Progress 05/12/05 to 09/30/09
Outputs
OUTPUTS: Progress (Risatti): Mapping Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) Genetic Determinants of Macrophage Host Range and Immune Modulation. New knowledge was derived from this research. Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) genetic determinants affecting the response of the host primary target cell, the macrophage, to infection are yet to be defined. We have used recombinant viruses encompassing ORF 1A to identify PRRSV determinants associated with growth and modulation of pro- and anti-inflammatory cytokine expression in primary pulmonary alveolar macrophages cultures (PAMs). Three genomic chimeras encompassing ORF 1A of PRRSV live attenuated vaccine Prime Pac (LAV SP) in the genetic background of pathogenic strain NVSL 97-7895 (FL12v) were characterized in vitro. Unlike parental viruses, two of the recombinant viruses encompassing the area of the genome encoding for NSP2 to NSP8 showed reduced growth in PAM cultures. The effect of virus infections on gene activation was studied for 25 immunomodulatory cellular genes in PAMs at 24 and 48 hours post-infection (hpi). Steady state mRNA levels in PAMs infected with recombinant viruses were compared to levels observed in cells infected with parental virus FL12v. Recombinant viruses induced patterns of transcriptional activation differing from patterns induced by parental FL12v, suggesting a regulatory role of PRRSV ORF1A on PAM gene expression. Progress (Garmendia): "Assessment of Virulence of PRRSV Isolates Based Both on their Sensitivity to IFNbeta and Ability to Induce Type I IFN Responses". The aims of the study are to determine the sensitivity to and induction of IFNbeta by the isolates, to identify mechanisms of evasion of host's innate immune responses by PRRSV and to determine correlations with virulence. We have phenotyped several PRRSV field isolates in their sensitivity to and ability to induce IFNbeta in vitro. A series of chimeric viruses derived from infectious clones of FL12 (virulent isolate) and a vaccine virus (attenuated), provided by Dr. Osorio (University of Nebraska at Lincoln, Nebraska), are also being tested. Preliminary data show significant differences in sensitivity to IFNbeta among different PRRSV isolates and between MARC-145 cells and porcine alveolar macrophages (PAM). The data on induction obtained thus far show that PRRSV isolates do induce IFNbeta in PAM but at variable levels. The sensitivity to and ability to induce IFNbeta in vitro will be the basis to formulate phenotypes for further testing. Selected isolates of different phenotypes will be utilized to identify segments in the type I IFN pathway that may be blocked by the virus. Efforts to test the effects of the virus on the signaling phase are under way. The pathogenicity of defined isolates will be investigated in swine. PARTICIPANTS: Guillermo Risatti (PI/PD), Inga Gudmunsdottir (Graduate student/graduated with a MS), Antonio E. Garmendia (PI/PD), Chris Overend (Graduate student/Ph.D. candidate), Ruj Maganti (Post-Doctoral fellow) TARGET AUDIENCES: The project is aimed to identify Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) genetic determinants associated with macrophage host range and with modulation of pro- and anti-inflammatory cytokine expression. This research will provide critical information needed for rational engineering of PRRS live attenuated vaccines (LAV) and/or antivirals. Mapping of these pathobiologically relevant PRRSV genes/determinants will permit rational design of differential vaccines of unprecedented safety, efficacy, and utility. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
Investigations on transcriptional activation in macrophages upon PRRSV infections have produced the following results (Risatti): Infection of primary porcine alveolar macrophages (PAMs) with virulent PRRSV FL12v resulted in transcriptional activation of 14 out of the 25 genes analyzed. A sustained transcriptional activation was observed for IL-1α, IL-6, TNF-α, IFN-β, IRF-7, PKR and Mx1, consistent with activation of an antiviral state within PAMs upon infection with virulent FL12v. At 24 hour post-infection (hpi), which corresponds to the peak logarithmic phase of FL12v infectious virus assembly and release, transcription of IL-1α, IL-10, IL-15, IRF-7, VCAM, and Mx-1 genes was significantly different in PAMs infected with cP5U.NSP12 or cPNSP3.8 relative to PAMs infected with FL12v. Live attenuated vaccine (LAV) SP infection induced a pattern of transcriptional activation at 24 hpi similar to that of FL12v. At 48 hpi, at the decline of virus yield in PAMs, the transcriptional activation profile differed from the profile observed at 24 hpi. IL-1α, IL-1β, IL-10, IL-15, TNF-α, MCP-2, IRF-7, and Mx1 mRNAs accumulation was significantly different in cells infected with the recombinant viruses or LAV SP relative to cells infected with FL12v. Sensitivity to IFNbeta and Ability to Induce Type I IFN Responses by PRRSV (Garmendia): Preliminary data show significant differences in sensitivity to IFNbeta among different PRRSV isolates and between MARC-145 cells and porcine alveolar macrophages (PAM). The data on induction obtained thus far, show that PRRSV isolates do induce IFNbeta in PAM but at variable levels. The sensitivity to and ability to induce IFNbeta in vitro will be the basis to formulate phenotypes for further testing. Selected isolates of different phenotypes will be utilized to identify segments in the type I IFN pathway that may be blocked by the virus. Efforts to test the effects of the virus on the signaling phase are under way. The pathogenicity of defined isolates will be investigated in swine.
Publications
- Gudmundsdottir, I., Risatti, G.R. 2009. Infection of Porcine Alveolar Macrophages with Recombinant Chimeric Porcine Reproductive and Respiratory Syndrome Virus: Effects on Cellular Gene Transcription and Virus Growth. Virus Research 145(1) 145-50
- Gudmundsdottir, I., Tulman, E., Rock, D.L., Borca, M.V., Risatti, G.R. 2008. Patterns of Gene Expression in Porcine Alveolar Macrophage Cells Upon Infection with Virulent, Attenuatted, and Recombinant Chimeric Porcine Reproductive and Respiratory Syndrome Virus. International PRRS Symposium, Chicago, IL, Dec. 5-6, 2008. P 36
- Overend, C., Maganti, R., Garmendia, A.E. 2008. Comparison of N Protein Nucleolar Localization Among Porcine Reproductive and Respiratory Syndrome Virus and Their Ability to Induce Type I IFN. International PRRS Symposium, Chicago, IL, December 5-6, 2008, P 73
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Progress 01/01/08 to 12/31/08
Outputs
OUTPUTS: Output for project: "Mapping Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) Genetic Determinants of Macrophage Host Range and Immune Modulation", PI: Guillermo Risatti. A poster was presented at the 27th Annual Meeting of the American Society for Virology (P7-11) held on July 12-16, 2008 at Cornell University, Ithaca, NY. Results of this research were presented by Ms Inga Gudmundsdottir (MS student) at the 2008 College of Agriculture and Natural Resources Graduate Student Council Research Forum of the University of Connecticut on March 2008. A poster was also presented during the 2008 International PRRS Symposium (P266) held on December 2008 in Chicago, IL. Ms. Inga Gudmundsdottir was mentored under this project and received a Master of Science from the University of Connecticut on July 2008 with a dissertation entitled "Mapping Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) Genetic Determinants of Macrophage Host Range and Immune Modulation". Output for project: "Assessment of Virulence of PRRSV Isolates Based Both on their Sensitivity to IFN and Ability to Induce Type I IFN Responses". Active participation in the NC229 meeting held in Chicago, Illinois December 2008. Active participation at the International PRRSV Symposium held in Chicago, Illinois, December 2008. Mr. Christopher Overend a doctoral candidate in Pathobiology and Veterinary Science who is working in the project had a poster presentation. Mentoring Dr. Raj Maganti a post-doctoral fellow and participant in the study. Data obtained in this project are discussed with our collaborator Dr. Marvin Grubman who shares interests in the area of type I IFN as it relates with viral diseases of swine. Data are also presented and discussed internally at seminars in our department. PARTICIPANTS: "Mapping Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) Genetic Determinants of Macrophage Host Range and Immune Modulation is aimed at identifying PRRSV genetic determinants associated with macrophage host range and with modulation of pro- and anti-inflammatory cytokine expression upon infection of swine pulmonary alveolar macrophages with the virus". Principal Investigator: Guillermo Risatti, University of Connecticut. Graduate Student: Inga Gudmundsdottir, University of Connecticut. Collaborators: Daniel L. Rock and Federico Zuckerman, University of Illinois. "Assessment of Virulence of PRRSV Isolates Based Both on their Sensitivity to IFN and Ability to Induce Type I IFN Responses". Principal Investigator: Antonio E. Garmendia, University of Connecticut, Storrs, Connecticut; Graduate Student: Christopher Overend, University of Connecticut, Storrs, Connecticut; Post-Doctoral Fellow: Raj Maganti, University of Connecticut, Storrs, Connecticut; Collaborator Marvin Grubman, Plum Island Animal Disease Center, USDA-ARS, Greenport, New York. TARGET AUDIENCES: PRRSV researchers, vaccine companies, pork producers PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
Outcomes for project: Mapping Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) Genetic Determinants of Macrophage Host Range and Immune Modulation, PI: Guillermo Risatti. New knowledge has derived from this research. Since Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) genetic determinants affecting the response of the host primary target cell, the macrophage, to infection are yet to be defined. We have used recombinant viruses encompassing ORF 1A to identify PRRSV determinants associated with growth and modulation of pro- and anti-inflammatory cytokine expression in primary pulmonary alveolar macrophages cultures (PAMs). Three genomic chimeras encompassing ORF 1A of PRRSV live attenuated vaccine Prime Pac (LAV SP) in the genetic background of pathogenic strain NVSL 97-7895 (FL12v) were characterized in vitro. Unlike parental viruses, two of the recombinant viruses encompassing the area of the genome encoding for NSP2 to NSP8 showed reduced growth in PAM cultures. The effect of virus infections on gene activation was studied for 25 immunomodulatory cellular genes in PAMs at 24 and 48 hours post-infection (hpi). Steady state mRNA levels in PAMs infected with recombinant viruses were compared to levels observed in cells infected with parental virus FL12v. Recombinant viruses induced patterns of transcriptional activation differing from patterns induced by parental FL12v, suggesting a regulatory role of PRRSV ORF1A on PAM gene expression. Outcomes for Project: "Assessment of Virulence of PRRSV Isolates Based Both on their Sensitivity to IFN and Ability to Induce Type I IFN Responses". The aims of the study are to determine the sensitivity to and induction of IFN by the isolates, to identify mechanisms of evasion of host's innate immune responses by PRRSV and to determine correlations with virulence. We have phenotyped several PRRSV field isolates in their sensitivity to and ability to induce IFN in vitro. A series of chimeric viruses derived from infectious clones of FL12 (virulent isolate) and a vaccine virus (attenuated), provided by Dr. Osorio (University of Nebraska at Lincoln, Nebraska), are also being tested. Preliminary data shows significant differences in sensitivity to IFN among different PRRSV isolates and between MARC-145 cells and porcine alveolar macrophages (PAM). The data on induction obtained thus far, shows that PRRSV isolates do induce IFN in PAM but at variable levels. The sensitivity to and ability to induce IFN in vitro will be the basis to formulate phenotypes for further testing. Selected isolates of different phenotypes will be utilized to identify segments in the type I IFN pathway that may be blocked by the virus. Efforts to test the effects of the virus on the signaling phase are under way. The pathogenicity of defined isolates will be investigated in swine.
Publications
- No publications reported this period
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Progress 01/01/07 to 12/31/07
Outputs
OUTPUTS: Output for Project: "Mapping PRRSV Genetic Determinants of Macrophage Host-Range and Immune Modulation".PI: Guillermo Risatti. Abstract submitted to American Society for Virology 27th Annual Meeting to be held on 12-16 July 2008 at Cornell University, Ithaca, New York. Title: "Mapping PRRSV Genetic Determinants of Macrophage Host Range and Immune Modulation, by Ingigerdur Gudmundsdottir (1), Edan Tulman (1), Daniel L. Rock (2), Manuel V. Borca (3), and Guillermo R. Risatti (1). (1) Univ. of Connecticut, Storrs, CT 06269. (2) Univ. of Illinois, Urbana, IL 61802. (3) Plum Island Animal Disease Center, ARS, USDA, Greenport, NY 11944. (Abstract# 566). Information produced with this research is being shared with collaborators, Dr Daniel L. Rock and Dr Federico Zuckerman, Department of Pathobiology, College of Veterinary Medicine at University of Illinois, Urbana. The PI participated in a scientific colloquium: "Prospects for Development of an Effective PRRS Virus Vaccine", at the
College of Veterinary Medicine, University of Illinois at Urbana-Champaign, June 1-2, 2007. Mentoring:Ms. Ingigerdur Gudmundsdottir is being mentored in a Master Thesis program under this project. Output for Project: "Assessment of Virulence of PRRSV Isolates Based Both on their Sensitivity to IFN-b and Ability to Induce Type I IFN Responses". PI: Antonio Garmendia. Participation in the NC229 meeting held in Chicago, Illinois December 2007. Attendance to the International PRRSV Symposium and the CRWAD meeting held in Chicago, Illinois, December 2007. Mentoring Mr. Christopher Overend for doctoral degree in Pathobiology and Veterinary Science who is working in the project. Mentoring Dr. Raj Maganti a post-doctoral fellow and participant in the study. Started an initiative of cooperation in research on PRRSV with Dr. Dongsheng He, Associate Professor, College of Veterinary Medicine, South China Agricultural University, Guangzhou, People's Republic of China. A proposal to the USDA's
Scientific Cooperation Exchange Program was submitted.
PARTICIPANTS: "Mapping PRRSV Genetic Determinants of Macrophage Host-Range and Immune Modulation is aimed at identifying PRRSV genetic determinants associated with macrophage host range and with modulation of pro- and anti-inflammatory cytokine expression". Principal investigator: Guillermo Risatti, University of Connecticut Graduate Student: Ingigerdur Gudmundsdottir, University of Connecticut Collaborator: Daniel L. Rock, University of Illinois Collaborator: Federico Zuckerman, University of Illinois. "Assessment of Virulence of PRRSV Isolates Based Both on their sensitivity to IFN beta and Ability to Induce Type I IFN Responses" Principal Investigator: Antonio E. Garmendia, University of Connecticut, Storrs, Connecticut; Graduate Student: Christopher Overend, University of Connecticut, Storrs, Connecticut; Post-Doctoral Fellow: Raj Maganti, University of Connecticut, Storrs, Connecticut; Collaborator Marvin Grubman, Plum Island Animal Disease Center, USDA-ARS, Greenport, New York.
TARGET AUDIENCES: Target audiences: PRRSV researchers, vaccine companies, pork producers.
Impacts
Project "Mapping PRRSV Genetic Determinants of Macrophage Host-Range and Immune Modulation is aimed at identifying PRRSV genetic determinants associated with macrophage host range and with modulation of pro- and anti-inflammatory cytokine expression". PI: Guillermo Risatti.This research will provide critical information needed for rational engineering of PRRS live attenuated vaccines (LAV). We have hypothesized that specific genes/genetic determinants of PRRSV are associated with macrophage host range and with the virus' ability to modulate host cytokine responses. Mapping of these pathobiologically relevant PRRSV genes/determinants will permit rational design of differential vaccines of unprecedented safety, efficacy, and utility. Eight chimeras (Ch1-Ch8) represented the entire SP genome in the genetic background of NVSL 97-7895. Chimeric viruses Ch2v and Ch5v, containing regions encoding for SP nsp2 protein and SP ORF1B, respectively, were largely defective for
replication relative to NVSL 97-7895, showing limited growth titers in both MARC-145 cells and swine pulmonary alveolar macrophage (PAMs) primary cell cultures. Five chimeric viruses (Ch1v, Ch4v, Ch6v-Ch8v) were able to replicate but demonstrated a small-plaque phenotype relative to parental virus in MARC-145 cells, consistent with a restriction in virus attachment and/or spread. Only Ch3v, a virus containing genes for nsp3 to nsp9 proteins from SP, showed plaque phenotypes resembling parental viruses. Notably, while other chimeric viruses induced a pattern of PAM gene expression (among 58 genes) similar to that of virulent NVSL 97-7895, Ch3v induced a pattern more similar to that induced by attenuated SP. These data suggest that, in the region encompassing genes for nsp3 to nsp9, PRRSV contains genetic determinants affecting host gene expression, and they indicate a potential role for these proteins in regulating macrophage inflammatory response during PRRSV infection. Project:
"Assessment of Virulence of PRRSV Isolates Based Both on their Sensitivity to IFN-b and Ability to Induce Type I IFN Responses". PI: Antonio Garmendia. Outcomes/Impacts: the study is aimed at determining mechanisms as to how PRRSV subverts the host's innate immune responses and establishes long lasting infections. In order to accomplish this goal we have initiated efforts to phenotype PRRSV isolates in terms of their sensitivity to IFN-b and ability to induce IFN-b both in vitro and in vivo. Virus isolates with opposing phenotypes will be tested comparatively in cell culture in attempts to identify potential steps in the IFN type I pathway that may be blocked. Differences in pathogenicity will be ultimately investigated in swine. Initial tests conducted in our laboratory indicate that there are significant differences in sensitivity to IFN-b among different PRRSV isolates. These results will be combined with their individual capacities to induce or block IFN-b in vitro to formulate
their phenotype for further testing.
Publications
- No publications reported this period
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Progress 01/01/06 to 12/31/06
Outputs
A set of eight recombinant chimeric Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) between a highly virulent strain and a live attenuated avirulent vaccine strain of PRRSV has been characterized by their in vitro growth patterns in MARC-145 cells. Chimeras are being used to identify genetic determinants of PRRSV macrophage host range. Unlike the parental strain viruses, which grow at the same level in MARC 145 cells, two chimeras have shown limited growth in these cells, four other viruses have shown deficient growth kinetics, while two chimeras have shown growth patterns comparable to those of the parental viruses. Next, the genomic integrity of viral chimeras and parental virulent and attenuated viruses was assessed by sequencing of the complete viral genomes. The recombinant nature of chimeric viruses was confirmed. Then, a comparative gene expression analysis between chimeric and parental viruses in primary alveolar macrophages is being carried on,
aimed to determine genes/regions within PRRSV genome involved in modulation of macrophages gene expression. Expression profiles of 38 immunomodulatory genes are being analyzed by means of quantitative real time PCR in porcine alveolar macrophages infected with eight recombinant and two parental viruses.
Impacts
This study is aimed to identify genetic determinants of PRRSV macrophage host range and immunemodulation.
Publications
- No publications reported this period
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Progress 01/01/05 to 12/31/05
Outputs
A set of eight recombinant chimeric Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) between a highly virulent strain and a live attenuated avirulent vaccine strain of PRRSV have been characterized by their in vitro growth patterns in MARC-145 cells. Chimeras are being used to identify genetic determinants of PRRSV host range. Unlike the parental strain viruses that grow at the same level in these cells, two chimeras have shown limited growth in MARC-145 cells, four other viruses have shown deficient growth kinetics in these cells, while two chimeras have shown growth patterns comparable to the parental viruses. To assess the integrity of the viral genome, and to rule out defects in the sequences, four chimeras showing defective in vitro growth kinetics in MARC 145 cells are being characterized by sequencing their entire genomes. The genome of each of these four viruses has been amplified by means of RT-PCR in 18 overlapping fragments. Each amplicon has
been cloned and will be sequenced.
Impacts
Work will contribute to fundamental understanding of mechanisms influencing virus-host cell interactions.
Publications
- No publications reported this period
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