Swine Viral Diseases Pathogenesis and Immunology

APPROACH: 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. BSL-2: Recert 10/17/07 #0243, #0244, #0255; 6/08/07 #0268; 7/28/07 #0273; 8/27/07 #0274; 3/13/07 #0279A. BSL-Exempt; Recert 10/17/07, #0254. <P>

PROGRESS: 2006/10 TO 2007/09<BR>
Progress Report Objectives (from AD-416) <BR>
Obj. 1: Identify mechanisms of PRRS virus (PRRSV) pathogenesis to develop vaccination strategies to enhance or improve immunity against PRRSV. <BR>
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. <BR>
Obj. 3: 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. <BR>
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. <BR>
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Approach (from AD-416) For improved PRRSV control, one approach will be to identify strategies for improved immunoprophylaxis by testing various priming strategies using recombinant adenoviruses expressing selected PRRS viral gene constructs to increase the safety and efficacy of PRRS MLV vaccines. For improved SIV control, one approach will be to identify mechanisms of SIV pathogenesis and develop vaccination strategies to enhance or provide broad 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 exploit these findings to identify specific regions within the identified genes that confer growth advantages. Another approach will be to 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 the use of better cross-reacting isolates, novel combinations of adjuvants and/or cytokines and possibly different routes of administration. 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. 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. Accomplishments Title: Isolation and characterization of a new influenza subtype circulating in swine Problem: Swine influenza virus is prone to genetic shift (mixing of gene segments between virus isolates) and genetic shift (mutations) leading to new subtypes of virus causing disease in swine because the new virus is not represented in commercial vaccines. Activity: H2N3 influenza viruses have not previously been isolated from swine. We characterized genetically similar reassortant H2N3 viruses isolated from pigs from two farms in the United States. Sequence analysis revealed the HA, NA, and PA gene segments are similar to those of avian influenza viruses of the American lineage, whereas other segments are similar to those of contemporary swine influenza viruses. A leucine at HA position 226 indicates preferential binding to the mammalian virus receptor. The H2N3 virus was able to replicate in pigs, mice, and ferrets and transmitted among pigs and ferrets. Our findings indicate these can infect various mammalian hosts without adaptation, suggesting they may be transmissible to humans. Impact: These H2N3 viruses appear to have pandemic potential and warrant close monitoring. <BR>
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A manuscript is submitted. Title: Isolation of viruses carrying polymerase genes derived from avian, human and swine influenza viruses Problem: Swine influenza virus is prone to genetic shift (mixing of gene segments between virus isolates) and genetic shift (mutations) leading to new subtypes of virus causing disease in swine because the new virus is not represented in commercial vaccines. Activity: A panel of H1N1 and H3N2 viruses were created using various polymerase gene combinations. These viruses were tested in mice for their pathogenicity. Impact: These studies will aid in understanding critical virulence factors enabling emergence of new SIV strains. Title: Establishment of an H1N1 virus with the NS1d126gene segment Problem: H1N1 swine influenza isolates are a major subtype of virus circulating among swine. Current vaccines are all killed vaccines and there is evidence that killed vaccines are less effective than modified live vaccines. Activity: A classical swine H1N1 virus carrying the NS1d126 gene segment and internal genes from the TX/98 H3N2 viruses was created. Impact: This will enable further studies to examine critical virulence factors in emerging SIV isolates and whether an H1N1 modified live vaccine induces similar heterologous immunity as we found with a similar H3N2 MLV. Title: Generation of specific immunologic reagents and engineering recombinant PCV constructs and infectious PCV clones Problem: Production of antibodies and infectious clones will provide specific reagents that are needed to investigate PCV2 pathogenesis in pigs. Activity: Antibodies against the capsid proteins of PCV1 and PCV2 have been obtained. Antibodies against the Rep and Rep' proteins are being made. Infectious genomic clones of PCV1 and PCV2 have been engineered using the head-to-tail tandem construct methodology. Impact: Antibodies will be used to localize the distribution of each protein in virus-infected cells and infectious clones will be used to investigate virulence factors of PCV2 that may be used in vaccines. Title: Construct stable vectors expressing high-levels of individual PRRSV proteins. Problem: Lack of protection of current vaccines against heterologous PRRSV isolates. Activity: In order to identify antigens that afford better cross protection, efforts from the previous CRIS (057) had engineered 2 PRRSV proteins into rhAd5 vectors. In this reporting period, work was done to establish levels of protein expression. Full-length nucleocapsid protein (N1) from ORF 7 appears to be toxic to cells used in the expression system, hence a deletion mutant (N2) was made that expresses well. An ORF 3 construct expressing GP3 has been made but it expresses a smaller than predicted protein. Both of these expressed proteins N2 and GP3 reacted with sera from pigs infected with the parent strain of PRRSV. Due to the need to re-engineer the N and extensive follow-up work with the smaller than predicted GP3, no attempt was made yet to express ORF 4. Impact: Data derived from evaluating these vectored proteins in future animal PRRSV challenge studies will provide information on the role of these proteins in disease virulence. This research indicates both proteins appear to be recognized by a pig's immune system during a PRRSV infection, thus suggesting a possible role in disease pathogenesis. <BR>
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During the reporting period two new category I scientists were hired to conduct research on PRRSV, thus completing our staffing for this project. The impact of this activity will be a much enhanced research effort towards addressing virulence and pathogenesis questions related to PRRSV in pigs. Addresses: National Program 103 Animal Health Action Plan Component 4: Countermeasures to prevent and control respiratory diseases. Technology Transfer Number of New CRADAS and MTAS: 2 Number of Active CRADAS and MTAS: 2 Number of Invention Disclosures submitted: 1 Number of Patent Applications filed: 1 Number of Non-Peer Reviewed Presentations and Proceedings: 10