NC_OLD229: Porcine Reproductive And Respiratory Syndrome (PRRS): Mechanisms Of Disease And Methods For The Detection, Protection And Elimination of the

(Multistate Research Project)

Status: Inactive/Terminating

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SAES-422 Reports

Annual/Termination Reports:

[12/06/2002] [02/03/2004]

Date of Annual Report: 12/06/2002

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Annual Meeting Dates: 11/09/2002 - 11/09/2002
Period the Report Covers: 11/01/2001 - 11/01/2002

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Accomplishments

Abbreviated. Objective 1. MN, with the Danish Veterinary Institute, produced an infectious clone of strain VR-2332. Cytopathic effect (CPE) was seen only after several passages in MARC145 cells. MO, focusing on different potential pitfalls as compared to MN, cloned isolate NADC8. Transfections gave viral growth after 2-3 blind passages. Transfection supernatants injected into naove pigs yielded weakly positive serum, but none of the pigs seroconverted. ND and SD, with Guelph University (Canada), generated a clone of SD23983 that was not infectious. Clones were exchanged and are being used to generate chimeras in an effort to create an infectious clone. Nonstructural protein (nsp)-specific sera were generated, and obtained from Leiden Univ, the Netherlands, for detection of early viral nonstructural proteins to aid in troubleshooting. An equine arteritis virus (EAV) infectious clone was provided by Leiden Univ as a positive control. SD and ND showed that field strains adapted to grow to high titers in MARC-145 cells were markedly reduced for growth in macrophages. Sequencing of MARC-145-adapted and nonadapted strains is underway to identify attenuation-related mutations. ND is using EAV as a surrogate to develop assays to compare replicative properties of more- and less-virulent PRRSV strains. Advancements were made to quantify transcript accumulation, transcript stability, replicase subunit accumulation, and replicase subunit stability. KA, with Guelph University, showed that nucleocapsid (N)-eGFP fusion protein localizes to the nucleolus. Individual functional domains were also identified for a cryptic nuclear localization signal (NLS), NLS-1, at amino acid 10, a legitimate NLS, NLS-2, at amino acid 41, a cytoplasmic localization domain adjacent to NLS-2, and a potential nuclear export signal (NES) at amino acid 106. The results suggest that intracellular trafficking of N involves nuclear import, export and cytoplasmic retention signal sequences. KA reported that amino acids 34-53 of N bind the 5-leader of PRRSV. The RNA-binding sequence is conserved and is unique to PRRSV. KA also reported interactions of cellular proteins from nonpermissive BHK-21 and permissive MARC145 cells with the positive strand of the 3-untranslated region (UTR) and ORF 7 RNA. Virus titer was reduced by 98% by a monoclonal antibody against PRRSV binding proteins of 210, 80, 55, and 30 kDa, indicating a possible receptor complex for PRRSV. CD151, a tetraspan family member, was identified by KA as binding PRRSV 3-RNA. It made BHK-21 cells permissive to PRRSV, suggesting that the CD151 is a susceptibility factor for PRRSV infection. To explore possible mechanisms for the emergence of acute PRRS in 1996-97, the ORF5 gene of eight acute PRRSV isolates from herds in Iowa and North Carolina was sequenced (VA). They shared 87-97% nucleotide sequence identities with North American PRRSV isolates and vaccines. Isolate 98-37120-2 from a non-vaccinated herd in Iowa was very close to Ingelvac PRRS MLV. The other seven isolates were more related to earlier PRRSV isolates and PrimePac vaccine. The data do not fully support the hypothesis that acute PRRS is due to reversion of vaccines to a pathogenic phenotype. IA and VA collaborated on comparison of recent PRRSV field isolates to Ingelvac PRRS MLV vaccine, and its parent strain, VR2332. The results indicated that vaccine-like isolate 98-38803 is a derivative of Ingelvac PRRS MLV and that the isolate is pneumovirulent. Low virulent isolates of PRRSV and porcine respiratory coronavirus together enhance respiratory disease and lesions (IA). IA and VA also showed that circovirus-2 infection increases the severity of PRRSV-induced interstitial pneumonia and that circovirus-2, but not PRRSV, induces pathology characteristic of postweaning multisystemic wasting syndrome (PMWS). Objective 2. Mechanisms of transmission that facilitate PRRSV area spread and persistence in the environment were studied by MN under conditions that replicated commercial swine management practices and building design. Airborne transmission occurred across an airspace of 2 meters, but did not occur from within the facility to swine housed 1 meter outside and directly in line with an exhaust fan. Routing exhaust air into a housing unit containing naive pigs also failed to produce infection or seroconversion. Neither Mallard ducks housed inside a facility with infected pigs, nor experimentally infected Mallard and Rouen ducks, seroconverted or become viremic (MN, SD, IA, NE). Mechanical transmission of PRRSV by mosquitoes and houseflies occurred when they were fed on infected pigs. Virus was retained in the midgut for 6-12 hr. Interruption of feeding, followed by exposure to naive pigs, resulted in transmission and infection in recipient pig. Fomites, including packed snow, also were demonstrated to mediate transmission of PRRSV into biosecure facilities. These findings demonstrate that PRRSV can be disseminated across considerable distances in a disconnected fashion in nonfreezing and freezing weather. Viral persistence in pigs was studied by IA, NE, SD, MS, and IL. Animals on commercial farms were persistently infected and re-infected, and multiple viral types coexisted on farms (IL). The contribution of viral evolution to persistence in animals was studied by IA and MN. Multiple variants in ORF 5, but not ORFs 1b (replicase) and 7, were present concurrently in every pig. Most nucleotide changes in ORF5 resulted in substitution of amino acid residues. Virus mutation may not play a role in persistence, since overall genetic change was and there was no evidence of antigenic change (IA). PRRSV was detected in the tonsil of 5 of 8 boars that were negative in serum, semen and PBMC for 2-3 weeks (SD), but in 1 boar at 88 days post-inoculation, PRRSV could not be found by VI or PCR in any of 22 tissues, serum or semen. Objective 3. To determine if multiple PRRSV exposure induced a state of nonresponsiveness in breeding age swine, pigs were infected with field virus following a 6/60 protocol widely used for sow vaccination (NC). 20% of 55 pigs returned to IDEXX ELISA seronegative status following six homologous wild-type virus infections, even though all pigs had initially seroconverted. Neutralizing antibody was detected in all animals throughout the study. 12 months after initial exposure, animals were infected with homolous (SD 23983) or heterologous (Powell) PRRSV. PRRSV was detected in some animals by PCR, suggesting that they were not fully protected. Only modest anamnestic ELISA and SN antibody responses were observed after homologous virus challenge, but marked increases occurred after heterologous challenge. Several lines of evidence suggest that the immune response to PRRSV is weak. MN and IL showed that the cytokine IL-12 enhanced interferon (IFN) gamma responses of lymphocytes to PRRSV, and the mucosal adjuvant, cholera toxin, induced a robust antibody response to GP5. Further studies by IL and NE showed that co-delivery of IL-12 or porcine IFN alpha cDNA with PRRS MLV vaccine increased the primary PRRS-virus specific IFN gamma T cell response. Addition of IL-12 to a killed vaccine (PRRomise, Intervet) modestly increased IFN gamma response and antibody titers (IA). IL also showed that most pigs develop low or no titers of virus-neutralizing antibodies. A positive correlation was observed between the frequency of PRRS virus-specific IFN gamma secreting cells and the number of live-born pigs in immune-challenged sows (IL). In vaccinated sows at a commercial facility, only animals with a frequency of >150 PRRS virus-specific IFN gamma secreting cells per million PBMC did not abort following a natural outbreak of PRRS. The significance of the IFN gamma response is not fully understood, since there is a large variation in the response of individual pigs (IL and MN). To evaluate the protective immune effect of structural proteins, pigs were immunized with Powell strain recombinant ORFs 2-5 (outer membrane proteins) and matrix protein (M) expressed in insect cells (NC). Vaccination with any of the recombinant proteins reduced levels of infectious virus, prevented fever, and prevented behavioral depression. Strong DTH reactions occurred against ORFs 4 and 5, the immunogens that reduced viral load most substantially. NE and IA identified an immunodominant, nonneutralizing epitope in GP5, and a different, neutralizing but non-immunodominant epitope that highly conserved among isolates. The nonneutralizing epitope may act as a decoy, eliciting most of the antibodies directed to GP5 and delaying the induction of neutralizing antibody. NE, IL, and SD collaborated on the construction and immunogenicity evaluation of Mycobacterium bovis BCG expressing GP5 and M proteins. Pigs developed specific immune response against the viral proteins, and 3 of 5 animals developed neutralizing antibodies. An IFN gamma response was not developed against the viral proteins. Following virulent challenge, immunized pigs showed shortened fever, lower viremia, and reduced virus in bronchial lymph nodes, consistent with protection. KA, with Thomas Jefferson University, identified putative genetic suppressor elements (GSEs) in the PRRSV genome that may render cells permanently resistant to virus infection. Four sequences were identified; one encodes an antisense RNA matching the 5 end of ORF1b, and the others encode sense RNA matching sequences in nsp 11, ORF 2 and ORF 6. Objective 4. SD and MN developed a blocking N ELISA. Sensitivity was 97.3% and specificity was 100%. CV was 4.12%. 147 of 4142 serum samples from PRRSV negative farms were unexpectedly positive by the IDEXX ELISA (SD, MN, IA). 100% of the 147 samples were negative by IFA and 97% were negative by blocking ELISA. Thus, blocking ELISA is an alternative to IFA for determining the PRRSV serostatus of individual animals. IA, SD, MN, KA, and NE determined that the rate of suspected-false- IDEXX-positive reactors is 0.5-2%. As IDEXX recently introduced a new version of PRRS ELISA, its diagnostic performance needs to be evaluated. Serodiagnosis of European-genotype PRRSV in the US was improved by SD. European-like PRRSV isolates were grown for improved IFA and virus neutralization assays. 60 monoclonal antibodies recognized Lelystad virus-like isolates. One monoclonal antibody against N protein recognized the US European-genotype PRRSV isolates but not the Lelystad virus or other European isolates. Phylogenetic analysis of the structural protein sequences indicated that these isolates are typical European genotypes. Some of the unique characteristics of these European-like isolates can have a significant impact on diagnostic approaches to detecting PRRSV in US herds. Current PRRS diagnostic tests were evaluated for detecting PRRSV infection in fetuses. PCR showed the best performance. Submission of whole litters and more than one litter is critical for accurate diagnosis as distribution of the virus among fetuses is not uniform (SD). Quantitative, real-time PCR assays were developed for the detection of PRRSV by SD and MN. Probes that differentiate US and European PRRSV field and vaccine strains were made (SD). A heteroduplex mobility assay for rapid differentiation of vaccine-like viruses from field viruses was developed and compared to sequencing (VA, IA). The assay was shown to effectively detect and differentiate field isolates from vaccine strains. Isolates with 2% sequence divergence from the vaccine were all determined to be wild-type. A porcine respiratory disease complex (PRDC) microarray is being developed to simultaneously diagnose PRRSV, influenza A, circovirus 2, Pasteurella multocida, Mycoplasma hyopneumoniae, Streptococcus suis, Actinobacillus pleuropneumoniae, Bordetella bronchiseptica, Haemophilus parasuis, Leptospira species, and porcine parvovirus (MN).

Publications

Does not include abstracts, proceedings, or lay publications. Bastos RG, Dellagostin O, Barletta R, Doster A, Nelson E, Lopez O, Osorio FA. 2002. Construction and immunological evaluation of M. bovis BCG expressing GP5 and M protein of porcine reproductive respiratory syndrome virus, Vaccine (In Press) Batista L, Dee SA, Rossow KD, Deen J, Pijoan C. 2002. An assessment of PRRV persistence and shedding in a large population of breeding age female swine. Can J Vet Res 66:196-200. Bautista EM, Faaberg, KS, MacGruder ED. 2002. Cloning and expression of PRRSV ORF1b and characterization of NTPase activity. Virology 298:258-270. Benson JE, Yaeger MJ, Christopher-Hennings J, Lager K, Yoon K-J. 2002. A comparison of virus isolation, immunohistochemistry, fetal serology, and reverse transcriptase-polymerase chain reaction for the diagnosis of porcine reproductive and respiratory syndrome in the fetus. J Vet Diagn Invest 14:8-14. Bierk MD, Dee SA, Rossow KD, Collins JE, Otake S, Molitor TW. 2001. Transmission of PRRS virus from persistently infected sows to contact controls. Can J Vet Res 65: 261-266. Boettcher TB, Thacker BJ, Halbur PG, Waters WR, Nutsch R, Thacker EL. 2002. Vaccine efficacy and immune response to Mycoplasma hyopneumoniae challenge in pigs vaccinated against porcine reproductive and respiratory syndrome virus and M. hyopneumoniae. J Swine Hlth Prod 10:259-264. Cancel-Tirado SM, Yoon K-J. 2002. Antibody dependent enhancement of virus infection and disease. Viral Immunol. (in press) Chang CC, Yoon KJ, Zimmerman JJ, Harmon KM, Dixon PM, Dvorak CMT, Murtaugh MP. 2002. Evolution of porcine reproductive and respiratory syndrome (PRRS) virus during sequential passages in pigs. J Virol. 76:4750-4763. Christopher-Hennings J, Faaberg KS, Mengeling WL, Murtaugh MP, Nelson EA, Roof MB, Vaughn EM, Yoon K-J, Zimmerman JJ. 2002. Porcine reproductive and respiratory syndrome (PRRS) diagnostics: Interpretation and limitations. J Swine Hlth Prod. 10:213-218. Christopher-Hennings J, Holler LD, Benfield DA, Nelson EA. 2001. Detection and duration of porcine reproductive and respiratory syndrome virus in semen, serum, peripheral blood mononuclear cells and tissues from Yorkshire, Hampshire and Landrace boars. J Vet Diagn Invest. 13:133-142. Cuatero L, Dee SA, Deen J, Ruiz A, and Pijoan C. 2002. Association between clinical signs and PRRSV viremia in nursery pigs under field conditions. Journal of Swine Hlthd Prod. 10:119-122. Daginakatte GC, Kapil S. 2001. Fine mapping of the RNA binding domains of the porcine reproductive and respiratory syndrome virus nucleocapsid protein. Adv Expt Med Biol. 494:547-552. Dee SA, Deen J. 2001. Establishment of a PRRS ELISA-negative boar population using previously exposed boars. Vet Rec 149: 678-680. Dee SA, Deen J, Rossow KD, Mahlum C, Otake S, Joo HS, C Pijoan. 2002. Mechanical transmission of porcine reproductive and respiratory syndrome virus throughout a coordinated sequence of events during cold weather. Can J Vet Res (In press). Dee SA, Torremorell M, Rossow K, Mahlum C, Otake S, and Faaberg K. 2001. Identification of genetically diverse sequences (ORF 5) of PRRSV in a swineherd. Can J Vet Res. 65:254-260. Fairbanks K, Chase C, Benfield DA. 2002. Tonsil biopsies and polymerase chain reaction assay for detection of breeding age gilts persistently infected with porcine reproductive and respiratory syndrome virus. J Swine Hlth Prod. 10:1-2. Feng W, Laster SM, Tompkins M, Brown TT, Xu J-S, Gomez W, Benfield D, McCaw MB. 2002. Thymocyte and peripheral blood T lymphocyte subpopulation changes in piglets following in utero infection with porcine reproductive and respiratory syndrome virus. Virology 302:363-372. Foss DL, Zilliox MJ, Meier W, Zuckermann F, Murtaugh MP. 2002. Adjuvant danger signals increase the immune response to porcine reproductive and respiratory syndrome virus. Viral Immunol. 15:557-566. Halbur PG, Pallares FJ, Rathje JA, Evans R, Hagemoser WA, Paul PS, Meng XJ. 2002. Effects of different U.S. isolates of porcine reproductive and respiratory syndrome virus (PRRSV) on blood and bone marrow parameters of experimentally infected pigs. Vet Rec 151:344-348. Hermann JR, Honeyman MS, Zimmerman JJ, Thacker BJ, Holden PJ, Chang CC. 2002. Effect of dietary Echinacea purpurea on viremia and performance in porcine reproductive and respiratory syndrome virus-infected nursery pigs. J An Sci (submitted). Horter D, Chang CC, Pogranichnyy R, Zimmerman J, Yoon KJ. 2001. Persistence of porcine reproductive and respiratory syndrome in pigs. Adv Exp Med Biol. 494:91-94. Horter DC, Pogranichniy RM, Chang C-C, Evans RB, Yoon K-J, Zimmerman JJ. 2002. Characterization of the carrier state in porcine reproductive and respiratory syndrome virus infection. Vet Microbiol. 86:213-228. Hurd HS, Bush EJ, Losinger W, Corso B, Zimmerman JJ, Wills R, Swenson S, Pyburn D, Yeske P, Burkgren T. 2001. Outbreaks of porcine reproductive failure: report on a collaborative field investigation. J Swine Hlth Prod. 9:103-108. Key KF, Haqshenas G, Guenette D, Swenson SL, Toth TE, Meng XJ. 2001. Genetic characterization of the major envelope gene of acute porcine reproductive and respiratory syndrome virus isolates. Vet Micro. 83:249-263. Key KF, Guenette DK, Yoon KJ, Halbur PG, Vaughn EM, Roof M, Toth TE, Meng XJ. 2002. Identification and differentiation of vaccine-like isolates of porcine reproductive and respiratory syndrome virus from field isolates using a heteroduplex mobility assay. J Clin Micro. In press. Kim TS, Benfield DA, Rowland RRR. 2002. Porcine reproductive and respiratory syndrome virus-induced cell death exhibits features consistent with a non-typical form of apoptosis. Virus Res. 85:133-140. Kleiboeker SB, Lehman JR, Fangman TJ. 2002. Concurrent use of reverse transcription-polymerase chain reaction testing of oropharyngeal scrapings and paired serological testing for detection of porcine reproductive and respiratory syndrome virus in sows. J Swine Hlth Prod. 10:251-258. Majhdi F, Kapil S. 2001. Interactions of cellular proteins with the positive strand of 3‘ untranslated RNA and the nucleoprotein gene of porcine reproductive and respiratory syndrome virus. Adv Expt Med Biol. 494:633-639. Meier WA, Galeota J, OsorioFA, Husmann RJ, Schnitzlein W, Zuckermann FA. 2003. Gradual development of the interferon-gamma and antibody responses of swine to porcine reproductive and respiratory syndrome virus. Virology In press. Murtaugh MP, Yuan S, Nelson EA, Faaberg KS. 2002. Genetic interaction between porcine reproductive and respiratory syndrome virus (PRRSV) strains in cell culture and in animals. J Swine Hlth Prod. 10:15-21. Murtaugh MP, Foss DL. 2002. Inflammatory cytokines and antigen presenting cell activation. Vet Immunol Immunopathol. 87:109-122. Murtaugh MP, Xiao Z, Zuckermann F. 2002. Immunological responses of swine to porcine reproductive and respiratory syndrome virus infection. Viral Immunol. 15:533-547. Murtaugh MP, Yaun S, Nelson EA, Faaberg KS. 2002. Genetic interaction between PRRSV strains in cell culture and in animals. J Swine Hlth Prod. 10:15-21. Murtaugh MP, Xiao Z, Rutherford MS, Zuckermann F. 2002. Immunology. The Porcine Reproductive and Respiratory Syndrome (Porcine Arterivirus) Compendium (2nd Edition). Section 5.3. 28 pp. National Pork Board, Clive, Iowa. In press. Nielsen HS, Liu GP, Nielsen J, Oleksiewicz MB, Bxtner 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. In press. Opriessnig T, Halbur PG, Yoon K-J, Pogranichniy RM, Harmon KM, Evans R, Key KF, Pallares FJ, Thomas P, Meng X-J. 2002. Comparative pathogenicity of a modified live PRRSV vaccine (Ingelvac PRRS MLV), the parent strain of the vaccine (ATCC VR2332), ATCC VR2385, and two recent field isolates of PRRSV. J Virol. 76:11837-11844. Osorio FA, Galeota JA, Nelson E, Brodersen B, Doster A, Wills R, Zuckermann F, Laegreid WW. 2002. Passive transfer of virus -specific antibodies confers protection against reproductive failure induced by a virulent strain of porcine reproductive and respiratory syndrome virus and establishes sterilizing immunity. Virology 302:9-20. Ostrowski M, Galeota JA, Jar AM, Platt KB, Osorio FA, Lopez OJ. 2002 .Identification of neutralizing and non-neutralizing epitopes in the porcine reproductive and respiratory syndrome virus GP5 ectodomain. J Virol. 76:4241-50. Otake S, Dee SA, Jacobson L, Torremorell M, and Pijoan C. 2002. Evaluation of aerosol transmission of porcine reproductive and respiratory syndrome virus under field conditions. Vet Rec. 150:804-808. Otake S, Dee SA, Rossow KD, Deen J, Joo HS, Molitor TW, Pijoan C. 2002. Transmission of porcine reproductive and respiratory syndrome virus by fomites (boots and coveralls). J Swine Hlth Prod. 10: 59-65. Otake S, Dee SA, Rossow KD, Deen J, Joo HS, Molitor TW, Pijoan C. 2002. Transmission of PRRSV by needles. Vet Rec. 150:114-115. Otake S, Dee SA, Rossow KD, Moon RD, Pijoan C. 2002. Mechanical transmission of porcine reproductive and respiratory syndrome virus by mosquitoes, Aedes vexans (Meigen). Can J Vet Res. 66:191-195. Otake S, Dee SA, Rossow KD, Moon RD, Trincado C, Pijoan C. 2003. Transmission of porcine reproductive and respiratory syndrome virus by houseflies (Linneaus). Vet Rec. In press. Pallares FJ, Halbur PG, Opriessnig T, Sorden SD, Villar D, Janke BH, Yaeger MJ, Larson DJ, Schwartz KJ, Yoon KJ, Hoffman LJ. 2002. Porcine circovirus type 2 (PCV-2) co-infections in U.S. field cases of postweaning multisystemic wasting syndrome (PMWS). J Vet Diagn Invest. 14:515-519. Pallares FJ, Halbur PG, Opriessnig T, Vaughn EM, Paul PS. 2002. Effects of dual infection of SPF pigs with low virulent isolates of porcine respiratory coronavirus (PRCV) and porcine reproductive and respiratory syndrome virus (PRRSV). Anaporc Cientifico 2:8-13. Plagemann PGW, Rowland RRR, FaabergKS. 2002. The primary neutralization epitope of porcine reproductive and respiratory syndrome virus strain VR-2332 is located in the middle of the GP5 ectodomain. Arch Virol. 147:2327-2347. Pogranichnyy RM, Yoon K-J, Harm PA, Sorden S, Daniels M. 2002. Case-control study on the association of porcine circovirus type 2 and other swine viruses in postweaning multisystemic wasting syndrome. J Vet Diagn Invest. 14:449-456. Rowland R, Schneider P, Fang Y, Wootton S, Yoo D, Benfield DA. 2003. Peptide domains involved in the trafficking of the porcine reproductive and respiratory syndrome virus nucleocapsid protein. Virology. In press. Wagstrom EA, Chang CC, Yoon KJ, Zimmerman JJ. 2001. Shedding of porcine reproductive and respiratory syndrome virus in mammary gland secretions of sows. Am J Vet Res. 62:1876-1880. Wills RW, Doster AR, Galeota JA, Sur JH, Osorio FA. 2003. Duration of infection and proportion of pigs persistently infected with porcine reproductive and respiratory syndrome virus (PRRSV). J Clin Micro. In press. Wills RW, Doster AR, Osorio FA. 2002. Transmission of porcine reproductive and respiratory syndrome virus (PRRSV) to age-matched sentinel pigs. J Swine Hlth Prod. 10:161-165. Wootton SK, Rowland RR, Yoo D. 2002. Phosphorylation of the porcine reproductive and respiratory syndrome virus (PRRSV) nucleocapsid protein. J Virol. 76:10569-10576. Yoon K-J, Christopher-Hennings J, Nelson EA. 2002. Diagnosis. In: Porcine Reproductive and Respiratory Syndrome (Porcine Arterivirus) Compendium (2nd Edition). National Pork Board, Clive, Iowa. In press. Yoon K-J, Stevenson G. 2002. Porcine reproductive and respiratory syndrome: Diagnosis. In: Morilla A, Yoon KJ, Zimmerman JJ (eds). Trends in Emerging Viral Infections of Swine, pp 347-354. Iowa State University Press, Ames, Iowa Yoon K-J. 2002. Porcine reproductive and respiratory syndrome: Virology. In: Morilla A, Yoon KJ, Zimmerman JJ (eds). Trends in Emerging Viral Infections of Swine, pp 339-346. Iowa State University Press, Ames, Iowa Yoon K-J. 2002. Virology. In: The Porcine Reproductive and Respiratory Syndrome (Porcine Arterivirus) Compendium (2nd edition). Zimmerman JJ (ed). National Pork Board, Des Moines Iowa (in press). Zimmerman J. 2002. Epidemiology. In: The Porcine Reproductive and Respiratory Syndrome (Porcine Arterivirus) Compendium (2nd edition). Zimmerman JJ (ed). National Pork Board, Des Moines Iowa (in press). Zimmerman J. 2002. A short history of porcine reproductive and respiratory syndrome. In: The Porcine Reproductive and Respiratory Syndrome (Porcine Arterivirus) Compendium (2nd edition). Zimmerman JJ (ed). National Pork Board, Des Moines Iowa (in press). Zimmerman JJ. 2002. Porcine reproductive and respiratory syndrome virus epidemiology. In: Morilla A, Yoon K-J, Zimmerman J (eds). Trends in Emerging Viral Infections of Swine. Iowa State Press, Ames, Iowa, pp. 331-337.

Impact Statements

An infectious clone will provide both a direct method for studying the role of viral genes in the pathogenesis of PRRSV as well as the development of a genetically-engineered marker vaccine.
Transmission studies that confirm previous observations of limited airborne and avian spread of PRRSV, combined with demonstrations of fomite and insect-born spread of virus to biosecure facilities, address producer concerns about the inability to effectively prevent PRRSV introduction to noninfected herds by showing that current practices may not be sufficient.
Studies of genetic variation during persistent infection of animals shows that PRRSV persistence is not dependent on immune evasion or neutralization escape. It increases the likelihood that persistence in animals is due, at least in part, to a weak immune response.
Antibody and T lymphocyte responses are both important for an effective immune response to PRRSV. More specific immune assays that measure functionally relevant responses to defined antigens are expected to identify improved correlates of protective immunity to acute and persistent infection. These findings will facilitate development of more efficacious vaccines.
The blocking ELISA to resolve false-positive serology directly addresses a critical producer concern. Reagent development for serological diagnosis of European-like field cases further enhances the accuracy and reliability of PRRS serology.
Direct, rapid and sensitive PCR tests for infection status that differentiate US and European genotypes and distinguish between field and vaccine isolates will provide producers and veterinarians will greatly improved tools to assess the infection or protection status of herds and individual breeding animals.
Plans for the group are to develop a public, international workshop on PRRS advances, publish literature reviews built around the four programmatic objectives, and to investigate development of an international scientific training program for PRRS research.

Date of Annual Report: 02/03/2004

Report Information

Annual Meeting Dates: 11/07/2003 - 11/08/2003
Period the Report Covers: 11/01/2002 - 11/01/2003

Participants

Participants Attachment:

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Brief Summary of Minutes

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Accomplishments

1. Define molecular and cellular mechanisms of pathogenesis of respiratory and reproductive syndromes caused by PRRSV. Infectious clones of PRRSV were produced by SD in collaboration with Dr. Dongwan Yoo at Guelph University, Ontario, Canada and also shared with KA. A full-length clone of SD23983 containing a consensus 5end based on Genbank sequences of North American PRRSV strains is currently being tested. The infectious clone of VR2332 reported by Neilsen et al. was generously provided to SD and MO by MN. At MO, two rescue attempts with the VR-2332 clone both resulted in successful recovery of fully infectious virus with cytopathic effect and positive fluorescent-antibody staining indistinguishable from that of other PRRSV isolates. An infectious clone of PRRSV was constructed by NE from a highly abortifacient strain. New 184 field isolates at MN have regions of radical dissimilarity all through the genome. SD and MN characterized European-like PRRSV isolates identified in the U.S. SD and KA focused on heterogeneity in the Nsp2 gene and a preliminary evaluation of phylogeny of these isolates. The ORF 1 of a European-like PRRSV isolate, SD 01-08, is organized the same as LV. The principal difference was a 17 aa deletion in Nsp2 of SD 01-08 that is not consistently observed. Evolutionary relationships among the European, North American and European-like isolates showed evidence for nucleotide diversity, including several deletions in the Nsp2 region. The European-like PRRSV isolates in the U.S. appear to be from a single Lelystad-like isolate. KA and SD determined that the ratio of 2b to N in virion preparations was estimated to be approximately 20 N protein molecules for each 2b molecule. Thus 2b is a minor component of the PRRSV virion. KA and Guelph University determined that a single nuclear localization signal (NLS) is involved in the transport of N from the cytoplasm and into nucleus. VA and IA collaborated on molecular analyses and biological characterizations of PRRSV field reisolates of vaccine and determined that the vaccine-like isolate 98-38803 induces microscopic pneumonia lesions similar in type to, but different in severity and time of onset from, those observed with virulent strains VR2385 and the parent strain of the vaccine. Quasispecies diversity was analyzed in PRRSV in tonsils from 2 chronically infected farms in IL. All animals in the study harbored multiple, distinct PRRSV variants at both the nucleic acid and amino acid levels. The results indicate that PRRSV exists during natural infection as quasispecies distributions of related genotypes, but that positive natural selection for immune evasiveness in ORF5 does not appear to contribute to maintaining this diversity. NE and IA conducted studies that indicated genetic variation in susceptibility to PRRSV. Future research with tissues collected will determine which genes are expressed differently in pigs with resistant and susceptible responses to PRRSV. 2. Determine the mechanism(s) and consequences of viral persistence. NC developed and tested a serologic monitoring method to estimate the point-in-time portion of animals that are resistant to PRRSV, the portion that are susceptible, and the portion of animals that are infective. SD and MN examined the role of lymphoid and non-lymphoid tissue in acute and persistent infections of PRRSV. PRRSV disseminated to most tissues and lymph nodes in 2 d and was detected out to 70 dpi in various tissues. Lymphoid tissues play a prominent role as primary and persistent sites for PRRS viral replication. MN detected PRRSV in flies in baited jug traps up to 1.66 km from an experimentally infected herd. Hence, transmission and persisttence of PRRSV in swine herds appears to involve flies and mosquitoes. Transport vehicles were also shown to effectively transmit PRRSV, but washing, disinfecting and drying eliminated PRRSV. Drying appeared to be an important component of a transport vehicle sanitation program for PRRSV biosecurity. Aerosolized PRRSV was transported 150 meters in a straight tube model and was infectious to susceptible pigs (MN). Attempts to infect mallard ducks with PRRSV via direct inoculation were not successful. 3. To characterize the different components of the immune response during acute and persistent infection and its implications in diagnostics and pathogenesis. KA determined that the thymus is the principal site of virus replication in the fetus. IFN gamma and TNF were induced in lung and lymph nodes. IL and USDA-BARC reported an IFN gamma response to PRRSV after 5 weeks. A significant up-regulation of TNF indicated the involvement of innate immunity to PRRSV starting early in the infection, yet there was limited up-regulation of IFN alpha. Co-administration of IFN alpha caused faster returns to pre-vaccination levels for IL-6, IL-8 and IL-10 after 4 weeks. Co-administration of IFN alpha enhanced the IFN gamma response to vaccine. NE and IA produced chimeric mouse X pig anti-PRRSV antibodies that reacted with GP5 in ELISA and Western blot. MN, NC, and IA collaborated to examine humoral immune responses to structural and nonstructural (nsp) proteins. PRRSV nsp 1, 2, and 4 were cloned. Direct ELISA to nsp 1 revealed that peak levels of antibody exceed anti-PRRSV nucleocapsid (N), and the levels were maintained for at least 120 days. Antibodies to nsp 4 appeared at low levels for a short time. MN and IA collaborated to report that PRRSV-specific T cell responses were transient and varied substantially among animals. Viral loads decreased 1,000-fold in persistent infections, with tonsil being the primary site of persistence. A weak CMI response appears to contribute to prolonged PRRSV infection and suggests that PRRSV suppresses T cell recognition of infected macrophages. IL monitored the kinetics of the PRRSV-specific IFN gamma response in PBMC for 9 months. A weak IFN gamma response within a few weeks after vaccination appeared to wane at 10 weeks, then rebounded and, with fluctuation, increased gradually in intensity after a period of months without a booster immunization. IL and NE exposed replacement gilts to a wild-type virus or a heterologous commercial vaccine. A higher IFN gamma response was associated with a lower proportion of stillborn pigs per litter. There was no relationship between humoral or cellular immune response and the recovery of PRRSV from the tonsil. ELISA antibodies were not predictive of any production variables. The use of a heterologous killed PRRS virus vaccine, following exposure to live virus, significantly boosted the cell-mediated immune response. The role of dendritic cells (DC) in PRRSV pathogenesis and persistence was studied by SD and MN. PRRSV grew in pig DC and reduced expression of MHC class I, CD14, CD11b/c, but not MHC II. MN found no change of expression MHC class I, class II or costsimulatory molecules CD80/86. However, infected DC were impaired in antigen presentation. PRRSV appears to suppress DC function. Clinical signs and cellular and humoral immune responses were examined at MN in gilts challenged with heterologous or homologous strains 120 days after primary infection. No clinical signs were observed following homologous challenge. Mild clinical signs of fever, anorexia, and depression were observed in all heterologous challenge groups. Viremia was detected in all previously exposed groups challenged with all heterologous strains only on day 3 pi, and not at days 7 and 14 pi. Following homologous challenge, a vigorous CMI response and a weak humoral response were observed. Following heterologous challenge, weak or no CMI responses and vigorous humoral responses were observed. Development of recombinant PRRSV vaccines using a herpes virus-based amplicon vector system containing the ORF 5 gene of PRRSV 23983 was performed by SD in collaboration with the U of Rochester. ORF 5 protein was detected by western blot using a PRRSV immune serum. IL established a correlation between the two allelic forms of IL-12R?2 and the intensity of the interferon IFN-? response of swine to immunization with a PRRS MLV vaccine. The IL-12R?2 allele represents the first genetic marker in pigs associated with variation in the degree of cell-mediated immune response to a pig virus. MO showed that viral growth on macrophages in the presence of interferon alpha was reduced in a manner dependent of viral isolate. PRRSV also suppressed IFN production in response to infection with TGEV, PRCV, polyI:C and VSV, all known IFN inducers. These results demonstrate that PRRSV field isolates differ both in IFN alpha sensitivity and induction. Furthermore, PRRSV field isolates differ in suppression of IFN induction by other viruses and double-stranded RNA. IA identified anti-PRRSV antibody responses that were enhancing, neutralizing and neither. Identification of the epitopes responsible for enhancement or neutralization may provide the basis for developing efficacious second-generation vaccines. Serological characterization of auto-anti-idiotypic antibodies indicated that the Aab-2s recognized a site within or near the antigen-combining sites of the anti-GP5 antibodies. Aab-2 antibodies may play a role in immunity to PRRSV infection. 4. Develop improved methods for the diagnosis of PRRSV clinical disease and detection of virus and/or antibodies in PRRSV. Real-time rapid PCR detection of PRRSV and vaccine in serum and semen was evaluated at KA and MO. North American and European ORF7 were amplified using a single set of PCR primers and distinguished by distinct melting peaks. Thus, detection of PRRSV RNA in semen is possible. At MO Lelystad strain and US/European PRRSV field isolates were successfully amplified. Multiplex assays incorporating both PRRSV genotypes and SIV also were developed. SD and NE, with Tetracore, Inc., validated a real-time PCR with primers specific for U.S. or European-like PRRSV. 427 semen and serum samples showed 95% agreement with nested RT-PCR. IA developed a multiplex RT-PCR assay for PRRSV, SIV and PCV2 in nasal swabs and lung lavage. VA and IA developed a heteroduplex mobility assay (HMA) for quickly identifying PRRSV isolates similar to vaccines. The HMA results also correlated well with the results of phylogenetic analyses and indicate that HMA could be used as a rapid and efficient method for large-scale screening of potential vaccine-like PRRSV field isolates. SD and MN validated a monoclonal antibody-based blocking ELISA using sera from 686 individual animals of known PRRSV status. Agreement with IDEXX ELISA and IFA for detection of seroconversion was demonstrated. IA also showed that blocking ELISA against nucleocapsid is sensitive and specific. NE, MS and SD determined that antibodies could be raised to PRRSV in ducks and rabbits. An experimental infection of Rouen and Mallard ducks showed no evidence of infection. NC and SD collaborated to evaluate antibody responses in mature swine following repeated exposure to a single isolate of PRRSv by IDEXX 2XR commercial ELISA. All animals developed solidly positive antibody responses initially on HerdChek ELISA, but not on the HerdChek2XR. ELISA antibody levels dropped substantially 4 months after initial seroconversion, even in the face of repeated injections with virulent SD 28983 PRRSV. All animals remained FFN antibody positive at the end of the study. Challenge of the multiply exposed seronegative animals with a heterologous strain of PRRSV resulted in marked anamnestic ELISA and SN antibody responses. IA, SD, and KA investigated PRRS ELISA false positive reactors. Ninety seven of 12,000 animals in PRRS-negative swine herds tested as false-positive by IDEXX ELISA. They were evaluated on 3 alternate ELISA-based serologic assays (developed at ISU, SDSU, and KSU) and were negative on at least one test. No test was 100% reliable. Western blotting did not reveal the cause of false positives.

Publications

Bastos, R.G., O.A. Dellagostin, R.G. Barletta, A.R. Doster, E.A. Nelson and F.A. Osorio. 2002. Construction and immumogenicity of recombinant Mycobacterium bovis BCG expressing GP5 and M protein of porcine reproductive and respiratory syndrome virus. Vaccine 21:21-29. Batista L, Dee S, Molitor T, Olin M, Joo HS, Pijoan C, Murtaugh MP, Xiao Z, Rossow K, Polson D. 2004. Detection of porcine reproductive and respiratory syndrome virus in pigs with low positive or negative ELISA sample-to-positive ratios. Vet Rec. 154:25-26. Batista L, Lwamba H, Pijoan C, Johnson CR, Murtaugh MP. 2004. Genetic diversity of porcine reproductive and respiratory syndrome virus (PRRSV) in two regions of Mexico. In press. Cafruny, WA, QA Jones, TR Haven, NL Zitterkopf, PGW Plagemann and RRR Rowland. 2003. Glucocorticoid regulation of lactate dehydrogenase-elevating virus replication in macrophages, Virus Res, 92:83-87. Cancel-Tirado SM, Yoon K-J. 2003. Antibody dependent enhancement of virus infection and disease. Viral Immunol 16:69-86 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 In press. Dee SA, Deen J, Rossow KD, Eliason R, Mahlum C, Otake S, Joo HS, and Pijoan C. 2003. Mechanical transmission of porcine reproductive and respiratory syndrome virus throughout a coordinated sequence of events during warm weather. Can J Vet Res 67:12-16. Dee SA. 2003. Approaches to prevention, control and eradication of PRRS. National Pork Board PRRS Compendium, 2nd edition, 119-130. Feng, W-H, Laster, SM, Tompkins, M, Brown, TT, Xu, J-S, Gomez, W, Benfield, D, and McCaw, MB. 2002. Thymocyte and peripheral blood T lymphocyte subpopulation changes in piglets following in utero infection with porcine reproductive and respiratory syndrome virus. Virology 302:363-372. Feng, W-H, Tompkins, M. Xu, J., Zhang, H-X., McCaw, MB. 2003. Analysis of constitutive cytokine expression by pigs infected in-utero with porcine reproductive and respiratory syndrome virus. Vet Immuno Immunopath. 94:35-45. Ferrin, N.H., Y. Fang, C.R. Johnson, M.P. Murtaugh, D.D. Polson and E.A. Nelson. 2004. Validation of a blocking ELISA for the detection of antibodies against porcine reproductive and respiratory syndrome virus. Clinical and Diagnostic Laboratory Immunology. In press. Foss, D.L., M.J. Zilliox, W. Meier, F. Zuckermann, and M.P. Murtaugh. 2002. Adjuvant danger signals increase the immune response to porcine reproductive and respiratory syndrome virus. Viral Immunol. 15:557-566. Goldberg, T. L., J. F. Lowe, S. M. Milburn, L. D. Firkins. 2004. Quasispecies diversity of porcine reproductive and respiratory syndrome virus during natural infection. Virology. 317:197-207. 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. 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. Key K, Guenette DK, Yoon K-J, Halbur PG, Toth TE, Meng X-J. 2003. Identification and differentiation of vaccine-like isolates of porcine reproductive and respiratory syndrome virus from field isolates using a heteroduplex mobility assay. J Clin Microbiol 41:2433-2439 Kleiboeker, S.B., Lehman, J.R., and Fangman, T.J. 2002. Concurrent use of reverse transcription-polymerase chain reaction testing of oropharyngeal scrapings and paired serological testing for detection of porcine reproductive and respiratory syndrome virus in sows. J Swine Health Production 10:251-258. Meier WA, Judy Galeota J, Osorio FA, Husmann R, Schnitzlein W, and Zuckermann FA. 2003. Gradual Development of the Interferon__ Response of Swine to Porcine Reproductive and Respiratory Syndrome Virus. Virol. 309:18-31 Murtaugh, M.P. Z. Xiao, and F. Zuckermann. 2002. Immunological responses of swine to porcine reproductive and respiratory syndrome virus infection. Viral Immunol. 15:533-547. Murtaugh, M.P., Z. Xiao, M.S. Rutherford and F. Zuckermann. 2003. Immunology. The PRRS (Porcine Arterivirus) Compendium (2nd Edition). Section 5.3. 28 pp. National Pork Board, Clive, Iowa. Nielsen, H.S., Liu, G.-P., Nielsen, J., Oleksiewicz, M. B., Bxtner, A., Storgaard, T. and K.S. Faaberg. 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. Opriessnig T, Halbur PG, Yoon K-J, Pogranichniy RM, Harmon KM, Evans R, Key KF, Pallares FJ, Thomas P, Meng X-J. 2002. Comparison of molecular and biological characteristics of a modified live PRRSV vaccine (Ingelvac PRRS MLV), the parent strain of the vaccine (ATCC VR2332), ATCC VR2385, and two recent field isolates of PRRSV. J Virol 76:11837-11844. Osorio, F.A., J.A. Galeota, E.A. Nelson, B. Broderson, A. Doster, R. Wills, F. Zuckermann and W.W. Laegreid. 2002. Passive transfer of virus-specific neutralizing antibodies confers protection against reproductive failure induced by a virulent strain of porcine reproductive and respiratory syndrome virus and establishes sterilizing immunity. Virology, 302:9-20. Otake S, Dee SA, Moon RD, Rossow KD, Trincado C and Pijoan C. 2003. Evaluation of mosquitoes, Aedes vexans, as biological vectors of porcine reproductive and respiratory syndrome virus. Can J Vet Res 67:265-270. Otake S, Dee SA, Moon RD, Rossow KD, Trincado C, and Pijoan C. 2003. Transmission of porcine reproductive and respiratory syndrome virus by houseflies (Musca domestica). Vet Rec 152:73-76. Otake S, Dee SA, Moon RD, Rossow KD, Trincado C, Farnham M, and Pijoan C. 2003. Survival of porcine reproductive and respiratory syndrome virus in houseflies . Can J Vet Res 67:198-203. Reilly, C., C. Wang, and M.S. Rutherford, 2003. A method for the normalizing microarrays using the genes that are not differentially expressed. J. Amer. Statistical Assoc., in press. Ropp, S. L., Mahlum Wees, C. E., Fang, Y., Nelson, E. A., Rossow, K. D., Bien, M., Arndt, B., Preszler, S., Steen, P., Christopher-Hennings, J., Collins, J. E., Benfield, D. A., and K. S. Faaberg 2003. Characterization of emerging European-like PRRSV isolates in the United States. J. Virol., accepted for publication. Rowland, R.R.R., S. Lawson, K. Rossow and D. Benfield. 2003. Lymphoid tissue tropism of porcine reproductive and respiratory syndrome virus replication during persistent infection of pigs originally exposed to virus in utero. Vet Microbiol. 96:219-235. Rowland, RRR and D Yoo. 2003. Nucleolar-cytoplasmic shuttling of the PRRSV nucleocapsid protein: a simple case of molecular mimicry or the complex regulation by nuclear import, nucleolar localization and nuclear export signal sequences. Virus Res, 95:23-33. Rowland, RRR, P Schneider, Y Fang, S Wootton, D Yoo, and DA Benfield. 2003. Peptide domains involved in the localization of the porcine reproductive and respiratory syndrome virus nucleocapsid protein to the nucleolus. Virology 316:135-145. Thanawongnuwech, R., Young, T.F., Thacker, B.J., Halbur, P.G., Thacker, E.L. 2003. Interleukin (IL) 10, IL 12, and interferon gamma levels in the respiratory tract following Mycoplasma hyopneumoniae and PRRS infection in pigs. Viral Immunol., 16:357-367, Wills R.W., Doster AR, Galeota JA, Sur JH, Osorio FA. 2003. Duration of infection and proportion of pigspersistently infected with porcine reproductive and respiratory syndrome virus (PRRSV). J Clin Micro 41:58-62. Xiao Z, Batista L, Dee S, Halbur P, Murtaugh MP. 2004. The level of virus-specific T-cell and macrophage recruitment in porcine reproductive and respiratory syndrome virus infection in pigs is independent of the virus load. J. Virol. In press. Yoo D, SK Wootton, G Li, C Song and RRR Rowland. 2003. Colocalization and interaction of the porcine arterivirus nucleocapsid protein with the small nucleolar RNA-associated protein fibrillarin. J. Virol. 77:12173-12183. Yoon K-J, Christopher-Hennings J, Nelson E. 2003. Diagnosis of PRRS. In: J.J. Zimmerman, K.-J. Yoon and E. Neumann (eds). 2003 PRRS Compendium: A reference for pork producers, pp. 55-67. National Pork Board, Des Moines, Iowa. Yoon K-J, Christopher-Hennings J, Nelson E. 2003. Diagnosis. In: J.J. Zimmerman JJ and K.-J. Yoon (eds). 2003 PRRS Compendium: A comprehensive reference on PRRS for pork producers, veterinary practitioners, and researchers (2nd edition), pp 59-74. National Pork Board, Des Moines, Iowa. Yoon K-J, Stevenson G. 2002. Porcine reproductive and respiratory syndrome: Diagnosis. In: A. Morilla, K.-J. Yoon and J.J. Zimmerman (eds). Trends in emerging viral infections of swine, pp 347-354. Iowa State University Press, Ames, Iowa Yoon K-J. 2002. Porcine reproductive and respiratory syndrome: Virology. In: A. Morilla, K.-J. Yoon and J.J. Zimmerman (eds). Trends in emerging viral infections of swine, pp 339-346. Iowa State University Press, Ames, Iowa Yoon K-J. 2003. Virology. In: J.J. Zimmerman JJ and K.-J. Yoon (eds). 2003 PRRS Compendium: A comprehensive reference on PRRS for pork producers, veterinary practitioners, and researchers (2nd edition), pp 163-184. National Pork Board, Des Moines, Iowa. Yuan S, Murtaugh MP, Schumann FA, Mickelson D, and Faaberg KS. 2003. Characterization of heteroclite subgenomic RNAs associated with PRRSV infection. Virology. In press. Zhou E-M, Jiang Z, Ameri-Mahabadi M, Zimmerman JJ. 2003. Detection and characterization of auto-anti-idiotypic antibodies specific for Idiotypic antibodies against envelope glycoprotein and matrix protein of porcine reproductive and respiratory syndrome virus. J Virol Methods (in press). Zimmerman J. Epidemiology and ecology. 2003. In: The Porcine Reproductive and Respiratory Syndrome Compendium: A comprehensive reference on PRRS for pork producers, veterinary practitioners, and researchers (2nd edition). Zimmerman JJ, Yoon K-J (eds). National Pork Board, Des Moines Iowa, pp.27-50. Zimmerman J. Historical overview. 2003. In: The Porcine Reproductive and Respiratory Syndrome Compendium: A comprehensive reference on PRRS for pork producers, veterinary practitioners, and researchers (2nd edition). Zimmerman JJ, Yoon K-J (eds). National Pork Board, Des Moines Iowa, pp.1-6. Zimmerman JJ, Yoon K-J (editors). 2003. PRRS Compendium: A comprehensive reference on PRRS for pork producers, veterinary practitioners, and researchers (2nd edition). National Pork Board, Des Moines, Iowa. 294 pages PROCEEDINGS AND ABSTRACTS: Callahan, J.D., J. Christopher-Hennings, T.A. Gay, M.E. Reos, Y.Fang, M. Dammen, A. Wasilk, J. Galeota, F.A. Osorio, M. Torremorell, W.M. Nelson and E.A. Nelson. 2003. Development, validation and commercialization of a real-time RT-PCR assay for the detection of Lelystad, European-like and US Porcine reproductive and respiratory syndrome viruses. 107th Annual Meeting of the American Assoc. of Vet. Lab. Diagnostitions, p. 142. Carlson, D.L., A.N. Wasilk, Y. Fang, S.A.E. Marras, E.A. Nelson and J. Christopher-Hennings. 2002. Detection, differentiation and quantitation of PRRSV strains by real-time RT-PCR with molecular beacons. 83rd Conf. of Research Workers in Animal Disease. Christopher-Hennings, J., J. Callahan, T. Gay, A. Wasilk, Y. Fang, M. Dammen, M. Torremorell, D. Polson, M. Mellencamp, E.A. Nelson and W.M. Nelson. 2003. Validation of a real-time, quantitative PCR assay to detect U.S. and Lelystad/European-like PRRSV in boar semen and serum. 84th Conf. of Research Workers in Animal Disease. Faaberg KS, Murtaugh MP, Mahlum-Wees CE, Johnson JE, Dwan C. 2003. Evolution of ORF5 in North America over 15 years. Proc. IXth International Symposium of Nidoviruses. 4.4, Egmond aan Zee, The Netherlands. Faaberg, K. S., Murtaugh, M. P., Mahlum-Wees, C. E., Johnson, J. E., and C. Dwan. 2003. PRRSV: Evolution of ORF5 in North America over 15 years. IXth International Symposium on Nidoviruses, Fang, Y., D. Kim, R.R.R. Rowland, S. Ropp, P. Steen, J. Christopher-Hennings and E.A. Nelson. 2003. Heterogeneity in the NSP2 gene of European-like PRRSV isolated in the United States. 84th Conf. of Research Workers in Animal Disease. Fang, Y., W.P. Zhang, R.R.R. Rowland, J. Christopher-Hennings and E.A. Nelson. 2003. Phylogeny of European-like PRRSV isolates in North America. 84th Conf. of Research Workers in Animal Disease. Ferrin, N.H., Y. Fang, D.D. Polson, M. Torremorell, M. Gramer, M.P. Murtaugh, C.R. Johnson, and E.A. Nelson. 2002. Validation of a blocking ELISA for antibodies against PRRSV. 83rd Conf. of Research Workers in Animal Disease. Goldberg, T. L., J. F. Lowe, S. M. Milburn, L. D. Firkins. 2003. Quasispecies diversity of porcine reproductive and respiratory syndrome virus during natural infection. CRWAD 2003 Proceedings. ISU Press. Abst 153. Hadley, L., Xu, J-S., McCaw, M.B., and Laster, S. 2002. Purified proteins from an acute strain of PRRSv provide protection via TH1-type immune response. In: Proceedings of 17th Congress of the International Pig Veterinary Society, Ames, Iowa, USA. Liu, G., Nielsen, H. S., Burkhart, K. M., Roof, M. B., Vaughn, E. M., and K. S. Faaberg. 2003. Porcine Respiratory and Reproductive Syndrome Virus (PRRSV) Infectious Clones Reveal Nucleotides Important for Genome Replication. IXth International Symposium on Nidoviruses, P.6.6, Egmond aan Zee, The Netherlands. Liu, G., Nielsen, H. S., Zhiming, O., and K. S. Faaberg. 2003. Infectivity of porcine respiratory syndrome virus genomic clones. 21th Annual Meeting of the American Society for Virology, W14-5, Davis, CA. Lowe J.F., L.D. Firkins, F. A. Zuckerman, T.L. Goldberg. 2003. Immune response and reproductive performance in swine following known infection with PRRS virus in commercial herds. CRWAD 2003 Proceedings. ISU Press. Abst 144. McCaw, M.B. Roberts, J, Laster, S.M., Hadley, L., and Erickson, G.A. 2003. Characterization of PRRSv antibody and rtPCR responses following repeated exposures and then challenge with homologous wild-type virus. In: Proceedings of 4th International Symposium on Emerging and Re-emerging Pig Diseases, Rome, Italy. pp 81-82. McCaw, M.B., J. Roberts, J.J. Zimmerman, Laster, S., Hadley, L.,G.A. Erickson. 2002. PRRSv ELISA response and PCR viremia following repeated homologous exposures to wild-type virus in adult swine. Conference in Research Workers in Animal Disease. St. Louis, MO. McCaw, M.B., Roberts, J., Laster, S., Hadley, L., and Erickson, G.A. 2002. PRRSv serologic assay limitations following repeated homologous exposures to wild-type virus in adult swine. In: Proceedings of 17th Congress of the International Pig Veterinary Society, 2002, Ames, Iowa, USA. p208. Mondaca-Fernandez E, Morrison RB, Murtaugh MP. 2003. Using GIS and sequencing to assess regional epidemiology of PRRSV. Proc. Allen D. Leman Swine Conf. 30:71-74. Murtaugh MP, Harding J, Faaberg KS. 2003. Genetic variation and biological safety of Ingelvac MLV modified live PRRS vaccine during seven years of continuous use in the field. 4th International Symposium on Emerging and Re-emerging Pig Diseases. Rome. Murtaugh MP, Harding J. 2003. PRRS virus genomics: application to a field investigation. Proc. Allen D. Leman Swine Conf. 30:41-43. Murtaugh MP, Wees CE, Johnson JE, Dwan C,Faaberg KS. 2003. Molecular evolution of porcine reproductive and respiratory syndrome virus (PRRSV) envelope glycoprotein 5. 4th International Symposium on Emerging and Re-emerging Pig Diseases. Abstract 57. Rome. Murtaugh MP, Xiao Z, Johnson CR, Dee SA Batista L. 2003. Porcine immunity to porcine reproductive and respiratory syndrome virus (PRRSV): systemic and local responses in acute and persistent infection. Proc. IXth International Symposium of Nidoviruses Petry DB, Holl JW, Weber J, Doster AR, Osorio FA, and RK Johnson. 2004. Different biological responses of pigs of two genetic populations to PRRSV challenge suggests underlying genetic variation in susceptibility/resistance to PRRSV, Nebraska Swine Report (In press) Roof, M. B., Vaughn, E. M., Burkhart, K. M., and K.S. Faaberg. 2003. Efficacy of modified live virus porcine reproductive and respiratory virus vaccines against heterologous respiratory challenge. 4th International Symposium on Emerging and Re-emerging Pig Diseases: PRRS, PMWS, Swine Influenza. Abstract 15, 45-16, Rome, Italy. Ropp, S.L., Y. Fang, M. Bien, B. Arndt, S. Preszler, P. Steen, J. Christopher-Hennings, D.A. Benfield and E. A. Nelson. 2002. Antigenic and genetic characterization of emerging European-like PRRSV isolates in the United States. 83rd Conf. of Research Workers in Animal Disease. Royaee A, F.A. Zuckermann, R. Husmann, G. Calzada-Nova, W. Schnitzlein, J.K. Lunney. 2003. T cell cytokine response and gene expression profile of porcine lymphoid cells in response to vaccination with PRRS virus. CRWAD 2003 Proceedings. ISU Press. Abst. 115. Thacker E, Kittikoon P, Vincent A, Hipple K, Nilubol D, Yu S, Janke B, Thacker B. 2003. Influence of PRRS virus infection on swine influenza vaccine efficacy. Emerging Disease Conference, Rome, Italy. Vincent A, Thacker B, Halbur P, Rothschild M, Thacker E. 2003. An investigation of susceptibility to porcine reproductive and respiratory syndrome virus between genetically diverse lines of pigs. CRWAD, Chicago, Il Nov 10, 2003. Waldner, D., D. Zeman, A. Kasuske, S. Ropp, B. Arndt, E. Nelson and D. Benfield. 2003. Lymphoid tissues may represent not only sites of persistence but early sites of replication of porcine reproductive and respiratory syndrome (PRRS) virus. 84th Conf. of Research Workers in Animal Disease. Waldner, D., D. Zeman, A. Kasuske, S. Ropp, B. Arndt, E. Nelson, Y. Fang and D. Benfield. 2003. Temporal studies on the replication of PRRS virus in conventional pigs from six hours to 126 days post-inoculation. 107th Annual Meeting of the American Assoc. of Vet. Lab. Diagnostitions, p. 141. Xiao Z, Murtaugh MP, Johnson CR, Batista L, Dee SA. 2003. Immunity to porcine reproductive and respiratory syndrome virus (PRRSV): systemic and local responses in acute and persistent infection. 4th International Symposium on Emerging and Re-emerging Pig Diseases. Rome. Yoon KJ, Chang CC, Zimmerman JJ, Harmon KH, Dixon PH, Murtaugh MP. 2003. Molecular evolution of PRRSV during prolonged in vivo replication. 4th International Symposium on Emerging and Re-emerging Pig Diseases. Rome. Zuckermann, F.A., Schnitzlein, W., Husmann, R., and Calzada-Nova, G. 2003. Association of porcine interleukin-12 receptor beta 2 (IL-12Rb2) gene alleles with the development of high or low interferon-? response to PRRS virus. CRWAD 2003 Proceedings. ISU Press. Abst 114.

Impact Statements

Full-length infectious clones of virulent and attenuated PRRSV strains of North American and European genotypes will be powerful tools for elucidation of the genetic similarities and differences in PRRSV isolates that control phenotypic variation in pathogenesis and immune sensitivity as well as assist the development of a genetically-engineered marker vaccine of PRRSV.
Understanding the genetic basis for host susceptibility to disease, and the role of concurrent infections in pathogenesis will help to develop disease resistant pigs based on genetically desirable traits.
Information of the various routes of PRRSV transmission is essential to protect commercial swine operation from becoming infected.
Knowing that lymphoid tissues play a prominent role as both primary and persistent sites for PRRS viral replication will improve studies of disease pathogenesis and immune protection.
PRRSV Nsp2 may be useful as a marker for diversity within the group of European-like and North American PRRSV isolates.
Understanding the mechanisms that regulate the development of anti-viral protective immunity will lead to the development of more effective vaccines and adjuvants, and assist in developing better methods to monitor immunity.
Knowledge of viral mechanisms of immunosuppression, especially suppression of the innate immune response, will help elucidate improve methods of PRRSV control through vaccination.
Studies with dendritic cells will provide insight into the mechanisms for viral persistence and immune modulation associated with PRRSV, while new vaccine strategies may eventually offer improved methods for the control of PRRS.
Application of real-time, quantitative PCR assay has broad potential for commercial use in boar studs, diagnostic laboratories and other facilities for monitoring PRRSV infection and biosecurity for the detection of U.S and Lelystad or European-like PRRSV.
The blocking ELISA offers an alternative to the IFA for follow-up of suspect PRRS serological results.
The use of cell culture adapted European-like PRRSV isolates of U.S. origin in routine diagnostic IFA and virus neutralization assays allows for improved serological monitoring without the restrictions associated with imported European isolates, such as LV.