, Polson DD, Torremorell M (2011). Terminology for classifying swine herds by porcine reproductive and respiratory syndrome virus status. J Swine Health and Prod 19(1):44-56. Holtkamp DJ, Polson DD, Torremorell M (2011). Terminology for classifying swine herds by porcine reproductive and respiratory syndrome virus status. Tierärztliche Praxis Großtiere 39 2: 101-112 Huang YW, Meng XJ. Novel strategies and approaches to develop the next generation of vaccines against porcine reproductive and respiratory syndrome virus (PRRSV). Virus Research. 2010;154(1-2):141-9. Irwin CK, Yoon K-J, Wang C, Hoff SJ, Zimmerman JJ, Denagamage T, O'Connor AM. 2011. Using the systematic review methodology to evaluate factors that influence the persistence of influenza in environmental matrices. Appl Environ Microbiol 77:1049-1060. Jiaqi Lu, Fei Gao, Zuzhang Wei, Ping Liu, Changlong Liu, Haihong Zheng, Yanhua Li, Tao Lin, Shishan Yuan. 2011. A 5-Proximal Stem-loop Structure of 5 Untranslated Region of Porcine Reproductive and Respiratory Syndrome Virus Genome Is Key for Virus Replication. Virology Journal 2011, 8: 172, doi: 10. 1186/ 1743-422X-8-172. Johnson CR, Griggs TF, Gnanandarajah J, Murtaugh MP. 2011. Novel structural protein in Porcine reproductive and respiratory syndrome virus encoded in an alternative open reading frame 5 present in all arteriviruses. J Gen Virol 92:1107-1116. PMID: 21307222 Kappes MA, Sandbulte MR, Platt R, Wang C, Lager KM, Henningson JN, Lorusso A, Vincent AL, Loving CL, Roth JA, Kehrli ME, Jr. (2011) Vaccination with NS1-truncated H3N2 swine influenza virus primes T cells and confers cross-protection against an H1N1 heterosubtypic challenge in pigs. Vaccine (e-pub ahead of print 07Nov2011) Kittawornrat A, Prickett J, Wang C, Panyasing Y, Ballagi A, Rice A, Main R, Johnson J, Rademacher C, Hoogland M, Rowland R, Zimmerman J. Detection of porcine reproductive and respiratory syndrome virus (PRRSV) antibodies in oral fluid specimens using a commercial PRRSV serum antibody ELISA. J Vet Diagn Invest (in press). Langenhorst Robert J., Steven Lawson, Apisit Kittawornrat, Jeffrey J. Zimmerman, Zhi Sun, Yanhua Li, Jane Christopher-Hennings, Eric A. Nelson, Ying Fang. Development of a fluorescent microsphere immunoassay for detection of antibodies against PRRSV using oral fluid samples as an alternative to serum-based assays. Journal of Clinical Vaccine and Immunology. Lawson, Steve, Yanhua Li, John Patton, Robert J. Langenhorst, Zhi Sun, Zhiyong Jiang, Jane Christopher-Hennings, Eric A. Nelson, David Knudsen, Ying Fang, Kyeong-Ok Chang. Interleukin-1² expression by a recombinant porcine reproductive and respiratory syndrome virus. Virus Research, in preparation. Lin K, Wang C, Murtaugh MP, Ramamoorthy S. 2011. Multiplex method for simultaneous serological detection of porcine reproductive and respiratory syndrome virus and porcine circovirus type 2. J Clin Micro 49:3184-3190. Linhares D, Rovira A, Torremorell M (2011). Evaluation of FTA cards for collection and transport of samples for PRRS virus diagnostics. Accepted for publication Linhares D, Torremorell M, Cano JP and Dee SA. Effect of modified live porcine reproductive and respiratory syndrome (PRRS) vaccine on the shedding of wild-type virus from an endemically infected population of growing pigs. Vaccine (accepted for publication). Lorusso A, Vincent AL, Harland ML, Alt D, Bayles DO, Swenson SL, Gramer MR, Russell CA, Smith DJ, Lager KM, Lewis NS (2011) Genetic and antigenic characterization of H1 influenza viruses from United States swine from 2008. The Journal of general virology 92:919-930 Lunney JK, DA Benfield DA, RR Rowland. 2010. Porcine reproductive and respiratory syndrome virus: an update on an emerging and re-emerging viral disease of swine. Virus Res. 154:1-6. Lunney JK, Rowland RRR. 2011. Understanding Genetic Disease Resistance. National Hog Farmer. Blueprint Immunology 101. Apr. 15, 2011. p.30-42. Lunney JK, Steibel JP, Reecy J, Rothschild M, Kerrigan M, Trible B, Rowland RRR. 2011. Probing genetic control of swine responses to PRRSV infection: Current Progress of the PRRS Host Genetics Consortium. Proceedings of the International Symposium on Animal Genomics for Animal Health (AGAH 2010). BMC Proceedings. 5 Suppl 4:S30. Nfon CK, Berhane Y, Hisanaga T, Zhang S, Handel K, Kehler H, Labrecque O, Lewis NS, Vincent AL, Copps J, Alexandersen S, Pasick J (2011) Characterization of H1N1 swine influenza viruses circulating in Canadian pigs in 2009. Journal of virology 85:8667-8679 Ni YY, Huang YW, Cao D, Opriessnig T, Meng XJ. Establishment of a DNA-launched infectious clone for a highly pneumovirulent strain of type 2 porcine reproductive and respiratory syndrome virus: identification and in vitro and in vivo characterization of a large spontaneous deletion in the nsp2 region. Virus Research. 2011;160 (1-2):264-73. Nicholson, T. L., D. Kukielka, A. L. Vincent, S. L. Brockmeier, Miller, L. C. and K. S. Faaberg. 2011. Utility of a Panviral Microarray for Detection of Swine Respiratory Viruses in Clinical Samples. J. Clin. Microbiol. 49:1542-1548. ONeill K, Shen HG, Lin K, Hemann M, Beach NM, Meng XJ, Halbur PG, Opriessnig T. 2011. Studies on PCV2 vaccination of 5-day-old piglets. Clin Vaccine Immunol [Epub ahead of print] Opriessnig T, Gomes-Neto J, Hemann M, Shen HG, Huang YW, Halbur PG, Meng XJ. 2011. An experimental live chimeric PCV1-2a vaccine is efficacious at decreasing PCV2b viremia when administered intramuscularly or orally in a PCV2b and PRRSV dual-challenge model. Microbiol Immunol [Epub ahead of print] Opriessnig T, Madson DM, Roof M, Layton S, Ramamoorthy S, Meng XJ, Halbur PG. 2011. Experimental reproduction of porcine circovirus type 2 (PCV2)-associated enteritis in pigs infected with PCV2 alone or concurrently with Lawsonia intracellularis or Salmonella typhimurium. J Comp Pathol (in press). Opriessnig T, Madson DM, Schalk S, Brockmeier S, Shen HG, Beach NM, Meng XJ, Baker, RB, Zanella EL, Halbur PG. 2011. Porcine circovirus type 2 (PCV2) vaccination is effective in reducing disease and PCV2 shedding in semen of boars concurrently infected with PCV2 and Mycoplasma hyopneumoniae. Theriogenology 76:351-360. Opriessnig T, Patel D, Halbur PG, Meng XJ, Stein DA, Zhang YJ. 2011. Inhibition of porcine reproductive and respiratory syndrome virus infection in piglets by a peptide-conjugated morpholino oligomer. Antiviral Res 91:36-42. Opriessnig T, Patterson AR, Madson DM, Pal N, Ramamoorthy S, Meng XJ, Halbur PG: 2010. Comparison of the effectiveness of passive (dam) versus active (piglet) immunization against porcine circovirus type 2 (PCV2) and impact of passively-derived PCV2 vaccine-induced immunity on vaccination. Vet Microbiol 142:177-183. Opriessnig T, Shen H-G, Pal N, Ramamoorthy S, Huang Y-W, Lager KM, Halbur PG, Meng X-J. 2011. A live-attenuated chimeric vaccine based on the new porcine circovirus subtype 2b (PCV2b) is safe and efficacious in a triple challenge co-infection model with PCV2b, porcine reproductive and respiratory syndrome virus (PRRSV), and porcine parvovirus. (in press). Opriessnig T, Shen HG, Pal N, Ramamoorthy S, Huang YW, Lager KM, Beach NM, Halbur PG, Meng XJ. 2011. A live-attenuated chimeric porcine circovirus Type 2 (PCV2) vaccine is transmitted to contact pigs but is not upregulated by concurrent infection with porcine parvovirus (PPV) and porcine reproductive and respiratory syndrome virus (PRRSV) and is efficacious in a PCV2b-PRRSV-PPV challenge model. Clin Vaccine Immunol 18:1261-1268. Opriessnig, T., Shen, H.G., Pal, N., Ramamoorthy, S., Huang, Y.W., Lager, K.M., Beach, N.M., Halbur, P.G., and Meng, X.J., A live-attenuated chimeric PCV2 vaccine is transmitted to contact pigs but is not upregulated by concurrent infection with PPV and PRRSV and is efficacious in a PCV2a-PRRSV-PPV challenge model. Clinical and Vaccine Immunology. 29(15):P.2712-9 Opriessnig, T., Shen, H.G., Pal, N., Ramamoorthy, S., Huang, Y.W., Lager, K.M., Beach, N.M., Halbur, P.G., and Meng, X.J. 2011. A Live-Attenuated Chimeric Porcine Circovirus Type 2 (PCV2) Vaccine Is Transmitted to Contact Pigs but Is Not Upregulated by Concurrent Infection with Porcine Parvovirus (PPV) and Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) and Is Efficacious in a PCV2b-PRRSV-PPV Challenge Model. Clin Vaccine Immunol 18, 1261-1268. PMID: 21653745 Oshansky, C.M., Pickens, J.A., Bradley, K.C., Jones, L.P., Saavedra-Ebner, G.M., Barber, J.P., Crabtree, J.M., Steinhauer, D.A., Tompkins, S.M., and R.A. Tripp. 2011 Avian influenza viruses infect primary human bronchial epithelial cells unconstrained by sialic acid ±2, 3 residues. PLoS ONE, 6(6):e21183. PMID: 21731666 Otake S, Dee S, Corzo C, Oliveira S and Deen J. Long distance airborne transport of viable PRRSV and Mycoplasma hyopneumoniae from a swine population infected with multiple viral variants. Vet Microbiol 2010; 145: 198-208. Patterson AR, Baker RB, Madson DM, Pintar AL, Opriessnig T. 2011. Disinfection protocols reduce the amount of porcine circovirus type 2 contaminated livestock transport vehicles. J Swine Health Prod 19:156-164. Patterson AR, Johnson JK, Ramamoorthy S, Hesse RA, Murtaugh MP, Puvanendiran S, Pogranichniy RM, Erickson GA, Carman S, Hause B, Meng XJ, Opriessnig T. 2011. Interlaboratory comparison of Porcine circovirus-2 indirect immunofluorescent antibody test and enzyme-linked immunosorbent assay results on experimentally infected pigs. J Vet Diagn Invest. 23:206-212.. Patterson AR, Madson DM, Schalk SD, Halbur PG, Opriessnig T. 2011. Establishment and maintenance of a porcine circovirus type 2 (PCV2)-free breeding herd on a site that experienced a natural outbreak of PCV2-associated reproductive disease. J Swine Health Prod 19:165-174. Patterson AR, Ramamoorthy S, Madson DM, Meng XJ, Halbur PG, Opriessnig T. 2011. Shedding and infection dynamics of porcine circovirus type 2 (PCV2) after experimental infection. Vet Microbiol 149:91-98. Pena L, Vincent AL, Ye J, Ciacci-Zanella JR, Angel M, Lorusso A, Gauger PC, Janke BH, Loving CL, Perez DR (2011) Modifications in the polymerase genes of a swine-like triple-reassortant influenza virus to generate live attenuated vaccines against 2009 pandemic H1N1 viruses. Journal of virology 85:456-469 Pitkin A, Otake S and Dee SA. A one-night downtime period prevents the spread of PRRSV and Mycoplasma hyopneumoniae by personnel and fomites (boots and coveralls)". Swine Health Prod (accepted for publication). Platt R, Vincent AL, Gauger PC, Loving CL, Zanella EL, Lager KM, Kehrli ME, Jr., Kimura K, Roth JA (2011) Comparison of humoral and cellular immune responses to inactivated swine influenza virus vaccine in weaned pigs. Veterinary Immunology and Immunopathology 142:252-257 Prickett JR, Johnson J, Murtaugh MP, Puvanendiran S, Wang C, Zimmerman JJ, Opriessnig T. 2011. Prolonged detection of PCV2 and anti-PCV2 antibody in oral fluids following experimental inoculation. Transbound Emerg Dis. 58:121-127. PMID: 21223532 Puvanendiran S, Stone S, Yu W, Johnson CR, Abrahante J, Garcia Jimenez L, Griggs T, Haley C, Wagner B, Murtaugh MP. 2011. High prevalence of porcine circovirus type 2 exposure and infection in swine finisher herds. Virus Res. 157:92-98. PMID:21352865 Ramirez A, Wang C, Prickett JR, Pogranichniy R, Yoon K-J, Main R, Rademacher C, Hoogland M, Hoffmann P, Johnson JK, Kurtz A, Kurtz E, Zimmerman J. 2011. Efficient surveillance of pig populations using oral fluids. Prev Vet Med (in press). Romagosa A, Gramer M, Joo HS, Torremorell M (2011). Sensitivity of oral fluids for detecting influenza A virus in populations of vaccinated and non-vaccinated pigs. J of influenza and other respiratory viruses. DOI:10.1111/j.1750-2659.2011.00276.x Rowland, RRR. 2010. The interaction between PRRSV and the late gestation pig fetus . Virus Res., 154:114-122. Shen H, Wang C, Madson DM, Opriessnig T. 2010. High prevalence of porcine circovirus viremia in newborn piglets in five clinically normal swine breeding herds in North America. Prev Vet Med 97:228236. Shen HG, Beach NM, Huang YW, Halbur PG, Meng XJ, Opriessnig T. 2011. Comparison of commercial and experimental porcine circovirus type 2 (PCV2) vaccines using a triple challenge with PCV2, porcine reproductive and respiratory syndrome virus (PRRSV), and porcine parvovirus (PPV). Vaccine 28:5960-5966. Shen HG, Schalk S, Halbur PG, Campbell J, Russell L, Opriessnig T. 2011. Commercially produced spray dried porcine plasma contains high levels of PCV2 DNA but did not transmit PCV2 when fed to naïve pigs. J Anim Science 89:1930-1938. Shi, M., T. T. Lam, C. C. Hon, R. K. Hui, K. S. Faaberg, T. Wennblom, M. P. Murtaugh, T. Stadejek, and F. C. Leung. 2010. Molecular epidemiology of PRRSV: A phylogenetic perspective. Virus Res. 154:7-17. Sinha A, Schalk S, Lager K M, Wang C, Opriessnig T. 2011. Singular PCV2a or PCV2b infection results in apoptosis of hepatocytes 3 in clinically affected gnotobiotic pigs. Res Vet Sci DOI:10.1016/j.rvsc.2010.10.013 Sinha A, Shen HG, Schalk S, Beach NM, Huang YW, Halbur PG, Meng XJ, Opriessnig T. 2010. Porcine reproductive and respiratory syndrome virus (PRRSV) infection at the time of porcine circovirus type 2 (PCV2) vaccination has no impact on vaccine efficacy. Clin Vaccine Immunol 17:19040-1945. Sinha A, Shen HG, Schalk S, Beach NM, Huang YW, Meng XJ, Halbur PG, Opriessnig T. 2011. Porcine reproductive and respiratory syndrome virus (PRRSV) influences infection dynamics of porcine circovirus type 2 (PCV2) subtypes PCV2a and PCV2b by prolonging PCV2 viremia and shedding. Vet Microbiol 152:235-246. Smith, J.H., Brooks, P., Johnson, S., Tompkins, S.M., Custer, K.M., Haas, D.L., Mair, R., Papania, M., and R.A. Tripp. 2010 Aerosol Vaccination Induces Robust Protective Immunity to Homologous and Heterologous Influenza Infection in Mice. Vaccine 29(14): 2568-75. PMID: 21300100 Smith, J.H., Nagy, T., Driskell, E.A., Brooks, P., Tompkins, S.M., and R.A. Tripp. 2011 Comparative Pathology in Ferrets Infected with H1N1 Influenza A Viruses Isolated from Different Hosts. J Virol. 85(15):7572-81. PMID: 21593156 Smith, J.H., Papania, M., Knaus, D., Brooks, P., Hass, D.L., Mair, R., Barry, J., Tompkins, S.M., and R.A. Tripp. Nebulized Live-Attenuated Influenza Vaccine Provides Protection in Ferrets at a Reduced Dose. In Press, Vaccine. Spronk G, Otake S and Dee S. Prevention of PRRSV infection in large breeding herds using air filtration. Vet Rec 2010; 166:758-759. Subramaniam S, Sur JH, Kwon B, Pattnaik AK, Osorio FA.(2011) A virulent strain of porcine reproductive and respiratory syndrome virus does not up-regulate interleukin-10 levels in vitro or in vivo.Virus Res. 2011 Feb;155(2):415-22. Epub 2010 Dec 17. Tiawsirisup S, Blitvich BJ, Tucker BJ, Halbur PG, Bartholomay LC, Rowley WA, Platt KB. 2010. Susceptibility of fox squirrels (Sciurus niger) to West Nile virus by oral exposure. Vector Borne Zoonotic Dis 10:207-209. Trible, BR, M Kerrigan, N Crossland, M Potter, K Faaberg, R Hesse, RRR Rowland. Porcine circovirus type 2 capsid protein epitopes associated with vaccination, infection and disease. Clin Vacc Immunol. In press. Trible, Kerrigan, M., Faaberg, K. S., and R. R.R. Rowland. 2011. Identification of an immunodominant region the PCV2 capsid protein recognized by naturally infected and vaccinated pigs. Clinical and Vaccine Immunology, 18:749-757. Vincent, A. L., K. M. Lager, K. S. Faaberg, M. Harland, E. L. Zanella, J. R. Ciacci-Zanella, M. E. Kehrli, Jr., B. H. Janke, and A. Klimov. 2010. Experimental inoculation of pigs with pandemic H1N1 2009 virus and HI cross-reactivity with contemporary swine influenza virus antisera. Influenza Other Respiratory Viruses. 4:53-60. Vu HL, Kwon B, Yoon KJ, Laegreid WW, Pattnaik AK, Osorio FA. (2011) Immune evasion of porcine reproductive and respiratory syndrome virus through glycan shielding involves both glycoprotein 5 as well as glycoprotein 3. J Virol. 2011 Jun;85(11):5555-64. Epub 2011 Mar 16. Wagner J, Kneucker A, Liebler-Tenorio E, Fachinger V, Glaser M, Pesch S, Murtaugh MP, Reinhold P. 2011. Respiratory function and pulmonary lesions in pigs infected with porcine reproductive and respiratory syndrome virus. Vet. J. 187:310-319. PMID: 20089425 Zhang H, Lunney JK, Baker RB, Opriessnig T. 2011. Cytokine and chemokine mRNA expression profiles in tracheobronchial lymph nodes from pigs singularly infected or coinfected with porcine circovirus type 2 (PCV2) and Mycoplasma hyopneumoniae (MHYO). Vet Immunol Immunopathol 140:152-158. Zhang H, Mohn U, Prickett JR, Schalk S, Motz M, Halbur PG, Feagins AR, Meng X-J, Opriessnig T. 2011. Different capabilities of enzyme immunoassays to detect anti-hepatitis E virus immunoglobin G in sera from pigs infected experimentally with hepatitis E genotype 3 or 4 and in sera from field pigs with unknown status of HEV infection. J Virol Methods (in press).

Publications Attachment:

Attachment

Impact Statements

Evaluation of PRRSV strains in vivo. USDA-ARS. Examination of several PRRSV strains allow us to survey the viral growth properties, the disease in swine, the commensal bacteria that may arise during infection, the innate response, the adaptive immune response and the host gene expression patterns that differ between PRRSV strains. We use the gained knowledge to better understand PRRSV pathogenesis. With this knowledge, we can then develop better vaccines and vaccination strategies.
Construction of a chimeric vaccine to protect against Asian HP-PRRSV. USDA-ARS. HP-PRRSV strains have proven to be a serious threat to our nations pork industry. Utilizing an infectious clone to a vaccine strain in combination with several structural genes from the Asian lineage, along with an identifiable foreign marker, we are constructing a vaccine to be used to protect swine in case such HP-PRRSV strains appear in the U.S.
Gene expression in lymph nodes of PRRSV-infected pigs. USDA-ARS. The goal of this discovery project is to identify changes that occur in gene expression in porcine lung lymph nodes following PRRSV infection. Knowledge derived from this study will more clearly define the negative effect of PRRSV on the pig immune system, and it may be used to design better cross-protective vaccines.
Progress in this reporting period continues to focus on non-PRRS viruses (e.g. influenza). While not directly related to PRRSV, this work is contributing directly to development of swine reagents and resources (e.g. primary swine epithelial cell lines). As we develop these tools for other (re-)emerging swine diseases, we plan to apply many of these approaches directly to PRRSV projects (UGA).
In regard in influenza as an emerging (re-emerging) disease of swine, we have made extensive advances in understanding features of the virus and host that influence infection, tropism, and potentially reassortment. We have also explored a number of vaccine and anti-viral therapies for influenza and developed a novel approach for rapid and sensitive detection of influenza virus; all of which may directly impact swine and/or human health (UGA).
Four PRRSV-related refereed papers involving our laboratories have been published in refereed journals during the period covered in this report (UNL).
US and European Patent Title: Methods and Compositions for Vaccination of Animals with PRRSV Antigens with Improved Immunogenicity. Inventors: Ansari, I, Osorio FA, and Pattnaik, AK Serial No. 12/064, Issued: October 27, 2009. During 2011 this patent is being explored by a veterinary biologics company (UNL)
. Provisional claim for invention: A reverse genetics method to develop a PRRSV attenuated live vaccine strain with DIVA differential capacity that would permit distinguishing naturally infected from non-infected, just vaccinated animals (Osorio, FA, Pattnaik, AK, Kwon, BJ, and Vu H.) filed on March 4, 2011, EFS ID: 9585692 Application No. 61449138 (UNL)
Elucidation of neonatal infection with minimal impact of maternal immunity illuminated the critical need to control and eliminate early influenza virus infection (UMN).
Commercial vaccines are not likely to benefit influenza control since homologous protection is required to prevent transmission.(UMN)
PRRSv MLV vaccines and air filtration interventions can aid in regional elimination of PRRSv (UMN).
New protein identification provides novel targets for immune protection (UMN).
PCV2 pathogenesis characterization increases knowledge of key pathogenic features.
Genomic markers for improved response to PRRS creates the opportunity to conduct marker-selected breeding of pigs (KSU).
Identification of a decoy epitope in PCV2 capsid is being used for assays that assess protective immunity following infection or vaccination (KSU).
Luminex is being developed as a substitute for standard ELISA approaches (KSU)
PRRS is a more complicated swine disease in China. A lot of work for completely controlling PRRS needs to be done in China. Vaccination is the primary choice for the majority of pig products for preventing and controlling PRRS. The viral replication and transcription mechanism and related foundation research should be conducted and a new generation of DIVA vaccines should be developed (SHVRI)
Shishan Yuan, Jian Lv, Xiangjian Li, Jianwu Zhang, Dandan Yu, Zhi Sun, Fei Gao, Zuzhang Wei, Jinshan Zhuang, Tao Tan, Haihong Zheng, Feifei Tan, Changlong Liu, Jiaqi Lu, Yanfang Cong, Xiaoming Wang, Hao Zheng. The construction of highly pathogenic PRRSV recombinant plasmid and genetic engineering vaccine. Patent NO. 200710172364.8; Announcement No.: CN101205539A; Publication No.: 2010062500313230
Output for project: Assessment of Virulence of PRRSV Isolates Based Both on their Sensitivity to IFNb and Ability to Induce Type I IFN Responses. Mr. Christopher Overend a doctoral candidate in Pathobiology and Veterinary Science working on the project has successfully completed his degree. Data obtained in this project are routinely discussed with our collaborator Dr. Marvin Grubman, a scientist from Plum Island Animal Disease, Center who shares interests in the area of type I IFN (UCONN).
Epidemiology/pathobiology/diagnostic studies provide ideas for detecting, preventing and eliminating viruses. Work has been done on genetic/antigenic variation during replication and persistence and new methods of surveillance for cost-effective methods of tracking infection and use in elimination/eradication. Advances in these areas linked with research in viral ecology/epidemiology and improvements in vaccines leads to possible elimination/eradication of viruses from farms and regions (ISU).
Presence of replicating PRRSV in pigs from very early days post-infection modulates the innate immune function resulting in subversion of immunity. As this study was performed in pigs maintained in natural commercial environmental settings, the outcome of this study has more practical implications in development of new generation protective vaccines. Our results on nanoparticles-based PRRSV-killed vaccine suggested that mucosal immunization has the potential to induce protective immunity to PRRS (OSU).
Understanding the role nsp2 in PRRSV virulence has important implication in developing better vaccines against PRRSV (VA).
Understanding the PRRSV-host miRNAs interaction provides new insight into the role of miRNAs in PRRSV pathogenesis. (VA)
A panel of monoclonal and polyclonal antibodies to non-structural proteins of PRRSV will be important tools in studies of PRRSV replication and pathogenesis. (SDSU)
Recombinant PRRSV can be produced whereby Interleukin 1 B expression or modifications in nsp2 can enhance viral specific immunity. These are important modifications to further vaccine design. (SDSU)
A multiplex FMIA for antibodies against PRRSV differentiates types I and II and can be used for multiplexing for serological profiling (SDSU).
The PRRS Host Genetics Consortium (PHGC) is helping to dissect the role of host genetics in resistance to PRRS and in effects on pig health and related growth effects. Results using a Nursery Pig Model of commercial pigs infected with PRRSV and followed for 42 days have affirmed that all pigs become PRRSV infected but they clear virus from serum at different rates with variable weight effects. Genome wide association studies (GWAS) have mapped pig PRRS responses (viral load and weight gain during infection) to multiple swine chromosomes. This result suggests that genetic progress can be made by selective breeding.(BARC)
Functional gene and protein transcriptomic analyses are underway with PHGC samples to identify gene networks and resistance associated biomarkers that differ in high versus low VL or WG PHGC pigs. Pathways have been identified and are being validated. Overall, the PHGC project will enable researchers to verify important genotypes and phenotypes that predict resistance/susceptibility to PRRSV infection.(BARC).
An FMIA has been developed to simultaneously quantify multiple porcine cytokines in serum using Luminex xMap" technology. It has been optimized to detect innate (IL-1b, IL-6, IL-8, IFN-a, TNF-a); regulatory (IL-10), T helper 1 (Th1) (IL-12, IFN-g) and Th2 (IL-4) cytokines. The assay has been tested for levels of porcine cytokines in oral fluids with positive results. This assay will be a useful tool to determine cytokine involved in genetic resistance to PRRSV using PHGC samples (BARC).
Antigenic/genetic variation in PRRSV is a major impediment to vaccine development. By distilling this diversity down to a manageable unit, we are hoping to provide guidance for the development of next-generation polyvalent vaccines that have maximum broad efficacy (BARC).
Our studies on PRRSV inhibition of interferon signaling showed that NSP1² of virulent VR2385 may be the viral protein that interferes with IFN signaling, while NSP1² of Ingelvac MLV has no effect. This result has a biological relevance on PRRS vaccine design (UMD).
Our finding of a lab mutant A2MC2 inducing interferons in cultured cells may be beneficial for vaccine development to induce protective immunity against PRRS. This isolate is expected to induce higher titer of neutralizing antibody in pigs (UMD).
PRRSV expresses proteins that circumvent the type I IFN response and other cellular processes and to compensate the small coding capacity of PRRSV, these proteins are multifunctional. Studies for Dr. Yoos laboratory suggest that PRRSV Nsp1± and NSP11 are a multifunctional nuclear protein participating in the modulation of the host IFN system (UIUC).
Studies from Dr. Zuckermanns lab suggest that PRRSV is inhibiting the ability of porcine macrophages to produce IFNa in response to infection by interfering with the activation of the transcription factor IRF-3 but not NFkB (UIUC).
. The data presented by Dr. Laegreids lab firmly confirms the important notion that GP3 may be involved in inducing neutralizing antibodies. Collectively, our work aimed at deciphering the transcriptional and cytokine response of cells as we as porcine alveolar macrophages to PRRSV infection will likely lead to the development of strategies to developed better vaccine against this costly disease.

Date of Annual Report: 12/02/2012

Report Information

Annual Meeting Dates: 12/02/2012 - 12/02/2012
Period the Report Covers: 10/01/2011 - 09/01/2012

Participants

NC229 Representatives:

;
Chair: Christopher-Hennings, Jane South Dakota State U. (SDSU) jane.hennings@sdstate.edu;Secretary: Osorio, Fernando A. University of Nebraska-Lincoln (UNL) fosorio@unl.edu;
Administrative Advisor Benfield, David, Ohio State University (OSU) benfield.2@osu.edu;
Rowland, Raymond R.R. Kansas State University (KSU) browland@vet.k-state.edu;
Enjuanes, Luis, Centro Nacional de Biotecnologia (CNB-CSIC), Spain, L.Enjuanes@cnb.csic.es;
Faaberg, Kay National Animal Disease Center (NADC) kay.faaberg@ars.usda.gov;
Goldberg, Tony. University of Wisconsin-Madison(UWM) tgoldberg@vetmed.wisc.edu;
Gourapura, Renukaradhya J. The Ohio State University (OSU) gourapura.1@osu.edu;
Johnson, Peter USDA, CSREES pjohnson@reeusda.gov;
Lunney, Joan USDA-ARS, BARC, joan.lunney@ars.usda.gov;
Murtaugh, Michael P University of Minnesota (UMN) murta001@umn.edu;
Pogranichniy, Roman, (Purdue), IN rmp@purdue.edu;
Risatti, Guillermo, University of Connecticut guillermo.risatti@uconn.edu;
Tompkins, S. Mark University of Georgia (UGA) smt@uga.edu;
Yang, Hanchun China Agricultural University, Beijing,yanghanchun1@cau.edu.cn;
Zhang, Yanjin University of Maryland zhangyj@umd.edu;
Zimmerman, Jeff Iowa State University (ISU) jjzimm@iastate.edu;
Zuckermann, Federico University of Illinois at Urbana-Champaign (UIUC) fazaaa@illinois.edu;
Meng, X.J. Virginia Polytechnic Institute and State University (VA Tech) xjmeng@vt.edu;

Other NC229 Scientists:

Abrams, Sam BARC,
Anderson, Tavis UWI,
Araujo, Karla BARC,
Arceo M Purdue,
Baker, RB ISU,
Blecha, Frank KSU,
Boddicker, Nick ISU,
Brockmeier, Susan NADC,
Calvert, Jay Pfizer Animal health,
Carman, Susy University of Guelph, Canada,
Chang, KC KSU,
Chen, Hongbo USDA-BARC,
Choi, Igseo, BARC,
Ciobanu, Dan, UNL,
Clark, A., Purdue University,
Davies, Peter, UMN,
Dee, Scott, UMN,
Dekkers, Jack, ISU,
Ernst, Cathy, MSU,
Fang, Ying, SDSU,
Garmendia, Antonio, UCONN,
Garrick, Dorian, ISU,
Gourapura, Aradhya, OSU,
Gramer, Marie, UMN,
Halbur, Patrick, ISU,
Haley, Charles, USDA-APHIS,
Harhay, Greg, NADC,
Harris, DL (Hank), ISU,
Hause, Ben, Newport Labs, MN,
Hesse, Dick, KSU,
Holtkamp, Derald J, ISU,
Huang T, Purdue,
Huang, Tinghua, ISU,
Jiang, Zhihua, WSU,
Johnson, John K, ISU,
Joo, Han Soo, UMN,
Karriker, Locke, ISU,
Kerhli, Marcus Jr., NADC,
Kerrigan M., KSU,
Kittawornrat Apisit, ISU,
Kuhar, D., USDA-USDA-BARC,
Laegried, Will, UIUC,
Lager, Kelly, NADC,
Lawson, Steve, SDSU,
Lazar V, Purdue,
Leung, Frederick, Hong Kong University,
LeRoith T., VA Tech,
Loving, Crystal, NADC,
Lunney, Joan, BARC,
Main, Rodger G, ISU,
McCaw, Monte B. (deceased), NCSU,
McKean, JD, ISU,
Moore, B, Purdue,
Morrison, Robert, UMN,
Nelson, Eric, SDSU,
Nerem, Joel, Pipestone Vet Clinic, MN,
Nicholson, Tracy, NADC,
Opriessnig, Tanja, ISU,
Pattnaik, Asit, UNL,
Prickett, J., ISU,
Ramamoorthy, Sheila, UGA,
Ramirez, Alejandro, ISU,
Ramirez-Nieto, Gloria, Universidad Nacional de Colombia,
Raney NE, Purdue,
Raney, Nancy, MSU,
Reecy, Jim, ISU,
Rossow, Kurt, UMN,
Roth, JA, ISU,
Rothschild, Max, ISU,
Rovira, Albert, UMN,
Rowland, R.R.R., KSU,
Sang, Yongming, KSU,
Schwartz, Kent J., ISU,
Sina, R, Purdue ,
Souza, Carlos, BARC,
Steibel, J.P., MSU,
Stevenson, Greg W., ISU,
Strait, Erin, ISU,
Srinivas, Jay CVB-PEL/APHIS/USDA,
Torremorell, Montserrat, UMN,
Trible B., KSU,
Tripp, Ralph, UGA,
Tuggle, Chris, ISU,
Waide, Emily, ISU,
Wang, Chong, ISU,
Wang, Xiuqing, SDSU,
Wyatt, Carol, KSU,
Wysocki, Michal, USDA-BARC,
Yoo, Dongwan, UIUC,
Yoon, Kyoung-Jin, ISU,
Zhang, C. , VA Tech,
Zhu, Xiaoping, UMD,
Zimmerman, Jeff, ISU

Brief Summary of Minutes

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.

Accomplishments

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.

Publications

To view Publications issued or "in press" Funding Sources for Research Work for Next Year see attachment below

Publications Attachment:

Attachment

Impact Statements

VA Tech developed a unique approach through DNA shuffling of viral genes to attenuate PRRSV. They found that DNA shuffling of the PRRSV GP3 produced a chimeric virus with improved cross-neutralizing activity against a heterologous PRRSV and that transgenic corns serve as an efficient vaccine production and delivery system have important implications in PRRSV vaccine development. The observed immunomodulatory role of PCV2/PRRSV co-infection helps understand the mechanism of polymicrobial infections.
Ohio reports results of interest in the area of PRRSV vaccinology. Their study has confirmed the need for a better PRRSV vaccine strain (possibly a genetically modified strain) that has the ability to elicit better anti-PRRSV immune responses in pigs. PRRSV killed vaccine study has confirmed the benefits of intranasal delivery of nanoparticle-based PRRSV vaccine to elicit cross-protective immunity in pigs.
UIUC Illinois reports the development of the ZMAC macrophage cell line as a major contribution towards the isolation and propagation of PRRSV field strains in a swine system that is 100 % homologous to the natural target cell. The cell line serves both for experimental immune-pathogenesis studies towards the mapping of the effect of PRRSV on swine innate immunity as well as a platform for development of more successful vaccines.
UMN 1)Farms had a significantly improved productivity after the implementation of filtration technologies .In most cases the pay-back period of the system was calculated to be between 2 and 3 years depending on the initial investment. 2) Analysis of risks of influenza transmission will facilitate development of effective methods to reduce transmission. 3) Whole genome sequencing is expected to reveal candidate elements associated with virulence and cross-protective immunity, an essential knowledge for treatment and prevention of PRRS. 4) Elucidation of mechanisms of induction of cross-protective antibody production is expected to provide a rational basis for development of improved vaccines.
UMD The UMD studies on PRRSV A2MC2 strain that induces interferons in cultured cells may be beneficial for vaccine development to induce protective immunity against PRRS. This isolate is expected to induce higher titer of neutralizing antibody in pigs. Likewise, the UMD finding that nsp1² of virulent VR2385 strain inhibits interferon signaling by interfering with STAT1 nuclear translocation, while nsp1² of Ingelvac MLV has no effect. This result has a biological relevance on PRRS vaccine design.
UGA we have developed an immortalized porcine cell line (iPSC) that supports PRRS replication which may greatly benefit the research community, particularly in areas of vaccinology and understanding the virus-host interface. Re. swine influenza, we have made advances in understanding infection, tropism, and re-assortment. We have also explored a number of vaccine and anti-viral therapies for influenza and rapid diagnosis. These studies directly impact swine and/or human health, and address the One Health paradigm.
KSU SCID is a model identifying components of immune protection that will be incorporated into the next generation of vaccines. PCV2 CP(169-180) epitope will permit serological assessment of vaccination and infection. SSC4 genomic marker will soon be adopted by industry for the development of marker-assisted selection. The Luminex serological assay technology is being transferred to a commercial kit. Understanding the effect of PRRSV infection on digestibility will be incorporated into the formulation of nutritional regimens that optimize growth during PRRSV infection.
USDA/BARC Significance of The PRRS Host Genetics Consortium (PHGC): the PHGC is helping to dissect the role of host genetics in resistance to PRRS and in effects on pig health and related growth effects. These results could have a major impact in the swine industry by enabling geneticists to develop plans for marker-assisted selection of pigs with improved response to PRRS. We have also demonstrated for the first study cytokine profiles in OF from pigs vaccinated against or infected with PRRSV.
SDSU Significant advances in diagnostic technology have been made at SDSU: Detection of PRRSV and SIV and FMIA PRRSV antibodies in oral fluids have been pioneered at SDSU. A multiplex swine immune effector molecule assay was licensed to Life Technologies, Inc. and is now commercially available. Research wise: SDSU has contributed significantly to the study of interaction between PRRSV nsp2 and host innate immune responses well as the use of IFN expression in PRRSv vectors as a strategy to contribute further of PRRSV pathogenesis and future therapeutic interventions.
UCONN The UCONN aimed at identifying PRRSV genetic determinants associated with disease caused by PRRSV will, in the long term, contribute information needed for rational engineering of PRRS live attenuated vaccines (LAV) and/or antivirals.
USDA/NADCThrough the research conducted by the team at NADC, a foreign highly pathogenic PRRSV strain has been reconstructed under controlled conditions and used for experimentation in vitro and in vivo under BSL3 safety conditions in this country. Through this effort we can now better understand what factors are at play during high virulence infections vs. low virulence infections. With this knowledge, we can then develop better vaccines and vaccination strategies.
UWM ( in collaboration with UIUC,UNL. and SDSU) CAP2 funded project on PRRSV strain diversity: Antigenic/genetic variation in PRRSV is a major impediment to vaccine development. By distilling this diversity down to a manageable unit, we provide guidance for the development of next-generation polyvalent vaccines that have maximum broad efficacy.
ISU Iowa has a well documented publication record. Extensive work has been done at ISU on the mechanisms of host-pathogen(s) interactions. Likewise new work on the ecology and epidemiology of these agents (PRRSV, PCV2 and SIV) provide insight into the mechanisms by which they maintain endemnicity. Continued assessment and research in diagnostic technology is contributing to the improvement and refinement of our ability to surveil, and diagnose PRRSV and other respiratory viral infections.

Date of Annual Report: 12/08/2013

Report Information

Annual Meeting Dates: 12/08/2013 - 12/08/2013
Period the Report Covers: 10/01/2012 - 09/01/2013

Participants

Chair: Christopher-Hennings, Jane; South Dakota State U. (SDSU); jane.hennings@sdstate.edu
Secretary: Osorio, Fernando A.; University of Nebraska-Lincoln (UNL); fosorio@unl.edu
Rowland, Raymond R.R.; Kansas State University (KSU); browland@vet.k-state.edu
Benfield, David, Ohio State University (OSU); benfield.2@osu.edu
Enjuanes, Luis, Centro Nacional de Biotecnologia (CNB-CSIC), Spain, L.Enjuanes@cnb.csic.es
Faaberg, Kay; National Animal Disease Center (NADC); kay.faaberg@ars.usda.gov
Goldberg, Tony.; University of Wisconsin-Madison(UWM) tgoldberg@vetmed.wisc.edu
Gourapura, Renukaradhya J.; The Ohio State University (OSU); gourapura.1@osu.edu
Johnson, Peter; USDA, CSREES; pjohnson@reeusda.gov
Lunney, Joan; USDA-ARS, BARC, joan.lunney@ars.usda.gov
Murtaugh, Michael P; University of Minnesota (UMN); murta001@umn.edu
Pogranichniy, Roman, (Purdue), IN; rmp@purdue.edu
Risatti, Guillermo, University of Connecticut; guillermo.risatti@uconn.edu.
Tompkins, S. Mark; University of Georgia (UGA); smt@uga.edu
Yang, Hanchun; China Agricultural University, Beijing,yanghanchun1@cau.edu.cn
Zhang, Yanjin; University of Maryland; zhangyj@umd.edu
Zimmerman, Jeff; Iowa State University (ISU); jjzimm@iastate.edu
Zuckermann, Federico; University of Illinois at Urbana-Champaign (UIUC); fazaaa@illinois.edu
Meng, X.J.; Virginia Polytechnic Institute and State University (VA Tech); xjmeng@vt.edu

Other NC229 Scientists:

Abrams, Sam; BARC
Anderson, Tavis; GSU
Arceo M; Purdue
Baker, RB; ISU
Bandara Kalpanie UCONN
Blecha, Frank; KSU
Boddicker, Nick (Gensus)
Brockmeier, Susan; NADC
Butler, John University of Iowa
Calvert, Jay; Pfizer Animal health
Carpenter, Susan; ISU
Chang, KC; KSU
Chen, Hongbo; USDA-BARC
Choi, Igseo; BARC
Ciobanu, Daniel, UNL
Clark, A., Purdue University
Clement, Travis (SDSU)
Cui, Junru (UCONN)
Culhane (formerly Gramer), Marie; UMN
Davies, Peter; UMN
Dee, Scott; Pipestone Vet Clinic, MN
Dekkers, Jack; ISU
Dunkelberger, Jenelle; ISU
Eisley, Chris; ISU
Ernst, Cathy; MSU
Ewen, Catherine; KSU
Fang, Ying; KSU
Fritz-Waters, Eric; ISU
Gabler, Nick; ISU
Garmendia, Antonio; UCONN
Garrick, Dorian; ISU
Gauger, Phillip C; ISU
Gourapura, Aradhya; OSU
Halbur, Patrick; ISU
Haley, Charles; USDA-APHIS
Harhay, Greg; NADC
Harris, DL (Hank); ISU
Hause, Ben; Newport Labs, MN
Hess, Andrew; ISU
Hesse, Dick; KSU
Ho, Chak-Sum (Sam); Gift of Life Michigan, Ann Arbor, MI
Holtkamp, Derald J; ISU
Jiang, Zhihua; WSU
Johnson, John K; ISU
Joo, Han Soo; UMN
Karriker, Locke; ISU
Kerhli, Marcus Jr.; NADC
Kerrigan Maureen.; KSU
Koltes, James, ISU
Laegried, Will; UIUC
Lager, Kelly; NADC
Lawson, Steve; SDSU
Lazar V; Purdue
Lazarus, William; UMN
LeRoith, Tanya, VA Tech
Leung, Frederick; Hong Kong University
Loving, Crystal; NADC
Madson, Darin; ISU
Main, Rodger G; ISU
McKean, JD; ISU
Miller, Laura; NADC
Molitor, Tom; UMN
Moore, B; Purdue
Morrison, Robert; UMN
Nelson, Eric; SDSU
Nerem, Joel; Pipestone Vet Clinic, MN
Nicholson, Tracy: NADC
Opriessnig, Tanja; ISU
Pattnaik, Asit, UNL
Polson, Dale; Boehringer Ingelheim (BI)
Prather, Randy, MO
Ramamoorthy, Sheila; NDSU
Ramirez, Alejandro; ISU
Ramirez-Nieto, Gloria; Universidad Nacional de Colombia
Raney NE; Purdue
Raney, Nancy; MSU
Reecy, Jim; ISU
Rock, Dan UIUC
Rossow, Kurt; UMN
Roth, JA; ISU
Rothschild, Max; ISU
Rovira, Albert; UMN
Sang, Yongming; KSU
Schroyen, Martine, ISU
Schwartz, Kent J.; ISU
Sina, R; Purdue
Singrey, Aaron (SDSU)
Smith Justin (UCONN)
Souza, Carlos; BARC
Spear, Allyn; NADC
Steibel, J.P.; MSU
Stevenson, Greg W.; ISU
Stricker, Amber; Suidae Health and Production, IA
Summerfield, Artur, Switzerland
Torremorell, Montserrat; UMN
Trible B.; KSU
Tripp, Ralph; UGA
Tuggle, Chris; ISU
Waide, Emily; ISU
Wang, Chong; ISU
Wang, Xiuqing; SDSU
Wilkerson, Melinda; KSU
Wyatt, Carol; KSU
Xiao, Zhengguo, UMD
Yoo, Dongwan; UIUC
Yoon, Kyoung-Jin; ISU
Zhang, C. ; VA Tech
Zhang, Chenming, VA Tech
Zhou, Lei, CAU
Zhu, Xiaoping, UMD
Zimmerman, Jeffery; ISU

SEE ATTACHMENT FOR ENTIRE REPORT

Participants Attachment:

Attachment

Brief Summary of Minutes

Minutes NC229 Meeting Chicago, IL. 12/08/2012

The meeting started at 1 PM. Reports were given by Dr. David Benfield (Administrative Advisor) and Dr. Peter Johnson (updates from USDA via teleconference). Dr. KJ Yoon (ISU) was elected the next Secretary for NC229. Additional presentations on the next 5 year grant were given by Dr. Fernando Osorio (UNL), Dr. Federico Zuckermann (UIUC) and Dr. Aradya Gourapura (OSU) for the 1st objective on PRRSV and for the 2nd objective on emerging viral diseases of swine presentations were given by Dr. KJ Yoon (ISU) (discussion on PED); Dr. Dan Rock (UIUC) on ASF and Dr. Amy Vincent (NADC). Meeting was adjourned by 5:30 PM.

Accomplishments

B. PROGRESS OF WORK AND PRINCIPAL ACCOMPLISHMENTS Objective 1. Elucidate the mechanisms of host-pathogen(s) interactions. 1. (UCONN ,Risatti): We have identified swine macrophage proteins that interact with PRRSV NSP3 using a Yeast Two-Hybrid screening system. We focused on the interaction of NSP3 with host cell protein FKBP38 a FK506 binding protein associated with cellular processes. PRRSV has mechanisms to prevent host cell apoptosis likely mediated by PRRSV NSPs. 2. (UCONN: Garmendia) The aims of the study are to determine the sensitivity to and induction of IFNb by PRRSV, to identify mechanisms of evasion of hosts innate immune responses and determine correlations with virulence. We have shown significant differences among different PRRSV in sensitivity to IFNb. Chimeric PRRSV induce variable synthesis bioactive IFNb. Anti-swine IFNb mAbs were developed and will now be used to test samples obtained from the swine experiment. Results from a recent vaccination and challenge showed that challenge exposure of pigs to PRRSV results in induction of IFN b in BAL fluids, regardless of their vaccination status. 3. (SDSU, X. Wang), Protein kinase R (PKR) is involved in anti-viral activities in response to many virus infections. Several recent studies, suggest the pro-viral properties of PKR, which may or may not be dependent on the catalytic activity of PKR. To reveal the role of PKR in the replication of PRRSV, we first examined the kinetics of PKR activation during infection. Results showed that PRRSV transiently activates PKR during 12- 24 h PI. eIF-2±, one of the downstream targets of PKR, was only significantly phosphorylated compared to mock-infected cells at late time points of infection. A reduced viral protein synthesis and virus titer were detected in cells transfected with PKR silencing RNA prior to infection, indicating the role of PKR in facilitating virus replication. This is further confirmed by the reduced virus titer in cells treated with a PKR specific inhibitor. Experiments are ongoing to verify these observations by using cells overexpressing PKR. 4. (Purdue + PHGC) The PRRS Host Genetics Consortium (PHGC) studies are aimed at identifying genes and pathways that are associated with pigs that clear PRRSV 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 four 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). All RNA samples were converted to cDNA and subjected to real time PCR using primers corresponding to markers important for immune system activations involved in Th1, Th2, and immunological tolerance pathways. 5. (UMD) During the past year, we continued the studies to determine the mechanism of PRRSV interference with IFN-activated JAK/STAT pathway. We found that PRRSV nsp1² blocks STAT1/STAT2 nuclear translocation by interfering with their interaction with karyopherin-±1 (KPNA1 or importin-±5). KPNA1 is a key molecule in facilitating nuclear transportation of IFN-stimulated STAT1/STAT2/IRF9 heterotrimers. A nucleotide substitution resulting in an AA change of nsp1² at residue 19 from valine to isoleucine diminished its ability to induce KPNA1 degradation and to inhibit IFN-mediated signaling. Infection of MARC-145 cells by PRRSV also resulted in KPNA1 reduction, but an avirulent strain Ingelvac PRRS MLV did not. These results indicate that nsp1² blocks JAK/STAT pathway via inducing KPNA1 degradation and that the valine-19 in nsp1² correlates with the inhibition. 6. (UMD) We examined the interference of IFN-activated signaling by PRRSV viral proteins and compare the effects of several PRRSV strains. Among eleven non-structural proteins (nsps) and eight structural proteins of VR-2385, three nsps (1², 7 and 12) and two structural proteins (GP3 and N) were found to significantly inhibit the expression of IFN-stimulated response element (ISRE) luciferase reporter. In MARC-145 cells, all the six PRRSV strains with the exception of MN184, blocked the activity of exogenous IFN-±. In primary porcine pulmonary alveolar macrophages (PAMs), all the six strains with the exception of MLV and NVSL inhibited the activity of IFN-±. 7. (UMD) Elevation of proinflammatory cytokines is thought to contribute to PRRSV pathogenesis. We found that PRRSV VR-2385 induces phosphorylation of signal transducer and activator of transcription 1 (STAT1) at serine 727 (pSTAT1-S727) in MARC-145 and PAM cells, which was interferon-independent. IngelVac PRRS MLV strain had a minimal effect on pSTAT1-S727. Compared to MLV-infected cells, VR-2385 infection caused significantly higher level of expression of proinflammatory cytokines, including interleukin 1 beta (IL-1beta) and IL-8. 8. (NADC, Lager) Conducted animal experiment to develop a PRRSV pathogenesis matrix 9. (KSU, Rowland, Sang) are performing a study characterizing the expression of interferon genes and cytokine proteins in the PRRSV-infected fetus. 10. (KSU, Wyatt, Ewen, Wilkerson, Rowland) are characterizing a newly discovered SCID pig as a model for understanding PRRSV immunity and pathogenesis. 11. (KSU, Sang) is performing an analysis of type 1 and type 2 macrophages in PRRSV immunity. 12. (KSU, Rowland and several outside collaborators) continue to work on marker on SSC4 linked to increased weight gain and reduced virus load during PRRSV infection. 13. (KSU, Rowland) performing an analysis of broadly neutralizing antibody. 14. (KSU, Hesse) investigated the response of pigs to PEDV infection 15. (KSU, Rowland and Prather (MU)) tested C169 knockout pigs for PRRSV infection. 16. (USDA-BARC) The PRRS Host Genetics Consortium (PHGC) was developed to determine the role of host genetics in resistance to PRRS and effects on pig health and growth. Pig resistance/susceptibility to PRRS was assessed. All pigs became PRRSV infected but some pigs cleared virus quicker with variable weight effects. Pig DNA was genotyped. Multivariate analyses of viral load and weight data identified PHGC pigs in different virus/weight groups. Ongoing serum cytokine and gene expression studies will compare PRRS resistant/maximal growth pigs to PRRS susceptible/reduced growth pigs. 17. (USDA-BARC) Genome wide association studies have identified genetic regions associated with resistance/susceptibility to primary PRRSV infection. Whole genome analyses focused on viral load (VL) and weight gain (WG). We identified a 38-SNP region on swine chromosome 4 (SSC4) that explained 14.6% and 9.1% of the genetic variance for VL and WG, respectively. The SSC4 marker may be useful for genetic selection of pigs for increased resistance or reduced susceptibility to PRRSV isolates that differ genetically and possibly pathogenically. 18. (USDA-BARC) Evaluation of differences in gene expression of whole blood RNA from PHGC pigs revealed a range of responses to PRRSV. RNA was extracted from blood from14 pigs at 7 time-points. An average of 58M high quality reads/sample was obtained and approx. 87% could be aligned to the pig reference genome (Sus scrofa 10.2). Additional analyses will decipher genetic mechanisms controlling host response to PRRSV infection. 19. (USDA-BARC) Swine genome studies have expanded our knowledge of genes involved in immune and disease responses. The Immune Response Annotation Group used computational curation and manual annotation of the swine genome assembly 10.2 (Sscrofa10.2) to refine the currently available automated annotation of 1,369 immunity-related genes through sequence-based comparison to genes in other species. Extensive annotation dramatically extends the genome-based knowledge of the molecular genetics and structure of a major portion of the porcine immunome. This phylogenetic analysis of the core immunome cluster confirms rapid evolutionary changes and such immune genes are important components of the pig's adaptation to pathogen challenge over time. Current efforts are aimed at using high-density SNP panels to infer MHC haplotypes to identify exact genetic alleles controlling anti-PRRS responses. These analyses should provide important tools for global analyses and data-mining of the porcine immune response. 20. (CAU) A series of full-length infectious cDNA clones with exchanged regions between highly virulent RvJXwn and phylogenetic close low-virulent RvHB-1/3.9 were constructed, and then the replication and pathogenicity of rescued chimeric virus were systematically compared. The results suggested that the Nsp9 and Nsp10 together contribute to the increased fatal virulence of HP-PRRSV emerging in China. 21. (CAU) The HP-PRRSV JXwn06 and low virulent HB-1/3.9 were confirmed to have distinct ability of TNF-± induction. By comparing the capability of all NSPs from these 2 different strains on inhibiting ERK signal pathway, we found that the HP-PRRSV could inhibit TNF-± through its Nsp1² and Nsp11, which may result in the increased virulence for piglets. 22. (UMN) Developed an experimental infectious disease model for the rapidly emerging new viral disease of swine, PED. 10-day-old pigs were used and Koch's postulates were fulfilled. The model is very sensitive to detect live virus and is currently used to assess infectivity of research samples. 23. (UMN) Whole genome sequencing of virulent field viruses was performed to evaluate potential genetic changes characteristic of novel strains associated with seasonal PRRS. Research was performed to analyze genetic variation in the population of PRRSV produced from permissive cells. MN developed a sequencing technique for PEDV based on the S gene, and applied it to farms. Variation among NA samples is very limited. The first whole genome of a PEDV detected in NA was sequenced. Several other whole-genome sequences are being generated. 24. (UMN) Studies investigated the NAb response in sows from herds exposed to virulent PRRSV. Research was performed to determine the role of plasmacytoid dendritic cells in anti-PRRSV host response. Effect of host age on macrophage permissiveness to PRRSV infection was examined. 25. (UIUC, Yoo Lab). Mutations that destroyed the PCP± activities (C76S, H146Y, and C76S/H146Y) in nsp1± did not affect the IFN suppressive activity of nsp1±, indicating that the cysteine protease activity did not participate in IFN suppression. The mutations of C70S, C76S, H146Y, and/or M180I, which coordinated the ZF2 motif, did not alter IFN suppression. The mutations of C8S, C10S, C25S, and/or C28S for the ZF1 motif impaired the IFN antagonism of nsp1±, showing that ZF1 was the essential element of nsp1± for IFN suppression. Wild-type nsp1± localized in the both nucleus and cytoplasm, but the ZF1 mutants that lost the IFN suppressive activity did not localize in the nucleus and remained in the cytoplasm. 26. (UIUC, Yoo Lab). Bayesian phylogeographic analyses of 7040 ORF5 sequences were used to reveal the recent geographical spread of Type2 PRRSV in NA. 27. (UIUC Yoo Lab). To discover the impact PRRSV infections on the cellular miRNAome, small RNA expression profiles were developed from PRRSV-infected swine alveolar macrophages in vitro using deep sequencing. A total of 40 cellular miRNAs were significantly differentially expressed within the first 48 hpi. Six miRNA, miR-30a-3p, miR-132, miR-27b*, miR-29b, miR-146a and miR-9-2, were altered at more than one time point. The most highly repressed miRNA at 24 hpi was miR-147. A miR-147 mimic was utilized to maintain miR-147 levels in PRRSV-infected SAMs. 28. (UIUC, Zuckermann/ Rock). A highly pathogenic PRRSV with a ORF5 1-22-2 RFLP was isolated in the porcine alveolar macrophage cell line ZMAC from a sow farm with 100% pre-wean mortality. A virus stock of the isolated virus (LTX1) was prepared after 1 passage in ZMAC cells. Inoculation of pigs with the LTX1 resulted in viremia with similar kinetics and viral load as those observed after inoculation with the atypical PRRS strain. The average viral load in the bronchoalveolar lavage collected at 14 DPC with LTX1 was 44-fold higher compared to that in pigs receiving the atypical virus. Analysis of the genome indicated that nsp2 of the LTX1 virus has the same three discontinuous deletions as the MN184, but also has a novel 5 AA deletion corresponding to positions 464-468 and numerous unique single mutations. 29. (UGA) We continued to explore the immune response to influenza virus and the contribution of the host tryptophan-catabolizing enzyme indoleamine 2,3-dioxygenase (IDO) in primary and memory T cell responses. We established a primary normal swine bronchoepithelial cell culture system to evaluate the host cell response to influenza virus infection and replication, and are evaluating influenza reassortment. Studies continue on assessing the potential for reassortment of H1 and H3 human and swine influenza viruses in NSBE cells. 30. (UGA) We developed a PRRS-susceptible immortalized porcine stem cell line and are characterizing PRRS persistence in these iPSC cells and potential for vaccine production. 31. (UGA) We are exploring the potential for swine, human and avian influenza viruses to reassort on the TRIG backbone in primary swine epithelial cells, and primary human epithelial cells. A goal of these studies is to elucidate the potential for reassortment and determine the contribution of virus and host to reassortment. These studies are ongoing and in preparation for publication. 32. (UGA) Wee explored the potential for influenza viruses to infect bats. We assessed the potential for low pathogenic avian influenza to infect and transmit in the ferret model. We found that LPAI viruses readily infected and transmitted in in ferrets without adaptation and despite avian-specific receptor specificity. We assessed the potential for these viruses to infect and cause disease in domesticated cats. We assessed the potential for pH1N1 and swine H1N1to infect starlings and sparrows. We established the guinea pig transmission model. 33. (UGA) We continue to test the potential for a novel vaccine vector (PIV5) to serve as a vaccine against influenza virus. Using the mouse model of H5N1 highly pathogenic avian influenza virus (HPAIV) infection, we demonstrated that PIV5 expressing the HA of H5N1 provided robust protection from lethal challenge when vaccinated intranasally, intramuscularly, with live or killed vaccine. Similarly, vaccination with PIV5 expressing the internal, conserved NP protein also protectd from challenge. Finally, we demonstrated the utility of moving the transgene within the PIV5 genome to optimize expression and immunogenicity. Objective 2. Understand the ecology and epidemiology of PRRSV and emerging viral diseases of swine. 1. (NADC, Faaberg/Spear/Lager), Compared and contrasted the pathogenesis in swine after challenge with DIVA-tagged PRRSV MLV 2. (NADC) Faaberg) Virion purification; PRRSV nonstructural protein 2 studies 3. (NADC, Faaberg) Construction of chimeric PRRSV 4. (NADC, Lager/Vincent) Animal experiment to characterize H5N1 influenza infection in swine with recombinant viruses provided by Dr. Richard Webby. 5. (NADC, Lager) Animal experiments to evaluate potential infectivity of putative single-strand circular DNA viruses that could interact with PCV2. 6. (NADC, Miller) - Acute transcriptomic response in HP-PRRSV infected gnotobiotic pigs. Searchlight completed. 7. (NADC, Miller) Established globinRNA removal protocol from whole blood for transcriptomics 8. (NADC, Miller) Hi-Seq collaboration with Dr. Gary Rohrer 9. (NADC, Brockmeier) Continued analysis of bacterial enhancement of PRRSV strains 10. (NADC, Loving/Lager) Animal experiment evaluating protection against homologous PRRSV challenge following primary exposure challenge. Loving evaluated T cell responses and mucosal antibody responses. 11. (UW-Madison, Goldberg) Research (via NIH) has elucidated the ecology of SHFV, a relative of PRRSV. This research is mentioned because of its relevance to the ecology of arteriviruses. 12. (UNL, Pattnaik) We conducted the characterization of a serologic marker epitope, so-called epitope-M201, which can be a potential target for development of a live-attenuated DIVA vaccine against PRRSV. Epitope-M201 is located at the carboxyl terminus of the M protein. The epitope is highly immunodominant and well-conserved among type-2 isolates. Rabbit polyclonal antibodies prepared against this epitope are non-neutralizing; thus, the epitope does not seem to contribute to the protective immunity against PRRSV infection. The immunogenicity of epitope-M201 can be disrupted through the introduction of a single AA mutation which does not affect viral replication. 13. (CAU) A genotype 1 PRRSV GZ11-G1 was isolated. The genomic sequences analysis and phylogenetic trees showed it evolved from the vaccine Amervac PRRS. The further pathogenicity analysis indicated that GZ11-G1 could cause clinical signs and lung lesions. It is different from Amervac PRRS or genotype 2 isolate HB-1/3.9 at both the antigenic level and lesions. This is the first pathogenicity study of genotype 1 PRRSV wild isolate in Mainland China. 14. (UMN) MN continued research on seasonal PRRSV transmission dynamics. Airborne influenza transmission was characterized and modeled in acutely infected farms. Influenza virus was quantified in aerosols collected inside and outside swine facilities and in 2 live animal markets in MN. Environmental contamination of influenza virus was confirmed in high hand-contact surfaces easily accessible to personnel working or visiting farms. Indirect routes of influenza transmission via people acting as fomites was shown to be significant in the spread of influenza despite the adoption of biosecurity measures. Mathematical modeling of influenza virus transmission within swine farms and evaluation of the effects of vaccination on influenza dissemination was completed. MN is performing studies on the survivability of PEDV under different conditions of temperature, relative humidity and different matrices (feed, slurry). 15. (UMN) The role of the neonatal pig on influenza epidemiology has been studied at MN. Neonatal pigs were identified as a source of genetically diverse influenza viruses to growing pig populations. About 45% of farms (out of 52) monitored for 6 months weaned pigs positive with genetically distinct influenza viruses. Genetic mutations in influenza virus were detected in pigs with and without passive immunity. Persistence of influenza virus in wean-to-finish populations was shown to be prolonged despite the belief that influenza infections are short lived. Over 25% of pigs in a wean-to-finish population were shown to test positive more than once in non-consecutive weeks indicating that one possible mechanism for virus maintenance in populations includes re-infection despite the presence of immunity. 16. (UMN) Research was conducted on active surveillance for variant influenza viruses among swine, the environment, patrons and employees at live animal markets in Minnesota. The diversity of influenza viruses in live animal markets and the interspecies transmission between pigs and people was documented, indicating that live animal markets play an important role in the transmission of variant influenza viruses to people. 17. (UMN) Surveillance of influenza virus was also extended to the air and environment of 3 agricultural fairs. Objective 3. Develop effective and efficient approaches for detection, prevention and control of PRRSV and emerging viral diseases of swine. 1. (UNL Ciobanu) Pigs from various crossbred lines were experimentally infected with a PCV2b strain similar to a PCV2b strains known to induce clinical signs of PCVAD and high mortality. During challenge, weekly measurements of ADG, viremia, and PCV2 specific antibodies were profiled. Common sources of variation were evaluated by estimating pair-wise correlations between phenotypic and genomic prediction values and by genome-wide associations across traits. Viremia was the best indicator of decreased ADG following infection; moderate phenotypic correlations between viremia and ADG were observed starting with viremia at 14 DPI and ADG during the last 2 wks of challenge. A genome wide association study that included 56,433 SNPs uncovered two major SNPs that explain, 12.4% and 3.7% respectively, of the genetic variation for viral load. One SNP is located next to the SLA II complex of genes known for their role in immune response. These SNPs partially explained the negative correlations between viremia and growth. 2. (SDSU) Substantial progress has been made in PED diagnostic development. PEDV was isolated from intestinal contents of diagnostic cases using Vero-76 cells with 2.5µg/ml TPCK-treated trypsin. The PEDV-CO isolate at passage 5 was also received from NVSL and further adapted to cell culture through 15+ passages. Consistent high-titer virus stocks approaching 7 logs/ml are produced. These virus stocks are being used in studies of PEDV environmental stability and sanitation efforts. 3. (SDSU) The mAbs, monospecific hyperimmune serum and related reagents produced in this project should prove of substantial value in the detection of PEDV following VI attempts and in a variety of diagnostic methods such as IHC, antigen capture assays and fluorescent antibody technologies. They are currently being utilized in PEDV environmental stability studies and in fluorescent focus neutralization (FFN) assays for assessment of neutralizing antibodies produced following PEDV infection. 4. (SDSU) Cell culture adapted PEDV was used to develop an indirect fluorescent antibody (IFA) test for PEDV serology. A serological ELISA using expressed and purified PEDV nucleoprotein (NP) was developed optimized and is in the final stages of validation. It has demonstrated good correlation with IFA results from known PEDV seropositive and naïve populations. 5. (UW) Work on genetic and antigenic diversity within PRRSV was completed. We developed a novel analytical approach to identify a small number of representative viral genotypes from among the diversity of viral sequences available in GenBank and PRRSVdb. Viruses represented by the top ranking sequences are valuable targets for future study and a polyvalent vaccine development. 6. (UW-Madison). A post-doctoral researcher, Dr. Tavis Anderson, was employed for these analyses, and we secured additional personnel support through an international exchange program with the University of Torino, Italy. Dr. Anderson is now an Assistant Professor at Georgia Southern University and he will continue work on bioinformatics and polyvalent vaccine development at USDA. 7. (OSU) We developed a biodegradable PLGA nanoparticle-entrapped killed PRRSV vaccine (Nano-KAg) and given IN to evaluate immune correlates. In Nano-KAg vaccinated homologous virus challenged pigs, complete clearance of viremia was observed associated with a significant increase in virus neutralizing titers in the lungs. Nano-KAg vaccinated pigs had increased levels of IFN-³ and decreased levels of TGF-². Restimulation of mononuclear cells of vaccinates secreted significantly increased IFN³ and IL12. Higher frequencies of CD3+CD8+, CD4+CD8+, and gd T cells and reduced frequency of Foxp3+ T-reg cells were observed in vaccinates. In vaccinated but heterologous PRRSV challenged pigs, reduction in pathology, reduced viremia and viral load in the lungs was seen. Enhanced frequency of CD4+ cells, increased IFN-± and IFN-³, reduction in Tregs population, and decreased secretion of IL-10 and TGF-² was detected. Increased virus specific IgG and IgA, and Nabs were detected in vaccinates. We showed benefits of IN delivery of a nanoparticle-based killed PRRSV vaccine in inducing cross protective immune response. 8. (OSU). We standardized PRRSV NA assay using oral fluid collected over a period of 3 months from modified live vaccinated pigs, and oral fluid and serum samples collected from individual boars vaccinated (PRRS-MLV) or infected with a virulent PRRSV strain. Our results suggested that PRRSV NA titer of greater than 8 in oral fluid samples is virus specific, and it is detected from 4 weeks after vaccination or infection. Our results also showed that PRRSV NA titers in oral fluid samples are correlated with serum titers, and maternally derived PRRSV specific NA titers are detectable in the litters at the time of weaning. We have standardized and validated pen-based oral fluid PRRSV NA assay, which has 94.3% specificity and 90.5% repeatability. 9. (PURDUE). A relatively new method has been implemented allowing the detection of a wide variety of PRRSV strains by utilizing multiple primer sets and rt PCR. We utilized a single primer set designed from the conserved region of the PRRSV genome using a rt PCR to establish a more cost effective alternative. All the cases submitted to the ADDL and identified positive for PRRSV by the PRRSV kit from Tetracore® during the 2010-2011 fiscal year were analyzed. The diversity of the PRRSV genome among the submitted cases was determined by phylogenetic analysis ranging around 40% difference from type 1 and 2. All cases which were positive by the Tetracore® method were identified as positive using a single primer set designed from the PRRSV conserved region by PCR. We demonstrated that by using a single primer set in, PRRSV was detected across a wide diversity of the viral genome and produced comparable CT to a similar commercial assay. 10. (UMD) We identified an atypical PRRSV strain, A2MC2, which is able to induce type I IFNs. A2MC2 induction of neutralizing antibodies in vivo was compared with the Ingelvac PRRS MLV and VR-2385. A2MC2 resulted in earlier onset and significantly higher levels of PRRSV NAbs than the MLV. The A2MC2-induced NAbs were capable of neutralizing VR-2385. Pulmonary alveolar macrophages collected during the necropsy in the A2MC2 group had higher level expression of IFN-³ than the MLV group. A2MC2 can be further explored for development of an improved vaccine against PRRS. 11. (CNB-CSIC) focus was in the improvement of rTGEV vectors stability and the generation of new antigenic structures that may confer protection against PRRSV. Several rTGEV vectors were generated, stably expressing different PRRSV antigenic structures: rTGEV-M, expressing M protein; rTGEV-GP5fr-M, co-expressing a 33 aa GP5 ectodomain fragment, containing the epitope recognized by NAbs, and full-length M protein; rTGEV-GP3fr, expressing a 54 AA fragment from GP3 ectodomain, containing the epitope recognized by NAbs: rTGEV-GP4fr, expressing a fragment GP4 ectodomain, containing the epitope recognized by NAbs; rTGEV-GP3fr-MNH2, expressing a chimeric protein, consisting in a GP3 fragment containing the epitope recognized by NAbs fused to the amino-terminus of M protein. 12. (CNB-CSIC) The protection induced by rTGEV vectors was evaluated. 45 piglets were divided in 3 groups and were oral and IN vaccinated with each rTGEV vector described above (Group A), or empty rTGEV vector (Groups B and C). 2 wks later, animals were boosted. 2 wks after boost, animals from Grps. A and B were IN challenged with virulent PRRSV. 20% of the vaccinates (A) had clinical respiratory symptoms vs 60% from non-vaccinatess (B). A decrease in lung lesions was seen in vaccinates. There was a 6-fold reduction in virus titers in vaccinates. NAbs in the vaccinates were lower than non-vaccinates. This data were indicative of a limited protection conferred by rTGEV vectors expressing PRRSV antigens. Pigs were seropositive for TGEV after vaccination. Humoral responses demonstrated no significant differences between Group A and B. After challenge, vaccinated animals showed a faster and stronger induction of antibodies recognizing GP5, indicating a recall response in vaccinated piglets that was not fully protective. 13. (VA-TECH) We utilized DNA shuffling, to attenuate PRRSV by DNA shuffling of the viral envelope genes from multiple strains. The GP5 genes of 7 genetically divergent PRRSV and the GP5-M genes of 6 different PRRSV were shuffled. 2 representative chimeric viruses, DS722 with shuffled GP5 genes and DS5M3 with shuffled GP5-M genes, were rescued. A comparative pathogenicity study in pigs revealed that pigs infected with the 2 chimeric viruses had significant reductions in viral-RNA loads and in lung lesions, indicating attenuation of the chimeric viruses. Pigs vaccinated with the chimeric virus DS722, but not pigs vaccinated with DS5M3 acquired protection against PRRSV challenge at a level similar to the parental virus. DNA shuffling of envelope genes rapidly attenuated the virus. 14. (NADC, Nicholson, Spear and Faaberg) Tested new diagnostic nucleotide array. 15. (NADC, Faaberg and Spear) Development of additional DIVA vaccines. 16. (NADC, Spear, Faaberg) Developed ELISA for analysis of animal samples with DIVA Tag 17. (VA-TECH) We molecularly bred PRRSV through DNA shuffling of the GP4 and M genes, separately, from 6 genetically different strains of PRRSV to ID chimeras with improved heterologous cross-neutralizing capability 18. (KSU, Rowland, Fang, Opriessnig (ISU)) developed a Luminex platform for the detection of antibodies against PRRSV, PCV2 and SIV. 19. (KSU, Gabler and Rowland) are performing a study to determine the effect of PRRSV infection on feed digestibility. 20. (USDA-BARC, SDSU) A multiplex FMIA was developed to quantify serum cytokines using Luminex xMap" ( IL-1b, IL-8, IFN-a, IL-10, IL-12, IL-4, CCL2). Pigs were defined to 4 groups; high viremia-high growth (HvHg), high viremia-low growth (HvLg), low viremia-high growth (LvHg), low viremia-low growth (LvLg). After PRRSV, all cytokine levels except IL-4 were altered . 21. (UMN) MN developed several quantitative RT-PCR protocols to detect PEDV; a protocol based on detection of the S gene performed the best. A multiplex real-time RT-PCR for clinical samples to detect PEDV and TGEV in the same sample. MN is in the process of comparing this newly developed multiplex assay to other commercially available PCRs for PEDV. MN developed an immunohistochemistry technique to detect PEDV antigen in formalin-fixed paraffin-embedded samples. MN adapted a protocol for the isolation of PEDV in Vero cells. 22. (UMN) Studies were completed on the efficacy and cost-effectiveness of air filtration of large sow farms in hog dense regions. Methods were developed to evaluate filter performance against PRRS. 23. (UMN) Efforts on controlling aerosol dissemination centered on evaluating the electromagnetic particle ionization system to decrease infectious aerosols of PRRSV and influenza virus were studied. 24. (UGA) We explored host gene requirements for influenza virus replication, and have addressed how microRNAs govern their expression. These studies have identified miRNAs and multiple cellular targets for influenza viruses. 25. (UGA) We are currently using historical swine influenza sequence data to assess the evolution rates of SIV in swine herds in the United States as compared other global locations and as compared to human influenza viruses of the same subtype. This work is extremely preliminary and no results are available at this time. 26. (UGA) Test the potential for a vaccine vector (PIV5) to serve as a vaccine against influenza virus. Using the mouse model of H5N1 (HPAIV) infection, we demonstrated that PIV5 expressing the HA of H5N1 provided robust protection from lethal challenge when vaccinated IN, IM with live or killed vaccine. Vaccination with PIV5 expressing the internal, conserved NP protein also protected. We are developing a novel bivalent, adjuvanted vaccine using the F protein of RSV to enhance HA-specific immunity while priming and F-specific immune response. 27. (UGA) We are developing a surface enhanced Raman spectroscopic assay for detection of influenza virus and PRRSV.

Publications

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Publications Attachment:

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Impact Statements

(VA-TECH) Molecular breeding via DNA shuffling has important implications for future development of a broadly protective vaccine against PRRSV and will generate broad general interest in the scientific community in rapidly attenuating other important human and veterinary viruses.
(UCONN: Risatti): Detecting PRRSV genetic determinants associated with disease caused by the virus may contribute with information needed for rational engineering of PRRS live attenuated viruses.
(UCONN: Garmendia): Investigating IFN beta will contribute to gain a better understanding of the innate response to PRRSV which in turn will be useful to the overall knowledge of mechanisms of general pathogenesis, immune evasion and protection or lack thereof.
(USDA-ARS-NADC, Faaberg, Spear, Lager, Brockmeier, Miller, Loving, Butler): Constructs of Ingelvac® PRRS MLV were evaluated for their growth in swine and to determine if they induce antibodies to an inserted foreign tag. We analyzed animal samples infected with several PRRSV strains with and without bacteria, using common pathogenesis indicators. We investigated the effects of different PRRSV strains in gnotobiotic pigs, to seek a reliable index of pathogenicity. This allows us to survey viral growth properties, disease pathogenesis in swine, secondary bacterial pathogens that may arise during infection, the immune responses and host gene expression patterns that differ between PRRSV strains to understand what factors determine high vs. low virulence infections for development of better vaccines and vaccine strategies.
(SDSU) Current PRRSV vaccines are not highly effective in preventing PRRSV infections. A better understanding of virus-host interaction will facilitate the development of novel vaccine candidates against PRRSV.
(SDSU) Availability of high-titer cell culture adapted PEDV is particularly valuable for virus stability and disinfectant studies as simple virus re-isolation can be used to assess presence of viable PEDV, rather than relying on time-consuming and expensive swine bioassay systems. These virus stocks and associated re-isolation procedures are being used to develop appropriate biosecurity protocols specific to PEDV.
(SDSU) MAbs and related reagents will prove very valuable in the confirmation of PEDV antigen in cell culture and tissue samples associated with diagnostic cases and research studies.
(SDSU) Diagnostic serology tests such as IFA, virus neutralization and ELISA will be of substantial value in the control of PEDV. Specialized adaptations of PEDV neutralization assays may also provide good indicators of which animals may be immune or protected against PEDV associated disease.
(UW) Antigenic/genetic variation in PRRSV is a major impediment to vaccine development. By distilling this diversity down to a manageable unit, we provide guidance for the development of next-generation polyvalent vaccines that have maximum broad efficacy.
(ISU) Research has expanded our understanding of PRRSV, PCV2, influenza A, and other emerging viral diseases of swine and provide new ideas for preventing, countering and/or eliminating these infections. New work on the ecology and epidemiology of these agents provide insight into the mechanisms by which they maintain endemnicity. Research in diagnostic technology is contributing to the improvement and refinement of our ability to surveil, detect, and diagnose respiratory viral infections to provide highly cost-effective methods of tracking infection and implementing area elimination/eradication programs. This research will make possible the eventual elimination and eradication of viral infections from individual farms and regions.
(OSU) Intranasal delivery of nanotechnology based inactivated PRRSV vaccine may be a suitable strategy to elicit anti-PRRSV immune response and to clear viremia in pigs.
(OSU) Conventional ELISA results help only in PRRS survey. In contrast, pen-based PRRSV NA assay could provide information on PRRS herd immune status in vaccinated and/or infected recovered pigs and could be used to evaluate the levels of cross protective immune response against variant PRRSV strains.
(UMD) PRRSV A2MC2 inducing interferons in cultured cells may be beneficial for vaccine development to induce protective immunity against PRRS. This isolate induces higher titer of neutralizing antibody in pigs than MLV.
(UMD). Nsp1² of virulent VR-2385 inhibits interferon signaling by interfering with STAT1 nuclear translocation, while nsp1² of Ingelvac MLV has no effect. This result has a biological relevance on PRRS vaccine design.
(UMD) PRRSV VR-2385 induces pSTAT1-S727 and the expression of proinflammatory cytokines contributes to the insight of PRRSV pathogenesis.
(UMD) IFN signaling showed that several PRRSV proteins are involved in the interference with IFN signaling and that some PRRSV strains, such as NVSL and MN184, have variable effects on IFN-activated signaling in MARC-145 and PAM cells. These results may benefit vaccine development.
(UNL) Provision of an important starting point for the development of a live-attenuated DIVA vaccine against type-II PRRSV.
(UNL) The influence of host genetics on PCVAD susceptibility could lead to increase knowledge of swine immune system, and identification of genes involved in PCVAD susceptibility. Selection based on DNA markers associated with PCVAD susceptibility has the potential to reduce economic losses, improve animal welfare and provide alternatives to vaccination.
(KSU) The SCID is model will identify components of innate and adaptive immune protection that will be incorporated into the next generation of vaccines.
(KSU) The genomic marker on SSC4 is in the process of being tested by the industry for the development of marker-assisted selection.
(KSU) The Luminex multiplex serological assay technology is being transferred to a company for the development of a commercial kit.
(KSU) Understanding the effect of PRRSV infection on digestibility will be incorporated into the formulation of nutritional regimens that optimize growth during PRRSV infection.
(KSU) Reagents developed from the PEDV study are distributed to other labs for the purpose of assay development.
(KSU) CD169 is not a receptor for PRRSV
(USDA-BARC) Studies continue on the role of host genetics in resistance to PRRS and in effects on pig health and growth. A genome-wide association study revealed regions on SSC4 and X for VL and on SSC1, 4, 7, and 17 for WG. Pig response to PRRSV has a strong genetic component with a major QTL on SSC4 explaining a substantial proportion of the genetic variance. These results could have a major impact in the swine industry by enabling geneticists to develop plans for marker-assisted selection of pigs with improved response to PRRS.
(USDA-BARC, SDSU) An FMIA was developed to simultaneously quantify porcine cytokines in serum and oral fluids. It detects IL-1b, IL-8, IFN-a, L-10, IL-12, IL-4 and CCL2. Serum IL-8, IFN-a and CCL2 are significantly altered after PRRSV infection. Changes in cytokine and chemokine levels reflect potentially different viral control mechanisms. Correlations of cytokine profiles with serum viral levels, growth performance and genetic background are continuing in hopes of revealing candidate biomarkers of PRRS responses.
(CAU) The works on HP-PRRSV pathogenicity (Objective 1-1, above) is not only the first unambiguous illumination about the key virulence determinant of Chinese HP-PRRSV, but it also provides an opportunity to better understand the pathogenic mechanism of this virus.
(CAU) The works on pathogenicity of genotype 1 PRRSV (Objective 2-1, above) is the first pathogenicity study on wild isolate in Mainland China.
(CAU) The works (Objective 1-4 above) revealed one of the important mechanisms of how HP-PRRSV significantly suppresses innate immune responses.
(UMN) Air filtration research provides producers with technical knowledge and economic data to facilitate implementation of effective methods for reduction of airborne viral infection, including PRRSV and influenza virus, in swine herds.
(UMN) Economic cost-benefit analyses demonstrates the advantage of air filtration technologies for disease reduction and prevention in sow herds.
(UMN) After taking into account the production improvement and the PRRS status of the weaned piglets from both types of farms, the pay-back period of air filtration was calculated to be between 2 and 3 years depending on the initial investment.
(UMN) Analysis of risks of influenza transmission within and between farms will facilitate development of effective methods to reduce transmission and identify factors that influence transmission between pigs and between humans and pigs.
(UMN) Whole genome sequencing is expected to reveal candidate elements associated with virulence and cross-protective immunity that will facilitate development of improved tools for treatment and prevention of PRRS.
(UMN) Elucidation of mechanisms of induction of cross-protective antibody production is expected to provide a rational basis for development of improved vaccines.
(UMN) Identification of live animal markets as a source of influenza virus diversity and transmission of variant influenza raises awareness of multiple transmission opportunities.
(UMN) Characterization of routes of influenza exposure to people in commercial farms, live animal markets and agricultural animal fairs identified aerosols and hand contact surfaces as possible routes of influenza infection in people.
(UMN) Indirect transmission of influenza viruses via fomites was possible despite the implementation of moderate biosecurity measures.
(UMN) Approximately 45% of breeding farms weaned influenza-positive pigs, increasing the awareness of influenza virus transmission dynamics in swine operations.
(UMN) The effectiveness of electromagnetic particle ionization in reducing influenza and PRRSV aerosols under experimental and field conditions provides producers with another tool for disease control.
(UIUC) Our results indicate that the ZF1 motif of nsp1± plays an important role for IFN regulation and further demonstrate that the CBP degradation is likely the key mechanism for IFN suppression mediated by the nsp1± subunit protein of PRRS virus.
(UIUC). The directions and intensities in our inferred virus traffic network closely mirror the hog transportation. Most notably, we reveal multiple viral introductions from Canada in to the United States causing a major shift in virus genetic composition in the Midwest USA that went unnoticed by the regular surveillance and field epidemiological studies. Overall, these findings provide important insights into the dynamics of Type 2 PRRSV evolution and spread that will facilitate programs for control and prevention.
(UIUC) The miRNA study revealed a subset of a large number of miRNAs that is being altered in PRRSV infected macrophages. Virus replication was negatively impacted by high levels of miR-147. Target gene identification suggests that these miRNAs are involved in regulating immune signaling pathways, cytokine and transcription factor production. Whether down-regulation of miR-147 is directly induced by PRRSV, or if it is part of the cellular response and PRRSV indirectly benefits remains to be determined. No evidence could be found of PRRSV-encoded miRNAs.
(UIUC) The appearance of similar deletions in nsp2 in field PRRS viruses of different lineages and levels of virulence suggests a role for this protein in pathogenicity. Contagion might be increased by a higher virus load in the airways.
(UGA) The majority of studies during this reporting period have been on emerging viral diseases of swine, i.e. influenza. However, we are now applying these platforms to PRRSV and other swine disease control.
(UGA) In regard to influenza as an emerging (re-emerging) disease of swine, we continue to make extensive advances in understanding features of the virus-host interface that influence infection, tropism, and reassortment. We have also explored a number of vaccine and anti-viral therapies for influenza and developed a novel approach for rapid and sensitive detection of influenza virus. These studies directly impact swine and/or human health, and address the One Health paradigm.