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United States Department of Agriculture

Agricultural Research Service

Research Project: SWINE VIRAL DISEASES PATHOGENESIS AND IMMUNOLOGY
2009 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 project plan involves 4 objectives:.
1)Identify mechanisms of PRRS virus (PRRSV) pathogenesis to develop vaccination strategies to enhance or improve immunity against PRRSV. .
2)Identify mechanisms of SI virus (SIV) pathogenesis and develop vaccination strategies to enhance or provide broad cross-protection for circulating subtypes of SIV. .
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. .
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. In meeting objective 1 of our project plan we have been reverse engineering various mutations in to selected strains of PRRSV to identify virulence factors contributing to the disease caused by this virus. To support these investigations we have developed immunological assessments of host gene expression, host cytokine production and B-cell activation to study the effects of these mutations on the pig's immune system as part of the pathogenesis of disease. In meeting objective 2 of our project plan, we have investigated the pathogenesis and transmissibility of the 2009 A/H1N1 influenza pandemic virus in pigs. We developed rapid differential diagnostic PCR tests to detect and differentiate the pandemic virus from endemic North American SIV strains. We have also assessed the efficacy of several commercial SIV vaccines and an autogenous vaccine against this novel virus. In addition, we made progress in developing a novel swine H1N1 vaccine and in assessing novel adjuvants with experimental SIV vaccines. In meeting objective 3 of our project plan, we have constructed 8 recombinant adenoviruses containing various constructs of the capsid and replicase genes of PCV that were engineered. These viruses will be used to generate antibodies and tested as potential vaccine candidates against PCV2. We also developed a full-length genomic clone of a new swine virus, PPV4, from lungs of pigs exposed to tissue inoculums derive from the 2005 USA PCVAD outbreak. Phylogenetic analysis showed that PPV4 is a novel virus with limited nucleotide sequence homology to other known parvoviruses. Additionally, because this virus has been recalcitrant to growth in tissue culture, we engineered a head-to tail tandem construct of PPV4 that has all the ORFs intact and is expected to yield infectious viruses after transfection into an appropriate cell line. In meeting objective 4 of our project plan, we assessed the capacity of the PRRSV nucleoprotein to induce polyclonal activation of porcine B-cells in vitro, additional studies are needed.


4.Accomplishments
1. Determined the Safety of Meat from Pigs Infected with the 2009 Pandemic A/H1N1 Influenza Virus. Following the announcement of 2009 Pandemic A/H1N1 influenza virus in humans, U.S. pork export markets were severely damaged as U.S. pork was banned in several countries. With >20% of U.S. pork marketed as exports this was a huge negative economic impact on U.S. pork producers. NADC scientists conducted viral pathogenesis studies with the 2009 Pandemic A/H1N1 influenza virus in pigs and found that the virus is restricted to tissues of the respiratory tract and is not present in muscle tissues. This research resulted in several countries lifting their ban on U.S. pork exports.

2. Developed Diagnostic Tests to Detect and Differentiate the 2009 Pandemic A/H1N1 Influenza Virus from Endemic North American Swine Influenza Virus Isolates. The 2009 Pandemic A/H1N1 influenza virus has gene segments of swine origin but some of these gene segments are quite genetically distinct from current circulating North American swine influenza strains, thus making some standard diagnostic tests used in veterinary diagnostic labs unable to detect this pandemic virus should it be present. The NADC scientists and collaborators at the USDA-ARS' Southeast Poultry Research Laboratory (SEPRL) developed three different diagnostic tests that are capable of both detecting and differentiating the 2009 Pandemic A/H1N1 influenza virus from current circulating endemic North American influenza viruses. These diagnostic tests have been transferred to the APHIS diagnostic laboratories and are already being used successfully in the testing of endemic influenza isolates to ensure the 2009 Pandemic A/H1N1 influenza virus is not circulating in U.S. swine.

3. Identification and Molecular Cloning of a Novel Porcine Parvovirus. In late 2005, sporadic cases of an acute onset disease of high mortality were observed in 10- to 16-week-old growing pigs among several swine herds across the United States. Following our initial investigations into epidemics of this syndrome where we discovered a PCV2 strain not previously recognized in North America, we were able to detect the presence of a novel parvovirus among tissues from pigs with this syndrome. ARS scientists in Ames, Iowa, in cooperation with scientists from Washington University in St. Louis, Missouri, identified and cloned a novel porcine parvovirus, PPV4. PPV4 was identified in a co-infection with porcine circovirus type 2 (PCV2) from the 2005 USA field cases of severe PCV2-associated disease (PCVAD). PCV2 has been associated with multiple disease syndromes in swine and is the primary agent leading to PCVAD. It has been reported that other pathogens enhance the severity of PCVAD during co-infection with PCV2. Phylogenetically, PPV4 is highly divergent from the three previously identified porcine parvoviruses and exhibits limited similarity to its closest relative bovine parvovirus 2. Potentially, PPV4 may cause disease on its own or it may enhance the disease phenomenon of PCVAD. Therefore, the detection of PPV4 among diseased pigs that exhibit PCVAD warrants further investigation into the pathogenic nature of PPV4.

4. Compared Pathogenicity of Pseudorabies Virus Strains Isolated from Feral Swine in the U.S. Against Former Domestic Swine Strains of Pseudorabies Virus. Feral swine in the United States are infected with pseudorabies virus; a virus that has been eradicated from commercial swine herds in the U.S. The introduction of potentially novel pseudorabies strains into commercial herds is a concern and diagnostic tests are needed. At the request of APHIS-Veterinary Services, NADC scientists and their collaborators conducted pathogenesis studies in swine using domestic and feral swine isolates of pseudorabies virus. Tissues obtained from these pathogenesis studies are being provided to APHIS to support validation of diagnostic tests to detect feral swine strains of pseudorabies should they spill over into commercial swine production systems in the United States.


6.Technology Transfer

Number of Active CRADAs1
Number of the New/Active MTAs (providing only)7

Review Publications
Miller, L.C., Lager, K.M., Kehrli, Jr., M.E. 2009. Role of Toll-Like Receptors in Activation of Porcine Alveolar Macrophages by Porcine Reproductive and Respiratory Syndrome Virus. Clinical and Vaccine Immunology. 16(3):360-365.

Vincent, A.L., Swenson, S.L., Lager, K.M., Gauger, P.C., Loiacono, C., Zhang, Y. 2009. Characterization of an Influenza A Virus Isolated from Pigs During an Outbreak of Respiratory Disease in Swine and People at a County Fair in the United States. Veterinary Microbiology. 137(1-2):51-59.

Cheung, A.K. 2009. Homologous Recombination within the Capsid Gene of Porcine Circovirus Type 2 Subgroup Viruses via Natural Co-Infection. Archives of Virology. 154(3):531–534.

Ludemann, L.R., Lager, K.M. 2008. Porcine Reproductive and Respiratory Syndrome. In: OIE Biological Standards Commission, editor. Manual of Diagnostic Tests and Vaccines for Terrestrial Animals 2008: Mammals, Birds and Bees. 6th edition. Paris: Office International Des Epizooties. Volume 2. Chapter 2.8.7. p. 1116-1127.

Weingartl, H.M., Albrecht, R.A., Lager, K.M., Babiuk, S., Marszal, P., Neufeld, J., Embury-Hyatt, C., Lekcharoensuk, P., Tumpey, T.M., Garcia-Sastre, A., Richt, J.A. 2009. Experimental Infection of Pigs with the Human 1918 Pandemic Influenza Virus. Journal of Virology. 83(9):4287-4296.

Han, J., Liu, G., Wang, Y., Faaberg, K.S. 2007. Identification of nonessential regions of the nsp2 replicase protein of porcine reproductive and respiratory syndrome virus strain VR-2332 for replication in cell culture. Journal of Virology. 81(18):9878-9890.

Wang, Y., Liang, Y., Han, J., Burkhart, K.M., Vaughn, E.M., Roof, M.B., Faaberg, K.S. 2008. Attenuation of porcine reproductive and respiratory syndrome virus strain MN184 using chimeric construction with vaccine sequence. Virology. 371(2):418-429.

Brockmeier, S., Lager, K.M., Grubman, M.J., Brough, D.E., Ettyreddy, D., Sacco, R.E., Gauger, P.C., Loving, C.L., Vorwald, A.C., Kehrli Jr, M.E., Lehmkuhl, H.D. 2009. Adenovirus-Mediated Expression of Interferon-Alpha Delays Viral Replication and Reduces Disease Signs in Swine Challenged with Porcine Reproductive and Respiratory Syndrome Virus. Viral Immunology. 22(3):173-180.

Loving, C.L., Brockmeier, S.L., Vincent, A.L., Lager, K.M., Sacco, R.E. 2008. Differences in clinical disease and immune response of pigs challenged with a high-dose versus low-dose inoculum of porcine reproductive and respiratory syndrome virus. Viral Immunology. 21(3):315-325.

Last Modified: 9/29/2014
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