Progress 10/01/09 to 09/30/14
Outputs
Target Audience: Swine veterinarians, pork producers, andresearch scientists working on viral diseases of swine. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? The personnel involved in this project acquired knowledge and experience in the methods of macrophage cell culture as well the methods to assess the replication of a virus in its natural host cell. How have the results been disseminated to communities of interest? The data was presented at scientific meetings and a manuscript for publication on a referred journal is being prepared. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
With the funds provided by this Multi-State project to Dr. Zuckermann, major progress was made on research efforts aimed at defining the mechanism(s) by which porcine reproductive and respiratory virus (PRRSV) is able to inhibit the host interferon (IFN)-a response. The great majority of the research by other investigators has focused on the effect of individual viral proteins on transcription factors involved in the type I IFN response. By using non-PRRSV natural host cells transfected with reporter gene constructs in combination with over-expressed viral proteins, these types of studies have indicated that several nonstructural proteins of PRRSV have the ability to negatively affect the activation of NFkB and IRF-3. Concurrently, there is convincing evidence indicating that live PRRSV is indeed able to hinder the ability of its natural host cell, namely porcine alveolar macrophages (PAMs), to produce IFN-a in response to their stimulation with strong agonists, such as the synthetic analog of dsRNA, poly(I:C). Efforts to ascertain the mechanism(s) by which the infection of PAMs with PRRSV hinders the type I IFN response have been stifled by the fact that only a small fraction of this cell population is susceptible to infection by this virus. Consequently, any effect that PRRSV might have on transcription factor activation resulting from stimulation with poly(I:C) is obscured by the majority of the cells that are responding to this agonist, but are not infected by PRRSV. For this project, we evaluated the effect of PRRSV on the poly(I:C) stimulated activation of the transcription factors IRF-3 and NFkB, IFN-a and IFN-b gene transcription as well as IFN-a secretion by ZMAC cells. The ZMAC cell line is a non-transformed PAM that, as a population, is readily and 100% susceptible to PRRSV infection and has an intact type I IFN response system. Our results demonstrate that infection of ZMAC cells with PRRSV does not inhibit the poly(I:C)-induced activation of NFkB, STAT-1 or IRF-3, nor does it inhibit IFN-a or IFN-ß gene transcription. Nevertheless, the secretion of IFN-a was inhibited by >60% by 9 hours after infection. Notably, PRRSV alone induced the phosphorylation of NFkB but not IRF-3. Accordingly, PRRSV did not induce secretion of IFN-a. Our results indicate that whatever the mechanism is by which PRRSV inhibits the secretion of IFN-a in PAMs, it occurs at the post-transcriptional level.
Publications
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2014
Citation:
Chen, W.-Y., Calzada-Nova, G., Schnitzlein, W. and Zuckermann, F.A. The PRRSV-mediated inhibition of IFN-Beta production by pig alveolar macrophages occurs at the post-transcriptional level via the activation of eIF-2 alpha. Pathobiology Department, University of Illinois at Urbana-Champaign. CRWAD, Chicago, Illinois, December 2014.
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Progress 01/01/13 to 09/30/13
Outputs
Target Audience: Swine veterinarians,pork producers, and scientists working with PRRS virus. Changes/Problems: Funding was significantly reduced for this project. What opportunities for training and professional development has the project provided? The graduate student personnel involved in this project acquired knowledge and experience on methods to measureand assess the biology of a virus during its replication in a host cell. Graduate students also presented their work at scientific meetings which contributed to their professional development. How have the results been disseminated to communities of interest? The data has been published in referred journals and presented at scientific meetings. What do you plan to do during the next reporting period to accomplish the goals? Funding for this project was cut significantly for next year. This will negatively impact the accomplishments of the goals for the next reporting period.
Impacts
What was accomplished under these goals?
(3) Mutations that destroyed the PCPα activities (C76S, H146Y, and C76S/H146Y) in nsp1α did not affect the IFN suppressive activity of nsp1α, indicating that the cysteine protease activity did not participate in IFN suppression. The mutations of C70S, C76S, H146Y, and/or M180I, which coordinated the ZF2 motif also did not alter IFN suppression. However, the mutations of C8S, C10S, C25S, and/or C28S for the ZF1 motif impaired the IFN antagonism of nsp1α, demonstrating that ZF1 was the essential element of nsp1α for IFN suppression. Wild-type nsp1α localized in the both nucleus and cytoplasm, but the ZF1 mutants that lost the IFN suppressive activity did not localize in the nucleus and remained in the cytoplasm. Consistent with their cytoplasmic distribution, CBP was not degraded by these mutants. Bayesian phylogeographic analyses of 7040 ORF5 sequences were used to reveal the recent geographical spread of Type2 PRRSV in North America. To discover the impact ofPRRSV infections on the cellular miRNAome, small RNA expression profiles were developed from PRRSV-infected swine alveolar macrophages (SAMs) in vitro using Illumina deep sequencing. A total of 40 cellular miRNAs were significantly differentially expressed within the first 48 hours post infection (hpi). Six miRNA, miR-30a-3p, miR-132, miR-27b*, miR-29b, miR-146a and miR-9-2, were altered at more than one time point. The most highly repressed miRNA at 24 hpi was miR-147. A miR-147 mimic was utilized to maintain miR-147 levels in PRRSV-infected SAMs. A highly pathogenic PRRS virus with a ORF5 1-22-2 RFLP was isolated in the porcine alveolar macrophage cell line ZMAC from a severe outbreak in a Midwestern sow farm characterized by 6 weeks of 100% pre-wean mortality. A virus stock of the isolated virus (LTX1) was prepared after one passage in ZMAC cells and used for experimental pig inoculation and full genome sequencing by Illumina. Inoculation of 11 week-old pigs with the LTX1 resulted in the development of viremia with similar kinetics and viral load as those observed after inoculation with the “atypical PRRS” strain NADC20. In contrast, the average viral load in the bronchoalveolar lavage collected at 14 days after challenge with LTX1 (3x10e5 TCID50/ml) was 44-fold higher as compared to that in pigs receiving the NDC20 virus (7x10e4 TCID50/ml). Analysis of the genome indicated that nsp2 of the LTX1 virus has the same three discontinuous deletions as the MN184 (corresponding to strain VR-2332 positions 324-434, 486, and 505-532), but also has a novel 5 amino acid deletion corresponding to positions 464-468 as well as numerous unique single mutations.
Publications
- Type:
Journal Articles
Status:
Accepted
Year Published:
2013
Citation:
Hicks, J.A., Yoo, D. and Liu, H.C. Characterization of changes in microRNA expression and function in porcine reproductive and respiratory syndrome virus (PRRSV)-infected macrophages. PLoS One (In press).
- Type:
Journal Articles
Status:
Accepted
Year Published:
2013
Citation:
Shi, M., P. Lemey, M.S. Brar, M.A. Suchard, M.P. Murtaugh, S. Carman, S. DAllaire, B. Delisle, M. Lambert, C.A. Gagnon, L. Ge, D. Yoo, E.C. Holmes and F.C. Leung. The spread of type 2 porcine reproductive and respiratory syndrome virus (PRRSV) in North America: A phylogeographic approach. Virology 447:146-154 (This article was chosen and featured as a virology highlight http://www.virologyhighlights.com/?p=158).
- Type:
Journal Articles
Status:
Accepted
Year Published:
2013
Citation:
Han, M., Y. Du, C. Song and D. Yoo. Degradation of CREB-binding protein and modulation of type I interferon induction by the zinc finger motif of the porcine reproductive and respiratory syndrome virus nsp-1 alpha subunit. Virus Res. 172: 54-65.
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Progress 01/01/12 to 12/31/12
Outputs
OUTPUTS: The Zuckermann laboratory performed experiments aimed at defining the mechanism(s) by which porcine reproductive and respiratory virus (PRRSV) is able to inhibit the interferon (IFN)-α response of its natural host cell, namely swine pulmonary alveolar macrophages. The effect of PRRSV on the poly(I:C) stimulated activation of the transcription factors IRF-3 and NFκB, IFN-α and IFN-β gene transcription as well as IFN-α secretion by ZMAC cells. The ZMAC cell line is a non-transformed porcine PAM that as a population is >99% susceptible to PRRSV infection and has an intact type I IFN response system. The results demonstrated that infection of ZMAC cells with PRRSV does not inhibit the poly(I:C)-induced activation of NFκB, STAT-1 or IRF-3, nor does it inhibit IFN-alpha or IFN-beta gene transcription. Nevertheless, the secretion of IFN-α was inhibited by >60% by 9 hours after infection. Notably, PRRSV alone induced the phosphorylation of NFκB but not IRF-3. Accordingly, PRRSV did not induce secretion of IFN-α. The Yoo laboratory investigated the role of the nucleocapsid (N) protein in induction of IL-10 and CD4+CD25+Foxp3+ lymphocytes (Treg). Transfection of porcine monocyte derived dendritic cells (MoDCs) and PAMs with a plasmid encoding N protein resulted in significant upregulation of IL-10 gene expression in the gene-transfected cells. In addition, the possibility that the PRRSV glycoprotein 4 (GP4) may be a lipid-anchored membrane protein was examined. Using the human decay-accelerating factor (DAF; CD55), a known glycosyl-phosphatidylinositol (GPI) lipid-anchored protein, chimeric constructs were made to substitute the GPI-anchor domain of DAF with the putative lipid-anchor domain of GP4, and their membrane association and lipase cleavage were determined in cells. The DAF-GP4 fusion protein was transported to the plasma membrane and was cleaved by phosphatidylinositol-specific phospholipase C (PI-PLC), indicating that the C-terminal domain of GP4 functions as a GPI anchor. Mutational studies for residues adjacent to the GPI modification site and characterization of respective mutant viruses generated from infectious cDNA clones were performed to examine the effect of these changes on the importance of GP4 for membrane association as it relates to virus viability and growth characteristics. Presentations of the work above were done at scientific meetings such as the International PRRS symposium and CRWAD. The Yoo laboratory published a review describing the viral strategies used by PRRS virus to modulate host's type I IFN responses. The review was aimed at other researchers working in the field. An understanding of the molecular basis for virus-mediated modulation of host innate immune signaling will help scientists design new generation vaccines for PRRS. PARTICIPANTS: Participants: Federico A. Zuckermann (PI); Dongwan Yoo (PI); Gabriela Calzada-Nova (Graduate student); Robert Husmann (Research associate); William Schnitzlein (Research associate); Y. Sun (Graduate student); M Han (Graduate student); and C.Y. Kim (Graduate student). Partner organizations: University of Nebraska, Lincoln and University of Wyoming. Collaborators: Assit K. Pattnaik; Fernando Osorio; and William Laegreid. TARGET AUDIENCES: Scientists working on the biology of PRRS virus and the immune response of swine to this virus as well as swine veterinarians and pork producers. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
The great majority of the research by other investigators has focused on the effect of individual viral proteins on transcription factors involved in the type I IFN response. By using non-PRRSV natural host cells transfected with reporter gene constructs in combination with over-expressed viral proteins, these types of studies have indicated that several nonstructural proteins of PRRSV have the ability to negatively affect the activation of NFκB and IRF-3. Concurrently, there is convincing evidence indicating that live PRRSV is indeed able to hinder the ability of its natural host cell, namely porcine alveolar macrophages (PAMs), to produce IFN-α in response to their stimulation with strong agonists, such as the synthetic analog of dsRNA, poly(I:C). Efforts to ascertain the mechanism(s) by which the infection of PAMs with PRRSV hinders the type I IFN response have been stifled by the fact that only a small fraction of this cell population is susceptible to infection by this virus. Consequently, any effect that PRRSV might have on transcription factor activation resulting from stimulation with poly(I:C) is obscured by the majority of the cells that are responding to this agonist, but are not infected by PRRSV. Our results are based on the effect of PRRSV on its natural host cells and indicate that whatever the mechanism is by which PRRSV inhibits the secretion of IFN-α in PAMs, it occurs at the post-transcriptional level. The presence of recombinant PRRSV N protein in cultured PBMCs increased the number of IL-10-producing lymphocytes. Strong induction of IL-10-producing cells and Treg was observed when using N protein-pulsed MoDCs, suggesting an important role of MoDCs in induction of IL-10 and Treg by the N protein. Neutralization of IL-10 by addition of an anti-IL-10 antibody in the culture system resulted in marked reduction of PRRSV-induced Treg in the cultured PBMCs. Together, the data demonstrate the immunomodulatory properties of the PRRSV N protein and the linkage between IL-10 production and development of PRRSV-induced Treg. Our results reveal an immunomodulatory function of the PRRSV N protein that may contribute to the unique immunological outcome observed following PRRSV infection. The DAF-GP4 fusion protein studies indicate that residues T158 (ω−2, where ω is the GPI moiety at E160), P159 (ω−1), and M162 (ω+2) of GP4 were determined to be important for virus replication, with M162 being of particular importance for virus infectivity. The complete removal of the peptide-anchor domain in GP4 resulted in a complete loss of virus infectivity. The depletion of cholesterol from the plasma membrane of cells reduced the virus production, suggesting a role of lipid rafts in PRRSV infection. Remarkably, GP4 was found to colocalize with CD163 in the lipid rafts on the plasma membrane. Since CD163 has been reported as a cellular receptor for PRRSV and GP4 has been shown to interact with this receptor, our data implicates an important role of lipid rafts during entry of the virus.
Publications
- Wongyanin, P., Buranapraditkul, S., Yoo, D., Thanawonguwech, R., Roth, J.A. and Suradhat, S. 2012. The role of porcine reproductive and respiratory syndrome virus (PRRSV) nucleocapsid protein on induction of IL-10 and PRRSV-specific regulatory T lymphocytes (Tregs). Journal of General Virology, 93: 1236-1246.
- Du, Y., Pattnaik, A., Song, C., Yoo, D. and Li, G. 2012. Glycosyl-phosphatidylinositol (GPI)-anchored membrane association of the porcine reproductive and respiratory syndrome virus GP4 glycoprotein and its co-localization with CD163 in lipid rafts. Virology, 424: 18-32.
- Calzada-Nova, G., Schnitzlein, W., Husmann, R. and Zuckermann, F.A. 2012. Effect of the host cell line on the vaccine efficacy of an attenuated porcine reproductive and respiratory syndrome virus. Veterinary Immunology Immunopathology. 148:116-125.
- Sun, Y., M. Han, C. Kim and D. Yoo. 2012. Interplay between type I interferon signaling and porcine reproductive and respiratory syndrome virus. Viruses (Basel) 4: 424-446.
- Parida, R., Choi I.S., Peterson, D.A., Pattnaik, A.K., Laegreid, W., Zuckermann, F.A. and Osorio, F.A. Location of T-cell epitopes in non-structural proteins 9 and 10 of type-II porcine reproductive and respiratory syndrome virus. Virus Res. 2012 Jul 5. [Epub ahead of print].
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Progress 01/01/11 to 12/31/11
Outputs
OUTPUTS: Dr. Yoo's laboratory presented results at scientific meetings suggesting that the suppression of NF-kB activation and sumoylation of PRRSV Nsp1α is mediated by protein inhibitor of activated STAT1 (PAIS1). In addition, we examined the role of PRRSV Nsp11, and endoribonuclease, on virus replication and interferon regulation. The Zuckermann laboratory presented data that the influence of PRSV on the IFN-α response of macrophages to infection with PRRSV is strain-dependent. Thus, while macrophages appeared non-responsive in the presence of either of two wild-type PRRSV isolates, copious amounts of this cytokine was released by ZMAC cells exposed to a third individual. This "unconventional" isolate was also unique in that it induced type I gene transcription and stimulated the phosphorylation of IRF3 to a greater extent than was observed in those ZMAC cells infected with either "conventional" virus. Since the phosphorylation of a second transcriptional factor, NFkb, also involved in the initiation of IFN-alpha/beta gene transcription was comparatively unaffected in any of the virus-infected cells, the "conventional" viruses may be blocking IFN-α production by interfering with a step(s) in the pathway leading to the activation of IRF3. Dr. Laegreid's lab in collaboration with Drs. Osorio and Pattnaik at the University of Nebraska, we published an article confirming previous findings that the N-glycan moieties in GP5 of type II PRRSV are important for the virus to escape the effect of neutralizing antibodies. In addition, they demonstrate for the first time that the N-glycan in GP3 of type II PRRSV is also important in protecting the virus from antibody neutralization. Likewise, the overall body of results herein presented firmly confirms the important notion that GP3 may be involved in inducing neutralizing antibodies, a concept that has already been suggested by previous investigators PARTICIPANTS: Federico A. Zuckermann (PI), Dongwn Yoo (PI), William Laegreid (PI), Gabriela Calzada-Nova (graduate student), Robert Husmann (Research associate), and William Schnitzlein (Research associate). The grant partially supported the graduate training of Gabriela Calzada-Nova. TARGET AUDIENCES: Scientists working on the immune response of animals to viruses as well as swine veterinarians. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Porcine reproductive and respiratory syndrome virus (PRRSV) expresses proteins that circumvent the type I IFN response and other cellular processes, and to compensate the small coding capacity of PRRSV, these proteins are multifunctional. Studies for Dr. Yoo's laboratory suggest that PRRSV Nsp1α and NSP11 are a multifunctional nuclear protein participating in the modulation of the host IFN system. Studies from Dr. Zuckermann's lab suggest that PRRSV is inhibiting the ability of porcine macrophages to produce IFN-a in response to infection by interfering with the activation of the transcription factor IRF-3 but not NFkB. The data presented by Dr. Laegreid's lab firmly confirms the important notion that GP3 may be involved in inducing neutralizing antibodies. Collectively, our work aimed at deciphering the transcriptional and cytokine response of cells as porcine alveolar macrophages to PRRSV infection will likely lead to the development of strategies to developed better vaccine against this costly disease.
Publications
- Faaberg, K.S., Balasuriya, U.B., Brinton, M.A., Gorbalenya, A.E., Leung, F.C.-C., Nauwynck, H., Snijder, E.J., Stadejek, T., Yang, H. and Yoo, D. 2011. Family Arteriviridae. In: Virus Taxonomy. 9th Ed., International Committee on Taxonomy of Viruses (ICTV). International Union of Microbiological Societies, Edited by: E. Carstens, E.J. Lefkowitz, and A.M.Q. King. Elsevier (ISBN: 978-0-12-384684-6). p.796-805.
- Du, Y., Yoo, D., Paradis, M.A. and Scherba,G . 2011. Antiviral activity of tilmicosin for type 1 and 2 porcine reproductive and respiratory syndrome virus in cultured porcine alveolar macrophages. J. Antivirals Antiretrovirals 3:28-33.
- Calzada-Nova, G., Schnitzlein, W., Husmann, R. and Zuckermann, F.A. 2011. North American porcine reproductive and respiratory viruses suppress the type I interferon response of activated porcine plasmacytoid dendritic cells. J. Virol. 85:2703-13.
- Hiep, L., Vu, X., Byungjoon, K., Kyoung-Jin, Y., LAegreid, W., Pattnaik, A.K. and Osorio, F.A. 2011. Immune evasion of porcine reproductive and respiratory syndrome virus through glycan shielding involves both Glycoprotein 5 as well as Glycoprotein 3. J. Virol. 85:5555-64.
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Progress 01/01/10 to 12/31/10
Outputs
OUTPUTS: Research related to pathogenesis/persistence: During the reporting period we published articles demonstrating that the PRRSV nonstructural protein 1α (Nsp1α) subunit of Nsp1 is a nuclear-cytoplasmic protein distributed to the nucleus and contains a strong suppressive activity for IFN-β production. This effect was apparently mediated through the retinoic acid-inducible gene I (RIG-I) signaling pathway. Nsp1α inhibited IκB phosphorylation and as a consequence NF-κB translocation to the nucleus was blocked, leading to the inhibition of NF-κB stimulated gene expression. Other studies indicated that Nsp1 blocked dsRNA-induced IRF3 and IFN promoter activities. This effect occurred through Nsp1 mediated the degradation of CBP, which inhibited its recruitment for enhanceosome assembly, leading to the block of IFN response. CBP degradation is a novel strategy for viral evasion from the host response, and Nsp1 may form a new class of viral antagonists for IFN modulation. Other studies indicated that the myristoylation of PRRSV E protein is non-essential for PRRSV infectivity but that it promotes the growth of the virus. In other studies the interaction of porcine reproductive and respiratory (PRRS) virus with porcine plasmacytoid dendritic cells (pDC) was examined. Although enveloped viruses typically trigger the prodigious secretion of interferon (IFN)-α by pDC, porcine pDC remain quiescent when exposed to PRRS virus. We demonstrated that this inactivity is likely due to virus-mediated suppression which occurred independently of virus viability or its dissociation in acidified endosomes but correlated with diminished levels of IFN-α mRNA associated with a decrease in the otherwise enhanced amounts of the requisite interferon regulatory factor (IRF)-7 whose gene expression, which in turn was the consequence of a lessened availability of nuclear-localized signal transducer and activator of transcription (STAT)1. Moreover, an augmented phosphorylation of NFκB seen in activated pDC was not only unaffected by PRRS virus but actually occurred in its presence. We propose that this pathogen may interact with a cell-surface protein(s) to selectively impede the completion of signaling cascades involved in IFN-α production by stimulated pDC. Research related to viral immunity and cross-protection: The goal is to associate immunologic parameters such as serum neutralization (SN) and T-cell responses with specific genomic signatures in the PRRSV genome. A major technical impediment to achievement of this goal has been the extraordinary genetic variability of PRRSV. To overcome this problem, we have developed a novel method of sequencing library construction which allows unbiased sequencing of full-length PRRSV genomes using various high-throughput next-generation sequencing platforms, including pyrosequencing. Utilizing this methodology, full genome sequencing of each of 8 serologic groups identified by Dr. Osorio's group at UNL was performed (i.e.: isolates of defined SN reactivity profile and that are different from already known reference strains). PARTICIPANTS: Participants: Zuckermann, Federico (PI), Laegreid, William (PI) and Yoo, Dongwan (PI). Partner Organizations/Individuals: Osorio, Fernando - University of Nebraska. Goldberg, Tony - University of Wisconsin, Madison. Jane Christopher-Hennings - South Dakota State University. Training or professional development: Chen, Wei Yu. Graduate Student. TARGET AUDIENCES: Pork producers, swine veterinarians, companies that produce veterinary biologics and researchers working on PRRS virus biology. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Porcine reproductive and respiratory syndrome virus (PRRSV) expresses proteins that circumvent the type I IFN response and other cellular processes, and to compensate the small coding capacity of PRRSV, these proteins are multifunctional. Studies suggest that PRRSV Nsp1α is a multifunctional nuclear protein participating in the modulation of the host IFN system. PRRSV is the first arterivirus demonstrated to repress the ability of pDC to mount an type I IFN and pro-inflammatory cytokine response to itself as well as to a known stimulatory virus, TGEV, and a TLR9 agonist, ODN D19. This functional impediment could have important ramifications regarding the host's innate immune responses to infection. Blocking against virus-mediated inhibition of the innate immune response may lead to the future development of effective vaccines. Analysis of full-length PRRSV genomes sequences will help identify regions of immunologic significance in the PRRSV genome.
Publications
- Kim, O., Sun, Y., Lai, F., Song, C. and Yoo, D. 2010. Modulation of type I interferon induction by porcine reproductive and respiratory syndrome virus and degradation of CREB-binding protein by non-structural protein 1 in MARC-145 and HeLa cells. Virology 402: 315-326.
- Du, Y., Zuckermann, F.A. and Yoo, D. 2010. Myristoylation of the small envelope protein of porcine reproductive and respiratory syndrome virus is non-essential for virus infectivity but promotes its growth. Virus Res. 147: 294-299.
- Calzada-Nova, G., Schnitzlein, W., Husmann, R. and Zuckermann, F.A. 2010. Characterization of the cytokine and maturation responses of pure populations of porcine plasmacytoid dendritic cells to porcine viruses and toll-like receptor agonists. Vet. Imm. Immunopath. Vet. 135(1-2):20-33.
- Lawson S., Lunney J., Zuckermann, F., Osorio, F., Nelson, E., Welbon, E., Clement, T., Fang, Y., Wong, S., Kulas, K. and Christopher-Hennings, J. 2010. Development of an 8-plex Luminex assay to detect swine cytokines for vaccine development: Assessment of immunity after porcine reproductive and respiratory syndrome virus (PRRSV) vaccination. Vaccine 28: 5356-5364.
- Song, C., Krell, P. and Yoo, D. 2010. Nonstructural protein 1α subunit-based inhibition of NF-κB activation and suppression of interferon-β production by porcine reproductive and respiratory syndrome virus. Virology 407: 268-280.
- Yoo, D., Song, C., Sun, Y., Du, Y., Kim, O. and Liu, H.C. 2010. Modulation of host cell responses and evasion strategies for porcine reproductive and respiratory syndrome virus. Virus Res. (On-line published, DOI: 10.1016/j.virusres.2010.07.019) (Invited Review).
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Progress 01/01/09 to 12/31/09
Outputs
OUTPUTS: We analyzed the expression of CD163 on PAMs and macrophages derived from CD14 positive blood monocytes (MDMs), in correlation with PRRSV replication. By flow cytometric analysis, we showed that the levels of CD163 expression correlated well with the overall level of PRRSV replication. We further examined the effects of modulators of macrophage function, including 12-O-tetradecanoylphorbol-13-acetate (TPA), lipopolysaccharide (LPS), and interleukin (IL)-10. Pre-treatment of PAMs or MDMs with TPA or LPS resulted in decreased expression of CD163 and reduction in PRRSV replication, while incubation with IL10 had the opposite effect. Utilizing a yeast two-hybrid screening we identified that the inhibitor of MyoD family-a (I-mfa) domain-containing protein (HIC) is a cellular partner for PRRS virus (PRRSV) N protein. This protein is a homolog of human HIC, a recently identified cellular transcription factor. The specific interaction of PRRSV N with HIC was confirmed in cells by mammalian two-hybrid assay and co-immunoprecipitation and in vitro by GST pull-down assay. HIC is a zinc-binding protein and confocal microscopy demonstrated co-localization of N with the HIC-p40 isomer in the nucleus and nucleolus, and in the cytoplasm with HIC-p32, which is the N-terminal truncation of HIC-p40. Recently, our laboratory developed a porcine alveolar macrophage cell line. This cell line, named ZMAC, was found to efficiently support the replication of a number PRRS virus isolates, often achieving high titers (>107 TCID50/ml). Given the apparent high permissiveness of ZMAC cells to PRRS virus, we set out to test the proficiency of this cell line to isolate field PRRS virus from clinical samples. The ZMAC line proved highly efficient (>90%) at isolating PRRS virus within 72 hours after exposing ZMAC cells to pig serum samples known to be positive to PRRSV by real-time PCR, from which attempts to isolate PRRS virus in MARC-145, and even primary alveolar macrophages, had failed. Furthermore, the ZMAC cell line allowed us to determine the infectious virus load from these field samples. Remarkably, we observed a significant variation between samples in the ratio of viral genome copy number and titer of infectious virus. Using an infectious cDNA clone of North American PRRSV strain P129, the viral genome was engineered to transcribe an additional subgenomic RNA initiating between non-structural and structural genes. The enhanced green fluorescent protein (GFP) gene was cloned between the unique sites such that the inserted gene was transcribed from TRS2 which was located upstream within ORF1b, while the copy of TRS6 drives ORF2a/b transcription. Cells infected with P129-GFP produce virus progeny and showed fluorescence and the inserted gene was phenotypically stable for at least 37 serial in vitro passages. Subsequently, a capsid (C) protein gene was cloned from porcine circovirus type 2 (PCV2) was inserted into the PRRSV infectious clone vector, generating virus "P129-PCV". Pigs immunized with either P129-GFP or P129-PCV2 produced antibodies specific for GFP or PCV2 capsid respectively. This work was presented at the International PRRS Symposium. PARTICIPANTS: Zuckermann, Federico (PI) UIUC Station Representative (email: fazaaa@illinois.edu) Yoo, Dongwan (PI)- UIUC Laegried, Will (PI)- UIUC Gabriela Calzada-Nova (Research Associate) William Schnitzlein (Research Associate) Robert Husmann (Research Associate) Collaborating scientists at other institutions: Osorio, Fernando - University of Nebraska Lincoln Goldberg, Tony - Univ. Wisconsin, Madison TARGET AUDIENCES: Pork producers Swine veterinarians Companies producing Veterinary Biologics Researchers working in virology and immunology of virus infections PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
The use of the porcine alveolar macrophage cell line ZMAC to isolate PRRS virus from field samples will enable us to better understand this virus and to development methods to control its spread. The use of PRRS virus as a vector for foreign gene expression is a first, which demonstrates the potential use of PRRSV as a vaccine vector for swine pathogens. Our studies on the replication of PRRS virus in rodents indicated that PRRSV replication in common laboratory rodent species is inefficient, and suggests that a rodent model for this virus is not appropriate. The studies on the modulation of CD163 receptor expression and the replication of porcine reproductive and respiratory syndrome virus in porcine macrophages data indicated that the expression of CD163 on macrophages in different microenvironments, in vivo, may determine the replication efficiency and subsequent pathogenicity of PRRS virus. The interaction of viral capsid with the cellular transcription factor implicates a possible regulation of host cell gene expression by the N protein during PRRSV infection.
Publications
- Wu, J., Li, J., Tian, F., Shi, J., Ren, S., Lan, Z., Zhang, X., Yoo, D. and Wang, J. 2009. Porcine high fever disease: Genetic variation and pathogenicity of porcine reproductive and respiratory syndrome virus in China. Arch. Virol. 154: 1579-1588.
- Mohammadi, H., Sharif, S., Rowland, R.R. and Yoo, D. 2009. The lactate dehydrogenase-elevating virus capsid protein is a nuclear-cytoplasmic protein. Arch. Virol. 154: 1071-1080.
- Patton, J.B., Rowland, R.R., Yoo, D. and Chang, K.C. 2009. Modulation of CD163 receptor expression and replication of porcine reproductive and respiratory syndrome virus in porcine macrophages. Virus Res. 140:161-171.
- Song, C., Lu, R., Bienzle, D., Liu, H.C. and Yoo, D. 2009. Interaction of porcine reproductive and respiratory syndrome virus nucleocapsid protein with the inhibitor of MyoD family-a domain containing protein. Biol. Chem. 390: 215-223.
- Pei, Y., Hodgins, D.C., Wu, J., Welch, S.K.W., Calvert, J.G., Li, G., Du, Y., Song, C. and Yoo, D. 2009. Porcine reproductive and respiratory syndrome virus as a vector: Immunogenicity of green fluorescent protein and porcine circovirus type-2 capsid expressed from dedicated subgenomic RNAs. Virology 389:91-99. :1530-5.
- Calzada-Nova, G., Schnitzlein, W., Husmann, R. and Zuckermann, F.A. 2009. Characterization of the cytokine and maturation responses of pure populations of porcine plasmacytoid dendritic cells to porcine viruses and toll-like receptor agonists. Vet. Imm. Immunopath. doi: 10.1016/j.vetimm.2009.10.26.
- Du, Y., Zuckermann, F.A. and Yoo, D. 2009. Myristoylation of the small envelope protein of porcine reproductive and respiratory syndrome virus is non-essential for virus infectivity but negatively affects its growth. Virus Res. (In Press).
- Rosenfeld, P., Turner, P.V., MacInnes, J.I., Nagy, E. and Yoo, D. 2009. Evaluation of porcine reproductive and respiratory syndrome virus replication in the laboratory rodents. Can. J. Vet. Res. 73: 313-318.
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Progress 01/01/08 to 12/31/08
Outputs
OUTPUTS: A study was performed aimed at identifying the site(s) of immunodominant T cell epitopes for this virus's glycoprotein 5 (GP5) by using a peptide library. In this case, the ability of individual, overlapping pentadecamer peptides designed to represent the entire PRRSV GP5 to induce interferon (IFN)-γ production (cell-mediated immune response) from PBMC obtained from PRRSV-infected pigs was assessed. The results of this study have been recently published in the journal Vaccine. Recently, a porcine alveolar macrophage cell line designated ZMAC-1 was developed and was found to not only be susceptible to infection by PRRS virus but also to release significant amounts of virus progeny. A study was performed to compare the efficacy of stocks of the MLV vaccine Prime Pac PRRS (Schering-Plough Animal Health) prepared in either ZMAC-1 or the simian cell line MARC-145, afterwards a standard immunization-challenge study was conducted. For this project, groups of pigs were either immunized with vaccine virus derived from one of the two cell lines or mock-vaccinated. Four weeks after vaccination, all of the immunized animals as well as three of the control pigs housed in one cubicle were challenged with 10E4 TCID50 of PRRS virus strain NADC-20. The results of this study were presented at the 2008 AASV meeting and described in a report submitted to the National Pork Board. PARTICIPANTS: Federico Zuckermann, Gabriela Calzada-Nova, Tony Goldberg, Kapil Vashisht, William Schnitzlein, Mauricio Villamar, and Robert Husmann. TARGET AUDIENCES: Swine veterinarians, pork producers, and scientists working with PRRS virus vaccine development. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
Ninety-six pentadecapeptides spanning glycoprotein 5 (GP5) of porcine reproductive and respiratory virus (PRRSV) were screened for their ability to elicit a recall interferon-γ response from peripheral blood mononuclear cells isolated from 22 pigs infected with up to two genetically divergent PRRSV strains. Two distinct regions (amino acid residues 117-131, LAALICFVIRLAKNC, and 149-163, KGRLYRWRSPVII/VEK) appeared to contain immunodominant T-cell epitopes based on their ability to stimulate above average numbers of interferon-γ secreting cells as compared to other GP5 peptides. A survey of PRRSV isolates indicated that these two immunodominant PRRSV GP5 epitopes are relatively conserved with at most a two amino acid variation and elicited a significant IFN-γ response in almost half of the pigs tested in this study. Thus, pending in vivo verification of their immungenicity, one or both should be considered for incorporation into a polyvalent, peptide-based vaccine against PRRS. In view of the inherent variability of the genome of PRRSV, other components could include novel GP5 epitopes recognized by pigs with different haplotypes as well as yet undiscovered T cell epitopes present in other PRRSV proteins such NSP2, which has been shown to contain a high frequency of immunodominant B cell epitopes. In addition, the identification of T cell epitopes in other PRRSV structural proteins such as GP2 and the matrix (M) protein, which are recognized by T cells, need to be identified in order to develop a potentially effective peptide-based vaccine expressing a multitude of conserved immunodominant B and T cells epitopes which could be engineered into a more immunogenic biologic. In the vaccination and challenge study listed above, the unvaccinated animals experienced an average body weight (BW) loss of 5 pounds by 7 days after the virulent virus challenge, the PRRS virus-naive controls had gained on average 19.7 lb during this time interval. In contrast, at 7 days post-challenge, the animals vaccinated with the MLV virus grown in either ZMAC-1 or MARC-145 cells exhibit average BW gains of 8.2 and 9.3, respectively. Thus, statistically the Prime Pac MLV vaccine grown in either cell line was equally effective at reducing the negative effect of the exposure of pigs to a highly virulent PRRS virus on their growth. Remarkably, analyses of the virus load in serum and lung lavage samples from PRRS virus-immunized and challenged animals revealed that the vaccine virus grown in ZMAC-1 cells was significantly (P=0.015) more effective at reducing the extent of viremia at 7 days post-challenge and also at eliminating virulent virus from their lungs by 10 days post-challenge. The observation that the type of cell line used to grow the PRRS MLV vaccine can improve the level of protective immunity elicited by this product against a genetically divergent virulent PRRS virus has implications for vaccine development against this pathogen. Namely, that the effectiveness of a PRRS virus vaccine is not only determined by its genetic similarity to the challenge virus, but is also influenced by how it is produced.
Publications
- Vashisht, K., Erlandson, K.R., Firkins, L.D., Zuckermann, F.A. and Goldberg, T.L. 2008. Evaluation of contact exposure as a method for acclimatizing growing pigs to porcine reproductive and respiratory syndrome virus. Journal American Veterinary Medicine Association. 232:1530-5.
- Vashisht, K., Goldberg, T.L., Husmann R.J., Schnitzlein, W. and Zuckermann, F.A. 2008. Identification of immunodominant T-cell epitopes present in glycoprotein 5 of the North American genotype of porcine reproductive and respiratory syndrome virus. Vaccine. 26:4747-53.
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Progress 01/01/07 to 12/31/07
Outputs
OUTPUTS: Experiments were conducted aimed at understanding the mechanism responsible for the inhibitory effect of PRRS virus on the ability of porcine plasmacytoid dendritic cells (PDC) to produce interferon alpha. Production of interferon alpha by PDC is the most important innate immune mechanism of protection of an animal against a virus infection. These experiments examined changes in the transcriptome and proteome of PDCs resulting from their exposure to PRRS virus. Other experiments were aimed at deciphering the PRRS virus genetic determinants associated with host range and inflammatory cytokine response. To address the former issue the growth of chimeric PRRS viruses of FL12 (macrophage growth competent, wild type virus) and PrimePac (PP; macrophage growth defective attenuated virus) were constructed and characterized. Culture supernatants from cells transfected with chimeric infectious clone constructs were obtained and the amount of virus determined in both simian cell line
MARC-145, and the porcine alveolar macrophage (PAM) cell line ZMAC-1. To examine the effect of PRRS virus genetic determinants associated with inflammatory cytokine responses the amount of interferon (IFN)-alpha produced by the ZMAC-1 cells exposed to these chimeras was also determined. In addition, the growth and cytokine responses elicited by several PRRS virus field isolates in ZMAC-1 cells were also examined. We also performed experiments aimed at understanding the association between the PRRS virus phenotype of innate cytokine stimulation and the protective immune response elicited by the respective virus in pigs. Several PRRS virus isolates that exhibit distinct innate cytokine response phenotypes in vitro were examined for their ability to elicit protective immunity. Experiments were also conducted to evaluate the effectiveness of contact exposure of naive pigs to other pigs previously inoculated with PRRS virus as a acclimatization strategy against this virus. Other
experiments were aimed at the protective immunity elicited by commercially available live and attenuated vaccines against PRRS virus. Results from the research activities listed above were presented at the 2007 International PRRS Symposium, the 2007 Conference of Research Workers in Animal Disease (CRWAD) and also included in the 2007 NC-229 Illinois Station report and published in scientific journals. In June 2007, a meeting was held at the University of Illinois College Of Veterinary Medicine to discuss the state of current knowledge about PRRS vaccination. The meeting was attended by invited experts in PRRS, virology, immunology and vaccinology and included clinical veterinarians, academics and vaccine industry scientists. Three general questions were posed to the group: [1] What is the efficacy of current PRRS vaccines? [2] What are the knowledge gaps that need to be filled to develop improved/novel vaccines? [3] What is the probability that successful PRRS vaccines can be
developed? A report of this meeting can be seen in the University of Illinois CVM website, the AASV website, ThePigSite and the September issue of the PRRS-CAP Newsletter.
PARTICIPANTS: Federico Zuckermann, PI; Tony Goldberg coPI; Daniel Rock, coPI; William Schnitzlein, Research Associate; Robert Husmann, Research Associate; Gustavo Delhon, Research Associate; Gabriela Calzada, Graduate student. Mauricio Villamar, academic hourly Collaborators: Fernando Osorio, University of Nebraska-Lincoln; Guillermo Risatti, University of Connecticut.
TARGET AUDIENCES: National Pork Board, American Association of Swine Veterinarians, swine veterinarians, pork producers, and companies producing vaccines for swine.
Impacts
Significant advances in studying the outcome of the interaction between PRRS virus and porcine PDCs were made possible by utilizing the rflection cell sorter from iCyt. This sophisticated instrument, which has a very high cell-sorting throughput provided enough quantity of PDCs at >98% purity that allowed for the determination of changes in the transcriptome and proteome of these cells resulting from their exposure to PRRS virus. These analyses have yielded significant insights into the possible mechanisms by which PRRS virus negatively affects the anti-viral function of the PDCs. We were also successful in identifying the data generated with the chimeric viruses, which suggested that there was no correlation between their ability to stimulate interferon alpha by porcine alveolar macrophage ZMAC-1 cells and their ability to replicate in them. The lack of correlation between interferon alpha inducing capacity and the replication of the chimeric and parental viruses in
the ZMAC-1 cell line was confirmed with the 13 PRRS virus field isolates examined. Remarkably, there was significant variation on the ability of the 13 field isolates to stimulate interferon alpha production in this cell line. The study of the degree of protective immune response elicited by PRRS virus isolates, which differ in the ability to stimulate an innate cytokine response, is ongoing. The results of the study comparing the ability of a killed vs. live PRRS vaccine indicated that the modified live virus vaccine was the only type of vaccine capable of establishing protective immunity, as measured by viral load in blood and tissues. The killed vaccine, in spite of this product evoking a spontaneous interferon-gamma response and post-challenge titers of virus-neutralizing antibody, evoked no measurable protective immunity. In the case of the modified live vaccine, the protection exhibited did not appear to be based on humoral but rather on cell-mediated immunity. The
acclimatization studies revealed that contact exposure of PRRS naive 6.5 and 10.5 week-old pigs to pigs inoculated as late as 4 weeks earlier with endemic PRRS virus may be an efficient acclimatization strategy for controlling PRRS outbreaks on commercial farms.
Publications
- Zuckermann, F.A., Alvarez Garcia, E., Diaz Luque, I., Christopher-Hennings, J., Doster, A., Brito, M. and Osorio, F.A. 2007. Assessment of the efficacy of commercial porcine reproductive and respiratory syndrome virus (PRRSV) vaccines based on measurement of serologic response, frequency of gamma-IFN-producing cells and virological parameters of protection upon challenge. Vet Microbiol. 2007 July 20;123(1-3):69-85.
- Vashisht, K., Erlandson, K., Firkins, L., Zuckermann, F. and Goldberg, T. 2008. Evaluating contact exposure to inoculated swine as a strategy for acclimatizing growing pigs to porcine reproductive and respiratory syndrome virus. JAVMA. 2008 (In Press).
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Progress 01/01/06 to 12/31/06
Outputs
Effect of differences in route and time of exposure to PRRS virus on the reproductive outcome of sows: This longitudinal prospective cohort study was designed to compare the reproductive performance of randomly assigned pigs inoculated with a farm strain of PRRSv to that of naturally infected pigs on an operational swine farm in the Midwestern United States. Cohort-1 consisted of 20 pigs exposed to an attenuated farm strain of PRRSv by intramuscular injection at 4 weeks of age and maintained in an isolation facility. Cohort-2 consisted of 20 pigs "contact exposed" by cohabitation with cohort-1 pigs at 4 weeks of age. Cohort-3 consisted of 40 animals co-mingled with pigs of cohorts 1 and 2 when all were moved to a grow-finish facility at 8 weeks of age (delayed contact exposure). Viral RNA could not be detected in tonsil of any pig by RT-PCR at 34 weeks of age (pre-breeding). At 17 weeks of age, the ELISPOT responses to VR2332 of pigs in cohorts 1, 2 and 3 were 96 +/-
48, 97 +/- 54, and 83 +/- 49 respectively. The total number of piglets weaned averaged 9.9 +/- 2.8, 8.9 +/- 3.6 and 9.7 +/- 3.3 in cohorts 1, 2, and 3 respectively. Wild-type as well as attenuated versions of porcine reproductive and respiratory syndrome virus selectively inhibit the ability of porcine plasmacytoid dendritic cells (PDC) to produce interferon alpha. PDC are the most potent source of interferon (IFN)- Alpha and thus are primarily responsible for the initial protective response elicited during a virus infection. We evaluated the behavior of porcine PDC exposed to PRRSv both in vitro and in vivo. When freshly purified PDCv were incubated with wild type PRRSv, the resultant IFN-Alpha response was meager and at least 100-fold less intense than that registered in the presence of transmissible gastroenteritis virus (TGEv). That PRRSv was actually affecting PDC function was established by the demonstrated ability of this entity to repress the otherwise vigorous IFN-Alpha
responsiveness to TGEv of an isolated porcine peripheral blood mononuclear cells population that was partially comprised of PDC. A similar impact of PRRSv on the performance of PDC was observed in vivo. This inhibitory ability of PRRSv may be rather unique since a similar type of impairment was not noticed when swine were infected with Pseudorabies virus. Correlation of cell-mediated immunity against porcine reproductive and respiratory syndrome virus (PRRS) with protection against reproductive failure in sows during outbreaks of PRRS in commercial herds: The objective of this study was to determine whether cell-mediated immunity against PRRS virus is correlated with protection against reproductive failure in sows during clinical outbreaks of PRRS in 4 commercial breeding herds. A negative association between the intensity of the CMI response and the number of pigs born dead per litter was detected on one farm. Evidence that a strong CMI response was correlated with protection against
clinical PRRS was detected in 3 of 4 farms. However, farms and sows within farms varied considerably in their immune responsiveness and in the degree to which they were protected clinically.
Impacts
A field study revealed that intentionally inoculated (acclimatized) pigs performed no better than contact exposed pigs. Thus, acclimatization via inoculation offered no ultimate production-related advantages in this setting over natural infection. In addition, wild-type and attenuated versions of PRRS virus inhibits the ability of porcine plasmacytoid dendritic cells to produce interferon alpha.
Publications
- Lowe, J.F., Husmann, R., Firkins, L.D., Zuckermann, F.A. and Goldberg, T.L. 2005. Correlation of cellular immunity to Porcine Reproductive and Respiratory Syndrome Virus and clinical disease during outbreaks of PRRS in commercial swine herds. J. Am. Vet. Med. Assoc. 226:1707-11.
- Lowe, J.F., Zuckermann, F.A., Firkins, L.D., Schnitzlein, W.M. and Goldberg, T.L. 2006. Immunological and clinical consequences of exposure to wild type or attenuated porcine reproductive and respiratory syndrome virus in swine under field conditions. J. Am. Vet. Med. Assoc.
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Progress 01/01/05 to 12/31/05
Outputs
A series of field studies have been conducted with the objective of determining the influence of humoral and cell mediated immunity (CMI) on the outcome of the infection with porcine reproductive and respiratory syndrome virus (PRRSV). Evidence that a strong cellular immune response correlates with protection against clinical PRRS was found in three of the four farms examined. However, farms and animals within farms varied considerably in their immune responsiveness and in the degree to which they were protected clinically. Increasing cellular immunity within infected herds has the potential to reduce clinical reproductive disease. The identification of the sources of intra- and inter-farm variation in the intensity of CMI to PRRSV could help increase the level of herd immunity. In addition our results indicate that poor immunity to PRRSV may facilitate re-infection and we also observed a positive correlation (r = 0.63) between the number of pigs born alive and the
intensity of the virus-specific IFN-gamma response, indicating that cellular immunity provides some protection from clinical disease even for pigs housed in an environment characterized by multiple, co-circulating viral strains.
Impacts
A series of field studies have been conducted with the objective of determining the influence of humoral and cell mediated immunity (CMI) on the outcome of the infection with porcine reproductive and respiratory syndrome virus (PRRSV). Evidence that a strong cellular immune response correlates with protection against clinical PRRS was found in three of the four farms examined. However, farms and animals within farms varied considerably in their immune responsiveness and in the degree to which they were protected clinically. Increasing cellular immunity within infected herds has the potential to reduce clinical reproductive disease. The identification of the sources of intra- and inter-farm variation in the intensity of CMI to PRRSV could help increase the level of herd immunity. In addition our results indicate that poor immunity to PRRSV may facilitate re-infection and we also observed a positive correlation (r = 0.63) between the number of pigs born alive and the
intensity of the virus-specific IFN-gamma response, indicating that cellular immunity provides some protection from clinical disease even for pigs housed in an environment characterized by multiple, co-circulating viral strains.
Publications
- Lowe, J.F., Zuckermann, F.A., Firkins, L.D., Schnitzlein, W. and Goldberg, T.L. 2006. Immunological and clinical consequences of exposure to wild type or attenuated porcine reproductive and respiratory syndrome virus in swine under field conditions. J. Am. Vet. Med. Assoc. (In Press).
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Progress 01/01/04 to 12/31/04
Outputs
The natural response of pigs to porcine reproductive and respiratory syndrome virus (PRRSV) infections and vaccinations needs to be altered so that better protection is afforded against both homologous and heterologous challenges by this pathogen. Studies conducted during the reporting period demonstrated the ability of cytokines and synthetic double-stranded RNA to augment the T helper1 (i.e., interferon-gamma) immune response of swine to PRRSV. Thus, exposure to PRRSV in the presence of a variety of Th1 polarizing molecules can positively influence the development of the cell-mediated immune response of swine to this pathogen. Conceivably, such intervention could be applied to improve the formulation of anti-PRRSV vaccines. To extend these observations related studies were aimed at deciphering the involvement of innate immune factors in the development of the host responses to PRRSV vaccination. In these studies, efforts to enhance Th1 immunity, by utilizing an
expression plasmid encoding porcine interferon (IFN)-alpha (pINA) as an adjuvant, resulted in a temporary increase in the frequency of PRRSV-specific IFN-gamma secreting cells (SC) but only minor changes overall in the expression of Th1 associated cytokine or innate immune marker mRNA by virus-stimulated PBMC. Administration of pINA, however, did correlate with decreased IL1B secretion by cultured, un-stimulated PBMC but had no effect on their ability to release IFN-gamma. Thus, while exogenous addition of IFN-alpha during PRRSV vaccination has an impact on the development of a Th1 immune response, other alterations will be required for substantial boosting of virus-specific protection. It has become increasingly evident that the link between innate and adaptive immunity in viral infections occurs through the interaction of dendritic cells with type I IFN and dendritic-cell controlled polarization of T-cell function. The production of IFN-alphaby plasmacytoid dendritic cells (PDC) has
an autocrine effect that promotes functional and phenotypic activation events necessary for their optimal expression of co-stimulatory molecules and subsequent ability to cause naive T cells to differentiate into IFN-gamma-SC. Our studies have now demonstrated that PRRSV is a poor stimulator of IFN-alpha production by porcine PDC. This limited response of PDC to PRRSV may be at least partially responsible for the weak initial IFN-gamma response against PRRSV in swine and has implications regarding the development of an effective vaccine against this pathogen. A field study was also conducted with the objective of determining if cell mediated immunity (CMI) to PRRSV correlates with protection against reproductive failure in sows on commercial swine farms during clinical outbreaks of PRRS. Evidence that a strong cellular immune response correlates with protection against clinical PRRS was found in three of the four farms examined.
Impacts
Several major advances were made at the University of Illinois in understanding the immunobiology of PRRS virus as well as the molecular epidemiology of this virus. We expect that these advances will contribute to the development of a more effective vaccine as well as the development of control measures to prevent the spread of this virus.
Publications
- Bastos, R.G., Dellagostin, O.A., Barletta, R.G., Doster, A.R., Nelson, E., Zuckermann, F. and Osorio, F.A. 2004. Immune response of pigs inoculated with Mycobacterium bovis BCG expressing a truncated form of GP5 and M protein of porcine reproductive and respiratory syndrome virus. Vaccine. 22: 467-74.
- Lowe, J.F., Zuckermann, F.A., Firkins, L.D. and Goldberg, T.L. 2004. Immunological and reproductive outcomes of controlled exposure of sows to either wild type porcine reproductive and respiratory syndrome virus or modified live vaccine in a commercial swine herd. CRWAD. Chicago, Illinois. November 14-16, Abst. 215, pp. 144.
- Royaee, A.R., Husmann, R.J., Dawson H.D., Calzada-Nova, G., Schnitzlein, W.M., Zuckermann, F.A. and Lunney, J.K. 2004. Deciphering the involvement of innate immune factors in the development of the host responses to PRRSV vaccination. Vet. Immunol. Immunopathol. 102:199-216.
- Meier, W.A., Husmann, R.J., Schnitzlein, W.M., Osorio, F.A., Lunney, J.K. and Zuckermann, F.A. 2004. The use of cytokines and synthetic double-stranded RNA to augment the T helper1 immune response of swine to porcine reproductive and respiratory syndrome virus. Vet. Immunol. Immunopath. 102:299-314.
- Lowe, J.F., Husmann, R., Firkins, L.D., Zuckermann, F.A. and Goldberg, T.L. 2004. Correlation of cellular immunity to Porcine Reproductive and Respiratory Syndrome Virus and clinical disease during outbreaks of PRRS in commercial swine herds. J. Am. Vet. Med. Assoc. (In Press).
- Calzada-Nova , G., Husmann, R. and Zuckermann F.A. 2003. Porcine CD4+/CD3- peripheral blood leukocytes have plasmacytoid morphology and express SWC3 - a member of the signal regulatory protein (SIRP) family. Autumn Immunology Conference. Chicago, Illinois. November 22-24. Vol. 32:164 pp.60.
- Zuckermann, F.A., Schnitzlein, W., Husmann, R. and Calzada-Nova, G. 2003. Association of porcine interleukin-12 receptor beta 2 (IL-12Rbeta2) gene alleles with the development of high or low interferon-gamma response to PRRS virus. CRWAD. Chicago, Illinois. November 9-11.
- Calzada-Nova, G., Husmann, R. and Zuckermann F.A. 2004. Porcine CD4+/CD3- peripheral blood leukocytes have plasmacytoid morphology and express SWC3 - alpha member of the signal regulatory protein (SIRP) family. 7th International Veterinary Immunology Symposium. July 25-30. Quebec City, Canada.
- Zuckermann, F.A. 2004 Regulation of the IL-12 receptor expression and the interferon gamma response of swine to allogeneic and viral antigens. 7th Internation Veterinary Immunology Symposium. July 25-30. Quebec City, Canada.
- Zuckermann, F.A. 2004. Ultimos avances sobre inmunologia frente a PRRS. Symposium Internacional de Porcinocultura, XXXVII Semana Nacional de Ganado Porcino (SEPOR 2004). Lorca (Murcia) Spain, pp. 95-103.
- Calzada-Nova , G., Husmann, R., Schnitzlein, W. and Zuckermann F.A. 2004. PRRS virus is a poor stimulator of interferon-alpha production by porcine plasmacytoid dendritic cells. CRWAD. Chicago, Illinois. November 14-16. Abst. P76. pp.101.
- Zuckermann, F.A., Schnitzlein, W. and Husmann, R.J. 2004. Regulation of the IL-12 receptor expression and the IFN-gamma response of swine to PRRS virus. CRWAD. Chicago, Illinois. November 14-16. Abst. 139. pp. 130.
- Calzada-Nova, G., Husmann, R.J., Schnitzlein, W. and Zuckermann, F.A. 2004. PRRS virus is a poor stimulator of interferon-alpha production by porcine plasmacytoid dendritic cells. 17th Annual Cell and Molecular Biology and Biophysics Research Symposium. Univ. of Illinois, Beckman Institute. Urbana, Illinois. November 4th. Abst. 59, pp. 42.
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Progress 01/01/03 to 12/31/03
Outputs
The immune response of pigs to infection with wild-type virus or vaccination with a conventional modified live virus (MLV) vaccine against the arterivirus Porcine Reproductive and Respiratory Syndrome virus (PRRSV) is characterized by an initially weak interferon-gamma (IFN-gamma) response that increases gradually over a period of months (Meier et al., 2003). However, a conflicting report has appeared (Batista et al., 2003, AASP). In order to solve this discrepancy, a group of 5 naive pigs was immunized once at 10 weeks of age with the PRRS MLV instead of twice to mimic the study by Batista et al. This new study has confirmed that a weak IFN-gamma response to PRRS virus was initially detectable after vaccination. Although similar to the report by Batista, the response appeared to wane at 10 weeks,the frequency of IFN-gamma-secreting cells then rebounded and, although with fluctuation, it increased gradually over months without a booster immunization. The kinetics and
fluctuation of the response, as measured by ELISPOT, were confirmed by ELISA by determining the concentration of IFN-gamma in cell-free supernatants of parallel bulk cultures, suggesting that the development of the IFN-gamma response is under complex regulation. To understand the mechanisms for the regulation of the immune response to PRRS virus, we examined the expression profile of genes of several immune-regulatory molecules by real time PCR in PBMC isolated from pigs vaccinated with PRRS MLV. In one study blood was collected from 9 pigs at week 0, 2, 5, 9, 11 and 13 following vaccination with PRRS MLV. Gene expression was calculated relative to week 0. Our data indicated that following vaccinations, it takes up to 5 weeks for pigs to develop a statistically significant (p<0.05) IFN-gamma protein and mRNA response to PRRSV. No changes in IL-12 p35/p40 gene expression were seen. A vaccination boost at week 7 further increased the frequency of virus-specific IFN-gamma-SC. A
significant up-regulation of TNF-alpha indicated the involvement of innate immunity to PRRSV starting early in the infection, yet there was limited up-regulation of IFN-alpha. A second experiment determined the effect of an exogenous source of IFN-alpha at the time of vaccination with a PRRS MLV on the cellular cytokine responses and the lymphoid cell mRNA changes. Co-administration of IFN-alpha resulted in changes in the cytokine gene expression. The administration of IFN-alpha resulted in a faster return to homeostasis (pre-vaccination levels) of the expression of mRNA for IL-6, IL-8 and IL-10. Remarkably, the spontaneous production of IL-10 (as determined by ELISA) at 2 weeks post vaccination was significantly lower in the PBMC isolated from pigs that received IFN-alpha along with the vaccine as compared to that of the pigs immunized with the vaccine alone. Co-administration of IFN-alpha resulted in an enhancement of the IFN-gamma response to the vaccine. These results indicate
that the mechanism of the enhancing effect of IFN-alpha on the IFN-gamma response to the PRRS MLV vaccine might be through the induction of changes in the cytokine response to this virus.
Impacts
Understanding of the mechanisms that regulate the development of anti-viral protective immunity will lead to the development of more effective vaccines and methods to control this pathogen. Discernment of how immunity impacts the severity of clinical disease on commercial farms will lead to the development of improved strategies for managing PRRS in reproductive herds using strategies such as vaccination.
Publications
- No publications reported this period
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Progress 01/01/02 to 12/31/02
Outputs
The immune response of pigs to infection with wild-type virus or vaccination with a conventional modified live virus (MLV) vaccine against the arterivirus Porcine Reproductive and Respiratory Syndrome virus (PRRSV) is characterized by an initially weak IFN-g response that increases gradually over a period of months. Our studies have shown that this response cannot be enhanced by the inclusion in the vaccine formulation of a conventional oil-in-water adjuvant which otherwise significantly increases the IFN-g response to the highly immunogenic MLV vaccine against pseudorabies (PR) virus, a herpesvirus of swine. In contrast, inactivated vaccines against either PRRS virus or PR virus containing a similar adjuvant were unable to elicit the development of a virus-specific IFN-g response. In contrast, the co-delivery of plasmid encoding porcine IL-12 or porcine IFN-a along with the PRRS MLV vaccine increased the intensity of the primary PRRS-virus specific IFN-g T cell
response to PRRSV at least three-fold. While the enhancing effect of the IL-12-containing plasmid on the intensity of the IFN-g response was transient, the enhancing effect of the IFN-a encoding plasmid was maintained for several weeks. The enhanced IFN-g response was also evident during the booster phase of the response upon challenge with virulent PRRSV at 9 weeks after vaccination. The results obtained indicate that the immune response to vaccination with PRRSV can be enhanced by exogenous cytokines. Injection of exogenous cytokines in the form of plasmids containing cytokine cDNA might have an application in the formulation of a new generation of highly effective vaccines. Passive immunization with sera containing PRRS virus-neutralizing antibodies has been shown to confer not only protective but also sterilizing immunity (Osorio et al, 2002). However, the production of neutralizing antibodies as a result of infection or vaccination with PRRS virus is not a consistent event and
most pigs either only develop low titers of virus-neutralizing antibodies or none at all. For this reason we have been examining the possible correlation between the intensity of the PRRS virus-specific IFN-g response and protective immunity. So far three independent studies have suggested a positive correlation between these two parameters. In the first experiment a positive correlation coefficient of 0.61 (P=0.0013) between the frequency of PRRS virus-specific IFN-g-SC in the lymphocyte population of previously MLV-immunized sows at the time of virulent PRRS virus challenge (90 days of gestation), and the number of subsequent live-born piglets was obtained. In the second experiment we observed that in a routinely vaccinated sows at a commercial facility, only those with a frequency of >150 PRRS virus-specific IFN-g SC/106 PBMC did not abort as a result of exposure to a natural outbreak of PRRS during their pregnancies. In the third experiment, in a group of pigs in a commercial farm
with endemic PRRS, a positive correlation was observed between the intensity of the IFN-g response to PRRS virus and reproductive performance.
Impacts
Understanding of the mechanisms that regulate the development of anti-viral protective immunity will lead to the development of more effective vaccines and methods to control this pathogen. Discernment of how immunity impacts the severity of clinical disease on commercial farms will lead to the development of improved strategies for managing PRRS in reproductive herds using strategies such as vaccination.
Publications
- Osorio, F.A., Galeota, J.A., Nelson, E., Brodersen, B., Doster A., Wills, R., Zuckermann, F.A. and Laegreid, W.W. 2002. Passive transfer of virus -specific antibodies confers protection against reproductive failure induced by a virulent strain of porcine reproductive and respiratory syndrome virus and establishes sterilizing immunity. 2002. Virology (In Press).
- Meier, W.A., Judy Galeota, J., Osorio, F.A., Husmann, R., Schnitzlein, W. and Zuckermann, F.A. 2003. Gradual development of the interferon-g response of swine to porcine reproductive and respiratory syndrome virus. Virol. (In Press).
- Foss, D.L., Zilliox, M.J., Meier, W., Zuckermann, F. and Murtaugh, M.P. 2002. Adjuvant danger signals increase the immune response to porcine reproductive and respiratory syndrome virus. Viral Immunol. 15:557-566.
- Murtaugh, M.P., Xiao, Z. and Zuckermann, F. 2002. Immunological responses of swine to porcine reproductive and respiratory syndrome virus infection. Viral Immunol. 15:533-547.
- Murtaugh, M.P., Xiao, Z., Rutherford, M.S. and Zuckermann, F. 2002. Immunology. The PRRS (Porcine Arterivirus) Compendium (2nd Edition). Section 5.3. 28 pp. National Pork Board, Clive, Iowa. (In Press).
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Progress 01/01/01 to 12/31/01
Outputs
The mechanisms of protective immunity against viruses, in particular porcine reproductive and respiratory syndrome (PRRS) virus are not understood. In an effort to clarify this issue we characterized the immune response induced by either infection with wild-type PRRS virus or immunization with a modified-live (MLV) PRRS virus vaccine. A striking observation resulting from this investigation is the temporal polarization of the humoral and cellular immune responses to this virus. While a strong virus-specific humoral immune response is quickly developed , the generation of interferon gamma (IFN-g)-producing T cells, is initially weak . Moreover, the brisk antibody response that follows either infection or vaccination with PRRS virus consists predominantly of non-neutralizing antibodies, and in some animals neutralizing antibodies might not develop at all. Within four months after infection or vaccination with PRRS virus the titer of antibodies declines significantly,
becoming even undetectable by ELISA in at least 40% of the pigs by 6 to 8 months. In contrast, the intensity and quality of the virus-specific IFN-g response gradually increases from 3 to 6 months after exposure to wild-type or attenuated virus. Although the reason for the initially weak PRRS virus-specific IFN-g response is unclear at this time, we have evidence indicating that interleukin (IL)-12 and interferon-alpha are capable of enhancing the intensity of the virus-specific cellular immune response in vivo.
Impacts
The rational development of effective PRRS virus vaccines will require an understanding of the mechanisms that regulate the kinetics, quality and intensity of the humoral and cellular immune responses directed against this virus. The results of these studies indicate that the IFN-g response to vaccination with PRRS virus can be enhanced by providing an exogenous cytokine such IL-12 or IFN-alpha. The ultimate goal of this project is the development of more effective vaccine against PRRSV. Research will continue to achieve this goal.
Publications
- No publications reported this period
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Progress 01/01/00 to 12/31/00
Outputs
Studies aimed at the identification of the mechanisms that regulate the development of strong cell-mediated immunity to PRRS virus in swine are continuing. Based on the biology of the virus, as well as experimental and field observations, we predict that the development of an adequate number of PRRS virus-specific memory T cells capable of secreting of interferon (IFN)-gamma plays a major role in the development of protective immunity and elimination of the viral infection. To facilitate the analysis of cell-mediated immune response in large number of samples for research as well as diagnostic purposes, we developed an assay that will streamline the measurement of this type of immunity in pigs. Two major limitations for the measurement of this type of immunity are the labor intensive process of the purification of mononuclear cells from the blood and the relatively low amount of IFN-gamma produced at least within the first few months after infection. To eliminate
these two problems, we have developed a "Whole blood IFN-gamma assay." This assay has a 3 log dynamic range that allows measurement of IFN-gamma ranging from low picogram up to high nanogram amounts per ml. This assay is based on the principle of time resolved fluorometry that utilizes a Europium conjugate instead of an enzyme labeled conjugate as in a conventional ELISA. Second, we have developed a simple whole blood culture system that will eliminate the need to purify the mononuclear cells from the blood. The whole blood IFN-gamma assay will facilitate the analysis of large number of pig blood samples to conduct field studies to examine the development of cellular immunity to PRRS virus. This assay also has the potential to be used as a tool to eliminate false-positive results that are being observed with the PRRS IDEXX ELISA.
Impacts
The objective of this research is to understand the development of immunity to infection with PRRS virus. It is likely that this research will lead to the development of better vaccines.
Publications
- No publications reported this period
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