Duration: 10/01/2009 to 09/30/2014

Administrative Advisor(s):

NIFA Reps:

Non-Technical Summary

Statement of Issues and Justification

The need as indicated by stakeholders.


Porcine reproductive and respiratory syndrome (PRRS) is the most devastating disease of swine in the USA. Successful completion of the project aims will result in enhanced control of PRRS. Benefits will include reduced animal suffering, thus enhancing health and well-being. Swine producers will experience improved economic gain due to reduction in animal losses and more efficient pork production.

The National Animal Health Monitoring System (NAHMS) 2006 report found that respiratory problems accounted for the highest percentage of all nursery deaths (53.7 %) and the majority of grower/finisher deaths (60.1 % in swine). (1). For all producer-identified causes NAHMS 2006 found that the percentage of grower/finisher deaths did not differ substantially by size of site. The earlier NAHMS 2000 report found that PRRS affected 21.4% of all breeding herd operations and, importantly, 58.3% of operations with greater than 500 sows (2). 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 (3). With rising feed costs these numbers ballooned to $664 million in 2007 (4). 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 (5) 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.

Since its discovery in 1991, the PRRS virus has proven itself as a significant pathogen of swine in nearly all production areas of the world (6,7). In 2006 reports of a highly pathogenic pig disease, "Pig High Fever Disease" in China emerged; this was characterized by prolonged high fever, red discoloration of the body, and blue ears associated with high mortality. PRRSV was identified as the single most prominent virus in the samples collected from affected pigs (8,9). This threat has reemphasized the need for effective PRRS control and the importance of finding predictably successful tools for managing or eliminating the virus from farms. In their 2007 annual report (10) the National Pork Board (NPB) noted that through the Checkoff's PRRS Initiative and the USDA, university researchers and Extension personnel, animal health companies, state and federal government agencies, swine veterinarians and producers are working together to map out a plan to successfully manage and eliminate PRRS from U.S. swine farms. The NPB's Swine Health Committee discussed the research results of their PRRS Initiative funding and the USDA PRRS Coordinated Agricultural Project (CAP) and based on that review chose 3 research objectives for 2008 funding: PRRSV Immunology, PRRSV Epidemiology and Ecology, and PRRSV Diagnostics.

Since the fall of 2005 there has been an increase in the incidence of the severe form of Porcine Circovirus Associated Disease (PCVAD) in the U.S. Clinical signs of the more severe form of post-weaning multisystemic wasting syndrome (PMWS) include anorexia, rapid weight loss, enlarged lymph nodes, and respiratory signs (11). PMWS, now referred to as PCVAD, is still spreading in the United States swine population, based on the case definition published by Sorden (12). The affected farms have historically been positive for Porcine Circovirus type 2 (PCV2). In the last 4 years research has proven that a new, more virulent isolate, now termed PCV2b, has emerged in Canada and the US (13-17) with similar problems in the EU (18). Thus in 2006 NPB assembled scientific researchers, veterinarians, allied industry representatives, and producers to decide on PCVAD research priorities (19); in 2008 their Special PCVAD call addressed producer concerns that have not yet been answered: Immunology, Epidemiology, Pathogenesis, Diagnostics, and Prevention and Treatment.

For their most recent, 2009 Research initiatives NPB is targeting projects addressing Porcine Respiratory Disease Complex (PRDC), and the organisms involved: PRRS, Mycoplasma, influenza, Pasteurella and other bacterial infections. For PRDC the NPB Animal Health Committee is requesting grant applications for projects addressing the role of these organisms in PRDC, the epidemiology of the individual agents and their interactions with other pathogens, the effect of strain variability on virulence, pathogenicity and herd immunity, identification of reservoirs of the organisms and proposed control or practical eradication strategies. Projects that link swine practitioners and research laboratories in field studies are especially encouraged. The NPB has noted that swine disease surveillance is critical for maintaining and expanding markets as well as work with producers to receive input on a comprehensive swine health surveillance system. These outcomes will be used in health management decisions to maintain and improve herd-health status, increasing competitiveness of U.S. Pork.

Overall these stakeholders' recommendations provide the relevance for this new NC-229 proposal. No other multistate project specifically addresses swine respiratory diseases. Producers have indicated the need for research that expands and enhances our knowledge of the complex respiratory diseases faced by swine and to design methods to eliminate PRRS and the pathogens involved in PRDC using novel vaccines, drugs and therapeutics.

Thus the present proposal will address "Detection and control of PRRSV and emerging viral diseases of swine."

The importance of the work and the consequences of failing to respond. The US swine industry is at a crucial economic crossroads. Increased production costs and a strained economy have severely impacted many swine operations; pork producers are further hampered by infectious disease problems that increase production costs. When the pseudorabies (PRV) 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, many members of the original NC229 project team affirmed their scientific and organizational abilities when PRV 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" (20). Since 2006 this threat has been exacerbated by the Chinese outbreaks of "Pig High Fever Disease."

When the viral etiology of PRRS was established by investigators in the Netherlands in 1991 (5) and shortly thereafter, in the U.S. (6), research has progressed towards understanding the disease and the associated virus. The release of the first live-attenuated commercial vaccine (MLV) 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. However, MLV have not fully met expectations; deficiencies including virus shedding, persistent infection, potential reversion to virulence and incomplete protection have been reported. (21-27) Moreover, there is no method to distinguish infected from vaccinated pigs. Because of vaccine failures many producers have sought controlled exposure or acclimation, i.e., the intentional infection of naive animals with wild-type live PRRSV either through contact with infected animals or exposure to infectious material (28,29). Thus, young pigs are exposed to farm-specific live virus in an attempt to induce immunity; however, this results in continuous spread of the virus and perhaps inadvertent spread of other diseases.

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 (22,23). Second, PRRS virus elicits a rather complicated and unique immune response that subverts the immune system and results in persistently infected swine (21,27). 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) (30,31). Fourth, anecdotal evidence strongly suggests that the virus can efficiently move between farms, even those that utilize rigorous biosecurity and good production practices (32). Finally, relatively few tools, including effective vaccines and surveillance techniques, are available to producers and veterinarians for managing the disease.

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 economic catastrophe. Even farms that survive a PRRS outbreak become re-infected despite all best efforts to protect the animals. The NC229 project will continue to improve an American Association of Swine Veterinarian's risk assessment tool for PRRSV. It will be used to determine why most sites fail to stay PRRS-free, addressing issues of pig farm density, of aerosols and biosecurity.

Continuous changes and refinements in food animal production techniques support the hypothesis that PRRS virus represented just the tip of the iceberg of emerging disease threats to the food animal industry. The emergence of PCV2b caused major losses in the industry with classic porcine respiratory disease complex (PRDC) in neonates and early finisher pigs (33,34). Questions raised include whether PCV2- infected pigs are more likely to be co-infected with other pathogens than non-PCV2-infected pigs; what are the most likely co-infectors; and how are animals affected by age and production system. Successful vaccination against PCV2 protects pigs against much of the clinical symptoms of infection, yet many questions remain (35-37). Issues such as the effect of concurrent PRRS or other infections remain to be fully addressed (38,39).

Worldwide there is great concern that the next pandemic will be caused by potential cross-species adaptation and spread of influenza viruses (40,41). Influenza viruses infect humans, swine, and avian species; they can exchange genetic sequences and produce new reassortant viruses. Thus besides the respiratory problems caused in swine by flu viruses, swine are a potential source of new influenza viruses that can infect humans. This is particularly problematic given the potential for reassortment of swine flu viruses with highly pathogenic avian influenza. Vaccination of sows protects young pigs through maternally derived antibodies but there are a number of genetically diverse viruses circulating in swine herds, providing a continuing source of virus on a herd basis. Current vaccines are inactivated and adjuvanted; research into newer DNA, live-virus, or vectored vaccines is needed (42). Influenza is just one of numerous transboundary viral diseases of concern to the swine industry. Foot-and-mouth disease and classical swine fever virus are each major foreign animal disease threats to US producers.

Overall there is a sense of urgency for this proposed NC229 project. It presents an animal health paradigm to address future disease threats to swine and to humans. It provides new technologies to control newly emergent diseases; PRRSV was unknown just 20 years ago and PCV2b emerged in 2004. Moreover, PRRS is in the same taxonomic order (Nidovirales) as the human pathogenic SARS coronavirus that caused major disease issues in 2003 (43). 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 pig virus.

Failing to solve these swine disease problems 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. The new NC229 project will provide substantial opportunities to address important swine respiratory disease issues based on inter-institutional cooperation at the national and international level.

The technical feasibility of the research. Successful realization of the study objectives will require basic and applied research studies, including immunology, functional genomics, 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 virus control and elimination. In working towards these goals, the NC-229 committee directed and coordinated the preparation, successful submission and recent completion of the $4.4 million USDA NRI Coordinated Agricultural Project (CAP) grant #2003-05164 on the "Integrated Control and Elimination of Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) in the U.S." [PRRS CAP1]. In 2008 the NC229 again participated in writing the successful $4.8 million USDA NRI CAP renewal grant on the "Porcine Reproductive and Respiratory Syndrome (PRRS) Control and Elimination" [PRRS CAP2]. Both PRRS CAP proposals described collaborative research, education, and outreach plans via the coordinated efforts of the NC-229 multi-state consortium of PRRS researchers, academic institutions, USDA ARS labs, the NPB and private industry. These projects provide a financial resource for the NC-229 technical committee to work with the PRRS CAP2 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 effective control of swine respiratory viruses 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. It will also continue to sponsor cutting edge research in vaccines with infectious clones and vectors, novel drugs and therapeutics, epidemiologic analyses, and host genomic control, being just some of the newer areas of NC229 sponsored research. While there is much expertise available from single entities, the best hope for the control and elimination of swine respiratory infections is a collaborative, multidisciplinary research program that focuses on specific aspects of PRDC. In 2008 the NC-229 has been expanded to 14 stations (CT, GA, IA, IL, KS, MD, MO, MN, NC, NE, OH, IN, SD, and VA), 3 ARS labs (USDA-BARC, USDA-MARC, USDA-NADC), and importantly, international groups in China, Mexico and Spain. NC229 has a history of productive and collaborative PRRS virus research and its researchers were actively involved in addressing the 2006 PCV2b outbreak (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 new paradigms for the control of swine viral respiratory infections. Progress toward this goal will proceed through the successful accomplishment of specific aims and milestones described later in this proposal. The creation and operation of a virtual university environment where investigators share data and ideas has been effected by the PRRS CAP programs. A major milestone for this NC229 renewal will be to expand this network to address the real industry problem of complex swine viral respiratory infections that result in PRDC. A second milestone will be risk assessment projects that will demonstrate new protocols and management techniques for the control and elimination from herds of swine viral respiratory infections.

This multistate collaborative work will be instrumental in expanding existing data, and generating new data. Important research outcomes include:

1. Contribute to the knowledge base on pathogenesis and immunobiology of PRRS and PRRS-associated viral diseases, PCV2 and influenza.

2. Elucidation of strategies to evade host responses, genetic determinants of host response, tissue tropism and replication efficiency, and mechanisms of immune modulation and prevention of viral persistence.

3. Understanding effects of viral protein structures on function.

4. Development of infectious clones and vectors that allow expression, mutation or deletion of individual PRRSV genetic epitopes and proteins, as well as emerging swine viral diseases.

5. Rationale for the design and development of effective control measures, including new vaccines that differentiate infected from vaccinated animals (DIVA vaccines) and novel anti-viral drugs.

6. Novel molecular adjuvants that boost recognition of PRRSV and emerging viral disease antigens and improved cross-protective immunity and adjuvant strategies.

7. Discovery and development of methods that facilitate elimination of ongoing infections and prevent re-establishment of PRRS virus infection on a pig farm.

8. Development and delivery of differential diagnostics 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.

9. Identification of factors involved in inter-farm transmission and the role of geography, the environment, and viral genetics in regional spread between area farms.

10. Design and implementation of eradication protocols in large field-based investigations.

11. Development of outreach and educational materials and real time delivery methods that provide biosecurity and compliance information.

References:

1. NAHMS Swine 2006 Part III. http://nahms.aphis.usda.gov/swine/swine2006/Swine2006_PartIII.pdf

2. NAHMS 2000 report Part II. http://nahms.aphis.usda.gov/swine/#swine2000

3. Neumann et al. J Am Vet Med Assoc. 227:385-39, 2005.

4. Johnson et al. Proc. IPRRSS. P.16, 2007.

5. Dee, Joo. Vet Microbiol. 55:347-53,1997.

6. Wensvoort et al. The Vet Q. 13:121-130, 1991.

7. Benfield et al. J Vet Diag Invest 4:127-133, 1992.

8. Tian et al. PLoS ONE. Jun 13;2(6):e526, 2007.

9. Zhou et al. Transbound Emerg Dis. 55: 152-64, 2008.

10. NPB 2007 annual report http://www.pork.org/NewsAndInformation/News/docs/2007AnnualReport.pdf

11. Segalés et al., Anim Health Res Rev. 6: 119-42, 2005

12. Sorden. J. Swine Health Prod. 8: 133-136, 2000.

13. Cheung et al. Arch Virol. 152: 1035-44, 2007.

14. Horlen et al. J Swine Health Prod. 15: 270277, 2007.

15. Opriessnig et al. J Vet Diagn Invest. 19: 591-615, 2007.

16. Carman et al. Can J Vet Res. 72: 259-68, 2008.

17. Ramamoorthy and Meng, Anim Health Res Rev. 2:1-20, 2008.

18. Wallgren et al. Vet Q. 29:122-37, 2007.

19. NPB. J. Swine Health Prod. 15.1, 47-51, 2007.

20. Rowland. Vet J. 174:451, 2007.

21. Murtaugh et al. Viral Immunol. 15: 533-47, 2002.

22. Lager et al. Can J Vet Res. 67: 121-7, 2003.

23. Mengeling et al. Vet Microbiol. 93: 13-24, 2003.

24. Yoo et al. Vet Immunol Immunopathol. 102: 143-54, 2004.

25. Meier et al. Vet Immunol Immunopathol. 102: 299-314, 2004.

26. Zuckermann et al., Vet Microbiol. 123: 69-85, 2007.

27. Mateu and Diaz. Vet J. 177: 345-51, 2008.

28. Fano et al. Can J Vet Res. 69: 71-4, 2005;

29. Vashisht et al. J Am Vet Med Assoc. 232: 1530-5, 2008

30. Thacker. Vet Clin North Am Food Anim Pract. 17: 551-65, 2001.

31. Fachinger et al. Vaccine. 26: 1488-99, 2008.

32. Cho and Dee, Theriogenology. 66: 655-62, 2006.

33. Horlen et al. J Swine Health Prod. 15: 270277, 2007.

34. Opriessnig et al. J Vet Diagn Invest. 19: 591-615, 2007

35. Horlen et al. J Am Vet Med Assoc 232: 906912, 2008.

36. Opriessnig et al. Clin Vaccine Immunol. 13: 923-9, 2006.

37. Opriessnig et al. Vet Microbiol. 131: 103-14, 2008.

38. Opriessnig et al. J Gen Virol. 89: 2482-91, 2008.

39. Ramamoorthy and Meng. Anim Health Res Rev. 2:1-20, 2008.

40. Webby et al. Curr Top Microbiol Immunol. 2007;315:67-83, 2007.

41. Thacker and Janke. J Infect Dis. 197 Suppl 1: S19-24, 2008

42. Vincent et al. Vaccine. 25:7999-8009, 2007.

43. Saif. Dev Biol (Basel). 119: 129-40, 2004.

Joan K. Lunney, Ph.D. (Chair)

X.J. Meng, M.D., Ph.D. (Secretary)

Related, Current and Previous Work

Contributions of NC-229 investigators.

NC-229 was founded in 1999 as a vehicle to facilitate progress in Porcine Reproductive and Respiratory Syndrome (PRRS) virus research and promote collaboration and communication. NC229 originally involved the participation of 8 state universities and USDA ARS labs, increasing to 11 for the 2003 renewal and to 18 for this 2009 renewal with the additional inclusion of numerous international sites. Participating institutions (names of the official NC229 representative at each location is noted): The Ohio State Univ. (R Gourapura), Univ. of Minnesota (M Murtaugh), South Dakota State Univ. (E Nelson), Univ. of Connecticut (G Risatti), Univ. of Georgia (M Tompkins), Univ. of Maryland (YJ Zhang), Univ. of Missouri (S. Schommer), North Carolina State Univ. (M McCaw), Purdue Univ. (IN, R Pogranichniy), Virginia Polytechnic Institute and State University (XJ Meng), Univ. of Nebraska (F Osorio), Kansas State Univ. (R. Rowland), Iowa State Univ. (J Zimmerman), Univ. Wisconsin (T Goldberg), and Univ. of Illinois (F. Zuckermann) as well as representatives from ARS-BARC (J Lunney), ARS-NADC (K Faaburg), ARS-MARC (G Rohrer). Most recently, strong international participation has been included: China (FC Leung, Hong Kong; S Yuan, Shanghai), Mexico (J Hernandez, Sonora; R Molina, Sonora; E Fano, National Univ. of Mexico) and Spain (L Enjuanes, Campus Univ. Autonoma, Cantoblanco; J Dominguez, INIA). Scientists at the National Pork Board (NPB L. Becton), USDA CSREES (P Johnson), and USDA APHIS (J Srinivas, Center for Veterinary Biologics; D Pyburn and J Korslund, Veterinary Services) are active participants. Representatives from a variety of industries (animal health and vaccine and diagnostic companies) and institutions continue to participate in NC-229 activities and meetings. An annual (public) meeting, the International PRRS Symposium, is held in December of each year immediately prior to the Conference of Research Workers in Animal Diseases (CRWAD) to discuss research findings and plan future collaborative activities. Dr. David Benfield, Ohio State Univ., a pioneer in PRRSV research, now serves as the Projects Administrative Advisor.

The NC-229 philosophy and approach to PRRS and respiratory disease issues 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 2008 the National Association of State Universities and Land-Grant Colleges recognized NC-229 as the recipient of its first annual national multi-state research award, for a project that best exhibits the ideals of multi-state research, such as high standards of scientific quality, relevance to a regional priority, multi-state collaboration, and professional leadership. The Experiment Station Section Committee on Organization and Policy (ESCOP) Science and Technology committee served as the review panel. 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 PubMed for papers published between 1998 and 2008 using only the terms PRRS and swine recovered 1225 unique English language references, up from 614 in 2003; a substantial number of these were written by members of NC-229 institutions. Relevant peer-reviewed papers (274 total) and abstracts and proceedings (418 total) published between 2003 and 2008 by the NC-229 participants are listed in Appendix A.

2. Success with joint proposals demonstrates that NC-229 has the capacity to coordinate ideas and resources, focus on specific problems and projects, and respond immediately to new information related to virus control and elimination. Over the last 5 years the NC-229 committee directed and coordinated the preparation, successful submission, and recent completion of the $4.4 million USDA NRI Coordinated Agricultural Project (CAP) grant #2003-05164 on the Integrated Control and Elimination of Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) in the U.S. [PRRS CAP1]. In 2008 the NC229 again participated in writing the successful $4.8 million USDA NRI CAP renewal grant #2008-55620-19132 on the Integrated strategies to control and reduce the impact of PRRS [PRRS CAP2]. Both PRRS CAP proposals described collaborative research, education, and outreach plans via the coordinated efforts of the NC-229 multi-state consortium of PRRS researchers, academic institutions, USDA ARS labs, USDA-APHIS experts, the NPB, the American Association of Swine Veterinarians (AASV), and private industry. These projects provide a financial resource for the NC-229 technical committee to work with the PRRS CAP2 project director to implement a coordinated multistate and multidisciplinary approach to the elimination of PRRS while NC229 expands its mission to include major porcine respiratory disease complex pathogens.

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 coordinated the 2004 special issue of the journal Veterinary Immunology and Immunopathology (volume 102 #3) on PRRS immunology and Immunopathology. The PRRS CAP website www.prrs.org/ has been transferred to NPB; NC-229 personnel will assist in the sites management. Interactions with professional organizations, such as the AASV, and with scientists at the 1890s and 1994 land-grant institutions will continue and will be enhanced through PRRS CAP and NPB Outreach activities. Moreover advances by NC229 will continue to be presented at local, national and international meetings, as evidenced by the list of abstracts in Appendix A and epitomized by the open scientific forum at the annual International PRRS Symposium www.prrssymposium.org/.

4. Successful pursuit of competitive grants to support swine respiratory disease research. In addition to the PRRS CAP grants numerous grants and contracts have been awarded to NC-229 participants for pig respiratory disease research. Grants awarded in 2003 to 2008 from government sources, such as USDA CSREES NRI, Chinese and Korean governments, local institutional and state grants (not Hatch or Formula), the National and State Pork Boards, and Private Industry are listed in Appendix B. Theses include 80 grants of which 24 were funded through PRRS CAP1. PRRS CAP2 grants have been submitted; awards will be announced shortly.

NC229 Project Research Background:

Porcine Reproductive and Respiratory Syndrome (PRRS) is the most important disease affecting US swine producers. A 2005 study put the average annual cost to the US swine industry at nearly $600 million; that number was updated to $700 million based on 2007 feed and energy prices (64, 94). The USDA funded PRRS CAP1 project in 2004 enabled the collective talents of the stakeholder community of scientists, veterinarians, pork producers, and allied industry researchers to develop innovative strategies to lessen the impact of PRRS and lead to the eventual elimination of the virus. In 2008 this collaboration successfully competed for a second CAP project (PRRS CAP2) to continue their PRRS research and outreach efforts. NC229 plans to expand these activities to address other common interacting pathogens of the porcine respiratory disease complex.

Since its discovery in 1991, the PRRS virus has proven itself as a significant pathogen of swine in nearly all production areas of the world (13, 134). Periodically, severe outbreaks result in abortion storms accompanied by high sow mortality (22). Chronically recurring illness, debilitation, and potentially high mortality occur in affected nursing and growing pigs. North American and European viruses share only about 67% nucleotide sequence identity. Hence, European isolates are designed as Type 1 genotype viruses and North American isolates Type 2. Type 1 viruses of European origin were first identified in U.S herds in 1999 and have since become endemic in the U.S. The presence of two distinct genotypes with diverse antigenic properties further complicates efforts to control and eliminate PRRSV infections (11, 19, 48, 49, 113, 114). In 2006 a highly pathogenic pig disease emerged in China which was characterized by prolonged high fever, red discoloration of the body, blue ears, and frequently a very high rate of mortality. Type 2 PRRSV was identified as the single most prominent virus in the samples collected from these Pig High Fever Disease cases (125, 141). This threat has reemphasized the need for effective PRRS control and the importance of finding predictably successful tools for managing or eliminating the virus from farms. [The inclusion of Chinese scientists in this NC229 project offers great opportunities to enhance scientific exchanges on these important isolates.]

Since the fall of 2005 there has been an increase in the incidence of the severe form of Porcine Circovirus Associated Disease (PCVAD) in the U.S. Clinical signs of the more severe form of post-weaning multisystemic wasting syndrome (PMWS) include anorexia, rapid weight loss, enlarged lymph nodes, and respiratory signs (122). PMWS, now referred to as PCVAD, is still spreading in the United States swine population, based on the case definition published by Sorden (124). The affected farms have historically been positive for Porcine Circovirus type 2 (PCV2). In the last 4 years research has proven that a new, more virulent isolate, now termed PCV2b, has emerged in Canada and the US (18, 21, 60, 61, 98, 111).

For the most part inoculation of pigs with PCV2 does not cause severe clinical signs. Additional factors are needed for the manifestation of severe disease in growing pigs; co-infections with other swine pathogens are likely triggers (72, 81, 110). The rates of occurrence of coinfecting pathogens in PCVAD include PRRSV [41%], Mycoplasma hyopneumoniae [27%], bacterial septicemia [10.0%], bacterial pneumonia [6%], and swine influenza virus in [4%]. PCV2 alone caused disease in only 1% of the cases (40, 42, 53, 99, 101, 102, 111). In addition to PRRSV, Porcine parvovirus (PPV) is a common pathogen of pigs that causes sporadic reproductive failure. In natural conditions, PPV and PCV2 have been co-isolated in about 15% of PMWS cases (42). Experimental co-infection of PPV and PCV2 resulted in reproduction of PMWS in gnotobiotic and colostrum- deprived pigs (3, 41, 67). PPV vaccination, however, did not reduce the severity of PMWS in co-infected pigs (67, 97). Some of the other pathogens that have been associated with PCVAD include Mycoplasma hyopneumoniae (3, 4, 70, 71, 96, 98, 99), Cryptosporidium in enteritis cases (95), Aujeszkys disease (82, 110), swine influenza and bacterial pneumonia (39, 50, 53, 66, 109). Clearly more research is required to understand the specific interactions between these insidious swine pathogens including PRRSV, PCV2 and swine influenza virus.

The PRRS Virus is an enveloped, positive polarity, non-segmented, single-stranded RNA arterivirus possessing a 15-15.5 kb genome, which contains at least 9 ORFs and two untranslated regions flanking the 5 and 3 ends of the genome (13, 44, 80, 137-140). The principal non-structural proteins (nsps), encoded by ORF1a and ORF1b, have protease and replicase functions. The major structural proteins, GP5, matrix (M), and nucleocapsid (N) are derived from ORFs 5, 6, and 7, respectively (7, 10, 14, 19, 20, 44, 47, 83, 87, 138). GP5 participates in the interaction with the viral receptor on macrophages and cell lines, and is considered a target for neutralizing antibodies (8, 100, 106-108). GP2, GP3 and GP4 are minor structural proteins whose functions remain unclear, but are critical for virus replication and may represent additional targets for neutralization (87, 88, 135).

The complexity of PRRS epidemiology is illustrated in Fig. 1 in Appendix C (from the PRRS CAP2 grant). PRRSV can efficiently enter a swine unit at any stage of production and be transmitted by intranasal, intramuscular, oral, or vaginal routes of exposure. Once animals are infected, virus is shed in all bodily secretions, including semen. Hence many breeding herds have been (re-)infected through contaminated semen (27, 113, 115, 116, 132). If a pregnant female is infected, the virus may cross the placenta and infect developing fetuses (12, 15, 73, 84, 118, 119). Pigs that survive in utero infection can become long-term PRRSV carriers; RNA has been detected in their tissues up to 250 days post-inoculation (9, 62, 84, 90, 119). PCR-based serum diagnostic tests are preferred for early detection of PRRSV infection (17, 25, 49, 56, 68, 69, 91). However, they are ineffective once the viremia is resolved for detecting and preventing addition of persistently infected animals since gilts and boars may still harbor PRRSV in lymphoid tissues (15, 45, 63, 115, 116, 119, 132). Fomites are also an effective means to spread the virus among pigs and farms, particularly during the winter (33-36, 38). Transmission by aerosols is an area of active investigation, but its role is still poorly understood (23, 24, 29-32, 37, 58, 59, 126-128).

Modified live virus vaccines (MLV) became available in the U.S. in 1994. Regrettably, vaccination alone has not achieved consistent control or elimination of PRRSV. It has been reported that vaccinated pigs can shed, and become persistently infected by, heterologous PRRSV, possibly due to incomplete cross-protection or reversion to virulence (16, 26, 27, 86, 142, 143). Strong PRRSV strain-specific (homologous) immune responses are developed in pigs following vaccination or natural infection, however these responses may provide only weak cross-protective immunity against heterologous PRRSV strains. Heterologous PRRSV are generated by both a high rate of mutation during replication and viral recombination events; both processes contribute to evolution of several viral genes which may allow the virus to persist in infected populations (9, 19, 48, 51, 52, 73, 79, 85, 91). Following infection some pigs remain persistently infected and shed virus for at least 112 days (9, 90, 119, 136) while other pigs clear the PRRSV rapidly (8, 9, 90, 119, 136, 140). The variability of pig responses to PRRSV infection suggests a genetic basis for the rapid clearance of virus by some pigs versus persistent infection in others; research indicates that pigs selected for improved traits, such as reproductive performance, could be more resistant to PRRSV (1, 55, 77, 79, 104, 105, 129, 130).

Albina et al. (2) was the first to describe the mechanisms that allow PRRSV to persist in infected farms: 1) incomplete infection of the susceptible population during the acute phase, 2) introduction of new susceptible pigs, 3) a persistent viral infection in individual pigs with the potential to excrete virus under certain conditions, 4) weak protective passive immunity, with young pigs becoming susceptible to infection or re-infection, and 5) lack of protective immunity, or variable periods of active immunity, in infected pigs.

PCV2 is a single-stranded circular DNA virus ~1.7 kb in length with 2 major ORFs; ORF1 is 945 bases and codes for replicase proteins; ORF2 is 702 nt and codes for the capsid protein (93). The capsid protein interacts with a heparin sulfate receptor on cells and is considered the principal target for the induction of protective antibody (89). A third gene, ORF3, is in a different reading frame embedded within ORF1 and may be important for pathogenesis (78). Recombination between PCV2a and 2b has now been reported from field samples (60). Unlike PRRSV PCV2 has a stable DNA genome and vaccines even with PCV2a protect against challenge PCV2a or PCVb infection (111). Interactions between PRRSV and PCV2 infection result in severely reduced average daily weight gain and reduced protective anti-PCV2 antibody levels (95).

Development of infectious cDNA viral clones and viral vectors has made it possible to begin research on a new generation of vaccines (46, 65, 74-76, 131, 133, 139). The goal is to deliver broad or heterologous protection in combination with the ability to Differentiate field virus-Infected from Vaccinated Animals (DIVA). Control and elimination of PRRS has been impeded by the inability to distinguish vaccinated from infected pigs (15, 24, 25, 85, 139). A companion DIVA diagnostic test would provide the means to meet vaccine compliancy standards, identify non-vaccinated pigs or to detect vaccine failures. Vaccines that protect breeding animals and block vertical transmission may prevent entry of virus into the nursery and downstream production stages. In addition to providing immunological protection through vaccination, another potential means for controlling virus would be terminating long-term infections.

Multiple studies have established that vaccination against PRRSV can result in protective immunity (11, 92, 143). Attenuated and inactivated vaccines have been described, the former are generally believed to be more efficacious. In considering MLV PRRSV vaccines, to achieve greatest success, a number of factors need to be taken into account, which include 1) MLV vaccine strains may persist in the host, 2) MLV vaccine strains may be transmitted from vaccinated to naïve pigs, 3) vaccine-induced protective immunity may be slow to develop, a feature that may be vaccine strain related, 4) immunity may be more robust against homologous and antigenically related strains than against more distantly related strains of PRRSV, and 5) some vaccinated animals may fail to seroconvert for PRRSV following vaccination. To overcome many of these issues, MLV vaccines, inactivated vaccines and other vaccine strategies that include subunit vaccines and live recombinant vectors have successfully used adjuvants to help boost and catalyze the immune response to protective antigens.

The discovery of toll-like receptors (TLRs) has shown the important role that stimulation of these cell receptors by microbial products plays in both innate and adaptive immune responses (121). TLR agonists to be considered include CpG ODN (ligand of TLR9), lipoteichoic acid (ligand of TLR2), RSV F protein or HSP70 (ligands of TLR4). Components of the complement system, in particular C3d, act as molecular adjuvants enhancing innate immunity and profoundly influencing acquired immune responses. Other promising molecular adjuvants to be examined include inflammatory cytokines. Encouraging studies indicate that cytokines such as granulocyte/macrophage colony-stimulating factor, interleukin-2 (IL-2), IL-12, IL-18, as well as IL-8, RANTES, CCL19, and CCL21 chemokines offer qualities as molecular adjuvants.

Virus proteins which spontaneously assemble into polymeric virus-like particles (VLPs) are increasingly used to present foreign antigenic sequences to the immune system of the host since VLPs represent a specific class of subunit vaccine that mimic the structure of authentic virus particles. VLPs are recognized readily by the immune system and present viral antigens in a more authentic conformation than other subunit vaccines, and can have improved immunogenicity (120). VLPs function as effective antigens but without viral genome or other potentially toxic viral gene products. Additionally, hybrid VLPs could be useful where self-assembly of other porcine capsid proteins with PRRSV proteins (or vice-versa), of different porcine viruses into a single VLP. The majority of VLPs have been synthesized using the baculovirus expression system (28). This insect-cell-based protein production system has many advantages for VLP production: 1) large amounts of recombinant proteins can be produced in high-density cell culture conditions in eukaryotic cells, resulting in high recovery of correctly folded antigen;2) insect cells used for vaccine production can be cultured without mammalian-cell-derived supplements, the risk of culturing opportunistic pathogens is minimized; 3) baculovirus is easily inactivated by simple chemical treatment, localized mainly in the nucleus, whereas VLPs are generally need to be purified from cytoplasmic extracts; and 4) the baculovirus system can be scaled-up for large-scale vaccine production.

Novel antiviral drugs that are based on RNA interference (RNAi) and silencing of PRRSV replication will be guided by small interfering RNAs (siRNAs) (57). Synthetic siRNAs can be readily developed that target conserved viral genes in PRRSV strains, e.g. nucleoprotein or polymerase. The siRNAs can be delivered to a cell and targeted to complementary viral mRNAs sequences where the viral mRNAs are precisely cleaved and undergo degradation thereby interrupting the synthesis of the targeted protein. Research has shown that naked or chemically modified siRNAs can show robust silencing activity in vitro when transfected into cells and such siRNA molecules can also demonstrate robust silencing in vivo (103).

Objectives

1. Elucidating the mechanisms of host-pathogen(s) interactions.
   
2. Understanding the ecology and epidemiology of PRRSV and emerging viral diseases of swine
   
3. Developing effective and efficient approaches for detection, prevention and control of PRRSV and emerging viral diseases of swine
   

Methods

Objective 1. Elucidate the mechanisms of host-pathogen(s) interactions. (GA, IA, IL, IN, KS, MD, NE, OH, TX, VA, WI, BARC, NADC, China, Mexico, Spain) Studies will establish underlying biological processes that determine the outcome of porcine encounters with viral respiratory pathogens. This objective will be achieved by research that seeks to provide a mechanistic understanding of: 1) host intrinsic, innate and adaptive immune responses; 2) disease pathogenesis at the molecular and cellular levels; and 3) viral co-infections on respiratory disease pathogenesis and host immune response. The expected results will provide a rational basis to design effective methods of protection and control, including vaccines and adjuvants, development of antivirals for treatment of acute disease, and improvement of diagnostic tools and methods for rapid, pen-side pathogen detection, and economical assessment of swine respiratory health, as well as environmental monitoring for disease surveillance. 1a. Mechanistic understanding of host intrinsic, innate and adaptive immune responses. Studies of immunological mechanisms of disease resistance are focused on identifying molecular pathways and cellular interactions that result in effective immune responses that prevent respiratory viral infections. Investigators will collaborate on discovery studies in innate responses to viral infection and vaccination, innate signaling pathways, development of humoral and cellular immunity locally and systemically, and mechanisms of protection and immunological memory. Experimental results will define mechanisms of immunological stimulation and antigenic targeting that guide vaccine development. Methods: Functional immunology and signal transduction pathways in cytokine production will be investigated in PRRSV-infected and uninfected pigs and in primary macrophage cell cultures. Specifically, cell proliferation and IFN-g production will be evaluated using proteins and antibodies relevant for immune cell identification and available through the Veterinary Immune Reagents Network. (www.vetimm.org) Flow cytometry facilities in support of the research were secured through PRRS CAP1 program funds. 1b. Molecular and cellular basis of viral pathogenic mechanisms. Studies will be directed toward elucidation of strategies pathogens use to evade host responses, genetic determinants of tissue tropism and replication efficiency, regulatory cell interactions, and mechanisms of immune modulation and persistence. Additional studies will be directed toward understanding effects of viral protein(s) on cell function. The long-term goal is to provide knowledge on viral biological characteristics that become targets for rational development of novel antiviral therapies and treatment modalities, and improved diagnostics. Methods: Animal challenge studies using infectious clones for different viruses (developed in Obj.3) will be conducted at selected institutions. Expression of cellular markers and cytokines will be measured during infections with different swine respiratory pathogens in vitro and in vivo. Different cell markers will be utilized to determine cell specificities for viral infection or viral receptors. Immunomodulation will be studied using immunological assays: flow cytometry, virus neutralization, cell proliferation and total white blood cell counts. Animals that are identified as persistently infected with the virus will be further analyzed using advanced immunological and genetic techniques. Major structural proteins of the viruses will be expressed and their structure and function analyzed to determine the nature of the reactions by which each is formed, and the mechanisms by which it enters new host cells, reproduces and promotes pathogenesis within host animals. The mechanism of virus replication, assembly and release in the infected cells will be studied for each respiratory swine viral pathogen in vitro. Collaborative animal challenge studies will help to validate these techniques and implement new diagnostic tests in the laboratories. 1c. Mechanistic aspects of viral co-infections on respiratory disease pathogenesis and host immune response. Polyvirological interactions that target the same tissue or cell can have synergistic consequences on disease and immunity. However, these interactions are poorly understood and confound control efforts because models of uncomplicated, single infections do not represent the field situation. Therefore, it is critical to characterize the interactions inherent in multiple infections and their consequences on disease control strategies. The NC229 team includes experts in multiple diseases; they will focus on the interaction of viruses that are widespread or emerging or both, but whose interactions in pigs are not fully known. Research investigations will focus on development of experimental animal models, determination of relevant endpoints that predict field conditions, elucidation of underlying mechanisms of enhanced disease outcome and reduced immunological efficacy and tests of peripheral and mucosal immune responses. Methods: To understand the immunological basis for viral co-infections the disease model proposed will incorporate a combination of two or more viral pathogens, such as PCV2 and PRRSV (the most common co-factor associated with PCV2) administered to gnotobiotic or specific pathogen free pigs. We will measure the functional properties of humoral and cellular responses during viral co-infection and development of disease in comparison to the single pathogen infection or as a response to vaccination for one or both pathogens. The analysis of serological responses will be performed by measuring virus specific antibody with ELISA, indirect fluorescent antibody and virus neutralization. Analysis of innate and cellular immune responses will be made by measuring cytokine expression in serum, peripheral blood mononuclear cells (PBMCs) and PBMC proliferation in response to re-stimulation with corresponding pathogen, purified antigens or their immunogenic peptides. The phenotypic properties of responding populations will be assessed with labeled antibodies specific for CD4 and CD8 T- cell populations. Viral load in serum and tissues will be evaluated by virus isolation and quantitative PCR. We will further correlate gross and microscopic lesions with immunohistochemistry test results to determine the significance of multiple pathogen infection in comparison to the single pathogen infection. This study will integrate experts from viral pathogenesis, immunology and vaccinology. Objective 2. Understand the ecology and epidemiology of PRRSV and emerging viral diseases of swine.IA, IL, IN, KS, NC, NE, VA, WI, BARC, NADC, China, Mexico, Spain) In these objective factors influencing virus transmission within and between swine farms will be determined to reduce economic losses caused by viral diseases. Identifying the mechanisms by which these pathogens enter, circulate and persist in swine herds is a critical step to devising methods that effectively prevent, control and/or eliminate these diseases. 2a. Investigate and identify factors that influence virus transmission within and between farms including: virus type, pig genetics, herd size, production system (number of sites), regional pig density and environmental factors. Additional research that estimates and evaluates whether susceptibility, transmissibility and persistence change with age, genetic strains, and various co-factors and management factors will be included. The role and frequency of vertical and horizontal transmission and their contribution to persistence of viral infections in swine dense areas will also be investigated. Methods: Methods used to study these components will include risk factor assessment using the AASV PRRSV risk assessment tool for large field-based investigations. Area spread (spread through air) has long been a confounding factor in PRRSV elimination. Meteorological risk factors for airborne spread of PRRSV between herds will be further characterized. A number of risk factors contribute to the inability to keep PRRSV negative herds negative. Determining the distance PRRSV can be transported via aerosols is an important part of maintaining negative herds in real-world situations. Field based studies are critical for this objective as experimental attempts to spread PRRSV by aerosols have not always correlated with field studies. Regional elimination studies have established a good basis to improve upon to fulfill this objective. 2b. Increase understanding of the risk factors for virus transmission to aid in preventing infection in a swine herd. Identify the mechanisms and factors that facilitate or inhibit the transmission of economically significant pathogens: individual pig, population, and pathogen factors and their role in host susceptibility, pathogen transmissibility, and persistence of the infection in pigs and herds. Investigate and quantify factors that affect virus transmission within and between farms including: virus characteristics, pig genetics, herd size, meta-populations, regional pig density, and environmental factors. Methods: Evaluation of the effectiveness of current and novel practices for preventing pathogen infection in a herd will be part of the research conducted. Enhancing aerosol biosecurity intervention and evaluation with field-based studies that can define the cost: benefit of these improvements to producers and other stakeholders will be devised. 2c. Virus Evolution. Expand current PRRSV database to link genetic sequences with clinical disease, infectious co-factors, management practices, chronology and geographic locale. Develop a community database using phylogenetic tools for the determination of viral evolution. Assure that full-length viral genome sequences are deposited in GenBank. Assess herd management and use of vaccines for effects on viral evolution and polymicrobial interactions. Methods: Determine the role and impact of virus diversity and evolution on the ecology, epidemiology, and virulence of and other emerging viral infections of swine in production settings and in different countries (USA, Mexico, China, Spain). Polymicrobial interactions will be investigated to assess the role of viral co-infections on pathogen levels and viral genetic mutations and to determine the effect of co-evolution and evolution of different viruses during co-infections. 2d. Quantify the effects of viral pathogens on swine health and productivity and the factors / interactions involved in the expression of clinical disease, e.g., virus virulence factors, co-infections, host factors, and herd/population factors. Methods: Develop models of infection and disease at the herd, production system, and meta-population levels for the purpose of identifying ecologic and epidemiologic critical control points that may be used to prevent infection, reduce their impact on the swine health, or eliminate them from the U.S. swine herd. Objective 3. Develop effective and efficient approaches for detection, prevention and control of PRRSV and emerging viral diseases of swine (GA, IA, IL, IN, KS, MD, NC, NE, TX, VA, WI, BARC, NADC, China, Mexico, Spain) 3a. Novel molecular adjuvants and adjuvant strategy. Devise and evaluate strategies that boost recognition of PRRSV and emerging viral disease antigens to improve cross-protective immunity: 1) Incorporate molecular adjuvants into existing modified-live vaccines; 2) Evaluate adjuvanted subunit vaccines; 3) Evaluate inclusion of novel immunostimulants such as TLR agonists; and 4)potential use of dendritic cells as natural adjutants. Methods: Humoral and cellular immunity associated with long-term protective immunological memory defines the efficacy of a given vaccine (assessed in Obj.1). However, few vaccines achieve this goal without the aid of an adjuvant. Molecular adjuvants offer a noninvasive means of enhancing the immune response against target antigens on PRRSV and PCV2. Infectious cDNA clones make it possible to construct modified-live vaccines (MLV) that include viral or cellular genes having adjuvanting qualities in the vaccine candidate. The potential of infectious cDNA clones as foreign gene expression vectors has been proven for PRRSV using a 9 amino acid epitope of influenza virus hemagglutinin protein inserted into ORF7 (54). New MLV candidates will be designed to be 1) avirulent and safe, not cause clinical disease; 2) genetically stable to prevent reversion to virulence; 3) broadly cross-protective; and 4) include a genetic marker to distinguish vaccinated animals from those with natural infection. Various strategies have been evaluated using arterivirus infectious clones to substitute particular amino acids, to delete regions of the genome, to insert foreign sequences, or to substitute parts of viral genes to achieve these desired features. A beginning strategy will be to delete central portions of PRRSV ORFs 2A, 3-6 structural genes, where individual deletions do not affect downstream TRS and coding sequences. A second strategy will be to delete non-structural proteins (NSP2) to potentially attenuate and/or provide a region for insertion of molecular adjuvants (65, 112). Subunit vaccines will be developed to target surface antigens that induce neutralizing antibodies. Examples of subunit vaccines for PRRSV could include: the N protein, that has common antigens for type 1 and 2 strains induce T helper cell responses; GP3 that is antigenic and protective; and GP4 induces neutralizing antibodies. The subunit vaccine studies will focus on PRRSV structural proteins that carry common and type-specific antigenic determinants. Molecular adjuvants will also be examined for their efficacy to enhance primary and memory immune responses to subunit antigens and vectored vaccines, such as transmissible gastroenteritis virus (TGEV) recombinant virus. The biometrics used for evaluation of novel immunostimulants such as TLR agonists and other molecular adjuvants involve the following criteria: 1) Vaccine projects will include a challenge experiment. Since the determinants of protective immunity are not known, there is no single immunological measurement that can be used to predict vaccine efficacy. 2) Challenge experiments will be performed with a virulent strain substantially different genetically from the vaccine strain and a homologous virus. 3) The respiratory challenge model must produce evidence of infection, viremia, and disease (clinical signs, lung lesions) so comparisons between vaccinated and naive control animals can be analyzed. 4) Challenge studies will include for comparison a positive control ( an existing commercial vaccine ). 3b. Novel vaccine strategies and platforms. Availability of infectious clones and vectors will allow expression, mutation or deletion of individual PRRSV or PCV2 genetic epitopes and proteins, as well as those from emerging swine viral diseases, and to test their efficacy as vaccine antigens; a DIVA strategy will be considered in all tests. Approaches will include: 1) Recombinant viral and bacterial vectors; 2) DNA vaccines with improved delivery technologies; 3) Rationally designed, improved MLV and killed vaccines, to reduce virulence and/or improve immune response; 4) Virus-like particles and glycoprotein fusion proteins; and 5) Vaccination strategies targeting immunity in the local mucosal tissue Methods: Full-length infectious cDNA clones are available for type 1 and 2 genotypes of PRRSV, so it is now possible to alter the PRRSV genome to create rationally defined vaccine candidates. The general format will be that adopted from previous work by NC229 scientists. The infectivity of the synthetic RNA will be evaluated; if low, the transcripts may be serially passaged to amplify the infectivity. The reconstituted construct(s) with genetic marker will be tested for its ability to produce clinical symptoms in infected pigs . Microparticle delivery systems and non-invasive plasmid DNA immunization strategies will be considered. DNA vaccines will include a gene encoding the target antigen under the transcriptional control of an effective viral/eukaryotic promoter, along with a poly-adenylation signal sequence (poly-A) and a bacterial origin of replication. As a first approach, the target antigen will be the PRRSV N protein DNA vaccine approach (117); improved immunogenicity using the above delivery strategies will be evaluated for other ORF proteins. Virus proteins which spontaneously assemble into polymeric virus-like particles (VLPs) represent a specific class of subunit vaccine that mimic the structure of authentic virus particles and are recognized readily by the immune system. Hybrid VLPs could be useful where self-assembly of other porcine capsid proteins with PRRSV proteins (or vice-versa), or different porcine viruses into a single VLP. VLPs can be antigen delivery systems to enhance antibody and cell mediated immunity to several porcine viruses. VLPs for PRRSV will be assembled using strategies published for related viruses that focus on expression of glycoprotein fusion proteins known to be important in humoral immune responses to PRSSV infection. Mucosal surfaces are prominent in the gastrointestinal, urogenital, and respiratory tracts and provide portals of entry for PRSSV and PCV2. Several approaches for mucosal vaccines will be tested: 1) co-administration of mucosal adjuvants including heat-labile enterotoxins and their non-toxic derivatives, cholera toxin (CT) and other bacterial products, and cytokines ; 2) coupling antigens to cell-binding proteins such as the B subunits of heat-labile enterotoxins; 3) of antigens into various microparticles or membrane-bound vesicles that promote uptake of the antigens and protect against digestion; 4) expression of antigens in attenuated or commensal bacteria that colonize mucosal tissues; 5) expression of antigens in engineered viruses that undergo limited replication in mucosal tissues; and combinations of these methods. An additional approach to elicit mucosal and systemic immunity to PRRSV is the development of a recombinant vector that stimulates both humoral and cell immune responses against PRRSV. A TGEV vector, developed by the Enjuanes group, induces strong mucosal and systemic immune responses to different PRRSV proteins expressed in this vector ; the cDNA can be propagated as a bacterial artificial chromosome (BAC) (5, 43, 123). Foreign gene expression levels were optimized and a set of transcription-regulating sequences (TRSs) ranging from intermediate to high foreign gene expression levels established (6). A combination of these TRSs could be used to drive the expression of two or three heterologous genes from just one infectious cDNA (i.e., dicistronic or tricistronic vectors). This multicistornic expression system allows for expression of multiple PRRSV antigens and/or coexpression of molecular adjuvants from the same viral vector. 3c. Novel antiviral drugs, therapeutics, and delivery methods. Approaches targeting both viruses and host will include: 1) Small molecule inhibitors; 2) RNA interference; 3) Therapeutic antibodies; 4) Anti-sense strategies and 5) Nucleoside analogues. Methods: The development of novel antiviral drugs and therapeutics will be based on RNA interference (RNAi); silencing of PRRSV replication will be guided by small interfering RNAs (siRNAs). As in vivo delivery of siRNA-based antiviral drugs may be affected by endogenous endo- and/or exonucleases, we will introduce phosphorothioates at the 3 ends of siRNA molecules. To protect against endonucleases, we will introduce 2-O-methyl groups at potential nuclease hot spots that are sequence-specific. Targeting conserved viral mRNA to inhibit replication will involve antisense phosphorodiamidate morpholino oligomers (PMO); peptide conjugation of PMO provides an in vivo delivery method. Small molecule inhibitors of viral replication will include the microtubule inhibitor, colchicine, to prevent cytoskeletal dependent virus transmission to infected cells, or the actin inhibitor, cytochalasin D, to suppress secondary virus spread. Small molecule inhibitors that inhibit key molecules critical to cytoskeletal function will be investigated, e.g. inhibitors of the Rho family of GTPases, which have conserved functions in rearrangements of microfilaments and microtubules. 3d. Improved diagnostics and surveillance methods. Approaches will include: 1) Improved specificity of antibody-based diagnostics for swine viral diseases; 2) Develop and evaluate pen-side diagnostics; 3) Develop and evaluate novel diagnostic tools, e.g. surface-enhanced Raman spectroscopy (SERS); and 4) Enhancing surveillance systems - improving sampling, diagnostic test selection, monitoring protocols, and system security for endemic and emerging viral diseases. Designing surveillance protocols for negative farms is difficult. It involves the selection of the sample specimen (serum, semen, blood, oral fluids, post-mortem samples), sample size, sampling frequency, pooling strategy, and diagnostic test, and will involve operations interactions with Obj.2 scientists. Optimum combination of factors might be different for different such as boar studs, sow farms, nurseries and finishers. The data needed to evaluate surveillance protocols can be obtained by: 1) experiments looking at the effects of individual factors; 2) computer modeling looking at the optimum combination of several well characterized factors; 3) field trials looking at the performance of alternative surveillance protocols compared side by side to current protocols; and 4) studies underway through Obj.2. Methods: While current diagnostic methods are generally very good, both PCR and ELISA assays require monitoring and modification of probes, diagnostic laboratory resources, and can be improved. Moreover, an assay to identify every PRRSV strain is not available. An ideal diagnostic test would not require target labeling, would provide molecular specificity, and would eliminate the need for an amplification step. The emergence of nanotechnology holds the promise of developing biosensors that will allow for the direct, rapid, and sensitive detection of PRRSV; recent evidence indicates that SERS is a novel, reagentless, sensitive, and specific approach for the direct detection of important viral and bacterial pathogens. SERS provides a unique molecular fingerprint of the nucleic acid and protein content, does not require modification to the virus for detection, and will be explored to enhance and facilitate pig virus diagnostics. It is possible to improve the specificity of antibody-based diagnostics for swine viral diseases, and use these improvements to develop and evaluate pen-side PRSSV diagnostics. Development of different assay formats will be based on competitive or noncompetitive principles. Furthermore, the introduction of antibody engineering and phage-displayed antibodies provides greater flexibility in assay and probe, e.g. antibody fragment design which holds great promise within the area of microarray technology. The recent development of non-biological alternatives to antibodies may create distinct opportunities for future improvements in immunoassay technology. Enhancing surveillance systems by improving sampling techniques, as well as diagnostic test selection and monitoring protocols will be evaluated as an aid for endemic and emerging porcine viral diseases.

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 International PRRS Symposium and meetings of the American Association of Swine Veterinarians, the International Pig Veterinary Society, the Iowa State Univ. Swine Health Conference, and the Allen D. Leman Swine Conference.
• Presentations in Spanish and Chinese at meetings, workshops, and symposia in the USA, Mexico and Spain and in China, respectively.
• Biological materials, including infectious clones, PRRS virus strains, purified proteins, monoclonal antibodies, and genetically characterized animals.
• Output 6: Standard methods, protocols, and reagents for serological, immunological, and virological assessments of virus-host swine interactions. <br> <li>Output 7: Bilingual (Spanish, simplified Chinese) educational and training manuals, CDs, pamphlets and literature related to biosecurity, biosecurity implementation and biosecurity monitoring. <br> <li>Output 8: Archived experimental data on a program website where it will be available for data sharing, subject to confidentiality agreements among all participants. <p> <b>The products of the research will affect stakeholders and end users as follows: </b> <br> 1. Swine producers and veterinarians will be able to rapidly apply knowledge, including recommendations and guidelines for PRRS disease prevention, elimination and control, in the field for improved economic outcomes and improved animal well-being. <br> 2. Reduced swine respiratory disease incidence and severity will improve economic well-being of producers. <br> 3. Reduced disease incidence will enhance swine well-being and improve the safety and nutritional value of pork to consumers. <p> <b>Critical points of achievement to indicate progress are:</b> <br> 1. Industry adoption and recommendation of group research findings for field use. <br> 2. Scientific publication of research findings indicative of scientifically advancement in methods of PRRS control. <br> 3. Annual progress reports to the public in the annual International PRRS Symposium. <p>

Outcomes or Projected Impacts

• Production of value-added swine and pork products through the diagnosis and control of PRRSV and emerging viral disease 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 disease agents most impactful on health 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)
• Outcome/Impact 6: Increased capacity of communities and families to enhance their own economic well-being through more profitable management of swine farms. (G5.1) <br> Outcome/Impact 7: Increased capacity of communities, families and individuals to improve their own quality of life and job satisfaction by raising healthier pigs. (G5.2) <p>

Milestones

(2009): <p> <b>Objective 1.</b> Develop in vitro systems for analysis of molecular pathways and cellular interactions relevant to response to and prevention of respiratory infection. <p> <b>Objective 2.</b> Establish methods to describe factors influencing virus transmission within and between swine farms. <p> <b>Objective 3. </b> Engineer novel vaccine constructs for expression of PRRSV ORFs (DNA vaccine and recombinant proteins).

(2010): <p> <b>Objective 1.</b> Validate in vitro systems relevant to dissection and description of effective host response to respiratory infection. <p> <b>Objective 2. </b>Evaluate factors influencing virus transmission within and between swine farms and their relative economic impact. <p> <b>Objective 3. </b> Express recombinant PRRSV antigens. Generation viral vectors expressing different PRRSV antigenic combinations. Evaluate host gene requirements for PRRSV replication

(2011): <p> <b>Objective 1. </b> Deduce innate cellular response mechanisms and pathways controlling PRRSV and PCV2 infection. <p> <b>Objective 2. </b> Identify mechanisms by which pathogens enter, circulate and persist in swine herds. <p> <b>Objective 3. </b> Test adjuvanted recombinant PRRSV antigens and viral vectors. Confirm gene and non-coding RNA targets

(2012): <p> <b>Objective 1. </b> Deduce PRRSV and PCV2 mechanisms of pathogenesis in relevant in vitro systems, and evaluate adaptive immune response mechanisms and pathways controlling PRRSV and PCV2 infection. <p> <b>Objective 2. </b> Identify methods to effectively prevent or control viral pathogens in swine herds. <p> <b>Objective 3. </b> Evaluate molecular adjuvants with PRRSV DNA or vectored vaccines. Evaluate pen-side diagnostics

(2013): <p> <b>Objective 1. </b> Analyze in the in vitro systems co-variable interactions of secondary viral and/or bacterial pathogens implicated in polymicrobial disease. <p> <b>Objective 2. </b> Identify methods to effectively prevent or control virus transmission within and between swine farms and quantitate their relative economic impact. <p> <b>Objective 3. </b> Test alternative delivery strategies and adjuvants with DNA vaccines. Develop host gene target therapies and/or genetic solutions for pig respiratory infections. Enhance surveillance systems.

Projected Participation

View Appendix E: Participation

Outreach Plan

The NC229 Committee members will develop educational outreach tools for disseminating information through established outreach and extension networks to producers, veterinarians, educators, and researchers. These will be coordinated and directed through the NPB and Kansas State Univ. using existing NPB and PRRS CAP2 educational resources and outreach channels. As specific knowledge is acquired on topics of PRRS and PCV2 control, elimination, diagnosis and prevention, 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 NC229 meeting and affiliated international symposium, particularly at the International PRRS Symposium. This renewal includes international participants in China, Mexico and Spain. This expansion will help to expand opportunities for material interchanges, research planning and sharing of resources, and outreach. These interactions will help to establish new collaborative international research programs on emerging threats of swine viral diseases.

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. A host response database will be available through the PRRS Host Genetics Consortium and CAP2 to store and interrogate data on host genetics and host phenotypic responses to infection or vaccination.

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 PRRS Coordinated Agricultural Program 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 of PRRS-CAP will provide advice, counsel and oversight on matters of management, direction, and science. These groups will meet and deliberate at the time of the NC-229 annual meeting.

Literature Cited

1. Ait-Ali T, Wilson AD, Westcott DG, Clapperton M, Waterfall M, Mellencamp MA, Drew TW, Bishop SC, Archibald AL. 2007. Innate immune responses to replication of porcine reproductive and respiratory syndrome virus in isolated Swine alveolar macrophages. Viral Immunol. 20:105-18.

2. Albina E, Madec F, Cariolet R, Torrison J. 1994. Immune response and persistence of the porcine reproductive and respiratory syndrome virus in infected pigs and farm units. Vet Rec. 134:567-73.

3. Allan GM, McNeilly F, Ellis J, Krakowka S, Meehan B, McNair, I, Walker I, Kennedy S. 2000b. Experimental infection of Colostrum Deprived Piglets with Porcine Circovirus 2 (PCV2) and Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) Potentiates PCV2 Replication. Arch Virol, 145: 2421-9.

4. Allan GM, McNeilly F, Ellis J, Krakowka S, Botner A., McCullough K, Nauwynck H, Kennedy S, Meehan B, Charreyre C. 2004. PMWS: Experimental Model and Co-Infections. Vet Microbiol, 98: 165-8.

5. Almazán F, González JM, Pénzes Z, Izeta A, Calvo E, Plana-Durán J, Enjuanes L. 2000. Engineering The Largest RNA Virus Genome As An Infectious Bacterial Artificial Chromosome. Proc. Natl. Acad. Sci. USA. 97: 5516-5521.

6. Alonso S, Izeta A, Sola I, Enjuanes L. 2002. Transcription Regulatory Sequences and mRNA Expression Levels in the Coronavirus Transmissible Gastroenteritis Virus. J Virol. 76: 1293-1308.

7. Andreyev VG, Wesley RD, Mengeling WL, Vorwald AC, Lager KM. 1997. Genetic variation and phylogenetic relationships of 22 porcine reproductive and respiratory syndrome virus (PRRSV) field strains based on sequence analysis of open reading frame 5. Arch Virol.142: 993-1001.

8. Ansari IH, Kwon BJ , Osorio FA, Pattnaik AK. 2006. Influence of N-Linked Glycosylation of PRRS Virus GP5 on Virus Infectivity, Antigenicity, and Ability to Induce Neutralizing Antibodies. J Virol. 80: 3994-4004. 31

9. Batista L, Dee SA, Rossow KD, Deen J, Pijoan C. 2002. Assessing the duration of persistence and shedding of porcine reproductive and respiratory syndrome virus in a large population of breeding-age gilts. Can J Vet Res. 66:196-200.

10. Batista L, Dee SA, Rossow KD, Polson DD, Xiao Z, Olin M, Molitor TW, Murtaugh MP, Pijoan C. 2004. Detection of porcine reproductive and respiratory syndrome virus in pigs with low positive or negative ELISA s/p ratios. Vet Rec 154:25-26.

11. Bautista EM, Molitor TW. 1997. Cell-mediated immunity to porcine reproductive and respiratory syndrome virus in swine. Viral Immunol. 10:83-94.

12. Benfield DA, Christopher-Hennings J, Nelson EA, et al. 1997. Persistent fetal infection of porcine reproductive and respiratory syndrome (PRRS) virus. Proc AASV 455-458.

13. Benfield DA, Nelson E, Collins JE, Harris L, Goyal SM, Robison D, Christianson WT, Morrison RB, Gorcyca D, Chladek D. 1992. Characterization of swine infertility and respiratory syndrome (SIRS) virus (Isolate ATCC VR-2332). J Vet Diag Invest 4:127-133.

14. Benson J E, Yaeger MJ, Christopher-Hennings J, Lager K, Yoon K-J. 2002. A comparison of virus isolation, immunohistochemistry, fetal serology, and reverse-transcription polymerase chain reaction assay for the identification of porcine reproductive and respiratory syndrome virus transplacental infection in the fetus. J Vet Diagnostic Investigation 14:8-14.

15. Bierk MD, Dee SA, Rossow KD, Collins JE, Guedes MI, Pijoan C and Molitor TW. 2001. Diagnostic investigation of chronic PRRS virus infection in a breeding herd of Pigs. Veterinary Record 148: 687-690.

16. Botner A, Strandbygaard B, Sorensen KJ, Have P, Madsen KG, Madsen S, Alexandersen S. 1997. Appearance of acute PRRS-like symptoms in sow herds after vaccination with a modified live PRRS vaccine. Vet Rec 141:497-499.

17. Cano JP, Murtaugh MP, Dee SA. 2007. Evaluation of the survival of porcine reproductive and respiratory syndrome virus in non-processed pig meat. Vet Rec. 160:907-8.

18. Carman S, Cai HY, DeLay J, Youssef SA, McEwen BJ, Gagnon CA, Tremblay D, Hazlett M, Lusis P, Fairles J, Alexander HS, van Dreumel T. 2008. The emergence of a new strain of porcine circovirus-2 in Ontario and Quebec swine and its association with severe porcine circovirus associated disease--2004-2006. Can J Vet Res. 72: 259-68. 12

19. Cha S-H, Chang C-C, Yoon K-J. 2004. Instability of ORF5 RFLP pattern of PRRS virus during sequential pig-to-pig passages. J Clin Microbiol 42:4462-4467.

20. Cha S-H, Choi E-J, Park J-H, Yoon S-R, Song J-Y, Kwon J-H, Song H-J, Yoon K-J. 2006. Molecular characterization of recent Korean PRRS (PRRS) viruses and comparison to other Asian PRRS viruses. Vet Microbiol 117: 248-257.

21. Cheung AK, Lager KM, Kohutyuk OI, Vincent AL, Henry SC, Baker RB, Rowland RR, Dunham AG. 2007. Detection of two porcine circovirus type 2 genotypic groups in United States swine herds. Arch Virol. 152: 1035-44. 9

22. Cho JG and Dee SA. 2006. Porcine reproductive and respiratory syndrome. Theriogenology, 66:655-662.

23. Cho JG, Dee SA, Deen J, Murtaugh MP, and Joo HS. 2007. An evaluation of isolate pathogenicity on the transmission of porcine reproductive and respiratory syndrome virus by aerosols. Can J Vet Res 71:23-27.

24. Cho JG, Dee SA, Deen J, Trincado C, Fano E, Murtaugh MP, Collins JE, Joo HS. 2006. An evaluation of different variables on the shedding of PRRS virus in aerosols. Can J Vet Res 70: 297-301.

25. Christopher-Hennings J, Dammen M, Nelson E, Rowland RRR, Oberst R. 2006. Comparison of RNA extraction methods for the detection of PRRS virus from boar semen. J Virol Methods 136: 248-253.

26. Christopher-Hennings J, Nelson EA, Althouse GC, Lunney JK. 2007. Comparative antiviral and proviral factors in semen and vaccines for preventing viral dissemination from the male reproductive tract and semen. Animal Health Research Reviews. 8: 1-11.

27. Christopher-Hennings J, Nelson EA, Hines RJ. 1995. Persistence of porcine reproductive and respiratory syndrome virus in serum and semen of adult boars. Journal Veterinary Diagn Invest 7:456-464.

28. Cox MM. 2008. Progress on baculovirus-derived influenza vaccines. Curr Opin Mol Ther. 10: 56-61.

29. Dee SA, Batista L, Deen J, Pijoan C. 2005. An evaluation of an air filtration system for the prevention of porcine reproductive and respiratory syndrome virus transmission by aerosols Can J Vet Res 69:293-298.

30. Dee SA, Batista L, Deen J, Pijoan C. 2005. An evaluation of an air filtration system for the prevention of PRRS virus transmission by aerosols. Can J Vet Res 69: 293-298.

31. Dee SA, Deen J, Batista L, Pijoan C. 2006. An evaluation of alternative systems for reducing the transmission of PRRS virus by aerosols. Can J Vet Res 70: 28-33.

32. Dee SA, Deen J, Cano JP, Batista L, Pijoan C. 2006. Further evaluation of alternative air filtration systems for reducing the transmission of PRRS virus by aerosols. Can J Vet Res 70: 168-175.

33. Dee SA, Deen J, Pijoan C. 2005. Evaluation of disinfectants for the sanitation of porcine reproductive and respiratory syndrome virus-contaminated transport vehicles at cold temperatures. Can J Vet Res 69:64-70.

34. Dee SA, Deen J, Pijoan C. 2006. An evaluation of an industrybased sanitation protocol for PRRSV-contaminated transport vehicles. Swine Health Prod 14: 126-132.

35. Dee SA, Deen J, Pijoan C. 2006. An evaluation of an industry-based sanitation protocol for full-size PRRSV-contaminated transport vehicles. Swine Health Prod 14: 307-311.

36. Dee S, Deen J, Rossow K, Wiese C, Otake S, Joo HS, Pijoan C. 2002. Mechanical transmission of porcine reproductive and respiratory syndrome virus throughout a coordinated sequence of events during cold weather. Can J Vet Res 66:232-239

37. Dee SA, Jacobson L, Rossow K, Pijoan C. 2005. Laboratory model to evaluate the role of aerosols in the transport of porcine reproductive and respiratory syndrome virus. Vet Rec 156:501-504.

38. Dee SA, Torremorell M, Deen J, Thompson B, Pijoan C. 2005. An evaluation of the Thermo-Assisted Drying and Decontamination (TADD) system for the elimination of porcine reproductive and respiratory syndrome virus from contaminated livestock transport vehicles. Can J Vet Res 68:208-214.
39. Dorr PM, Baker RB, Almond GW, Wayne SR, Gebreyes WA. 2007. Epidemiologic Assessment of Porcine Circovirus Type 2 Coinfection with Other Pathogens in Swine. J Am Vet Med Assoc, 230: 244-50.

40. Drolet R, Larochelle R, Morin M, Delisle B, Magar R. 2003. Detection Rates Of Porcine Reproductive And Respiratory Syndrome Virus, Porcine Circovirus Type 2, and Swine Influenza Virus In Porcine Proliferative and Necrotizing Pneumonia. Vet Pathol, 40: 143-8.

41. Ellis J, Krakowka S, Lairmore M, Haines D, Bratanich A, Clark E, Allan G, Konoby C, Hassard L, Meehan B, Martin K, Harding J, Kennedy S, McNeilly F. 1999. Reproduction of Lesions of Postweaning Multisystemic Wasting Syndrome in Gnotobiotic Piglets. J Vet Diagn Invest, 11: 3-14.

42. Ellis JA, Bratanich A, Clark EG, Allan G, Meehan B, Haines DM, Harding J, West KH, Krakowka S, Konoby C, Hassard L, Martin K, McNeilly F. 2000. Coinfection by Porcine Circoviruses and Porcine Parvovirus in Pigs with Naturally Acquired Postweaning Multisystemic Wasting Syndrome. J Vet Diagn Invest, 12: 21-7.

43. Enjuanes, L., I. Sola, Et Al. 2005. Coronavirus Reverse Genetics And Development Of Vectors For Gene Expression. Coronavirus Replication And Reverse Genetics. L. Enjuanes, Springer. 287: 161-197.

44. Faaberg KS. 2007. Arterivirus Structural Proteins and Assembly. In: The Nidoviruses, chapter 17. Edited by Snjder E, Perlman S, Gallaher T. ASM Press. In Press.

45. 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 Health Production 10:87-88.

46. Fang Y, Faaberg KS, Rowland R, Christopher-Hennings J, Pattnaik AK, Osorio FA, Nelson EA. 2006. Construction of a full-length cDNA infectious clone of a European-like Type 1 PRRSV isolated in the U.S. In: The Nidoviruses: The Control of SARS and Other Nidovirus Diseases. Edited by S. Perlman and K. Holmes, pp. 605-608.

47. Fang Y, Kim D-Y, Ropp S, Steen P, Christopher-Hennings J, Nelson EA, Rowland RRR. 2004. Heterogeneity in Nsp2 of European-like porcine reproductive and respiratory syndrome viruses isolated in the United States. Virus Res 100:229-235.

48. Fang Y, Schneider P, Zhang WP, Faaberg KS, Nelson EA, Rowland RR. 2007. Diversity and evolution of a newly emerged North American Type 1 porcine arterivirus: analysis of isolates collected between 1999 and 2004. Arch Virol. 152:1009-17.

49. Fetzer C, Pesch S, Ohlinger VF. 2006. High risk of false positive results in a widely used diagnostic test for detection of the porcine reproductive and respiratory syndrome virus (PRRSV). Vet Microbiol. 115:21-31.

50. Gagnon CA, Tremblay D, Tijssen P, Venne MH, Houde A, Elahi SM. 2007. The Emergence of Porcine Circovirus 2b Genotype (PCV-2b) in Swine in Canada. Can Vet J. 48: 811-9.

51. Goldberg TL, Hahn NC, Weigel RM, Scherba G. 2000. Genetic, geographical, and temporal variation of porcine reproductive and respiratory syndrome virus in Illinois. J General Virology 81: 171-179.

52. Goldberg TL, Lowe JF, Milburn SM, Firkins LD. 2003. Quasispecies variation of porcine reproductive and respiratory syndrome virus during natural infection. Virology 317:197-207.

53. Grau-Roma L, Segales J. 2007. Detection of Porcine Reproductive And Respiratory Syndrome Virus, Porcine Circovirus Type 2, Swine Influenza Virus and Aujeszky's Disease Virus In Cases Of Porcine Proliferative And Necrotizing Pneumonia (PNP) In Spain. Vet Microbiol, 119: 144-51.

54. Groot Bramel-Verheije MH, Rottier PJM, Meulenberg JJ. 2000. Expression Of A Foreign Epitope By Porcine Reproductive And Respiratory Syndrome Virus. Virology. 278: 380-389.

55. Halbur, P, Rothschild M, Thacker B. 1998. Differences in susceptibility of Duroc, Hampshire, and Meishan pigs to infection with a high-virulence strain (VR2385) of porcine reproductive and respiratory syndrome virus (PRRSV). J Anim Breed Genet. 115:181-189.

56. Have P, Alban L, Drew T, Le Potier M-F, Murtaugh, MP, Nauwynck HJ, Wellenberg G, Wierup M, Sanchez Vizcaino J. 2005. The probability of transmission of Porcine Reproductive and Respiratory Syndrome virus (PRRSv) to naive pigs via fresh meat. The EFSA Journal. 239:1-85.

57. He YX, Hua RH, Zhou YJ, Qiu HJ, Tong GZ. 2007. Interference of porcine reproductive and respiratory syndrome virus replication on MARC-145 cells using DNA-based short interfering RNAs. Antiviral Res. 74: 83-91.

58. Hermann JR, Hoff SJ, Muñoz-Zanzi C, Yoon KJ, Roof M, Burkhardt AC, Zimmerman JJ. 2007. Effect of temperature and relative humidity on the stability of infectious porcine reproductive and respiratory syndrome virus in aerosols. Vet Res 38:81-93.

59. Hermann JR, Hoff SJ, Yoon KJ, Burkhardt AC, Evans RB, Zimmerman JJ. 2006. Optimization of a sampling system for the recovery and detection of airborne PRRS virus and swine influenza virus. Appl Environ Microbiol 72:4811-4818.

60. Hesse R, Kerrigan M, Rowland RR. 2008. Evidence for recombination between PCV2a and PCV2b in the field. Virus Res. 132: 201-7.

61. Horlen KP, Schneider P, Anderson J, et al. 2007. A cluster of farms experiencing severe porcine circovirus associated disease (PCVAD): clinical features and association with the PCV2b genotype. J Swine Health Prod. 15: 270277. 10

62. Horter DC, Pogranichniy RC, Chang CC, Evans R, Yoon K-J, Zimmerman J. 2002. Characterization of the carrier state in porcine reproductive and respiratory syndrome virus infection. Vet Microbiol 86:213-218.

63. Horter DC, Yoon K-J, Zimmerman JJ. 2004. A review of porcine tonsils in immunity and disease. Animal Health Research Reviews 4:143-155.

64. Johnson C, Kliebenstein J, Zimmerman J. 2007. Re-assessment of the economic impact of PRRS on swine production in the USA in the context of rising feed costs. Proceedings International PRRS Symposium. P.16. 2

65. Kim D-Y, Calvert JG, Chang KO, Horlen K, Kerrigan M, Rowland RR. 2007. Expression And Stability Of Foreign Tags Inserted Into Nsp2 Of Porcine Reproductive And Respiratory Syndrome Virus (PRRSV). Virus Research. 128: 106-114.

66. Kim J, Chung HK, Chae C. 2003. Association of Porcine Circovirus 2 with Porcine Respiratory Disease Complex. Vet J, 166: 251-6.

67. Kim J, Ha Y, Chae C. 2006. Potentiation of Porcine Circovirus 2-Induced Postweaning Multisystemic Wasting Syndrome by Porcine Parvovirus is Associated with Excessive Production of Tumor Necrosis Factor-Alpha. Vet Pathol, 43: 718-25.

68. 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 Health and Production 10:251-258.

69. Kleiboeker SB, Schommer SK, Lee S-M, Watkins S, Chittick W, Polson D. 2005. Simultaneous detection of North American and European Porcine reproductive and respiratory syndrome virus using real-time quantitative RT-PCR. J Vet Diagn Invest 17:165-170.

70. Krakowka S, Ellis JA, McNeilly F, Ringler S, Rings DM, Allan G. 2001. Activation of the Immune System is the Pivotal Event in the Production of Wasting Disease in Pigs Infected with Porcine Circovirus-2 (PCV-2). Vet Pathol, 38: 31-42.

71. Krakowka S, Ellis J, McNeilly F, Waldner C, Rings DM, Allan G. 2007. Mycoplasma Hyopneumoniae Bacterins and Porcine Circovirus Type 2 (PCV2) Infection: Induction Of Postweaning Multisystemic Wasting Syndrome (PMWS) in the Gnotobiotic Swine Model of PCV2-Associated Disease. Can Vet J, 48: 716-24.

72. Kyriakis SC, Saoulidis K, Lekkas S, Miliotis ChC, Papoutsis PA, Kennedy S. 2002. The Effects of Immuno-Modulation on the Clinical and Pathological Expression of Postweaning Multisystemic Wasting Syndrome. J Comp Pathol, 126: 38-46.

73. Lager KM, Mengeling WL, Wesley RD. 2003. Strain predominance following exposure of vaccinated and naive pregnant gilts to multiple strains of porcine reproductive and respiratory syndrome virus. Can J Vet Res. 67: 121-7. 16

74. Lee C, Calvert JG, Welch SK, Yoo D. 2005. A DNA-launched reverse genetic system for porcine reproductive and respiratory syndrome virus reveals that homodimerization of the nucleocapsid protein is essential for virus infectivity. Virology 331:47-62.

75. Lee CD, Hodgins G, Calvert JG, Welch SK, Jolie R, Yoo D. 2006. The nuclear localization signal of the PRRS virus nucleocapsid protein modulates viral replication in vitro and antibody response in vivo. Adv. Exp. Med. Biol. 581:145-148.

76. Lee CD, Hodgins G, Calvert JG, Welch SK, Jolie R, Yoo D. 2006. Mutations in the nuclear localization signal of the PRRS virus nucleocapsid protein attenuate virus replication. Virology 346: 238-250 (Cover page article in volume 346, issue 1).

77. Lewis CR, Ait-Ali T, Clapperton M, Archibald AL, Bishop S. 2007. Genetic perspectives on host responses to porcine reproductive and respiratory syndrome (PRRS). Viral Immunol. 20:343-58.

78. Liu, J., Chen, I., Kwang, J., 2005. Characterization of a previously unidentified viral protein in porcine circovirus type 2-infected cells and its role in virusinduced apoptosis. J. Virol. 79, 82628274.

79. Lowe JF, Zuckermann FA, Firkins LD, Schnitzlein WM, Goldberg TL. 2006. Immunological and clinical consequences of exposure to wild type or attenuated PRRS virus in swine under field conditions. J. Am. Vet. Med. Assoc. 228: 1082-8.

80. MacLachlan NJ, Balasuriya UB, Murtaugh MP, Barthold SW, Lowenstine, LJ. 2007. Arterivirus pathogenesis and immune response. In The Nidoviruses, ed. S. Pearlman, ASM Press.

81. Madec F, Rose N, Grasland B, Cariolet R, Jestin A. 2008. Post-weaning multisystemic wasting syndrome and other PCV2-related problems in pigs: a 12-year experience. Transbound Emerg Dis. 55: 273-83.

82. Maldonado J, Segales J, Martinez-Puig D, Calsamiglia M, Riera P, Domingo M, Artigas C. 2005. Identification of Viral Pathogens in Aborted Fetuses and Stillborn Piglets from Cases of Swine Reproductive Failure in Spain. Vet J, 169: 454-6.

83. Meng XJ, Paul PS, Halbur PG. 1994. Molecular cloning and nucleotide sequencing of the 3'-terminal genomic RNA of the porcine reproductive and respiratory syndrome virus. XJ Gen Virol. 75:1795-1801

84. Mengeling WL, Lager KM, Vorwald AC. 1998. Clinical effects of porcine reproductive and respiratory syndrome virus on pigs during the early postnatal interval. Am J Vet Res 59:52-55.

85. Mengeling WL, Lager KM, Vorwald AC, Koehler KJ. 2003. Strain specificity of the immune response of pigs following vaccination with various strains of porcine reproductive and respiratory syndrome virus. Vet Microbiol. 93: 13-24. 17

86. Mengeling WL, Lager KM, Wesley RD, Clouser D F, Vorwald AC, Roof M B. 1999. Diagnostic Implications of concurrent infections with attenuated and virulent strains of porcine reproductive and respiratory syndrome virus. American Journal Veterinary Res. 60: 119 122.

87. Meulenberg JJ, Petersen-den Besten A, De Kluyver EP, Moormann RJ, Schaaper WM, Wensvoort G. 1995. Characterization of proteins encoded by ORFs 2 to 7 of Lelystad virus. Virology. 206:155-63.

88. Meulenberg JJ, Petersen den Besten A, de Kluyver E, van Nieuwstadt A, Wensvoort G, Moormann RJ.1997. Molecular characterization of Lelystad virus. Vet Microbiol. 55:197-202.

89. Misinzo, G., Delputte, P.L., Meerts, P., Lefebvre, D.J., Nauwynck, H.J., 2006. Porcine circovirus 2 uses heparan sulfate and chondroitin sulfate B glycosaminoglycans as receptors for its attachment to host cells. J. Virol. 80, 34873494.

90. Molina R, Hermann J, Rowland RRR, Christopher-Hennings J, Nelson E, Lunney J, Yoon K-J, Zimmerman J. 2005. Management of PRRS persistence: Identification of persistently infected swine. Proc 86th CRWAD, Abstr #172, and Proc 2005 International PRRS Symposium, p. 66.

91. Mondaca-Fernández E, Meyns T, Muñoz-Zanzi C, Trincado C, Morrison RB. 2007. Experimental quantification of the transmission of Porcine reproductive and respiratory syndrome virus. Can J Vet Res. 71:157-6

92. Murtaugh MP, Xiao Z, Zuckermann F. 2002. Immunological responses of swine to porcine reproductive and respiratory syndrome virus infection. Viral Immunol. 15: 533-47. 15

93. Nawagitgul, P., Morozov, I., Bolin, S.R., Harms, P.A., Sorden, S.D., Paul, P.S., 2000. Open reading frame 2 of porcine circovirus type 2 encodes a major capsid protein. J. Gen. Virol. 81, 22812287.

94. Neumann EJ, Kliebenstein JB, Johnson CD, Mabry JW, Bush EJ, Seitzinger AH, Green AL, Zimmerman JJ. 2005. Assessment of the economic impact of porcine reproductive and respiratory syndrome on swine production in the United States. J Am Vet Med Assoc. 227:385-39. 1

95. Nunez A, McNeilly F, Perea A, Sanchez-Cordon PJ, Huerta B, Allan G, Carrasco L. 2003. Coinfection by Cryptosporidium parvum and Porcine Circovirus Type 2 in Weaned Pigs. J Vet Med B Infect Dis Vet Public Health, 50: 255-8.

96. Opriessnig T, Halbur PG, Yu S, Thacker EL, Fenaux M, Meng XJ. 2006b. Effects of the Timing of the Administration of Mycoplasma Hyopneumoniae Bacterin on the Development of Lesions Associated with Porcine Circovirus Type 2. Vet Rec, 158, 149-54.

97. Opriessnig T, Madson DM, Prickett JR, Kuhar D, Lunney JK, Elsener J, Halbur PG. 2008b. Effect of porcine circovirus type 2 (PCV2) vaccination on porcine reproductive and respiratory syndrome virus (PRRSV) and PCV2 coinfection. Vet Microbiol. 131: 103-14.

98. Opriessnig T, Meng XJ, Halbur PG. 2007. Porcine circovirus type 2 associated disease: update on current terminology, clinical manifestations, pathogenesis, diagnosis, and intervention strategies. J Vet Diagn Invest. 19: 591-615. 11

99. Opriessnig T, Thacker E L, Yu S, Fenaux M, Meng XJ, Halbur P G. 2004b. Experimental Reproduction of Postweaning Multisystemic Wasting Syndrome in Pigs by Dual Infection with Mycoplasma Hyopneumoniae and Porcine Circovirus Type 2. Vet Pathol, 41: 624-40.

100. Ostrowski, M., Galeota, J.A., Jar, A.M., Platt, K.B., Osorio, F.A. and Lopez, O.J. 2002. Identification of neutralizing and nonneutralizing epitopes in the porcine reproductive and respiratory syndrome virus GP5 ectodomain. J. Virol. 76, 4241-4250.

101. 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) Coinfections in US Field Cases of Postweaning Multisystemic Wasting Syndrome (PMWS). J Vet Diagn Invest, 14: 515-9.

102. Park JS, Kim J, Ha Y, Jung K, Choi C, Lim JK, Kim SH, Chae C. 2005. Birth Abnormalities in Pregnant Sows Infected Intranasally with Porcine Circovirus 2. J Comp Pathol, 132: 139-44.

103. Patel D, Opriessnig T, Stein DA, Halbur PG, Meng XJ, Iversen PL, Zhang YJ. 2008 Peptide-conjugated morpholino oligomers inhibit porcine reproductive and respiratory syndrome virus replication. Antiviral Res. 77: 95-107.

104. Petry DB, Hall JB, Weber J, Doster AR, Osorio FA, Johnson RK. 2005. Biological responses to PRRSV in pigs of two genetic populations to PRRSV. J An Sci 83:1494-1502.

105. Petry DB, Lunney J, Boyd P, Kuhar D, Blankenship E, Johnson RK. 2007. Differential immunity in pigs with high and low responses to porcine reproductive and respiratory syndrome virus infection. J Anim Sci. 85:2075-92.

106. Plagemann PG. 2006 Neutralizing antibody formation in swine infected with seven strains of porcine reproductive and respiratory syndrome virus as measured by indirect ELISA with peptides containing the GP5 neutralization epitope. Viral Immunol. 19:285-93.

107. Plagemann PGW, Faaberg KS, Han J. 2006. Neutralizing antibody responses of pigs infected with natural GP5 N-glycan mutants of PRRS virus. Arch. Virol., 19: 294-304.

108. Plagemann PG, Rowland RR, Faaberg KS. 2002. The primary neutralization epitope of porcine respiratory and reproductive syndrome virus strain VR-2332 is located in the middle of the GP5 ectodomain. Arch Virol. 147(12):2327-47.

109. Pogranichniy RM, Yoon KJ, Harms PA, Sorden SD, Daniels M. 2002. Case-Control Study on the Association of Porcine Circovirus Type 2 and Other Swine Viral Pathogens with Postweaning Multisystemic Wasting Syndrome. J Vet Diagn Invest, 14: 449-56.

110. Quintana J, Segales J, Rosell C, Calsamiglia M, Rodriguez-Arrioja GM, Chianini F, Folch JM, Maldonado J, Canal M, Plana-Duran J, Domingo M. 2001. Clinical And Pathological Observations on Pigs with Postweaning Multisystemic Wasting Syndrome. Vet Rec, 149: 357-61.

111. Ramamoorthy S, Meng XJ. 2008. Porcine circoviruses: a minuscule yet mammoth paradox. Anim Health Res Rev. Sep 2:1-20.13

112. Ran Z, Chen X, Guo X, Ge XN, Yoon KJ, Yang HC. 2008. Recovery of Viable Porcine Reproductive and Respiratory Syndrome Virus from an Infectious Clone Containing a Partial Deletion within the Nsp2-Encoding Region. Archives Virology 153: 899-907.

113. Reicks DL, Munoz-Zanzi C, Mengeling B, Christopher-Hennings J, Lager K, Polson D, Dee S, Rossow K. 2006. Timeline of PRRS virus detection in boars during the first six days after infection. J Swine Health Prod. 14: 35-41.

114. Ropp SL, Fang Y, Mahlum Wees CE, Nelson EA, Rossow KD, Bien M., Arndt B, Preszler S, Steen P, Christopher-Hennings J, Collins JE, Benfield DA, Faaberg KS. 2004. Characterization of emerging European-like PRRSV isolates in the United States. J Virol 78:3684-3703.

115. Rovira A, Clement T, Christopher-Hennings J, Thompson B, Engle M, Reicks D, Muñoz-Zanzi C. 2007. Evaluation of the sensitivity of reverse-transcription polymerase chain reaction to detect porcine reproductive and respiratory syndrome virus on individual and pooled samples from boars. J Vet Diagn Invest. 19:502-9.

116. Rovira A, Reicks D, Muñoz-Zanzi C. 2007. Evaluation of surveillance protocols for detecting porcine reproductive and respiratory syndrome virus infection in boar studs by simulation modeling. J Vet Diagn Invest. 19:492-501.

117. Rompato GE, Ling HX, Chen Z, Van Kruiningen H, Garmendia AE. 2006. Positive inductive effects of IL2 on virus specific cellular responses elicited by a PRRSV ORF7 DNA vaccine in swine. Vet Immunol Immunopathol. 109: 151-60.

118. Rowland RRR, Even C, Anderson GW, Chen Z, Hu B, Plagemann PGW. 1994. Neonatal infection of mice with lactate dehydrogenase-elevating virus (LDV) results in suppression of humoral antiviral immune response but does not alter the course of viremia, polyclonal activation of B cells and immune complex formation. J Gen Virol 75:1071-1079.

119. Rowland RRR, Lawson S, Rossow K and Benfield DA. 2003. Lymphotropism of porcine reproductive and respiratory syndrome virus replication during persistent infection of pigs originally exposed to virus in utero. Vet Micro 96: 219-235.

120. Roy P, Noad R. 2008. Virus-like particles as a vaccine delivery system: myths and facts. Hum Vaccin. 4: 5-12.

121. Sang Y, Ross CR, Rowland RR, Blecha F. 2008. Toll-like receptor 3 activation decreases porcine arterivirus infection. Viral Immunol. 21: 303-13.

122. Segalés J, Allan GM, Domingo M. 2005. Porcine circovirus diseases. Anim Health Res Rev. 6: 119-42. 7

123. Sola I, Alonso S, Zúñiga S, Balasch M, Plana-Durán J, Enjuanes L. 2003. Engineering Transmissible Gastroenteritis Virus Genome As An Expression Vector Inducing Lactogenic Immunity. J. Virol. 77: 4357-4369.

124. Sorden, S.D., 2000. Update on porcine circovirus and post-weaning multisystemic wasting syndrome (PMWS). J. Swine Health Prod. 8: 133-136. 8

125. Tian K, Yu X, Zhao T, et al. 2007. Emergence of fatal PRRSV variants: unparalleled outbreaks of atypical PRRS in China and molecular dissection of the unique hallmark. PLoS ONE. Jun 13;2(6):e526. 5

126. Trincado C, Dee SA, Jacobson L, Otake S, Pijoan C. 2006. An evaluation of an all glass impinger (AGI-30) for the detection of PRRS virus in natural and artificial aerosols. Vet Rec 158:206-208.

127. Trincado C, Dee SA, Jacobson L, Otake S, Rossow K, Pijoan C. 2004. Attempts to transmit porcine reproductive and respiratory syndrome virus by aerosols under controlled field conditions. Vet Rec 154:294-297.

128. Trincado C, Dee SA, Rossow KD, Halvorson D, Pijoan C. 2004. Transmission of porcine reproductive and respiratory syndrome virus by non-porcine vectors: A re-evaluation of Mallard ducks. Vet Rec 154:233-237.

129. Vincent A, Thacker BJ, Halbur PG, Rothschild MF, Thacker EL. 2005. In vitro susceptibility of macrophages to porcine reproductive and respiratory syndrome virus varies between genetically diverse lines of pigs. Viral Immunology 18(3):506-512.

130. Vincent AL, Thacker BJ, Halbur PG, Rothschild MF, Thacker EL. 2006. An investigation of susceptibility to porcine reproductive and respiratory syndrome virus between two genetically diverse commercial lines of pigs. J Animal Science 84:49-57.

131. Wang Y, Liang Y, Han J, Burkhart KM, Vaughn EM, Roof MB, Faaberg KS. 2008. Attenuation of porcine reproductive and respiratory syndrome virus strain MN184 using chimeric construction with vaccine sequence. Virology. 371: 418-29.

132. Wasilk A, Callahan J, Christopher-Hennings J, Gay BT, Fang Y, Dammen M, Torremorell M, Polson D, Mellencamp M, Nelson EA, Nelson W. 2004. Detection of U.S. and Lelystad/European-like porcine reproductive and respiratory syndrome virus and relative quantitation in boar semen and serum by real-time PCR. J Clin Micro 42:4453-4461.

133. Welch SK, Jolie J, Pearce DS, Koertje WD, Fuog E, Shields SL, Yoo D, Calvert JG. 2004. Construction and evaluation of genetically-engineered replication-incompetent porcine reproductive and respiratory syndrome virus vaccine candidates. Vet Immunol Immunopathol 102:277-290.

134. Wensvoort G, Terpstra C, Pol JMA, et al. 1991. Mystery swine disease in the Netherlands: the isolation of Lelystad virus. The Vet Quarterly 13:121-130. 3

135. Wieczorek-Krohmer M, Weiland F, Conzelmann K, Kohl D, Visser N, van Woensel P, Thiel HJ, Weiland E. 1996. Porcine reproductive and respiratory syndrome virus (PRRSV): monoclonal antibodies detect common epitopes on two viral proteins of European and U.S. isolates. Vet Microbiol.51:257-66.

136. 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 Microbiology 41(1):58-62.

137. Wootton S, Yoo D. 2000. Full-length genomic sequence of the Canadian PA8 porcine reproductive and respiratory syndrome virus (PRRSV). Archive Virology 145:2297-2323.

138. Yang L, Frey ML, Yoon K-J, Zimmerman JJ, Platt KB. 2000. Categorization of North American porcine reproductive and respiratory syndrome viruses: Epitopic profiles of the N, M, GP5, and GP3 proteins and susceptibility to neutralization. Archive Virology 145: 1599-1619.

139. Yoo D, Welch SK, Lee C, Calvert JG. 2004. Infectious cDNA clones of porcine reproductive and respiratory syndrome virus and their potential as vaccine vectors. Vet Immunol Immunopathol. 102: 143-54.

140. Yuan S, Murtaugh MP, Faaberg KS. 2000. Heteroclite subgenomic RNAs are produced in porcine reproductive and respiratory syndrome virus infection. Virology. 275: 158-69.

141. Zhou YJ, Hao XF, Tian ZJ, Tong GZ, Yoo D, An TQ, Zhou T, Li GX, Qiu HJ, Wei TC, Yuan XF. 2008. Highly virulent porcine reproductive and respiratory syndrome virus emerged in China. Transbound Emerg Dis. 55: 152-64. 6

142. Zimmerman J. 2005. PRRS research update. Proceedings 13th ISU Swine Disease Conference for Swine Practitioners, pp. 146-148.

143. Zimmerman J. 2006. An update on PRRSV diagnosis, prevention, and control. Proceedings 19th IPVS, 1:18-25.

Attachments

Land Grant Participating States/Institutions

CT, IA, IL, KS, MD, MN, ND, NE, OH, SD, VA, WI, WY

Non Land Grant Participating States/Institutions

China, CIAD, A.C., CSIC, Iowa State University - College of Vet Med, Spain, University of Georgia, University of Missouri - Columbia, USDA-APHIS, USDA-ARS, USDA-ARS Beltsville Agricultural Resarch Center, USDA-ARS/Iowa, USDA/NADC