Duration: 10/01/2004 to 09/30/2009

Administrative Advisor(s):

NIFA Reps:

Non-Technical Summary

Statement of Issues and Justification

The need as indicated by stakeholders. According to the National Pork Board (NPB), Porcine Reproductive and Respiratory Syndrome (PRRS) is the most economically significant disease facing the industry today, costing U.S. pork producers at least $600 million annually. The NAHMS 2000 report (Part II: Reference of Swine Health & Health Management in the United States, 2000) found that PRRS affected 21.4% of all breeding herd operations and, importantly, 58.3% of operations with greater than 500 sows. Similarly, 16.6% of all grow/finish operations had PRRS during the previous months, including 50.7% of large sites with 10,000 or more pigs. The estimated monetary losses due to PRRS virus outbreaks range from $100 to $510 per inventoried female (209). Thus a small sow or gilt herd of 250 animals would loose approximately $25,000 to $127,500, whereas, a herd of 1000 sows or gilts suffers a monetary loss of $100,000 to $500,000. Losses are not only due to reduced reproduction capacity in gilts or sows, but to other aspects of production. Typically, PRRS appears prior to breeding and continues to exert its negative economic impact through the farrowing, nursery, and finishing phases of production. For example, Dee and Joo (210) estimated that PRRS virus infection delayed marketability for 14 to 30 days at an additional cost of $7.50 to 15.00 per pig marketed.

Swine producers have been extremely frustrated by the economic losses resulting from PRRSV infections and the lack of effective protocols for the control and/or prevention of this disease in herds. In July 2002, the NPB, through the Pork Checkoff Swine Health Committee, recommended an increased sense of urgency among those activities involved in control, elimination, vaccine development, and PRRS basic science research. In June 2003, the NPB, in collaboration with a broad-based stakeholder panel that included university researchers and extension personnel, biopharmaceutical companies, state and federal government agencies, and swine veterinarians, outlined a national initiative with broad objectives to create tools and strategies for the successful management of PRRS on U.S. swine farms (see the June 5, 2003, news release, Pork Checkoff Coordinating National PRRS Initiative, at www.porkboard.org/News/default.asp and the supplemental information therein; also Journal of Swine Health and Production 11(5):253-254). This panel identified at least 11 critical needs for research, technology transfer, and communication of research on PRRS:

1. Understanding the persistently infected pig, specifically, the mechanisms for persistence and testing strategies for identifying persistently infected pigs.

2. Cooperative vaccine development, including new and novel candidate vaccines.

3. Investigation of immune and antiviral therapies.

4. Implementation of PRRS virus typing systems for the purposes of vaccine development and diagnosis.

5. Creation of a national PRRS virus genomic sequence database for the purpose of cataloging old strains and tracking new strains during outbreaks.

6. Design and implementation of a national epidemiologic survey for the purpose of performing risk factor analyses to provide a fundamental understanding of the measures necessary for successful on-farm and regional PRRS elimination programs.

7. Understanding mechanisms of inter-farm virus transmission.

8. Design and implementation of regional PRRS virus elimination demonstration projects.

9. Engagement of international PRRS researchers into problems faced by U.S. producers.

10. Collaboration with researchers of related (non-swine) viruses for the purpose of developing model systems for understanding the disease.

11. Creation of real-time PRRS information/education systems for disseminating updates and information to producers and veterinarians.

These stakeholders' recommendations provide the relevance for this new NC-229 proposal. No other multistate project specifically addresses PRRS. Producers have indicated the need for research that enhances our knowledge to eliminate PRRS as the highest research priority of the NPB and NC-229 has embraced these stakeholder suggestions in the present proposal.

The importance of the work and the consequences of failing to respond. The United States swine industry is at a crucial economic crossroads. Increased production costs and declining prices have severely impacted many swine operations. In addition to market and price factors, pork producers are hampered by infectious disease problems that increase production costs. When the pseudorabies eradication programs were implemented in 1989, it was thought that one of the most devastating diseases in the U.S. swine industry would be quickly eradicated. In fact, our scientific and organizational abilities were affirmed when pseudorabies was declared eliminated from U.S. domestic swine herds on January 14, 2002. However, the expectation of reducing economic losses in swine due to viral diseases was shattered with the appearance in 1987 of PRRS, then known as mystery swine disease (190). Since 1991, when the viral etiology of PRRS was established by investigators in Europe (192) and then in the U.S.,(193) research has progressed towards understanding the disease and the associated virus. 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 PRRS virus. A proportion of producers have reported satisfactory results using modified live vaccines, but several deficiencies in performance have been noted. In particular, virus shedding, transmission, and persistent infection of vaccine virus have been documented in the field and in controlled experiments (25; 193; 211). There are also efficacy issues related to the failure of vaccines to provide protective immunity against heterologous virus isolates and reversion to virulence has been alleged from off-label use (202). These issues have highlighted the adaptability of the virus, the difficulty in achieving progress in control of the disease and the frustration of producers and veterinarians, who cannot prevent, moderate or eliminate the disease. From the perspective of the level of scientific understanding and after 14 years of study, the name Mystery Disease is still an appropriate description of PRRS. An unfortunate consequence of our gaps in knowledge is the increasing use of alternative, but scientifically untested and potentially hazardous methods (such as using live field virus to vaccinate pigs) to control this disease.

There are several reasons why PRRS virus infections are difficult to control. First, mutation of the virus creates strains with unique antigenic profiles that result in poor cross-protective immunity. Second, PRRS virus elicits a rather complicated and unique immune response that subverts the immune system and results in persistently infected swine. Third, PRRSV synergizes with ubiquitous infectious agents of low virulence to produce clinically and economically significant disease syndromes, such as porcine respiratory disease complex (PRDC). Fourth, anecdotal evidence strongly suggests that the virus can efficiently move between farms, even those that utilize rigorous biosecurity and good production practices. Finally, relatively few tools, including effective vaccines and surveillance techniques, are available to producers and veterinarians for managing the disease.

In the final analysis, if ignored and left untreated, PRRS virus becomes entrenched in all phases of a production system, setting the stage for a biological train wreck and an economic catastrophe. Even farms that survive a PRRS outbreak become re-infected despite all best efforts to protect the animals. Failing to solve the PRRS problem jeopardizes foreign trade in swine breeding animals, semen, and pork products; places a secure, nutritious, and wholesome food supply for the U.S. consumer at risk; and continues the downward economic spiral as farmers lose their livelihood.

Continuous changes and refinements in food animal production techniques support the hypothesis that PRRS virus represents the tip of the iceberg of emerging disease threats to the food animal industry. This brings a sense of urgency to this proposed project, as it represents a new animal health paradigm to address future disease threats. Moreover, PRRS is a newly emergent disease, unknown 20 years ago. It is in the same taxonomic order (Nidovirales) as the SARS coronavirus. Currently, PRRS is not pathogenic for human beings. However, three characteristics of the ecology of this virus (its uncertain origin, rapid evolution rate and human-pig contact), increase its potential to become a human pathogen. Thus, perhaps another reason to work towards elimination of this virus.

The technical feasibility of the research. Successful realization of the study objectives will require basic and applied research studies, including functional genomics, immunology, epidemiology, genetics, and molecular biology. Within this framework, NC-229 has the capacity to coordinate ideas and resources, focus on specific problems and projects, and respond immediately to new information related to PRRS virus control and elimination. In working towards these goals, the NC-229 committee directed and coordinated the preparation and submission (July 30, 2003) of a $4 million grant proposal to the USDA National Research Initiative Integrated Program office on the Integrated Control and Elimination of PRRS virus in the U.S. This proposal describes collaborative research, education, and outreach plans via the coordinated efforts of the NPB and the NC-229 multi-state consortium of PRRS researchers, academic institutions, USDA, and private industry. This project has been recommended by the USDA for funding and will provide a financial resource for the NC-229 technical committee to work with the USDA-NRI project director to implement a coordinated multistate and multidisciplinary approach to the elimination of PRRS.

The advantages of a multi-state research effort. The NPB, NC-229 and other swine health experts have concluded that complete elimination of PRRS virus is the only viable long-term strategy for alleviating the economic impact of the virus. Successful elimination of the PRRS virus will not rely on a single technology or solution, but on multiple strategies applied to all levels in the swine production system. Within this framework, NC-229 will continue to build on the capacity it has developed to coordinate ideas and resources, focus on specific problems and projects, and respond quickly to new information related to PRRS virus elimination. While there is much expertise available from single entities, the best hope for the control and elimination of PRRS is a collaborative, multidisciplinary research program that focuses on specific aspects of the disease. The NC-229, composed of personnel from eleven stations (IA, IL, KS, MN, MO, MS, NC, NE, OH, SD and VA), plus USDA-BARC, USDA-NADC, and the University of Guelph (Canada), has a history of productive and collaborative PRRS virus research (see Related, Current, and Previous Work).

Likely impact of successfully completing the work. The greatest impact of the successful conclusion of this research will be a new paradigm for the control of PRRS virus infections. Progress toward this goal will proceed through the successful accomplishment of specific aims and milestones described later in this proposal. A principal milestone is the creation and operation of a virtual university environment where investigators share data and ideas. A second milestone is a regional PRRS demonstration project that will demonstrate new protocols and management techniques for the elimination of PRRS virus from herds. Other important research outcomes include;

1. The discovery and development of methods that prevent establishment of PRRS virus infection on a pig farm and facilitate elimination of ongoing infections.

2. The identification of factors involved in inter-farm transmission and the role of geography and viral genetics in regional spread between area farms.

3. The development and delivery of differential immunodiagnostics capable of determining animal infection status, rapidly identifying virus strains and detecting and differentiating animals exposed to field versus vaccine viruses, including newly developed marker vaccines.

4. The design and implementation of eradication protocols in relevant ecological settings.

5. The development of outreach and educational materials and real time delivery methods that provide biosecurity and compliance information and training to producers, veterinarians, and swine health specialists, including multi-lingual biosecurity manuals and protection protocols. This material will also be readily available through the internet.

Related, Current and Previous Work

A description of previous and current work related to PRRS reflects a history of collaboration between NC-229 institutions. These collaborative efforts were formalized in 1999 with the successful implementation of the first PRRS NC proposal. References 1-155 are representative of the accomplishments of NC-229 institutions during the past four years. A detailed summary of past work is presented in Attachment E.

PRRS was initially described in the 1980s as Mystery Swine Disease and associated with reproductive failure in adult females and respiratory disease in nursing pigs (13; 190; 191; 192). With time, the clinical definition of the syndrome was expanded to include pneumonia in nursery and grow/finish pigs. Clinical signs range from subclinical to severe, depending on the virulence of the PRRS virus isolate, the presence of concurrent (polymicrobial) infections (116), genetic differences in pig susceptibility, environmental/management factors, the level of herd immunity, and other factors that are still poorly defined. The evolution of our understanding of PRRS symptomology reflects increased knowledge combined with the continued genetic evolution of the virus in the field.

The causative agent of PRRS is a recently emerged, single-stranded, positive-sense RNA virus, first isolated and identified by investigators in the Netherlands (Lelystad virus ) (192) and shortly thereafter in the U.S. (193). PRRS virus is a member of the family Arteriviridae in the order Nidovirales (194). In addition to PRRS virus, the arterivirus group consists of lactate dehydrogenase-elevating virus (LDV) of mice, equine arteritis virus (EAV), and simian hemorrhagic fever virus (SHFV) (195). The RNA of arteriviruses is 5capped and 3polyadenylated with two large open-reading frames (ORFs), 1a and 1b, that code for protease and viral replicase proteins followed by ORFs 2-7 at the 3 end of the genome. Viral proteins are translated from a nested 3'-coterminal set of subgenomic mRNAs possessing a common leader derived from the 5end of the genome. These subgenomic mRNAs encode four glycoproteins (GP 2 to 5, encoded by ORFs 2 to 5) and non-glycosylated matrix (M, ORF 6) and nucleocapsid (N, ORF 7) proteins (196). An additional structural protein, called 2b, is translated by a small ORF embedded within ORF 2 (139). The recent development of several infectious PRRS virus clones, including one to strain VR2332 (84) provides a new genetic tool to assist in delineating the role of viral proteins in the genesis of disease and immunity.

PRRS virus transmission and endemic infection. The complex nature of PRRS virus entry and circulation within the production system is illustrated in Figure 1 (Attachment A). Virus can be efficiently introduced into a pig by several routes, including intranasal, intramuscular, oral, and vaginal. Exposure to 10 or fewer PRRS virus particles by intranasal or intramuscular injection results in full-blown infection (148). Once animals are infected, virus is shed in saliva, nasal secretions, urine, semen, mammary secretions in pregnant females, and perhaps feces (193). Dee et al. (33) demonstrated that PRRS virus could be transported on fomites in the field, particularly during the winter. In a similar manner, flies and mosquitoes are capable of mechanical transmission under experimental conditions, but are not biological vectors for the virus (88; 90; 94). Transmission by aerosols has been difficult to prove experimentally and aerosolization of PRRS virus is still poorly characterized at this time (89). Aerosols are hypothesized to play a role in area spread. Because all mechanisms of PRRS virus entry into a herd have yet to be discovered, the problem is being actively investigated by NC-229 participants (see Previous Activities).

PRRS virus is able to efficiently exploit a variety of routes to enter and circulate within a herd (see Figure 1, Attachment A). A central target of PRRS virus control is to either prevent entry of the virus into uninfected breeding herds or to stabilize and eliminate circulation of the virus in infected breeding herds. Virus can enter the breeding herd through contaminated semen, the introduction of acute or persistently infected replacement sows/gilts, virus-contaminated fomites, and perhaps other unknown mechanisms. It is clear that current biosecurity protocols are inadequate to prevent entry of PRRS virus into a herd. In a typical scenario, the introduction of PRRS virus into a PRRS virus-free breeding herd leads to the rapid and stealthy spread of infection by asymptomatic animals. Routine herd screening, e.g., serology, is not useful or adequate because of the delay in the development of detectable levels of antibody; the inability of serology to differentiate antibodies against vaccine, as opposed to field infection; and because some persistently infected animals have low levels of antibodies. Often, the presence of PRRS virus is not apparent until the appearance of aborted, stillborn, and weak-born pigs. PRRS virus is easily transmitted vertically, infecting late gestation fetuses. Pigs that survive in utero infection (congenital PRRS) often become long-term carriers and index sources of this virus.

Congenital PRRS. Unfortunately, the consequences of congenital PRRS virus infections do not end at farrowing. Maternal antibody, delivered to the piglet through colostrum and milk, does not prevent the infection of suckling pigs. The surviving PRRS virus-infected neonates exhibit a severe form of clinical disease with mortality sometimes reaching 100% within three weeks after birth (13). Besides interstitial pneumonia, viral lesions are easily identified in several organ systems (197; 198). After recovery from acute infection, congenitally-infected pigs continue to exhibit signs of infection, but in the form of slower development and growth, increased susceptibility to secondary infections, and persistent infection (13; 102; 160). The complex pathology following exposure to PRRS virus in utero represents a syndrome known as congenital PRRS. The protracted clinical effects of congenital PRRS virus infections are responsible for the highest pre-weaning mortalities and have the greatest economic impact on swine producers. These pigs also tend to become carriers that can transmit virus to naove penmates for an extended period of time (102).

Persistence and endemic infection. The ability of PRRS virus to persist in pigs and the lack of diagnostic methods to accurately identify persistently infected pigs are major factors in our inability to eliminate this virus. Once a herd is infected, PRRS virus becomes endemic and may circulate indefinitely. Investigators have reported isolation of PRRS virus from nursery pigs up to 2.5 years after the initial PRRS outbreak (200). At this time, the mechanism for endemic PRRS virus infections is not fully understood; however, the key components appear to be persistent PRRS virus infection in clinically normal carrier animals, the inability of passive immunity to block congenital infection and the continued introduction of naove replacement pigs into the breeding herd (186).

The mechanisms of persistence with RNA viruses are varied, but generally relate to biological properties that allow the virus to evade host defense mechanisms combined with the capacity to establish a state of persistent replication inside a cell or within an immunoprivileged site (201). The arteriviruses establish a persistent infection in all organs and tissue, but contrary to the accepted paradigm, replicate cytopathogenically in a subpopulation of macrophages (195). PRRS virus has been identified in pigs well over 150 days after infection (132) and perhaps as long as 250 days, i.e., more than the length of time that a pig is present in the typical production system. Therefore, in the context of a production pig, PRRS virus produces a life-long infection. In addition to supporting virus replication for an extended period, persistently infected pigs can efficiently transmit virus for at least 112 days (102). Collectively, these data show that persistent infection occurs regardless of pig age and immune status at the time of infection (14; 16; 99; 132; 158; 161; 163; 189).

Conclusions. The complex pattern of transmission of PRRS virus between swine herds and the ability to establish a subpopulation of asymptomatic carrier animals makes the prevention, control, and elimination of PRRS virus a daunting task. Vaccines do not produce satisfactory levels of protective immunity against many strains of virus. Current serum-based tests do not detect all infected pigs and molecular based alternatives, such as reverse transcriptase-PCR, are not yet cost effective as general screening tools. Finally, if elimination is achieved within an individual herd, re-infection with PRRS virus is almost certain, particularly in regions of high swine density. The above issues have been identified by stakeholders as major obstacles to the control and elimination of PRRS virus (see p. 1 and 2 of this proposal). The Supporting Objectives identified in this proposal address stakeholders needs by implementing a program that rapidly and efficiently develops the technology necessary to eliminate the virus.

Contributions of NC-229 investigators. NC-229 was founded in 1999 as a vehicle to facilitate progress in PRRS virus research and promote collaboration and communication and originally involved the participation of 8 state universities but this number increased to 11: The Ohio State University (D. Benfield), University of Minnesota (M. Murtaugh), Mississippi State University (R. Wills), South Dakota State University (E. Nelson), University of Missouri (S. Kleiboeker), North Carolina State University (M. McCaw), Virginia Tech (X.J. Meng), University of Nebraska (F. Osorio), Kansas State University (R. Rowland), Iowa State University (J. Zimmerman) and University of Illinois (F. Zuckermann). Most recently, representatives from ARS-BARC (J. Lunney), ARS-NADC (K. Lager), University of Guelph (D. Yoo) and NPB (E. Neuman) have been added to the group. In addition, scientists from the Roman L. Hruska U.S. Meat Animal Research Center (MARC) and representatives from a variety of industries and institutions continue to participate in NC-229 activities and meetings. An annual (public) meeting is held immediately prior to the Conference of Research Workers in Animal Diseases (CRWAD) to discuss research findings and plan future collaborative activities.


The NC-229 philosophy and approach to PRRS is to address research problems that cannot be answered through traditional, single-investigator-initiated grants. The complexities of the problems addressed by NC-229 require a multi-state, multi-disciplinary, and multi-investigator research approach. In essence, NC-229 activities are an embodiment of progress made towards understanding and eliminating PRRS. Examples of sustained endeavors by NC-229 participants include:

1. A history of extensive publication in the scientific literature on PRRS. A search of Medline and CAB abstracts for papers published between 1998 and 2003 using only the MESH or CAB Thesaurus terms for PRRS or PRRS virus recovered 613 unique references. Removal of 114 general or applied papers left 499 papers, which includes references from foreign research groups. Of these, 160 (32%) were written by members of NC-229 institutions. PRRS virus papers published between 1999 and 2003 by the NC-229 participants are listed as references 1-155 in the proposal reference list.


2. Submission of a joint NC-229 proposal to the USDA CSREES Initiative for Future Agriculture and Food Systems (IFAFS) in 2001 entitled Molecular Immunology and Immunodiagnostics of Acute and Persistent PRRS requested $4,236,010 for four years. Although the proposal was not funded, the exercise resulted in new interactions and laid the foundation for the USDA-NRI proposal and this project rewrite. Recently, the NC-229 committee directed and coordinated the preparation and submission (July 30, 2003) of a $4 million grant proposal to the USDA National Research Initiative Integrated Program office on the Integrated Control and Elimination of PRRS virus in the U.S. This proposal has been recommended for funding in 2004.


3. Collaboration with the NPB in producer education and other special publications. The PRRS Compendium (ISBN 0-9722877-1-X), published in 2003 by the NPB, includes chapters written by 12 authors from 5 NC-229 institutions. The NC-229 is also coordinating a special publication related to PRRS immunology and immunopathology for a 2004 edition of Veterinary Immunology and Immunopathology (project print publication November 2004).


4. Successful pursuit of competitive grants to support PRRS virus research. Grants and contracts awarded to NC-229 participants for PRRS virus research in years 2000 to 2001 from USDA-NRI, institutional grants (not Hatch or Formula), National Institutes of Health, the National and State Pork Producers Councils, and Private Industry totaled more than $1.2 million. Active grants from USDA-NRIGCP are listed below in Table 1 (Appendix B).

Objectives

1. Implement a virtual laboratory infrastructure through the development and open distribution of resources, materials, protocols, and data among participating researchers. Our goal is to allow all participating stations in this project to have a "level playing field" of information. The implementation of a "virtual laboratory" provides the medium to synergize, coordinate, and optimize intellectual and economic assets by pooling knowledge and laboratory resources of geographically dispersed scientists. Therefore, we will create a virtual laboratory of PRRS protocols, standard operating procedures, reagents banks, virus collections, experimental sample banks, and biological materials. Ideas, data, and future experimental protocols will be shared in real time through a secure electronic network (maintained by NPB) that protects data ownership and confidentiality.
   
2. Achieve biosecurity within herds by preventing the spread of virus within a herd and facilitating its elimination from endemically infected herds. The three tools for preventing PRRS virus infections in herds and enhancing the elimination of the virus are: genetically resistant animals, effective anti-virals and efficacious vaccines. Efforts to achieve Objective 2 will focus on functional genomics of PRRS virus resistance, mechanisms of protective immunity for PRRS virus prevention, evaluation of immune modulators to stimulate and/or enhance antiviral immunity, and agents that target virus replication within the pig.
   
3. Achieve biosecurity among herds by preventing viral spread between sites. Molecular and field data collected through longitudinal and/or cross-sectional surveys will be used to understand PRRS virus virulence, evolution, immunity/cross protection, persistence, and transmission. These results, in combination with studies on the stability of PRRS virus in the environment, will be used to understand and predict the risk of PRRS virus transmission between sites.
   
4. Improve diagnostic assays and create on-farm monitoring systems. There is no single test or combination of tests that can identify all PRRS virus-infected pigs. This shortfall has made PRRS virus elimination nearly impossible. There is a need for the standardization of current diagnostic assays among laboratories, the development of improved assays, and implementation of novel sensor technology to detect virus in the environment or infected animals.
   
5. Develop and test PRRSV virus eradication protocols under various ecological settings. Historically, elimination of swine diseases in the U.S. are best achieved and sustained if done on a regional basis (hog cholera and pseudorabies eradication are two examples). The same principle of regional elimination applies to PRRS. Areas of North America will be targeted to initiate regional eradication of PRRS virus. These areas will be required to have natural barriers that serve as physical borders, a manageable number of farms, cooperative producer groups positioned for long-term presence in the industry, and local veterinarians who support the concept.
   
6. Develop educational outreach tools for disseminating information through established outreach and extension networks to producers, veterinarians, educators, and researchers. <p>The NC-229 committee will collaborate with the NPB to develope educational outreach materials for training swine producers and veterinarians in biosecurity methods and procedures for management and elimination of PRRS. The NPB has an excellent history of transferring scientific discoveries and technology developed by university scientists to producers and consumers. Therefore, in collaboration with NPB and their producer committees, we will develop tools directed at education, outreach and communication.
   
7. Create an information network to ensure rapid and efficient communication of PRRS virus research protocols and results through a web-based communication ne
   

Methods

Objective 1. Implement a virtual laboratory infrastructure through the development and open distribution of resources, materials, protocols, and data among participating researchers and states (IA, IL, MN, MO, NE, NC, SD, VA, U. Guelph, BARC, NPB, NADC). As discussed above, this is an effort to standardize research protocols among laboratories and to "level the playing field" by increasing the accessibility to all PRRS researchers of research methods, reagents and other materials. This will be done through the creation of a virtual laboratory environment within which PRRS virus researchers will share reagents, protocols, standards, experimental samples, and biological materials. Objective 1.1. Standards (IL and MO to coordinate; all states and participants). Protocols, methods, procedures, and standards for research performance will be established to enable investigators to directly compare research results. This will be achieved by depositing laboratory procedures on the program website, establishing control standards for laboratory assays, reviewing experimental protocols in advance, and sharing data across the program website. This is performed in coordination with communication tools described in Objective 7. Objective 1.2. Shared Materials (MN to coordinate; all states and participants). Research materials, including viral infectious clones, recombinant proteins and nucleic acids, standard PRRS virus challenge strains, and experimental animals, including commercial pig populations and cloned pigs, will be validated and made available to participants. Objective 2. Achieve biosecurity within herds to prevent the spread of virus within a herd and facilitate its elimination from endemically infected herds. The purpose of Objective 2 is to develop methods to prevent the establishment of PRRS virus infections in herds and to facilitate the elimination of the infections in infected herds. Stakeholders and scientists recognize that the perfect tool for preventing the spread of a virus is a completely resistant animal or a completely effective vaccine. Given the impact that host genetic resistance to PRRS virus would have on the swine industry, it is essential to initiate well-designed genetics experiments for this purpose. For a susceptible animal, the perfect tool for protection is a vaccine that is safe, inexpensive, effective against all viral strains, and induces sterile immunity in pigs at all stages of swine production. Therefore, collaborative multidisciplinary research into mechanisms of protective immunity and potentiation of anti-viral immunity will be conducted. New concepts in antiviral therapy may also reveal novel insights into mechanisms of viral replication. For these reasons, under Objective 2 we propose to: 1. Establish a resource population of pigs for analysis of genetic resistance to PRRS. 2. Investigate mechanisms of protective immunity for PRRS prevention. 3. Investigate mechanisms of immune stimulation that might increase anti-PRRS immunity and prevent persistence. 4. Evaluate novel antisense RNA reagents that target viral replication. Objective 2.1. Functional genomics of PRRS virus resistance (IA, MN, NE, NPB, Pig Improvement Corporation, BARC, MARC). The host functional genomic studies are directed at identifying genes and gene pathways involved in PRRS virus disease and resistance. The long-term goal is to identify candidate genes or markers that may be used for selecting PRRS virus-resistant pigs. The initial structural genomics investigation related to PRRS virus infection has two aspects. The first is the use of genotyping and gene expression analysis on tissues collected from pigs (provided by Pig Improvement Corporation) that show increased resistance to PRRS virus infection. Second, a quantitative trait loci (QTL) study will be initiated to eventually identify a family whose offspring differ measurably in traits associated with improved resistance or susceptibility to PRRS virus. Objective 2.2. Mechanisms of protective immunity for PRRS virus prevention (IA, IL, KS, NC, NE, MN, SD, BARC).( Objective 2.3. Evaluation of immune modulators to stimulate and/or enhance antiviral immunity (IA, IL, KS, MN, NE, BARC).( Low expression of interferon-α during the initial stages of infection is hypothesized to play a role in the pigs weak immune response to PRRS virus. Strategies to stimulate interferon-α production will be tested for their ability to reduce viral infection of macrophages, viremia, duration of viremia, viral loads in lymphoid tissues, and duration of persistent infection. Objective 2.4. Agents that target virus replication (KS, IA, U. Guelph). Specific antiviral drugs will provide a complement to vaccines for PRRS intervention and control, especially in critical areas of production, such as boar studs. The effectiveness of antiviral drugs in blocking HIV infection and the recent interest in antivirals for SARS virus (like PRRS virus, SARS virus is a nidovirus) makes this area of research especially relevant and topical. Experiments will be conducted in two main areas. First, virus replication will be studied for the purpose of identifying drug targets. Second, the response to currently available antivirals will be tested, e.g., phosphoramidite morpholino antisense oligomers (PMOs), which have shown to be efficacious in the inhibition of RNA viruses such as hepatitis C and West Nile. Objective 3. Achieve biosecurity among herds by preventing viral spread between herds. Objective 3.1. Molecular epidemiology of PRRS virus (IA, IL, MN, MS, NE, SD, VA, U. Guelph). Comprehensive longitudinal and cross-sectional surveys of North American commercial swine herds will be conducted for the purpose of estimating the variability and rate of change in viral genomic sequences. This information has direct relevance and utility for our understanding of viral virulence, viral evolution, immunity/cross protection, persistence, and transmission. The resultant database of virus isolates, contemporary viral genetic sequence information, and pig clinical/population data will be available to NC-229 basic and applied researchers. Objective 3.2. Environmental stability and transmission of PRRS virus (IA, MN). Area spread, i.e., herd-to-herd transmission of PRRS virus in the absence of an apparent cause is widely recognized but poorly understood. The stability of infectious PRRS virus under different environmental conditions will be determined and used to develop models to predict the spread of PRRS virus. This information will also be incorporated into regional elimination strategies and on-farm biosecurity protocols. Objective 4. Improve diagnostic assays and create on-farm monitoring systems (IA, IL, MN, MO, NC, SD, VA, NPB, NADC). At present, there is no single test or combination of tests that can accurately identify all PRRS virus-infected pigs. This shortfall has made PRRS virus elimination nearly impossible. Diagnostic tests that reliably identify persistently infected pigs or rapidly distinguish vaccine from wild-type infection are also not available. Thus, the goals of this objective are to: 1. Improve existing diagnostic testing protocols. 2. Explore novel diagnostic and/or detection strategies, such as gene chips and biosensors. 3. Construct recombinant PRRS virus strains that stably integrate marker tags for use in future vaccines. Objective 4.1. Standardize comparisons of current and future diagnostic assays (IA, MN, MO, NADC, NC, NPB, SD). At present, most diagnostic laboratories use PCR diagnostic assays developed in-house. The majority of these assays have not been independently validated and diagnostic performance established. For that purpose, a bank of virus isolates and samples will be established and used to compare, validate, and standardize current diagnostic PCR assays used by the NC-229 institutions. The protocols of the assays that show the best reproducibility, specificity, and sensitivity will be available to all participants. Objective 4.2. Differential serology for use in the preparation of recombinant vaccines (IL, MN, MO, NE, NPB, SD). There are no tests that can readily distinguish a vaccine-derived immune response from antibodies produced following infection with a wild-type virus. The availability of a marker vaccine and complementary differential serological assay (HerdChek Anti-PRV g1 assay, IDEXX Laboratories Inc., Westbrook, Maine) was critical to the success of the U.S. pseudorabies eradication campaign. Therefore, studies will be conducted for the purpose of identifying genes that can stably accommodate either insertion or deletion of nucleotides sufficient to alter antibody recognition of existing PRRS virus antigens. If successful, this marker technology will be further developed in collaboration with interested sponsors to discriminate between antibody responses to vaccine and wild-type virus. Objective 4.3. PRRS virus gene chips (MN). Successful implementation of microarray-based PRRS virus diagnosis may provide more rapid genotypic analysis and classification of viruses than the current molecular-based diagnostic tests. This project received initial funding from the NPB and the Biotechnology Research Development Corporation. Eventually, arrays are envisioned for the characterization of specific genetic-based properties of single isolates, such as the capacity to cause reproductive/respiratory disease, the probability of becoming endemic, and virus susceptibility to immune protection. Objective 4.4. Biosensor monitoring of PRRS virus (MO). Diagnostic tools that can continuously sample the environment for the presence of PRRS virus would be invaluable in any biosecurity protocol. Biosensor diagnostic technologies for PRRS virus based on an innovative optical sensing technique incorporating virus-specific monoclonal antibodies will be examined in an interdisciplinary project combining PRRS virus diagnostics with electrical/chemical engineering. Initial studies will focus on identifying monoclonal antibodies that can be incorporated into a sensor, and demonstrating the feasibility of measuring viral antigens in the blood. Subsequent efforts will extend the application to detect PRRS virus in aerosols and dust within swine facilities. Objective 5. Develop and test PRRS virus eradication protocols under various ecological settings (IA, MN, MS, NC, NE, SD, NPB). Historically, it is apparent that sustainable PRRS virus elimination will best be achieved when applied on a regional basis. Areas of North America will be targeted to initiate regional eradication of PRRS virus. These areas will be required to have natural barriers that serve as physical borders, a manageable number of farms to address the objectives, cooperative producer groups positioned for long-term presence in the industry, and local veterinarians who support the concept. Objective 6. Develop educational outreach tools for disseminating information through established outreach and extension networks to producers, veterinarians, educators, and researchers. The NPB will work with the NC-229 to create educational and instructional materials for producers, workers, veterinarians, and consumers. External outreach is directed towards the development of educational outreach materials for training swine producers and veterinarians in biosecurity compliance issues and methods, and in procedures for management and elimination of PRRS. Objective 6.1. Publication and distribution of publications on PRRS-related topics (IA to coordinate regional sites; MN, NE, NC, SD, NPB). The 2003 PRRS Compendium (2nd Edition), Producer Edition, and CD Edition are examples of educational materials produced by NC-229 and NPB. Additional publications will be directed at educating and motivating federal, state, and private resources to support research directed at PRRS virus elimination. Additional activities include the incorporation of PRRS virus-related information into existing outreach activities at NPB, including distance learning modules (interactive web-based, and CDs) and a traveling swine health seminar series done at the state level. Objective 6.2. Development of web-based communication networks (NPB). A website providing producers, veterinarians, extension veterinarians, and researchers with updated information regarding PRRS virus research and elimination, including the results generated through research conducted within this proposal will be emphasized. For example, the NC-229 public meeting held on November 8, 2003 was taped for delayed Webcast on the NPB website.

Objective 6.3. Effective educational materials for use on swine farms (NPB, IA, MN). This project area will develop comprehensive biosecurity educational materials for swine farm employees, evaluations of the employees capacity to comprehend and apply guidelines, and continued refinements in educational materials that improve compliance. Materials will be available in several formats, including written, online, and CD. Objective 7. Create an information network to ensure rapid and efficient communication of PRRS virus research. Objective 7.1. Development of a web-based communication network (NPB). A secure area for internal communication tools will be set aside on an NPB server within the NPB external communication website. This secure network will be used for the sharing of information between PRRS virus investigators. Objective 7.2. On-line catalog of PRRS virus research-related resources (NPB). The online catalog will greatly reduce the cost of future projects by avoiding the re-development of reagents and experimental protocols. This catalog will be posted on the web and will be available to all researchers. Objective 7.3. Creation of a national PRRS Epidemiological Registry and Database (IA, MN, NE, SD, NPB). While a great deal of information is known about PRRS outbreaks on individual farms and even more information is being collected on the genomic sequence of various PRRS virus isolates, the data are not being collected in a uniform manner. Systematic collection of these data, including outbreak parameters, geospatial location, sequence information, and variables identified as potential risk factors will permit analyses that will be vital to regional PRRS virus elimination projects, risk factor mitigation, and understanding inter-farm viral transmission. Virus isolate and nucleotide sequence information collected under the molecular epidemiology project will be included in the database. The information collected will support a national PRRS Risk Factor Study conducted in collaboration the USDA Center for Epidemiology and Animal Health. Objective 7.4. Annual meeting and program review (All stations). This meeting will be held over a two-day period in conjunction with the annual Conference of Research Workers in Animal Diseases meeting. The first day of the meeting will involve a critical review of past, current, and future work with additional time being devoted to strategic planning. The second day of the meeting will be an International PRRS Symposium with attendance open to all interested individuals. A format of keynote speakers with smaller workshop sessions is anticipated (a workshop session related to standardization of immunology and virology protocols related to the study of PRRS virus is planned for 2004). Information developed both through this project and outside of this project will be highlighted. International guests and speakers are expected to participate.

Measurement of Progress and Results

Outputs

• Data published in peer-reviewed scientific literature.
• Data and interpretations published in industry newsletters and other publications targeted to the swine industry and allied providers.
• Presentations at meetings, workshops, and symposia attended by swine veterinarians and members of the swine industry. Examples include the annual meeting of the American Association of Swine Veterinarians, the International Pig Veterinary Society meeting, the Iowa State University Swine Health Conference, and the Allen D. Leman Swine Conference.
• Real-time posting of experiments and reporting of results on NPB website open to all program participants.
• Archiving of experimental data on a program website where it will be available for data sharing, subject to confidentiality agreements among all participants. The program website will be created and maintained by the National Pork Board.
• Annual meeting and International PRRS Workshop held in conjunction with the Conference of Research Workers in Animal Diseases. Development and sharing of biological materials, including infectious clones, PRRS virus strains, purified proteins, monoclonal antibodies, and genetically characterized animals. Standard methods, protocols, and reagents for serological, immunological, and virological assessments of the PRRS virus infection status of an animal and its level of protection against PRRS. Bilingual educational and training manuals, CDs, pamphlets and literature related to biosecurity, biosecurity implementation and biosecurity monitoring.

Outcomes or Projected Impacts

• Production of value-added swine and pork products through the elimination of PRRS in breeding herds and genetic stocks. (GPRA obj G1.1)
• Increased global competitiveness of the U.S. swine industry by eliminating the cost of PRRS and by producing PRRS-free pigs. (obj. G1.2)
• Improved access to affordable and healthful pork and pork products. (obj. G2.1)
• Improved food safety by elimination of the disease agent most important for secondary infections and multifactorial disease in swine. (G2.2)
• Promotion of greater harmony between agriculture and the environment by the development of more efficient and sustainable swine production practices through elimination of the most significant health hazard to swine. (G4.1)
• Promotion of greater harmony between agriculture and the environment by the development of more efficient and sustainable swine production practices through elimination of the most significant health hazard to swine. (G4.1) Increased capacity of communities and families to enhance their own economic well-being through more profitable management of swine farms. (G5.1) Increased capacity of communities, families and individuals to improve their own quality of life and job satisfaction by raising healthier pigs. (G5.2)

Milestones

(2004): Objective 1 = Establish "virtual laboratory" website for posting of protocols, reagents and sample banks and other biological materials. Convene a Workshop for the Establishment of Virological and Immunological Standards. Objectives 2-5 = Project Director of USDA-NRI grants solicits projects for funding from NC-229 participants. Objectives 6-7 = Establish Website for outreach materials and "virtual university" per Objective 1.

(2005): Objectives 2 -5 = Identify pigs genetically resistant to PRRS virus and begin generational breeding of these lines. Immunology nad virology standards from prior year workshop used to implement studies on role of T-cells in immunity to PRRS virus. Ztudies on molecular epidemiology and transmission of PRRS virus and antiviral drugs ongoing. Begin standardization of diagnostic assays per Objective 4.

(2006): Objectives 2-5 = Establish PRRS Epidemiological Registry and Database. Potential sites for insertion of marker genes identified for marker vaccine and diagnostic kit develoment.

(2007): Begin regional elimination studies per Objective 5 based on Milestones accomplished in Years 2004 to 2006.

(2008): Objectives 2-5 = Complete ongoing studies related to immunology, virology, diagnosis and epidemiology of PRRS. Recommend protocols for regional elimination of PRRS virus or at least herd elimination of PRRS virus. Assess accomplishments of USDA-NRI funded projects and other NC-229 contributions in anticipation of submitting new Integrated Programs grant and new NC proposal.

(0):al milestones 2004-2009 related to Objectives.</b> 1. Infrastructure implemented in Objective 1 is on-line and available to all participants in project. Standard controls identified and on deposit with National Veterinary Services Laboratory (national depository). A list of shared reagents and other biologic materials is on-line beginning in 2004 and continues with updates annually. 2. Beginning 2004, External Stakeholder and Scientific Advisory Boards meet to evaluate research proposals and budgets and to meet with project director, investigators and NC-229 Executive Committee. See appendices C and D. 3. Beginning 2004, External Stakeholder and Scientific Advisory Boards review annual reports from project directors and make recommendations to continue, modify, cease and add to the specific aims in the following year. Approved research will continue with program funds. 3. Initiated in 2003, development of outreach, extension and communication activities continues to 2009. 4. International PRRS Symposium to be held in fall of 2004 and each year thereafter.

Projected Participation

View Appendix E: Participation

Outreach Plan

Communication to the public and external stakeholders, as described under Objectives 6 and 7, will be coordinated and directed through the offices of the NPB using existing educational resources and outreach channels. As specific knowledge is acquired on topics of PRRS control, elimination, spread within herds, and spread among herds, bilingual educational materials and operation manuals will be prepared and distributed via print and electronic media under the direction and coordination of the NPB. Scientific communication will be managed by the NC-229 committee to assure full reporting of research findings in peer-reviewed scientific literature, abstracts and proceedings of relevant meetings and symposia, book chapters, and review articles. Timely communication will occur through the annual meeting and affiliated international symposium. Lists, descriptions, and sources of control standards, assay protocols, planned experiments, critical reagents, clones, and so forth will be permanently available for PRRS virus researchers on an NPB server. A centralized virus database with information about field isolates with clinical histories, geographic information, nucleic acid sequence information and other relevant information will be constructed and maintained on the NPB website.

Organization/Governance

The program will be directed by the chair of NC-229 working with an executive committee comprising the chair, the past chair, project director for the USDA-NRI grant and the secretary. Elections for chair and secretary are held every two years. Members of NC-229 and the NPB will be responsible for organization and management of individual specific aims. An external stakeholder advisory board has been formed to provide advice, counsel and oversight (Attachment C). A scientific advisory board comprised of internationally renowned experts will review the program as a whole and individual projects and activities, and make recommendations for funding of all research and outreach activities throughout the life of the program (funding available through the USDA-NRI Integrated Activities Program 2004; Attachment D). They will be assisted in review of proposed projects by an ad hoc grant review board organized by the NPB. Program direction and funding decisions related to the USDA-NRI proposal will be determined by the project director with recommendations from the scientific and stakeholders advisory boards and the NC-229 committee. These groups will meet and deliberate at the time of the NC-229 annual meeting.

Literature Cited

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Attachments

Land Grant Participating States/Institutions

CT, IA, IL, KS, MN, MO, NE, SD, VA

Non Land Grant Participating States/Institutions

CIAD, A.C., Iowa State University - College of Vet Med, National Pork Board, USDA-ARS Beltsville Agricultural Resarch Center, USDA/NADC