Duration: 10/01/1999 to 09/30/2004

Administrative Advisor(s):

NIFA Reps:

Non-Technical Summary

Statement of Issues and Justification

The United States swine industry is at a crucial economic crossroads. Increased production costs and declining prices have severely impacted many swine operations in recent months. In addition to market and price factors, pork producers are continually hampered with infectious disease problems that continue to increase production costs due to morbidity, mortality and treatment. Viral diseases of swine (transmissible gastroenteritis virus, pseudorabies, rotavirus, swine influenza, porcine reproductive and respiratory syndrome virus and the emerging circovirus) represent the greatest challenges in the control and prevention of infectious diseases in this species. When pseudorabies eradication programs were implemented in the 1980s, it was thought that one of the most devastating diseases in the U.S. swine industry would shortly be eradicated. Although the process has taken longer than expected, the U.S. is on a timetable to eradicate this disease in the next few years. However, our confidence in reducing losses in swine due to viral diseases was shaken with the appearance in 1987 of porcine reproductive and respiratory syndrome (PRRS), which is now the most important viral disease of swine in North America.

Twelve years have elapsed since the first report of PRRS in 1987 (Keffaber, 1989) and more than seven years since the discovery of the virus that causes PRRS (Wensvoort et al, 1991; Collins et al, 1992; Benfield et al, 1992). Despite the resolution of the etiology of PRRS, many aspects of the basic mechanisms of pathogenesis, immunity and protection against this virus remain unknown. Research on PRRSV in the U.S. has also been uncoordinated, isolated and influenced by aggressive industry interests that succeed in dividing and isolating efforts of individual research and academic groups through implementation of exclusive confidentiality agreements, proprietary and patent rights between individuals and institutions. Hopefully, this regional project proposal represents an alternative to this trend. This project involves scientists from eleven AES (IL, IA, KS, MI, MN, MO, NC, ND, NE, OH and SD) conducting collaborative and prioritized research on various aspects of PRRSV.

JUSTIFICATION: Since the initial description of "mystery swine disease", now known as porcine reproductive and respiratory syndrome (Keffaber, 1989), this viral disease has become the most economically devastating disease in the U.S. and other swine- producing countries in the world. Reproductive failure and respiratory disease are the principal outcomes following PRRSV infection (reviewed in Benfield et al, 1999; Rossow, 1998; Zimmerman et al, 1998). PRRS appears prior to breeding and continues to exert its negative economic impact through farrowing, nursery, and finishing units. Respiratory disease in infected neonates is severe, frequently resulting in acute respiratory distress and death. The reproductive form of PRRS appears following the infection of pregnant gilts or sows and results in abortions, stillbirths and weak, live-born pigs. Mortality in weak, live-born pigs and pre-weaned pigs can reach 100% within 3 weeks after infection. PRRSV can also retard growth and lengthen the time to market weight in grow/finishing pigs. Estimated monetary losses due to PRRSV outbreaks range from $100 to $510 per inventoried female (Hoefling, 1992; Poison et al, 1994) or $25,000-$127,500 and $100,000-$510,000 in a 250 and 1,000 sow herd, respectively. Dee and Joo (1993) estimated that PRRSV infection delayed marketability for 14-30 days at an additional cost of $7.50 to $15.00/pig marketed. Since PRRSV is a worldwide problem its overall economic impact is considerable.

Even after 12 years of study the name "Mystery Disease" is still an appropriate description of PRRS. Once the viral etiology was initially established in 1991 by investigators in Europe (Wensvoort et al, 1991) and later in the United States (Collins et al, 1992; Benfield et al, 1992) research progressed significantly towards finding a resolution to this problem. The release of the first live-attenuated commercial vaccine in June 1994 was hailed as a significant achievement and a hoped for solution for an industry that was experiencing acute and chronic infections of PRRSV in breeding, weaned and finishing pigs. However, the recent outbreak of severe "abortion storms" in southeastern Iowa in 1996-1997 were also believed to be caused by a "new and perhaps different strain of PRRS virus" (Epperson and Holler, 1997; Halbur and Bush, 1997). This new outbreak of PRRS and the continuing endemic persistence of PRRSV in some herds frustrated producers and veterinarians and sent many looking for alternative but untested methods to control this disease.

There is a renewed demand within the swine industry for the development of management practices that will prevent, control or eliminate PRRSV, including improved methods of prophylaxis and immunoprophylaxis, as well as, methods to detect acute and persistently infected swine within large herds. Meeting these producer demands requires us to increase our knowledge of the PRRSV including: 1) molecular characterization of translated and untranslated regions of the genome, 2) understanding interactions between host and virus that lead to pathogenesis, persistence, and immunity, 3) understanding of the epidemiology of PRRS and 4) development of better methods to diagnose and detect the disease and the virus.

PRRS is a complicated disease and a difficult virus to characterize and understand. We realize the best hope for the control, and elimination of PRRS is collaborative, multidisciplinary research on various aspects of the disease. Thus, there is a need to formalize cooperation and collaboration between AES sites to maximize these research efforts. The accelerated elimination of pseudorabies in most North Central and other states offers an opportunity to shift resources and personnel from a disease that is controlled and almost eliminated to one that has emerged as the most economically important viral disease of swine. The Regional Project format is the prototype for the organization of cooperative projects between AESs. The eleven stations in this proposal have a history of collaboration or are planning future collaborative efforts to answer questions related to basic mechanisms of the pathogenesis and control of PRRS. The investigators from each participating AES stations have many years of experience in PRRS research and have published widely on the topic.

Related, Current and Previous Work

Much of the early work on PRRSV originated with AES scientists in the North Central Region (IA, IL, IN, MN, NE and SD). Since the discovery of the PRRSV in the U.S. (joint effort by MN and SD), research has focused primarily on seven areas of investigation: 1) molecular characterization of the PRRSV, especially the genome and proteins; 2) replication of PRRSV in macrophages and simian cell lines; 3) pathogenesis of the respiratory and reproductive forms of the disease; 4) viral persistence in herds and individual pigs; 5) mechanism of immunity to PRRSV infections and vaccines; 6) diagnosis of PRRSV infections and methods to detect the virus; and 7) epidemiology and management protocols to control PRRSV in the field. These areas of research, even though distinct, are interrelated. For example, understanding the epidemiology of PRRSV requires an ability to diagnose the disease and detect the virus. Diagnosis of infection cannot be made without understanding the nature of viral proteins and viral genome sequences. Viral proteins also play an important role in the pathogenesis of PRRSV and the immune response of pigs to PRRSV infection. The design and use of new vaccines cannot be made without knowledge of how the virus interacts with its host to cause disease and induce a protective immune response.

The molecular basis for PRRSV virulence. The gene organization of PRRSV consists of a short leader segment followed by 8 open reading frames. ORFsIa and Ib cover three-fourths of the 15-kb genome and code for the RNA polymerase and replicase proteins. The remaining ORFs, 2-7, are overlapping and code for structural proteins. On the 3' end of the genome is a short untranslated region followed by a poly-A tail. During replication a nested set of subgenomic mRNA's is produced, each contains a common leader and poly-A tail. Each subgenomic mRNA codes for a single individual viral protein (Plagemann, 1996). Excluding the genomic RNA, there are a total of 6 subgenomic mRNAs produced during PRRSV replication. Recently, Nelson et al (1999) reported at least 2 ORF7 transcripts produced during infection. These different ORFs reflect the use of a different leader-body splice site upstream from the ORF7 start codon.

The principal structural proteins of the PRRS virion are the nucleocapsid (N), matrix (M), and envelop (E) proteins (Meulenberg et al, 1995). The N protein (ORF7) forms the nucleocapsid core, which packages the RNA genome inside the virion. Outside the nucleocapsid is the integral M protein (ORF6). The E glycoprotein, also known as GP5, is translated from ORF5 and forms the major protein on the viral surface. Presumably, GP5 forms the principal interaction with the viral receptor on MARC-145 cells and macrophages (Conzelmann et al, 1993; Morozov et al, 1995) and is the principal target of neutralizing antibody (Gonin et al., 1999). The remaining glycoproteins, GP2, GP3, and GP4 are coded for by ORFs2, 3, and 4, respectively. GP2 is a minor surface protein and probably forms an association with GP5. The functions for GP3 and GP4 are less well understood, but may form additional structural components of the virion.

Investigators at IA, KS, MN, ND, NE and SD are elucidating the molecular basis for virulence. One approach is the molecular comparison of virulent and attenuated forms of the virus. SD and NE have prepared attenuated PRRSV isolates by serial passage on MARC-145 cells. One property of these attenuated strains is poor growth on porcine alveolar macrophages, possibly the result of change in the ORF5 sequence that codes for GP5, which interacts with the cell receptor for PRRSV on macrophages (Kauers et al., 1998). Sequence analysis of GP5 in virulent and avirulent isolates has failed to identify a significant mutation. In fact the attenuated phenotype has yet to be mapped to a single structural gene. Additional studies with mutant PRRSV and infectious clones, proposed by MN, NE and SD in this project, are necessary to elucidate the roles of viral structural and non-structural proteins, and untranslated regions of the PRRSV genome in the pathogenesis. Host factors may also interact with the PRRSV genome and regulate replication of PRRSV as indicated by recent experiments by investigators at KS (Majhdi and Kapil, 1999). There is virtually no knowledge on the role of nonstructural PRRSV proteins in the pathogenesis of disease. Such proteins play very important roles in the virulence of other viruses, such as poliovirus. Currently, investigators at SD and ND are examining the role of the viral proteases and viral polymerases coded by the ORF1 gene. It is crucial to understand the role of these enzymes in the pathogenesis of PRRSV, because these proteins are potential targets for pharmaceutical or other intervention to moderate virus replication and disease.

MN and NE have sequenced the entire genome of the prototype VR-2332 (Murtaugh et al, 1995; Murtaugh and Faaberg, personal communication, 1999) and another North American strain (NE 16244B, Allende et al, 1999), respectively. SD is completing the sequence of a third strain of PRRSV, SDSU-23983. These sequence databanks are or will be available in the public domain and are necessary to understand the molecular basis of PRRSV pathogenesis. Efforts are also underway at IA, KS, MN, NE and SD to sequence field isolates of "acute isolates of PRRSV" from recent outbreaks of abortion storms in southeastern Iowa. NE has completed the sequence on the IA 142 strain of acute PRRS (Allende et al, unpublished). Current efforts are also focused on producing "infectious clones" of the entire PRRSV genome for mutagenesis studies and the possible production of "marker vaccines". While an infectious clone has been produced to the European LV isolate of PRRSV (Meulenberg et al, 1998) and industries in the U.S. are pursuing this goal; most of these clones are proprietary and not available to the PRRS research community. The infectious clone to the LV isolate also has limited use to understanding the North American isolates of PRRSV, because of the genotypic differences between European and North American isolates.

See attached "Related, Current" for additional information.

Objectives

1. Define the molecular and cellular mechanisms of pathogenesis of respiratory and reproductive syndromes caused by PRRSV.
   
2. Determine the mechanism(s) and consequences of viral persistence.
   
3. Characterize the different components of the immune response during acute and persistent infection and the implications of this response in the pathogenesis and diagnosis of disease.
   
4. Develop improved methods for the diagnosis of PRRSV clinical disease and detection of virus and/or antibodies to PRRSV.
   
5. 
   

Methods

Objective 1. Define molecular and cellular mechanisms of pathogenesis of respiratory and reproductive disease. There is little knowledge on the biological mechanisms used by the PRRSV to induce respiratory or reproductive disease. This information is essential to: 1) understand how PRRSV causes disease and lesions, 2) the design of molecular probes, which can be used to assess the virulence of a particular field isolate, and 3) the development of new vaccines and therapies. The first goal is to compare virulent and attenuated strains of PRRSV to identify genes and nucleotide sequences involved in virulence. The second goal is to develop a cDNA copy of the PRRSV genome (U.S. strain) that would produce infectious virus when transfected into cells (an infectious clone). The information gained from these two studies will make it possible to study the pathogenesis of PRRSV using site directed mutagenesis. Another important benefit of the infectious clone is the modification of strains for use as vaccines, especially marker vaccines for differential serologic tests. Such vaccines and diagnostic assays will be crucial to eradication programs in the future. Little is also known as to how PRRSV causes abortion. Does this occur directly by virus replication in the fetus or indirectly through the effects of virus-induced alterations at the fetal-maternal interface. To answer this question, participating stations will identify sites of PRRSV replication at the maternal-fetal interface; determine when fetuses become infected in utero; and determine if cytokines are involved in fetal alterations caused by PRRSV.

a. Identification of molecular components of PRRSV responsible for disease. The identification of specific nucleotide and amino acid changes, which correlate with virulence of a particular PRRSV isolate will be made by comparing nucleotide sequences of wild type and attenuated strains of PRRSV. SD has attenuated several strains of PRRSV isolates (VR-2332; SDSU-23983: SDSU 2367; SDSU 26663, a neurovirulent strain and an atypical PRRSV) by extensive serial passage on MARC-145 cells. The initial benchmark for attenuation is the decreased ability to replicate in cultures of alveolar macrophages and gnotobiotic pigs. KS, MN, NE and SD will continue to identify field strains of PRRSV with unusual virulence patterns in an attempt to identify isolates with altered sequences. KS, NE and SD will also perform sequence analysis of the untranslated regions and structural and non-structural genes. NE will contribute sequences obtained from attenuated (commercial vaccines) and vimlent strains that correspond to the non-structural protein-coding region of ORF la and 1b regions. Each one of the sequenced strains will be made available to KS and SD. The results between the two stations will be done to identify candidate mutations that might be important in the regulation of virulence. Comparisons between stations are important, because variations in PRRSV may be geographically isolated and not detected in all swine herds.

It is also anticipated that there is a genetic basis for resistance to PRRS in pigs. Investigators at IA have previously reported that susceptibility to PRRSV of various breeds is different (Halbur et al, 1998). However, studies documenting host cell molecular changes associated with infection are not completely nor accurately linked to mechanisms of resistance/susceptibility or to resistance phenotypes. Identifying and characterizing a specific disease resistance gene is prerequisite for breeding and transgenic approaches aimed at delineating mechanisms of pathogenesis and for genetic manipulation of host disease resistance. Investigators at MN will identify and map porcine alveolar macrophage expressed sequence tags (ESTs), which are associated with virus-infected cells. This will involve cloning and sequencing macrophage cDNAs that demonstrate altered expression in macrophages infected by PRRSV. A mRNA fingerprinting approach to directly capture porcine ESTs reflective of macrophage status during PRRSV infection will do this. These ESTs will specifically reflect infectious disease events and provide starting points for biologic dissection of PRRSV pathogenesis. These experiments represent necessary first steps in describing the underlying genetic and biologic mechanisms by which macrophages respond to and are affected by PRRSV and expression of relevant PRRSV-regulated genes can be screened across animals, which show differential PRRSV susceptibility.

b. Construction of an infectious clone. This work will be done in collaboration between MN, SD and NE. MN has already sequenced the complete genome ofVR2332, the prototype strain of PRRSV (Nelson et al, 1999) and is constructing an infectious clone using this isolate. NE has already sequenced the complete genome of a highly pathogenic strain NEB 16442B (Allende et al., 1999) and this sequence is available in GenBank (Accession No.AF 046869). NE will make available the sequenced viral strain, primers and initial clones (n= 14) encompassing the entire genome, for work to be initiated in SD. The actual construction of the clone will be made in a manner similar to the construction of aLV infectious clone, reported by Meulenberg et al. (1998). Infectious clones allow us to convert the entire RNA of the PRRSV into a cDNA that can eventually be used to create mutants in the laboratory. Currently, we can only identify viral mutations and variant PRRS viruses by the expensive and time-consuming process of screening hundreds of isolates to detect changes in virulence or other properties. With the infectious clone, the process of creating mutant viruses to characterize genes for virulence, host specificity, and markers is more economical and more accurate. Development of such clones also allows for construction of either marker vaccines or vaccines lacking virulent traits.

c. Define molecular, cellular, and virological mechanisms of respiratory and reproductive disease. These studies are performed primarily at IL, MN, NC, NE and SD. The use of attenuated viruses combined with the infectious clone will make it possible to dissect the individual elements of PRRSV pathogenesis. One important goal is the identification of viral protein(s) that interact with the receptor on macrophages (NC, SD). Another is the role that viral and host factors play in pathogenesis (KS, NE, SD) and immunity (IL, KS, NE). Much work has been done on the pathogenesis of the respiratory disease but little is known about the pathogenesis of the reproductive syndrome. The overall design of these experiments is to infect pregnant sows with both virulent and attenuated forms of PRRSV isolates described under la. Techniques, including virus isolation (VI), quantitative PCR, and in situ hybridization will be used to assess virus replication in both maternal and fetal tissues at different times after infection. The effect of PRRSV may not be totally dependent on virus replication, but the ability of virus infection to induce abortagenic cytokines such as TNF and IFN (Raghupathy, 1997). Pathogenesis may also be influenced by the ability of a particular strain to support virus replication in macrophages. SD and NE will conduct the animal experiments and in collaboration with IL, the IFN-y ELISPOT assay will be used to look at cells in both maternal and fetal tissues that produce IFN-y. Other cytokines will be measured using a quantitative RT-PCR technique that will detect cytokine mRNA levels in tissues (Benavides, et al., 1995; Reddy et al, 1998). Identification of the mechanisms of how PRRSV induces reproductive disease will aid in the design of potential pharmaceutical mediators of cytokines or design of better vaccines for use in pregnant animals. At present, there is no method of treatment for pregnant animals infected with PRRSV or at risk for exposure to PRRSV.

d. PRRSV and co-infections with other viruses and agents. Previous investigators have reported that secondary infections with bacteria and viruses are not uncommon in herds with PRRSV infections (reviewed by Rossow, 1998; Zimmerman et al, 1998). Recently, controversy has been stirred as to the interaction of an emergent porcine circovirus and PRRSV (Ellis et al, 1999). Investigators at IA and NE have also reported that prior infection of pigs with PRRSV enhances the pathogenicity of Salmonella choleraesuis in pigs (Wills et al, submitted for publication). Investigators at NC have also reported that pigs infected in utero are more susceptible to Streptococcus suis infections (McCaw et al, 1998). Additional studies at IA indicate that prior infection of pigs with Mycoplasma hyopneumoniae contributes to the severity of PRRSV infections (Thacker et al, 1998). Investigators at OH (collaborative with SD) have determined that co-infection of pigs with porcine respiratory coronavirus and PRRSV may contribute to a more severe pneumonia (Hayes et al, 1998).

Investigators at IA, NC, NE, OH and SD will investigate the interactions between bacteria (IA, NC and NE) and viruses (OH and SD) with the PRRSV in pigs co-infected with multiple agents. IA and NE will continue to examine the interaction between PRRS and Mycoplasma and PRRS and Salmonella. OH and SD will study the interactions between PRRSV and porcine respiratory coronavirus (OH) and the porcine circovirus (OH and SD). These studies will be done in conventional, SPF and gnotobiotic pigs using protocols described previously in publications from these stations. Investigators at NC will use the in utero infection model developed by SD to investigate the role of in utero infection upon piglet susceptibility to secondary bacterial infections. Specifically, pigs will be infected in utero by inoculating gilts at 90 days gestation. Piglets farrowed by these gilts will be reared in feeding cages on artificial milk. Conventional methods of flow cytometry and RT-PCR for cytokines will be used to determine if fetal infection results in immune alterations. Other agents, to develop better methods of diagnosing co-infections and to determine the mechanism via PRRSV enhances infections by other agents will use results from these co-infection studies to determine if PRRSV does enhance infections.

Objective 2. Determine the mechanism and consequences of viral persistence. Persistent infection is a property shared by all arteriviruses. It is clear that the presence of a long-term low-level infection is responsible for the uncontrolled spread of PRRSV and is the major impediment to successful control of the disease. Preliminary studies at IA, MN, NE and SD have identified morphologic sites of PPRS virus replication during acute and persistent infection and yet the mechanism for persistence remains largely unknown. The participating stations will define how long pigs remain infected, how long pigs can shed PRRSV and identify anatomical sites of PRRSV persistence.

a. Sites of virus replication during persistence. Preliminary studies at IA, MN, NE and SD indicate that PRRSV replication during acute infection is initially present in all organs and tissues. However, as the pig recovers, replication is restricted to specific anatomical sites, including lymph nodes, tonsil, and testes (MN, NE and SD; Benfield et al, 1997; Christopher-Hennings et al, 1994; 1995; Shin et al, 1997; Sur et al., 1997). KS, NE and SD will continue to determine the importance of virus replication in the reproductive tract of the male pig and the effects of PRRSV infection on sperm production. IA, MN, NE and SD will also determine potential sites of persistence in gilts and sows. MN and SD will determine if there are potentially new sites of persistence (immune-privileged sites such as the eye). It is essential that sites of virus replication in persistent infections be identified to determine if these sites represent a biologically significant portal for virus shedding to susceptible contacts.

b. Determine the length of virus replication and viral shedding. Research in this area will focus on how long PRRSV replicates in pigs. KS, MN, NE and SD will focus on the length of virus replication in adult pigs, whereas SD will focus on virus replication in pigs exposed as neonates. Virus isolation, PCR, quantitative PCR, in situ hybridization and use of contact sentinel pigs will be used to detect persistence and shedding of PRRSV.MN and SD will study persistence in breeding age pigs and pigs exposed to PRRSV in utero and in the nursery. MN will examine the prevalence of PRRSV persistent infection over a statistically significant sample of breeding swine, SD and MN will both evaluate the use of antemortem samples (tonsil biopsies) to traditional postmortem samples for the diagnosis of persistently infected animals. SD will continue to determine the mechanism of persistence in pigs infected in utero. MN will determine what stressors or risk factors induce increased replication and transmission from infected swine to contact controls. In conclusion, this information is necessary to enhance PRRS control measures and assess the feasibility of PRRS eradication. Data from each animal model system will be compared to determine potential sites for viral persistence in young and adult pigs.

c. Identify mutants that arise during persistent infection. One explanation for why the PRRSV persists in pigs is that populations of persistent viruses arise following selection of escape variants by neutralizing antibody. RT-PCR will be used to amplify PRRSV from tissues of persistently infected pigs. SD will make available to NE and other stations all primer sets that have been used successfully to amplify ORFs2-7. NE will make available primers to amplify ORFs la and 1b. Each of these stations and IA will determine the role of RNA quasispecies (populations of virus selected during the course of acute infection or persistence) in viral persistence. Molecular evolution of the virus through animal passages will be assessed by sequencing, monoclonal antibody analysis and serological assays to determine genotypic and phenotypic changes of the virus, rates and locations of changes, and biological significance of changes. A mathematical modeling will be applied to predicting the PRRSV evolution.

Objective 3. To characterize the different components of the immune response during acute and persistent infection, its implications in diagnostics and pathogenesis. The unsolved mystery of PRRSV is the inability of pigs to initiate an appropriate immune repose that can eliminate infection. Both humoral and cell-mediated immunity has been documented during infection, but the roles of each in the protection of pigs are not known (Bautista and Molitor, 1997). NE and IL will examine both the qualitative and quantitative components of anti-PRRSV immune responses during infection. SD will provide samples to NE and IL to determine if the humoral and cell-mediated immune responses of acute and persistent infections are different.

a. The role of neutralizing antibody and identification of neutralizing epitopes. As indicated earlier, PRRSV may escape neutralizing antibody through mutation of viral proteins. SD and NE will pursue studies to characterize the specificity and quantity of neutralizing antibody during infection.

b. Cell-mediated immunity. The laboratories at NE and IL have good records of collaborative studies in the study of the pathogenesis, virulence and immunology of both the Pseudorabies Virus (PRV) and now PRRSV. These two laboratories are currently cooperating in the characterization of the host response to PRRSV infections. With recent joint funding from NPPC, both laboratories conducted a study to ascertain the immunogenic efficacy of the current PRRSV vaccines against the strains involved in the new virulent forms of the disease (Osorio et al, 1998). IL has also developed methods to study swine T-cell responses to PRRSV by quantitative ELISPOT (IFN-y) on populations of swine PBMNCs as originally described for Pseudorabies Virus (PRV) (Zuckermann et al, 1998).

Based on these results IL and NE will assess T-cell reactivity as a clinical protection correlate and extend studies to assess the IFN-y response in single, purified cells, using the complete array of markers that IL has available for positive or negative enrichment of the T- and NK cells from PBMNC suspensions. At the same time NE plans to focus on the significance of the humoral neutralizing immune response that occurs during late the post-infection period and determine if this late serum-neutralizing response is a correlate of clinical protection. This will be done by passive transfer of PRRSV-neutralizing immunoglobulin to serological naive pregnant gilts that will be challenged with PRRSV in late gestation.

NC, NE and SD will clone the ORF 2-7 into expression vectors to evaluate the humoral and CMI response to various viral proteins. The primary objective of these studies is to identify the viral proteins responsible for the induction of a protective immune response in swine. Investigators at NC will examine a variety of different vaccine strategies using PRRSV cDNA.

Objective 4. Develop improved methods for the diagnosis of PRRSV clinical disease and detection of virus and/or antibodies to PRRSV. Currently, diagnosis of PRRSV infection relies on clinical history; lesions observed by histopathology; viral antigens detected by immuno fluorescence or immunohistochemistry; detection of antibodies by fluorescent antibody, neutralization or ELISA tests; isolation of virus; and the demonstration of viral RNA by PCR (reviewed by Benfield et al, 1999; Zimmerman et al, 1998). IA and SD are collaborating on development of a monoclonal antibody panel to differentiate various field isolates of PRRSV. These stations will attempt to establish a serological marker system using these monoclonal antibodies as an epidemiological tool for profiling field isolates. These monoclonal antibodies can also be used to identify epitopes associated with ADE and virus neutralization.

KS and MN are currently comparing virus isolation and RT-PCR for sensitivity and specificity for the diagnosis of PRRSV in swine herds and plan to continue this collaboration in the present project. Several states (IA, NE, SD and NC) participated in a recent study to standardize the virus neutralization assay for antibody to PRRSV and these results with a recommended standard operating procedure for PRRSV viral neutralizing assays has been proposed for approval by the American Association of Veterinary Laboratory Diagnosticians.

Measurement of Progress and Results

Outputs

Outcomes or Projected Impacts

• <b>General outcomes.</B> PRRS continues to be a source of frustration for pork producers and veterinarians. The proposed collaborative effort between the eleven AES stations will for the first time provides a blueprint for a coordinated effort of PRRSV research. These collaborative efforts will contribute to the design of improvements in management protocols for the control of PRRS, improved diagnostics, and more efficient and efficacious vaccines. The long-range goals are to determine the means by which PRRSV is perpetuated in domestic swine, how it causes disease, and how it can be eliminated from commercial swine herds. Information on pathogenesis and immunobiology of PRRSV is scant. Variation of PRRSV that apparently exists among field isolates is a significant obstacle to development of efficacious vaccine(s). Persistence of PRRSV has become a significant issue for trade business, artificial insemination, and PRRS control. All study projects described above will address these important issues. Better utilization and interpretation of PRRS infection and virulence status will aid in improved diagnostics and better control recommendations.
• <B>b. Technical transfer and outreach.</B> The individual states in this proposal have an excellent history of transferring information to the field for use by clientele. As an example, SD maintains a Website (www.vetsci.sdstate.edu/prrs/prrs.htm) for PRRSV. Scientists from IA, IN and MN recently released the 1998 PRRS Compendium to pork producers with funding from the National Pork Producers Council. This Compendium is the most up-to-date summary of PRRS literature. Scientists from SD, IA and MN also reviewed this Compendium. To continue in this position, SD will continue to maintain the PRRSV Website with input contributed to this Website on a quarterly basis by the states participating in this project. This Website contains information for the education of producers and swine veterinarians. The long range plan is to transfer the maintenance of the Website to the NPPC with the Regional PRRS Committee as the oversight committee to screen and summarize information to be placed on this Website. The Regional PRRS Committee will publish an up-to-date listing of papers on a yearly basis on this Website.
• The Compendium will continue to be produced by the current authors and NPPC with reviews provided by members of the Regional PRRS committee and the NPPC. Research findings will also be reported at regional, national and international scientific, producer and other meetings and in popular and refereed publications. The stations initiating this project have a proven record in releasing scientific information with field applications in a timely matter. We recognize that unraveling the mysteries of PRRS V will require new experimental models that don't necessarily fit established paradigms in animal virology. Therefore, we are presented with the opportunity to contribute fundamentally new information into understanding viral-host relationships.

Milestones

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Projected Participation

View Appendix E: Participation

Outreach Plan

Organization/Governance

The technical committee shall consist of one technical representative from each cooperating state AES or other agency as appointed or otherwise designated by the respective organization; an administrative advisor appointed by the Association of North Central Experiment Station Directors, and a representative of the Cooperative State Research, Education and Extension Service (CSREES).

The executive committee shall consist of a chairperson, vice chairperson/secretary of the technical committee and three additional members elected annually from the appointed technical representatives. The chairperson, vice-chairperson/secretary will serve two-year terms with the vice-chairperson assuming the chair after two years. A committee of individuals from cooperating states will be appointed by the chairperson to oversee the outreach activities and the Website.

Meetings will be held annually at that time and place mutually agreed upon by the technical committee, or as designated by the executive committee with the approval of the administrative advisor. The vice-chairperson /secretary will record the minutes of the annual meeting for distribution to the Administrative Advisor and appropriate individuals.

The chairperson will prepare the annual report summarized from material supplied by technical committee members) from each participating station or agency. The administrative advisor will approve the final report.

Coordination states are identified for the cooperative procedures within each objective. The executive committee is responsible for an overall coordination of the project. Such coordination will occur continuously throughout the project period. The annual meeting of the technical committee will serve as a monitoring session for coordination.

Literature Cited

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Attachments

Land Grant Participating States/Institutions

IA, IL, MN, MO, NC, ND, NE, SD, VA

Non Land Grant Participating States/Institutions

Iowa State University - College of Vet Med