2008 Annual Report 1a.Objectives (from AD-416) Obj. 1: Identify mechanisms of PRRS virus (PRRSV) pathogenesis to develop vaccination strategies to enhance or improve immunity against PRRSV. Obj. 2: Identify mechanisms of SI virus (SIV) pathogenesis and develop vaccination strategies to enhance or provide broad cross-protection for circulating subtypes of SIV. Obj. 3: Identify the host-pathogen interactions and environmental factors that lead to PCVAD and discover effective measures to prevent, control, and eliminate this emerging disease from U.S. swine herds. Obj. 4: Develop methods of modulation of innate and adaptive immune responses to swine viral pathogens with an emphasis on modulating the effects of innate immunity on pathogenesis of viral diseases. 1b.Approach (from AD-416) For improved PRRSV control, one approach will identify strategies for improved immunoprophylaxis by testing vaccine strategies with recombinant adenoviruses expressing selected PRRS viral gene constructs to increase safety and efficacy of PRRS vaccines. For improved SIV control, one approach will identify mechanisms of SIV pathogenesis and develop vaccination strategies to enhance cross-protection for circulating subtypes of SIV. We will investigate the role of avian polymerase genes in adaptation of novel reassortant SIVs to pigs. We will study specific regions within identified genes that confer growth advantages. Another approach will maintain a contemporary repository for emerging SIV subtypes and genotypes and combine with novel vaccine approaches for improved SIV vaccines. Vaccine strategies will be developed that have broader subtype coverage through by use of better cross-reacting isolates, novel combinations of adjuvants and/or cytokines and different routes of vaccination. Specific aims are: A) Genetic, antigenic and pathogenic characterization of novel isolates; B) Evaluation of new inactivated vaccines against current isolates; and C) Evaluation of genetically engineered, modified-live vaccines against current isolates. For improved control of PCV type 2, we will conduct research to identify mechanisms of PCV type 2 (PCV2) pathogenesis in PMWS and perform genetic analysis of the replication and virulence mechanisms of PCV2 to develop vaccination strategies against porcine circoviruses. The goal is to develop recombinant virus vaccines against PMWS by attenuation of the viral replication and virulence mechanisms. In addition we will develop and evaluate multiplex diagnostic assays to detect pathogens involved in PCVAD, determine the role of endemic and novel swine viruses in inducing PCVAD, and finally evaluate genetic and biological determinants that lead to PCVAD. Our approach to develop methods for modulation of innate and adaptive immune responses to swine viral pathogens will focus on modulating the effects of innate immunity on pathogenesis of viral diseases. We will evaluate whether the early serum IFN-gamma response is caused by the interaction of PRRSV structural proteins with components of the hosts' immune system. Another approach will be to ameliorate clinical disease through prophylactic or metaphylactic administration of granulocyte-colony stimulating factor in an attempt to reduce the severity or duration of viral pneumonia associated with PRRSV and SIV. Another approach will be to investigate the B cell response to these swine viruses with a focus on immunoglobulin class switch recombination and diversification of the VDJ repertoire. These changes in B cells correlate with the appearance of neutralizing antibody, understanding the virulence mechanisms contributing to the delayed development of neutralizing antibody against PRRSV may provide essential insights into the improved control of PRRSV shedding in vaccinated and infected pigs. 3.Progress Report The overall objective is to identify mechanisms of swine viral pathogenesis to develop strategies to enhance or improve immunity against disease caused by current and emerging pathogens. In addition to accomplishments summarized in section 4, we also made additional progress in swine virology. We increased by 2 scientists our staff focused on porcine reproductive and respiratory virus (PRRSV) research. Field isolates of PRRSV with natural deletions in the Nsp2 region have been associated with severe outbreaks of disease. To study the role of Nsp2 as a virulence trait associated with this observation, 4 PRRSV strain VR-2332 mutants that contained unique deletions mimicking field isolates were inoculated into pigs. When compared with wild-type VR-2332 virus, we found that as much as 400AA can be deleted from the Nsp2 region without loss of viability, and deletions in Nsp2 do not always result in a more virulent virus. These findings suggest that if wild-type Nsp2 deletions are associated with an increase in virulence, then there may be mutations elsewhere in the viral genome that contribute to virulence. To improve our infrastructure to perform high-throughput molecular diagnostics, we compared and contrasted DNA/RNA isolation from clinical samples by different methods. Use of magnetic particle processors for this purpose dramatically improves our efficiency in producing molecular information from our directed studies, thus improving our ongoing efforts to develop new, multiplexed diagnostic tests for swine diagnostics. We continue to investigate the effects of porcine viral infections on the host immune response and have increased our focus on the effects of infection on leukocyte transcriptomes. Recently, a H5N1 highly pathogenic avian influenza virus has emerged in waterfowl in other countries that is very deadly in poultry and humans. Swine are susceptible to infection with avian flu viruses raising a question about the consequences of swine becoming infected with such an H5N1 virus. When compared to swine influenza virus isolates, pigs infected with one of four H5N1 HPAIV viruses developed minimal to mild disease. These results suggest swine have a low susceptibility to these H5N1 viruses and may not play a role in their transmission. The U.S. domestic swine herd is free of pseudorabies virus (PRV), a virus that can cause severe disease and death. Feral swine can be infected with PRV and are found in 44 states, thus posing a risk or infection to domestic swine. In order to improve PRV diagnostics, a real-time PCR assay was developed to detect this virus that has the potential to improve current PRV surveillance techniques. We are conducting ongoing collaborative studies with Animal and Plant Health Inspection Service using domestic and feral PRV isolates in experimental studies in pigs to provide necessary tissues for validating this real-time PCR assay, and continue to investigate a novel swine virus recently identified through the use of DNA-microarray technology. Relates to NP103 Animal Health Components 3, 4, and 5: Countermeasures to Prevent and Control: Zoonotic, Respiratory; and Reproductive and Reproductive and Neonatal Diseases. 4.Accomplishments 1. Inactivated influenza vaccine fails to cross-protect against a variant virus challenge. The ability of swine flu vaccines to cross-protect against different field viruses is unpredictable. A killed-virus vaccine prepared from the IA30 virus isolate was used to study cross-protection. Vaccinated pigs were not protected against challenge with the MN03 virus. Moreover, some of the vaccinated pigs had more severe lesions than the non-vaccinated pigs that received challenge virus. When pigs were given IA30 live virus as a vaccine, they were able to mount a protective immune response against MN03 challenge. This study suggests immunity to influenza virus is complex and the type of vaccine virus may be an important factor to improving cross-protection. NP 103 – Animal Health Program Component 4: Prevention and Control of Respiratory Diseases, Problem Statement 4B: Porcine Respiratory Diseases. 2. Demonstrating porcine reproductive and respiratory virus (PRRSV) can negatively modulate the swine immune response. Porcine reproductive and respiratory syndrome (PRRS) is the number one disease concern of the U.S. swine industry. It is caused by the PRRS virus (PRRSV). PRRSV vaccines are commercially available for use in control of this disease; however, they do not fully cross-protect vaccinated swine against all PRRSV strains. Why vaccines do not fully cross-protect is not understood and this phenomenon is one focus of research at the National Animal Disease Center. Animal studies involving germ-free pigs have been conducted to study the direct effect of PRRSV on a pig's immune response to this virus. Our findings show that PRRSV can modulate the host response by diverting the specific antibody response. This diversion takes energy away from the development of a protective immune response; presumably, the diversion is an advantage to the virus since it delays the pig's specific immune response against the virus. Another research focus is to identify the properties of the virus that cause this diversion to occur. The anticipated goal of this work will be to understand the biology of the virus to an extent where specific mutations can be made that disable the diversionary tactics of PRRSV resulting in the improvement of vaccines. NP 103 – Animal Health Program Component 4: Prevention and Control of Respiratory Diseases, Problem Statement 4B: Porcine Respiratory Diseases. 3. Bioinformatic selection and sequence analysis of new PRRSV strains. In 2007 highly pathogenic U.S. PRRSV Type 2 isolates with a novel genetic pattern were detected in North Carolina and southwestern Minnesota. The genetic pattern of both isolates was compared to each other, and to the PRRSV database (http://prrsv.org), a collection of over 8500 field isolates from different areas of the US and Canada. Surprisingly, the two novel US viruses with the same pattern were only 85% similar when evaluated with a more detailed analysis, and each showed no close similarity to PRRSV sequences in the database. This shows the practice of using the genetic pattern to relate viruses has limitations. We also worked with a team of scientists to establish a new protocol for full-length nucleotide sequencing of up to 20 RNA viruses simultaneously. This new protocol, which does not need sequence-specific primers, will be used to unravel the genomes of the novel PRRSV isolates from Vietnam, North Carolina, and Minnesota. The technology will dramatically decrease the time needed for full-length genome nucleotide sequencing of RNA viruses, the only method which can accurately delineate genetic differences. NP 103 – Animal Health Program Component 4: Prevention and Control of Respiratory Diseases, Problem Statement 4B: Porcine Respiratory Diseases. 4. Identified novel changes in gene expression in porcine alveolar macrophages (PAM) infected with PRRSV-VR2332 in vitro. Porcine reproductive and respiratory syndrome virus (PRRSV) is believed to evade a protective immune response by modulating how the pig normally responds to an infection. This presumption is supported by a study that used Serial Analysis of Gene Expression (SAGE) to measure gene expression in porcine alveolar macrophages, the cells in which PRRSV replicates. This research identified gene expression pathways affected by PRRSV infection forming a basis for current research investigating virulence mechanisms of PRRSV. The SAGE library sequence has been submitted to National Center for Biotechnology Information (NCBI) Gene Expression Omnibus. NP 103 – Animal Health Program Component 4: Prevention and Control of Respiratory Diseases, Problem Statement 4B: Porcine Respiratory Diseases. 5. Established a quantitative PCR assay to measure the amount of porcine circovirus (PCV) type 1 and PCV type 2 in a test sample. Two types of Porcine Circovirus (PCV) have been identified, PCV1 and PCV2. Type 1 PCV is non-pathogenic and is found associated with the porcine kidney (PK-15) cell line. PCV2 is widely found in populations of diseased pigs that display a wide variety of clinical signs and lesions. A real-time RT-PCR assay was developed to distinguish PCV1 from PCV2 in the same sample. This PCR assay was found to be a rapid and sensitive technique, which might prove useful to veterinary diagnostic laboratories for routine testing and surveillance of PCV infection in pigs. NP 103 – Animal Health Program Component 4: Prevention and Control of Respiratory Diseases, Problem Statement 4B: Porcine Respiratory Diseases. 6. Nucleotide sequence analysis of PRRSV isolates from Vietnam. PRRSV was isolated from multiple swine tissues submitted to the Foreign Animal Disease Diagnostic Laboratory on Plum Island from Vietnam. We were consulted to assist in the genetic analysis of these viruses. Results showed the viruses isolated had 98.6% or greater genetic identity to recent Chinese PRRSV isolates associated with porcine high fever disease (PHFD) in Asia. After comparison with the PRRSV database we direct (http://prrsv.org), it was clear the PHFD strain of PRRSV has not been detected in the US. The strain characterization and database comparison was extremely beneficial and encouraging knowledge for swine veterinarians and producers. NP 103 – Animal Health Program Component 4: Prevention and Control of Respiratory Diseases, Problem Statement 4B: Porcine Respiratory Diseases. 7. Investigation of a swine influenza virus (SIV) isolated from pigs and people during a 2007 Ohio county fair. Swine influenza viruses in North America have undergone rapid mutation over the last ten years. We characterized an SIV isolate from pigs that also infected at least 2 people during a county fair in 2007. The virus caused severe respiratory disease in pigs, spread from pig to pig, and the pigs were shown to be contagious for up to 7 days after infection. The characteristics of this virus suggest opportunity for exposure of people in contact with pigs infected with this virus. The characteristics of the virus as well as the documented human transmission warrant close monitoring of the spread of this virus in pig and human populations. Subsequent investigations have demonstrated the spread of the Ohio-like viruses to other hog-producing states. NP 103 – Animal Health Program Component 4: Prevention and Control of Respiratory Diseases, Problem Statement 4B: Porcine Respiratory Diseases. 8. PCV2 capsid protein localization in tissue culture cells differ between PCV2 Group-1 and Group-2 viruses. The genetic basis for PCV2 pathogenicity is not known. This work is the first description of two naturally occurring recombinant viruses with chimeric Group-1 and Group-2 capsid proteins. These two recombinants exhibited reciprocal capsid protein localization patterns. This work provides information on the possible mechanism PCV2 pathogenicity and potential targets for generating vaccine candidates. NP 103 – Animal Health Program Component 4: Prevention and Control of Respiratory Diseases, Problem Statement 4B: Porcine Respiratory Diseases. 9. Comparison of a truncated NS1 modified live influenza virus vaccine and inactivated vaccine in pigs with maternally acquired antibody. One of the challenges to control and eliminate swine influenza virus (SIV) in the U.S. is that the currently used inactivated vaccines do not provide adequate cross-protection against multiple antigenic variants of SIV in the field, especially when administered in the presence of maternally derived antibody (MDA). We previously demonstrated that a recombinant A/SW/TX/4199-2/98 virus (TX98), TX98-NS1delta126, was attenuated in swine and showed potential for use as a modified live-virus vaccine (MLV) after intranasal application in pigs. We then compared 1 dose of an intranasal administration of the MLV with 2 doses of an intramuscular administration of an inactivated TX98 vaccine (KV). In addition we compared the efficacy of the two vaccines when given to piglets with TX98 MDA or naïve piglets. Pigs were challenged with wild type homologous TX98 H3N2 virus or with a genetic and antigenic variant H3N2 SIV (influenza A/SW/CO/23619/99 virus, CO99). When 2 doses of inactivated TX98 vaccine were given to piglets with MDA and challenged with the heterologous CO99 virus, a significant enhancement of macroscopic lung lesions was demonstrated at 5 days post infection. This enhancement was not seen in the pigs given MLV in the presence of MDA and challenged with CO99. Although the efficacy of the single-dose MLV was reduced by MDA, it appeared to be safer when compared to inactivated vaccine in our swine influenza model. These results may have implications not only to pigs, but to other influenza virus host species as well. NP 103 – Animal Health Program Component 4: Prevention and Control of Respiratory Diseases, Problem Statement 4B: Porcine Respiratory Diseases. 5.Significant Activities that Support Special Target Populations None. 6.Technology Transfer Number of New CRADAS 1 Number of the New MTA (providing only) 1 Number of New Patent Applications Filed 1 Number of Non-Peer Reviewed Presentations and Proceedings 1 Review Publications Butler, J.E., Wertz, N., Weber, P., Lager, K.M. 2008. Porcine reproductive and respiratory syndrome virus subverts repertoire development by proliferation of germline-encoded B Cells of all isotypes bearing hydrophobic heavy chain CDR3. Journal of Immunology. 180(4):2347-2356. Vincent, A.L., Ma, W., Lager, K.M., Janke, B.H., Webby, R.J., Garcia-Sastre, A., Richt, J.A. 2007. Efficacy of intranasal administration of a truncated NS1 modified live influenza virus vaccine in swine. Vaccine. 25:7999-8009. Ma, W., Vincent, A.L., Gramer, M.R., Brockwell, C.B., Lager, K.M., Janke, B.H., Gauger, P.C., Patnayak, D.P., Webby, R.J., Richt, J.A. 2007. Identification of H2N3 influenza A viruses from swine in the United States. Proceedings of the National Academy of Sciences. 104(52):20949-20954. Quan, P.L., Palacios, G., Jabado, O.J., Conlan, S., Hirschberg, D.L., Pozo, F., Jack, P.J., Cisterna, D., Renwick, N., Hui, J., Drysdale A., Amos-Ritchie, R., Baumeister, E., Savy, V., Lager, K.M., Richt, J.A., Boyle, D.B., Garcia-Sastre, A., Casas, I., Perez-Brena, P., Briese, T., Lipkin, W.I. 2007. Detection of respiratory viruses and subtype identification of influenza A viruses by GreeneChipResp oligonucleotide microarray. Journal of Clinical Microbiology. 45(8):2359-2364. Wesley, R.D., Lager, K.M. 2006. Overcoming maternal antibody interference by vaccination with human adenovirus 5 recombinant viruses expressing the hemagglutinin and the nucleoprotein of swine influenza virus. Veterinary Microbiology. 118:67-75. Vincent, A.L., Lager, K.M., Janke, B.H., Gramer, M.R., Richt, J.R. 2008. Failure of protection and enhanced pneumonia with a US H1N2 swine influenza virus in pigs vaccinated with an inactivated classical swine H1N1 vaccine. Veterinary Microbiology. 126(4):310-323. Lipatov, A.S., Kwon, Y., Sarmento, L., Lager, K.M., Spackman, E., Suarez, D.L., Swayne, D.E. 2008. Domestic pigs have low susceptibility to H5N1 highly pathogenic avian influenza viruses. Public Library of Science for Pathogens [serial online]. 4(7):e1000102. Available: http://www.plospathogens.org/article/info%3Adoi%2F10.1371%2Fjournal.ppat.1000102. Ma, W., Lager, K.M., Richt, J.,A. Stoffregen, W.C., Zhou, F., Janke, B.H., Yoon, K.J.. 2008. Development of real-time polymerase chain reaction assays for rapid detection and differentiation of wild-type pseudorabies and gene-deleted vaccine viruses. Journal of Veterinary Diagnostic Investigation. 20(4):440-447. Childs, J.E., Richt, J.A., Mackenzie, J.S. 2007. Introduction: conceptualizing and partitioning the emergence process of zoonotic viruses from wildlife to humans. In: Childs, J.E., Mackenzie, J.S.; Richt, J.A., editors. Wildlife and Emerging Zoonotic Diseases: The Biology, Circumstances and Consequences of Cross-Species Transmission. Series: Current Topics in Microbiology and Immunology. Berlin Heidelberg: Springer-Verlag. 315:1-31. Webby, R.J., Webster, R.G., Richt, J.A. 2007. Influenza viruses in animal wildlife populations. In: Childs, J.E., Mackenzie, J.S., Richt, J.A., editors. Wildlife and Emerging Zoonotic Diseases: The Biology, Circumstances and Consequences of Cross-Species Transmission. Series: Current Topics in Microbiology and Immunology. Berlin Heidelberg: Springer-Verlag. 315:67-83.