Brief Summary of Minutes of Annual Meeting

NC-229 Business Meeting Minutes. Friday 11/12/04. The meeting was brought to order at 1:12 pm by chair Jeff Zimmerman. An overview of the meeting agenda was presented. Financial support of the meeting was acknowledged (Boehringer Ingelheim Vetmedica, IDEXX Laboratories, Inc., Intervet, Inc., National Pork Board, Pig Improvement Company). Names of attendees were collected. Old Business The minutes from the last meeting were presented to the group and accepted without corrections. New Business The Annual Report for October 2003 to September 2004 (in progress) was presented for the purpose of soliciting corrections and/or additions. None were indicated at this time. Peter Johnson (NC-229 USDA Representative) discussed the latest activities related to USDA. Dr. A. Palmisano was hired as a new administrative director and a new web site is under development. Investigators will be submitting grants this year by paper; next year, submission will be electronic. Michael Murtaugh (Minnesota) presented the progress report for the PRRS Integrated Program Project, including a detailed analysis of expenditures. In summary, expenditures were $2.5M for Year One (ending Jan 15, 2005). A total of 17 proposals received $3.8M with $1.4M to 9 awards and 2 contracts for services. Murtaugh suggested that we convene at six-month intervals, to discuss issues related to funding. The special issue of Veterinary Immunology and Immunopathology on PRRS virus immunology has been published. Steve Gylling (President, Gylling Data Management, Inc., Brookings, South Dakota) gave a presentation on ARM Research Management Software. Handouts were provided and an overview of the software presented. Essentially, ARM software would provide NC-229 researchers web-based means to share data in a Good Laboratory Practice environment. Eric Neumann (National Pork Board) presented the NPB report. NPB funding for the previous year consisted of $2M for 50 proposals. Eric identified two projects funded through Leverage and Discovery: 1) PRRSV protein expression, using Mike Murtaughs techniques will be performed by a private company; 2) a chamber for use in aerosol stability studies will be built at Iowa State University and subsequently made available for other projects. NPB is interested in feedback from NC-229. Priorities for next year are similar, e.g., investment into vaccine research, regional eradication, outreach, and education. Funding for next year will include two calls for proposals. Following budget approval, the NPB may have another $2M in support of PRRS research. Proposal format for next year has been modified and includes a pre-proposal format. The principal message from the NPB proposal review committee is that researchers need to improve experimental design, focus on research that provides answers, and pay close attention to details in assembling their proposals. NPB will consider multi-year proposals, but Year One should be described in detail. Eric Neumann handed out a draft annual report on PRRS and suggested bundling NPB, NRI, and NC-229 reports into a single PRRSV Research Report. This idea was well received. Target date is February 15, 2005. Eric Neumann described the PRRS website and communications, including a link to the PRRS initiative. He described several new features, including a message board. The areas of Education and Outreach are currently in the process of development. Camile Moore (IPP Scientific Review Board) was asked to comment on the IPP. Overall, he was impressed by project selection and teamwork. He suggested that the next step is to build greater collaboration on projects; i.e. combine proposals into a collaborative network. He also suggested that the IPP needs to seek input from outside the current group of scientists. Open Discussion Host genetics related to resistance to PRRS was the first topic. The discussion was lively and participation was extensive among those present. The general consensus was that identification of host genes related to resistance to PRRS represented a long-term, difficult, and expensive project. A number of experimental approaches are possible, but it is not certain which would be most efficient in terms of arriving at a solution. Suggestions included: 1) additional input from geneticists, 2) additional input from the external advisory board, and/or 3) the formation of a committee on disease resistance. This was followed by a discussion on the Annual Meeting of NC-229 / International Workshop on PRRS. Given the large amount of information being generated by NC-229 participants and IPP researchers, has it become necessary to appoint a Program Committee? In the context of active multi-station research programs, do we need to discuss station reports? The need for a scientifically strong program was expressed. The idea of extending the meeting was raised, but not received with enthusiasm. The addition of poster presentations was suggested as a method to accommodate the diversity and depth of current research. The next issue discussed was financial issues related to funding. In particular, the question considered was whether PI salaries should be included on IPP grants. Although there are concerns regarding salaries, the general consensus was that reasonable requests are acceptable and excessive requests can be addressed during the proposal review process. The final discussion concerned the planning of the next meeting of the PRRS Integrated Program Project in conjunction with the Annual Meeting of the American Association of Swine Veterinarians (March 2005, Toronto). The suggestion was made to expand the stakeholder meeting to include fundees of NC-229. This idea was well received. The meeting was adjourned at 5:00 pm. NC-229 reconvened on Saturday 11/13/04. 5TH ANNUAL MEETING OF NC-229 / INTERNATIONAL WORKSHOP ON PRRS 08:00 am Opening remarks. Jeff Zimmerman, Chair NC-229; 08:15 am Comments. David Benfield, Administrative Advisor NC-229; 08:30 am Comments. Michael Murtaugh, Director, PRRS Integrated Program Project. Overview of Integrated Program progress and forecast for Year Two; 08:45 am Comments. Eric Neumann, Director, Swine Health Information and Research, National Pork Board. NPBs perspective.; 09:00 am Creation of a Virtual Laboratory infrastructure. Moderator: Steve Kleiboeker; Immunological reagents: What and how? Joan Lunney. USDA/ARS/BARC; Protein reagents: A test case for common reagent use. M. Murtaugh. University of Minnesota; A proposed listserv to facilitate communication. Steve Kleiboeker. University of Missouri; NC-229 / NPB website Eric Neumann. National Pork Board; ARM7 data management software Jeff Zimmerman. Iowa State University; 10:00 am Break 10:30 am The PRRS viral genome. Moderator: Dongwan Yoo; Evolution and quasispecies of PRRSV in cells in vitro Susan Schommer. University of Missouri; Evolution and quasispecies of PRRSV in pigs. K-J Yoon. Iowa State University; Emergence of European-like PRRSV in North America Ying Fang. South Dakota State University; Infectious clones and manipulation of PRRSV genome Dongwan Yoo. University of Guelph; 11:30 am Juxtaposition: Equine arteritis virus vs PRRSV - James MacLachlan 12:00 pm Lunch 01:00 pm Pathogenesis. Moderator: RRR Rowland; Gene expression analysis of PRRSV pathogenesis Elisabetta Giuffra. Roslin Institute; Engineering the PRRSV genome as an infectious bacterial artificial chromosome. Young-Min Lee. Chungbuk National University; Heteroclite RNAs - potential role in pathogenesis. Kay Faaberg. University of Minnesota; The PRRSV-macrophage molecular interface. Mark Rutherford. University of Minnesota; 02:00 pm The immune response. Moderator: Fernando Osorio; PRRSV adaptive immunity M Murtaugh. University of Minnesota; Effect of repeated PRRSV exposure on immune response Eileen Thacker. Iowa State University.; Suppression of the humoral immune response to PRRSV M McCaw. North Carolina State Univ; Mechanisms of immune subversion by PRRSV. F Zuckermann. University of Illinois; Innate immune response to PRRS virus J Lunney. USDA / ARS / BARC / ANRI; PRRSV humoral immunity F Osorio. University of Nebraska-Lincoln; 03:00 pm Break 03:30 pm Viral ecology, epidemiology, and eradication. Moderator: Locke Karriker; PRRSV immunoepidemiology (?) on commercial farms Tony Goldberg. University of Illinois; PRRSV transmission in nurseries Cate Dewey. University of Guelph; Considerations for monitoring rate of transmission in populations C Muñoz-Zanzi. University of Minnesota; PRRSV circulation in commercial nursery/finisher flows Locke Karriker. Iowa State University; 04:30 pm Defining the role of host genetics in disease control - Steve Bishop;

Objective 1. Implement a virtual laboratory infrastructure through the development and open distribution of resources, materials, protocols and data among participating researchers. A workshop on diagnostics, reagents, immunology methods standardization and host genetics vis-à-vis PRRSV infection was held on March 5, 2004. The 5th Annual Meeting of the NC-229 hosted a demonstration of Agricultural Research Management (ARS version 7) software. This data management software facilitates web accessibility and sharing of databases under Good Laboratory Practice standards. Objective 2. Achieve biosecurity within herds by preventing the spread of virus within a herd and facilitating its elimination from endemically infected herds. 2.1 Cells of the immune system. Kansas State University (KSU) determined the effects of PRRSV infection on immunophenotypes of lymphoid cells. North Carolina State University (NCSU) is using a training grant to adapt and validate immune cell proliferation measurement by flow cytometry. The University of Guelph reported changes in host cell gene expression by PRRSV glycoproteins, GP4 and GP5. No apoptosis-related genes were regulated in cells expressing GP5, indicating that GP5 is not the protein responsible for apoptosis in PRRSV-infected cells. The University of Minnesota (UMn) evaluated gene expression patterns in macrophages infected with either virulent (VR-2332) vs vaccine strains of the virus using the Qiagen 70-meroligonucleotide array system. Replication in dendritic cells was studied by South Dakota State University (SDSU) reported that PRRSV underwent a productive replication in pig monocyte-derived dendritic cells (Mo-DC). University of Missouri (UMo), Ghent University, and the UMn examined the interaction of PRRSV with macrophage-matured DCs. 2.2 Cytokines. University of Illinois (UI) showed that IL-12 receptor (IL-12R) transcripts were only marginally induced in alloantigen-stimulated cultures of PBMC from PRRSV immune pigs, the IL-12R gene response increased on addition of recombinant porcine IL-18. One important defect during PRRSV infection is the apparent failure to initiate an appropriate IFN response. Institutions involved in the study of IFN responses included USDA:ARS:MARC, UMo, and UI. In MARC-145 cells, both IFN-alpha and -beta transcript abundance were unaffected by PRRSV infection. Further studies suggest that PRRSV infection directly interferes with type I IFN transcriptional activation early in its pathway, at the level of IFN-beta gene transcription. UMo extended these studies to include the IFN response in cultured macrophages (PAMs). PRRSV isolates were differentially sensitive to porcine recombinant IFN-alpha (rIFN-alpha) and varied in their ability to induce IFN-alpha. The relative number of IFN-alpha transcript copies did not correlate with IFN-alpha protein levels, suggesting a post-transcriptional mechanism of IFN suppression. Using ELIspots for the measurement of IFN-gamma-specific T cells, the UI, found that the T helper 1 response was variable and generally weak. UI studied the ability of DCs cells to produce IFN. Depending on the virus stock, PRRSV was at least 10-100 times less efficient than transmissible gastroenteritis virus (TGEV) or type-A CpG oligonucleotides at stimulating the secretion of IFN-alpha from DC. 2.3 Antibodies. The role of neutralizing antibody in the control of PRRSV remains under active study. University of Nebraska (UNL) previously demonstrated that transfer of antibodies highly enriched in neutralizing activity to PRRSV protected pregnant sows and conferred sterilizing immunity in sows and offspring. Augmentation of the neutralizing antibody response in a PRRSV-infected herd was investigated by NCSU. Antibody responses to nonstructural proteins was investigated at the UMn. Iowa State University (ISU) investigated auto-anti-idiotypic antibodies (Aab-2s) specific for antibodies against GP5 and M surface proteins. The early and late Aab-2s possessed different idiotype-binding specificities. 2.4. Persistent infection in pigs. SDSU and Ohio State University (OSU) examined the role of lymphoid and non-lymphoid tissue in acute and persistent infections of PRRSV for the detection and elimination of PRRSV infected pigs. 2.5. Viral genome. UNL used reverse genetics to identify viral genes involved in virulence. Using an infectious clone, they developed a series of chimeric constructs containing structural genes of a well studied PRRSV attenuated vaccine strain within the genomic context of a highly virulent PRRSV strain. The role of heteroclite RNAs in pathogenesis was studied at the UMn. It was found that there was a differential effect of sequential heteroclite transcript addition to transcripts of the infectious clone of full-length virus, in that varying amounts of added heteroclite transcripts could enhance or abolish viral replication. Studies were conducted on the evolution of virus at ISU. Three independent lines of in vivo replication were maintained for 2 years. Plaque-cloned viruses were obtained at each passage and sequenced for all major ORFs (1b, 2 to 7). All ORFs except 1b and 7 co-evolved, although at different rates. ISU examined the immunobiological significance of genetic variation among PRRSV. Correlation between the level of genetic divergence and cross-neutralization (both in vitro and in vivo) was studied. Isolates with less than overall 95% homology did not cross neutralize well. The UMo is using an infectious clone to evaluate in vitro quasispecies evolution. 2.6. Pathogenesis (virus factors). USDA:ARS:NADC looked at viral properties of pathogenesis. Preliminary data indicate the highest virus load in sera and lung tissue occurs with the most virulent wild-type virus. The UMn, SDSU, UMo, and Boehringer Ingelheim Vetmedica collaborated on studies to understand the in vivo growth properties of virulent field isolates and attenuated PRRSV isolates. Virulent PRRSV isolates were found to exhibit longer and more elevated levels of viremia that correlated with faster and more intense humoral immune responses. A microarray and semi-quantitative reverse-transcription polymerase chain reaction (sqRT-PCR) approaches were used by USDA:ARS:MARC to evaluate the gene response of cells to infection. Twenty-six apoptosis-related genes were examined during the first 24 h of infection and found to be unaltered, indicating that apoptotic induction was not occurring in PRRSV-infected cells. RNA silencing is being used at the UMn to study PRRSV-host cell interactions in cells infected in vitro. Conditions for siRNA transfection of primary porcine alveolar macrophages and MA-104 cells have been optimized and siRNA silencing of PRRSV replication demonstrated. 2.7. Pathogenesis (host factors). Research at KSU is directed at understanding the short and long-term consequences of congenital infection through assessment of virus replication and the induction of inflammatory/antiviral cytokines. UNL studied the response of pigs from either the NE Index Line (I) or Hampshire-Duroc cross pigs (HD) infected with PRRSV at 26 d of age. Pigs in each line responded differently to PRRSV challenge, indicating an underlying genetic variation. 2.8. Vaccines / Vaccination. USDA:ARS:BARC and UI used a novel cytokine adjuvant, IFN-alpha and molecular tests to evaluate immune factors that influence vaccine efficacy. Results showed that PRRSV vaccination, with or without IFN-alpha, stimulated low levels of protective IFN-gamma and only limited amounts of innate immune markers, interleukin-1 beta, IL-6 and IL-8 that should enhance immunity. At ISU, subunit preparations are being evaluated for their ability to induce neutralizing antibody and prevent disease due to PRRSV (subunit vaccine). Glycoproteins present in the envelope of the PRRSV will be modified by deglycosylation to prepare various subunit vaccine candidates. ISU is evaluating commercial killed and MLV vaccines to acclimate PRRSV negative gilts prior to breeding. ISU found that the degree of genetic homology of the ORF 5 between MLV PRRSV vaccines and the isolate that pigs are challenged with is not a good predictor of vaccine efficacy. Objective 3. Achieve biosecurity among herds by preventing viral spread between sites. 3.1. Virus diversity. Genetic variation among PRRSVs in pigs and farms was investigated at the UI. ORF5 from tonsils of naturally infected swine was amplified, cloned, and individual clones sequenced to characterize viral diversity in nine animals from two farms. All animals harbored multiple PRRSV variants at both the nucleic and the amino acid levels. Using viruses isolated from diagnostic samples submitted to the South Dakota ADRDL, UMn, SDSU, KSU, and OSU collaborated in an effort to understand the emergence of Type 1 PRRSV isolates in the U.S. Research interpreting genomic sequencing in the context of spatial analysis is being done to assess the regional epidemiology of PRRSV (UMn). Current results suggested that genomic variability correlated only with geographic and not temporal distance. 3.2. Transmission. PRRSV dose:response curves were produced by researchers at ISU. The ID50 for young pigs exposed orally and by the intranasal route to PRRS virus is approximately 5.0 TCID50/ml and 3.8 TCID50/ml, respectively. Work in progress at the UMn will provide quantitative estimates of PRRSV shedding by individual animals via the aerosol route for 25-and 120-kg pigs. ISU is also working to model virus transmission between herds under specific atmospheric conditions (temperature, relative humidity, sunlight, wind). 3.3. Herd immunity. The UI examined cellular immunity and protection against reproductive failure in sows on commercial swine farms during clinical outbreaks of PRRS. Evidence that a strong cellular immune response correlated with protection against clinical PRRS was found in 3/4 farms, but farms and animals within farms varied considerably in their immune response and the degree to which they were protected clinically. UMn is evaluating the effect of repeated immunization on persistence and transmission of a related virus in a population of pigs. A field study of PRRSV circulation in nursery and finisher phases of commercial herds indicated that persistent viremia and/or re-infection was found in 36 pigs post 20 weeks of age on the basis of PCR screening of serum (ISU). Objective 4. Improve diagnostic assays and create on-farm monitoring systems. 4.1 PCR-based assays. At the UMo, real-time (TaqMan) RT-PCR assays were developed for multiplex detection, differentiation, and quantification of NA and EU PRRSV field isolates. A multiplex fluorogenic PCR for PRRSV that detects NA and EU PRRSV in a differential manner was developed at ISU and used to monitor PRRS incidence. The efficiency of a SYBR green real-time PCR for detecting PRRSV in boar semen and serum was evaluated at KSU. The final stages of validation for a new NA/EU PRRSV TaqMan RT-PCR are in progress at the UMn. This test will replace two separate, NA and EU TaqMan, and will also provide increased sensitivity for EU PRRSV strains. A commercial, single-tube, real-time PCR assay was developed for the detection of U.S. and Euro/LV PRRSV isolates by UNL, SDSU, Pig Improvement Company (PIC), Syngen, Inc., Tetracore, Inc., and Boehringer Ingelheim Vetmedica. 4.2. Other work re diagnostics. NCSU characterized antibody and rtPCR responses following challenge with a high dose of homologous wild-type PRRSV in pigs initially immunized with multiple low doses of virus. Antibody levels to rORF 5-6 ectodomain chimera followed the SN antibody temporal response curve and to rORF7 followed the response curve observed with the commercial ELISAs. UMn, SDSU, PIC and Boehringer Ingelheim Vetmedica are developing and evaluating blocking ELISAs for the detection of antibodies against North American and EU-like PRRSV. This test appears to lack sensitivity in detecting antibodies produced against EU PRRSV. Virginia Polytechnic Institute and State University and ISU have collaborated on the development of a heteroduplex mobility assay (HMA) to identify PRRSV isolates with significant nucleotide sequence identities (>/=98%) with the modified live-attenuated vaccines. Objective 5. Develop and test PRRSV virus eradication protocols under various ecological settings. Research directed by the UMn is looking at the feasibility of controlling PRRS within a selected area (Rice County, MN). Semi-annual sampling of all sites will be conducted, seeking 95% confidence in detecting PRRSV pigs at various production stages (sows, nursery, finish) having at least 20% seroprevalence. ISU examined the question of how long herds that eliminated PRRSV remain free of infection. Information from 84 farms indicated that the estimate of probability of maintaining negative status for 2-years ranged 47 to 70%. Objective 6. Develop educational outreach tools for disseminating information through established outreach and extension networks to producers, veterinarians, educators, and researchers. A special issue (Dec 2004) of Veterinary Immunology and Immunopathology focused on PRRS was edited by KSU and the UMn, with contributions from participants from other Stations. In the past year, NC-229 participants have disseminated 71 refereed publications, 87 abstacts/proceedings, and 4 book chapters related to PRRSV. Objective 7. Create an information network to ensure rapid and efficient communication of PRRSV research.A national PRRS epidemiological registry and database was funded by The National Pork Board (NPB). Work-in-progress involves transferring the existing sequence database to a MySQL language and building a new web page for producers/veterinarians/ researchers to easily access the UMn data. New software tools are currently being created that will enable individuals to submit simple queries to the database. Communication activities were initiated in collaboration with the NPB (www.porkboard.org/prrs) for public dissemination of information. This website included an on-line catalog of a variety of PRRS-related resources, including The PRRS Compendium. Approximately 97 PRRSV researchers and scientists from the U.S., the U.K., Italy, France, Korea, and Mexico were in attendance at the annual meeting.

Refereed publications 1. Bassaganya-Riera J, Thacker B, Yu S, Strait E, Wannemuehler M, Thacker E. 2004. Impact of immunizations with porcine reproductive and respiratory syndrome virus on lymphocyte recall responses of CD8+ T cells. Viral Immunol 17:25-37. 2. Bastos RG, Dellagostini OA, Barletta RG, Doster AR, Nelson,E Zuckermann F, Osorio FA. 2004. Immune response of pigs inoculated with Mycobacterium bovis BCG expressing a truncated form of GP5 and M protein of porcine reproductive and respiratory syndrome virus. Vaccine 22:467-474; 3. Batista L, Dee SA, Rossow KD, Polson DD, Xiao Z, Olin M, Molitor TW, Murtaugh MP, Pijoan C. 2004. Detection of porcine reproductive and respiratory syndrome virus in pigs with low positive or negative ELISA s/p ratios. Vet Rec 154:25-26.; 4. Batista L, Dee SA, Rossow KD, Xiao Z, Olin M, Molitor TW, Murtaugh MP, Pijoan C. Virological and immunological responses to porcine reproductive and respiratory syndrome virus (PRRSV) in a large population of breeding age female swine. Can J Vet Res (In press).; 5. Batista L, Pijoan C, Dee S, Olin M, Molitor T, Xiao Z, Murtaugh M. Virological and immunological features of homologous and heterologous protection to porcine reproductive and respiratory syndrome virus (PRRSV) in gilts. Can J Vet Res (In press).; 6. Batista L, Pijoan C, Ruiz A, Utrera V, Dee SA. 2004. Assessment of the transmission of Mycoplasma hyopnuemoniae by personnel. J Swine Health Prod 12:75-77.; 7. Batista L, Pijoan C, Lwamba H, Johnson CR, Murtaugh MP. 2004. Genetic diversity and possible avenues of dissemination of PRRSV in two geographic regions of Mexico. J Swine Health Prod 12:170-175.; 8. Cancel-Tirado S, Evans R, Yoon K-J. 2004. Identification of PRRSV epitopes associated with antibody-dependent enhancement and neutralization of virus infection using monoclonal antibodies. Vet Immunol Immunopathol (in press).; 9. Cha S-H, Chang C-C, Yoon K-J. 2004. Instability of ORF5 RFLP pattern of PRRSV during sequential pig-to-pig passages. J Clin Microbiol (in press).; 10. Dawson HD, Royaee AR, Nishi S, Kuhar D, Schnitzlein WM, Zuckermann F, Urban JF, Lunney JK. 2004. Identification of key immune mediators regulating T helper 1 responses in swine. Vet Immunol Immunopathol 100:105-111.; 11. Dee SA, Boorman J, Moon RD, Fano E, Trincado C, Pijoan C. 2004. Transmission of porcine reproductive and respiratory syndrome virus under field conditions during a putative increase in the fly population. J Swine Health Prod 12:242-245.; 12. Dee SA, Deen J, Burns D, Douthit G, Pijoan C. 2004. An assessment of sanitation protocols for commercial transport vehicles contaminated with porcine reproductive and respiratory syndrome virus. Can J Vet Res 68:208-214; 13. Dee SA, Deen J, Otake S, Pijoan C. 2004. An assessment of transport vehicles as a source of porcine reproductive and respiratory syndrome virus transmission to susceptible pigs. Can J Vet Res 68:124-133.; 14. Dee SA, Deen J, Pijoan C. 2004. An evaluation of four intervention strategies to prevent mechanical transmission of porcine reproductive and respiratory syndrome virus. Can J Vet Res 68:19-26.; 15. Dee SA, Deen J, Pijoan C. Evaluation of disinfectant efficacy for sanitizing porcine reporductive and respiratory syndrome virus-contaminated transport vehicles. Can J Vet Res (in press).; 16. Dee SA, Jacobson L, Rossow K, Pijoan C. A laboratory model to evaluate the role of aerosols in the transport of porcine reproductive and respiratory syndrome virus Vet Rec (in press).; 17. Dee SA, Martinez BC, Clanton CJ. Survival and infectivity of porcine reproductive and respiratory syndrome virus in swine lagoon effluent. Vet Rec (in press).; 18. Dee SA, Torremorell M, Deen J, Thompson B, Pijoan C. An evaluation of the Thermo-Assisted Drying and Decontamination (TADD) system for the elimination of porcine reproductive and respiratory syndrome virus from contaminated livestock transport vehicles. Can J Vet Res (in press).; 19. Dee SA. 2004. Elimination of porcine reproductive and respiratory syndrome virus by test and removal on 30 farms. J Swine Health Prod 12:129-133.; 20. Dongwan Y, Wootton SK, Li G, Song C, Rowland RRR. 2003. Colocalization and interaction of the porcine arterivirus nucleocapsid protein with the small nucleolar RNA-associated protein fibrillarin. J Virol 77: 12173-12193.; 21. Fang Y, Kim DY, Ropp S, Steen P, Christopher-Hennings J, Nelson EA, Rowland RRR. 2004 Heterogeneity in Nsp2 of European-like porcine reproductive and respiratory syndrome viruses isolated in the United States. Virus Res 100:229-235.; 22. Fano E, Pijoan C, Dee SA. Evaluation of aerosol transmission of a mixed infection of Mycoplasma hyopneumoniae and porcine reproductive and respiratory syndrome virus. Vet Rec (in press).; 23. Ferrin NH, Fang Y, Johnson CR, Murtaugh MP, Polson DD, Torremorell M, Gramer ML, Nelson EA. 2004. Validation of a blocking enzyme-linked immunosorbent assay for the detection of antibodies against porcine reproductive and respiratory syndrome virus. Clin Diagn Lab Immunol 11:503-514.; 24. Gerrits RJ, Lunney JK, Johnson LA, Pursel VG, Rohrer GA, Dobrinsky JR. 2004. A vision for artificial insemination and genomics to improve the global swine population. Theriogenology. (in press); 25. Goldberg TL, Lowe JF, Milburn SM, Firkins LD. 2003. Quasispecies variation of porcine reproductive and respiratory syndrome virus during natural infection. Virology 317:197-207.; 26. Hermann JR, Honeyman MS, Zimmerman JJ, Thacker BJ, Holden PJ, Chang CC. 2003. Effect of dietary Echinacea purpurea on viremia and performance in porcine reproductive and respiratory syndrome virus-infected nursery pigs. J An Sci 81:2139-2144.; 27. Horter DC, Yoon K-J, Zimmerman JJ. 2004. A review of porcine tonsils in immunity and disease. Animal Health Research Reviews 4:143-155.; 28. Hyland K, Foss DL, Johnson CR, Murtaugh MP. 2004. Oral immunization to induce local and distant mucosal immunity in swine. Vet Immunol Immunopathol 102:331-340.; 29. Jiang Z, Zhou E-M, Ameri-Hahabadi M, Zimmerman JJ, Platt KB. 2003. Identification and characterization of auto-anti-idiotypic antibodies specific for antibodies against porcine reproductive and respiratory syndrome virus envelope glycoprotein (GP5). Vet Immunol Immunopathol 92:125-135.; 30. Johnson W, Roof M, Vaughn E, Christopher-Hennings J, Johnson CR, Murtaugh MP. 2004. Pathogenic and immunological responses to porcine reproductive and respiratory syndrome virus (PRRSV) are related to viral load in acute infection. Vet Immunol Immunopathol 102:235-250.; 31. Kleiboeker SB, Schommer SK, Lee S-M, Watkins S, Chittick W, Polson D. Simultaneous detection of North American and European porcine reproductive and respiratory syndrome virus using real-time quantitative RT-PCR. J Vet Diagn Invest (in press).; 32. Lee C, Bachand A, Murtaugh MP, Yoo D. 2004. Differential cellular gene expression regulated by the porcine reproductive and respiratory syndrome virus GP4 and GP5 glycoproteins. Vet Immunol Immunopath 102:189-198 (Published on-line Oct 18, 2004).; 33. Lee C, Bachand A, Murtaugh MP, Yoo D. Yoo. 2004. Differential host cell gene expression regulated by the porcine reproductive and respiratory syndrome virus GP4 and GP5 glycoproteins. Vet Immunol Immunopathol 102:179-188.; 34. Lee C, Calvert JG, Welch SK, Yoo D. A DNA-launched reverse genetic system for porcine reproductive and respiratory syndrome virus reveals that homodimerization of the nucleocapsid protein is essential for virus infectivity. Virology (in press); 35. Lee C, Rogan D, Erickson L, Zhang J, Yoo D. 2004. Characterization of the porcine reproductive and respiratory syndrome virus glycoprotein 5 (GP5) in stably expressing cells. Virus Res 104: 33-38.; 36. Lee S-M, Schommer SK, Kleiboeker SB. Porcine Reproductive and Respiratory Syndrome Virus Field Isolates Differ in in vitro Interferon Phenotypes. Vet Immunol Immunopathol (in press).; 37. Lemke CD, Haynes JS, Spaete R, Adolphson D, Vorwald A, Lager K, Butler JE. 2004. Lymphoid hyperplasia resulting in immune dysregulation is caused by PRRSV infection in neonatal pigs. J Immunol 172:1916-1925.; 38. Lopez OJ, Osorio FA. The role of antibodies in PRRSV protective immunity, a short survey. Vet Immunol Immunopathol (in press); 39. Lowe JF, Husmann R, Firkins LD, Zuckermann FA, Goldberg TL. Correlation of cellular immunity to porcine reproductive and respiratory syndrome virus and clinical disease during outbreaks of PRRS in commercial swine herds. J Am Vet Med Assoc (in press).; 40. Martens GW, Lunney JK, Baker JE, Smith DM. 2003. Rapid assignment of swine leukocyte antigen (SLA) haplotypes in pedigreed herds using a polymerase chain reaction based assay. Immunogenetics 55:395-401.; 41. Meier WA, Husmann RJ, Schnitzlein WM, Osorio FA, Lunney JK, Zuckermann FA. 2004. Cytokines and synthetic double-stranded RNA augment the T helper 1 immune response of swine to porcine reproductive and respiratory syndrome virus. Vet Immunol Immunopathol 102:299-314. ; 42. Miller LC, Fox JM. 2004. Apoptosis and porcine reproductive and respiratory syndrome virus. Vet Immunol Immunopathol (Published on-line October 18, 2004); 43. Miller LC, Laegreid WW, Bono JL, Chitko-McKown CG, Fox JM. 2004. Interferon type-I response in porcine reproductive and respiratory syndrome virus-infected MARC-145 cells. Arch Virol (Published on-line May 26, 2004); 44. Nielsen HS, Liu G-P, Nielsen J, Oleksiewicz MB, Bøtner A, Storgaard T, Faaberg KS. 2003. Generation of an infectious clone of VR-2332, a highly virulent North American-type isolate of porcine reproductive and respiratory syndrome virus. J Virol 77:3702-3711.; 45. Nilubol D, Harris DL, Thacker BJ, Thacker EL. Duration and protection of maternally derived antibodies in pigs farrowed from PRRSV-exposed sows following killed PRRSV vaccine administration. Viral Immunol (submitted).; 46. Nilubol D, Harris DL, Thacker BJ, Thacker EL. Immune responses and protection against high and low virulence PRRSV in pigs vaccinated with modified live and killed PRRSV vaccine, either alone or in combination. Vaccine (submitted).; 47. Nilubol D, Harris DL, Thacker BJ, Thacker EL. Responses of pigs following vaccination with a commercial killed porcine reproductive and respiratory syndrome virus (PRRSV) vaccine containing interleukin-12 and a combination vaccination with modified-live and killed PRRSV vaccines. Vaccine (submitted).; 48. Nilubol D, Platt KB, Halbur PG, Torremorell M, Harris DL. 2004. The effect of a killed porcine reproductive and respiratory syndrome virus (PRRSV) vaccine treatment on virus shedding in previously PRRSV-infected pigs. Vet Microbiol 102:11-18.; 49. Olin MR, Batista L, Lee J, Xioa Z, Dee SA, Murtaugh MP, Pijoan C, Molitor TW. Gamma-delta lymphocyte response to porcine reproductive and respiratory syndrome virus. Virus Immunity (in press).; 50. Opriessnig T, Pallarés FJ, Nilubol D, Vincent AL, Thacker EL, Vaughn EM, Roof M, Halbur PG. Genomic homology of ORF 5 gene sequence between modified live vaccines and porcine reproductive and respiratory syndrome virus challenge isolates is not predictive of vaccine efficacy. J Swine Health Prod (in press).; 51. Otake S, Dee SA, Moon RD, Rossow KD, Trincado C, Pijoan C. 2004. Studies on the carriage and transmission of porcine reproductive and respiratory syndrome virus in an individual housefly (Musca domestica, Linnaeus). Vet Rec 154:80-85.; 52. Ropp SL, Fang Y, Mahlum Wees CE, Nelson EA, Rossow KD, Bien M., Arndt B, Preszler S, Steen P, Christopher-Hennings J, Collins JE, Benfield DA, Faaberg KS. 2004. Characterization of emerging European-like PRRSV isolates in the United States. J Virol 78:3684-3703.; 53. Ropp SL, Mahlum Wees CE, Fang Y, Nelson EA, Rossow KD, Bien M, Arndt B, Preszler S, Steen P, Christopher-Hennings J, Collins JE, Benfield DA, Faaberg KS. 2004. Characterization of emerging European-like PRRSV isolates in the United States. J Virol 78:3684-3703.; 54. Rowland RRR, Lawson S, Rossow K, Benfield DA. 2003. 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