Progress 10/01/09 to 09/30/14
Outputs
Target Audience: Data were published in peer-reviewed scientific literature, as well as data and interpretations published in industry newsletters and other publications targeted to the swine industry and allied providers. We gave a number of presentations at meetings, workshops, and symposia attended by swine veterinarians and members of the swine industry. These include the annual International/North American PRRS Symposium, the Annual NC229 meeting and CRWAD swine diseases and viral pathogenesis sessions, as well as meetings of the American Association of Swine Veterinarians and National Pork Board (Commodity Group) We maintained cross-talk and increase partnership with NC-229 equivalent groups in other countries and continents ( i.e COST Action network EuropRRS.net, Simposio Internacional PRRS Mexico 2012-2013 ), IPVS 2014 Cancun Mexico, OIE intenational course on swine exotic and emerging diseases, etc Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? Four PhD students grdauted in this period (2009-2014), and 2 post docs were mentored, How have the results been disseminated to communities of interest? Data were published in peer-reviewed scientific literature, as well as data and interpretations published in industry newsletters and other publications were targeted to the swine industry and allied providers. We gave a number of presentations at meetings, workshops, and symposia attended by swine veterinarians and members of the swine industry. These include the annual International/North American PRRS Symposium, the Annual NC229 meeting and CRWAD swine diseases and viral pathogenesis sessions, as well as meetings of the American Association of Swine Veterinarians and National Pork Board (Commodity Group), Interntional Pig Veterinary Socity, OIE, • We maintained cross-talk and increase partnership with NC-229 equivalent groups in other countries and continents ( i.e COST Action network EuropRRS.net, Simposio Internacional PRRS Mexico 2012-2013 -2014) What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
The success of research conducted at Nebraska mirrors well the sucess of the NC-229 project at the national level. NC-229 national::Successful pursuit of competitive grants to support PRRSV and swine respiratory disease research in general. In addition to the PRRS CAP II grant, numerous industry grants and contracts have been awarded to NC-229 participants for pig respiratory disease research (not only for PRRSV but also including other infections such as swine influenza virus and PCV2) since 2003. Most recently several laboratories were awarded competitive funds made available by NPB to respond to the emergency posed by the appearance in the US, during the Spring 2013, of PEDV. Likewise selected laboratories located in Mid West (and other "non-PIADC" areas of mainland US) have received funding for ASFV. All those labs recently awarded on emerging viruses of swine. Most importantly, it should be mentioned that during the last five years different participants of NC229 were awarded 21 competitive USDA-AFRI-NIFA grants, amounting to a total of $ 7,827,252, thus exceedingly more than duplicating the amount granted to the entire CAPII during the same period of time. Nebraska: The Nebraska PIs working in NC-229 at Nebraska were able to attract , during the like of this project (2009-2014) a total of 2.1 million dollars in competitive extramural funds ( including AFRiNIFA foundational, NPB and USDA CAPII, having published a total of 19 papers in refereed journals Eloquent example of the accomplihsments obtained at Nebraska during the life of this project is provisional patent filed by the PIs: Provisional claim for invention: "A method for development of a porcine reproductive and respiratory virus vaccine strain capable of inducing broad protection"(Hiep, V, Osorio, FA, Laegreid, W, Pattnaik, AK, Ma F) filed on January 24, 2014 EFS ID: Pending Application approved on march 24, 2014 )and currently (january 2015) being explored by at least two companies while forwarded by Nutech Ventures nebraska towards full certification for patenting.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2014
Citation:
Lee JA, Kwon B, Osorio FA, Pattnaik AK, Lee NH, Lee SW, Park SY, Song CS, Choi IS, Lee JB 2014. Protective humoral immune response induced by an inactivated porcine reproductive and respiratory syndrome virus expressing the hypo-glycosylated glycoprotein 5. Vaccine. 2014 32(29):3617-22. PMID: 24814552
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Progress 10/01/12 to 09/30/13
Outputs
Target Audience: Data published in peer-reviewed scientific literature, as well as data and interpretations published in industry newsletters and other publications were targeted to the swine industry and allied providers. We gave a number of presentations at meetings, workshops, and symposia attended by swine veterinarians and members of the swine industry. These include the annual International/North American PRRS Symposium, the Annual NC229 meeting and CRWAD swine diseases and viral pathogenesis sessions, as well as meetings of the American Association of Swine Veterinarians and National Pork Board (Commodity Group) We maintained cross-talk and increase partnership with NC-229 equivalent groups in other countries and continents ( i.e COST Action network EuropRRS.net, Simposio Internacional PRRS Mexico 2012-2013) Changes/Problems: No major changes What opportunities for training and professional development has the project provided? One PhD graduatedin molecular virology and vaccinology of PRRSV: current position post-doctoral research associate working in PRRS vaccinology How have the results been disseminated to communities of interest? Following presntations to proefessional forums: Laegreid W.W., Pires-Alves, M, Vu, HL, and osorio FA Variation in Search of a Theme: The Extraordinary Diversity of Porcine Reproductive and Respiratory Syndrome Virus Schultz Lecture Twenty-first Annual Swine Disease Conference for Swine Practitioners, November 14-15, 2013 Engle,TB, E.E. Jobman, A.M. McKnite, T.W. Moural, J.W. Bundy, J.K. Tart, T.P. Bohnert, S.Y. Barnes, E.H. Davis, J.K. Qiu, K.L. Lucot, J.A. Galeota, S.P. Harris, M.F. Rothschild, G.S. Plastow , R.K. Johnson, S.D. Kachman, D.C. Ciobanu (2014) Genome-wide Analysis of Susceptibility to Porcine Circovirus 2b, ASAS Midwest Conference, March 17 – 19, 2014 (submitted). Engle, TB, E.E. Jobman, A.M. McKnite, T.W. Moural, J.W. Bundy, J.K. Tart, T.P. Bohnert, S.Y. Barnes, E.H. Davis, J.K. Qiu, K.L. Lucot, J.A. Galeota, S.P. Harris, M.F. Rothschild, G.S. Plastow , R.K. Johnson, S.D. Kachman, D.C. Ciobanu (2013) Genome-wide Analysis of Susceptibility to Porcine Circovirus 2b, Nebraska Virology Symposium, October 14, 2013. Engle, T. B., Jobman, E. E., McKnite, A. M., Moural, T. W., Tart, J. K., Bundy, J. W., Bohnert, T. P., Barnes, S. Y., Davis, E. H., Qiu, J. K., Galeota, J. A., Harris, S., Rothschild, M. F., Plastow, G. S., Johnson, R., Kachman, S., Ciobanu, D. (2013) Genomic Analysis of the Susceptibility to Porcine Circovirus 2b. Plant and Animal Genome XXI Conference, San Diego, January 12 - 16, 2013. Kreikemeier, C, Thomas Burkey, Daniel Ciobanu (2013) Genome-wide Analysis of Indicators of Oxidative Stress and Immune Response in Pigs Challenged with Porcine Circovirus 2b, Plant and Animal Genome XXI Conference, San Diego, January 12 - 16, 2013. Dekkers, JCM, N.J. Boddicker, A.M McKnite, J.K. Lunney, R.R.R. Rowland, D.C. Ciobanu, J.C.S. Harding, R.A. Kemp, J. Wilkinson, G.S. Plastow (2012) The role of host genetics on the impact of viral disease in pigs: large scale projects in North America, Canadian Swine Health Board Forum, October 17 – 18, 2012. Jobman, E. E., McKnite, A. M., Moural, T. W., Tart, J. K., Bundy, J. W., Bohnert, T. P., Barnes, S. Y., Kreikemeier, C. A., Engle, T. B., Davis, E. H., Qiu, J. K., Galeota, J. A., Harris, S., Rothschild, M. F., Burkey, T., Johnson, R., Kachman, S., Ciobanu, D. (2012). Genomic analysis of the differential response in experimental infection with Porcine Circovirus 2. International PRRSV Symposium and NSIF Annual Joint Meeting, Kansas City, November 30, 2012. What do you plan to do during the next reporting period to accomplish the goals? 1)To lead on the preparation of the national project NC229, coordinating the writing of the next five year renewal proposal (2014-2019) 2) To serve as Chair of the executive Board of NC-229 for the period 2014-2015 3) To approach cross-protective immunity against PRRSv by : a) subunit vaccines to induce broadly neutralizing antibodies, b) vectored platforms containing conserved epitopes, or c) synthetic PRRSV designed as a consensus antigenic sequence
Impacts
What was accomplished under these goals?
In the period reported ( 10/01/2012 to 09/30/2013) : 1) We provided an important starting point for the development of a live-attenuated DIVA vaccine against type-II PRRSV( preliminary disclosure filed with Nutech Ventures, Univ of Nebraska). 2) The research of the influence of host genetics on PCVAD susceptibility could lead to increase knowledge of swine immune system, as well as identification of genes involved in PCVAD susceptibility. Selection based on DNA markers associated with PCVAD susceptibility has the potential to reduce economic losses, improve animal welfare and provide alternatives to vaccination.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2013
Citation:
Vu, H. L., Kwon, B. J., M, d. L., Pattnaik, A., Osorio, F. (2013). Characterization of a serologic marker candidate for development of a live-attenuated DIVA vaccine against porcine reproductive and respiratory syndrome virus. Elsevier Vaccine, 31, 4330-4337.
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Progress 10/01/11 to 09/30/12
Outputs
OUTPUTS: For this period 10/2011 to 09/2012 NE reports active ongoing research on objectives 1 and 3 of the Multistate proposal. Objective 1. Elucidate the mechanisms of host-pathogen(s) interactions. At Nebraska RES we have continued focused on the role that certain non-structural proteins of PRRSV have on the innate immunity established against this viral infection. We have specifically studied the NSP1. Using reverse genetics (alanine-scanning mutagenesis) we have identified amino acid residues in NSP1 important for anti-IFN activity of porcine reproductive and respiratory syndrome virus non-structural protein 1. We were able to obtain a NSP1β mutant that presented an in vitro and in vivo distinct phenotype with relieved anti-IFN effect. However, the mutant reverted in vivo within the first week post-infection. The results indicate a strong selection pressure towards maintaining the IFN-inhibitory property of PRRSV for successful propagation in pigs.We also studied in detail the molecular determinants of anti-TNFα mediated by PRRSV NSP1. Using the same approach as above, two mutant viruses, with mutations at Nsp1α Gly90 orNsp1β residues70-74, generated from infectious cDNA clones exhibited attenuated viral replication in vitro and TNF-α was found to be up-regulated in infected macrophages. In infected pigs,theNsp1β mutant virus was attenuated in growth. These studies provide insights into how PRRSV evades the effector mechanisms of innate immunity during infection. ( UNL Osorio/Pattnaik, UIUC Zukermann/Laegreid) This collaborative project consisted of mapping, using pep-scan technology, T-cell epitope candidates contained in NSP9 and NSP10, both of which are highly conserved. The peptides were probed for their ability to elicit a recall proliferative and interferon-gamma response in peripheral blood mononuclear cells obtained from pigs immunized against the type-II PRRSV strain FL-12. These studies led to the identification of four peptides, two from each NSP9 and NSP10 that appear to contain seemingly highly conserved T-cell epitopes. The identified epitopes may be important for the formulation of immunogens ( i.e peptide vaccines) to provide broad cross-protection against diverse PRRSV strains. Objective 3. Develop effective and efficient approaches for detection, prevention and control of PRRSV and emerging viral diseases of swine This collaborative project (UNL/UIUC) consisted of mapping, using pepscan technology, T-cell epitope candidates contained in NSP9 and NSP10, both of which are highly conserved. The peptides were probed for their ability to elicit a recall proliferative and interferon-gamma response in peripheral blood mononuclear cells obtained from pigs immunized against the type-II PRRSV strain FL-12. These studies led to the identification of four peptides, two from each NSP9 and NSP10 that appear to contain seemingly highly conserved T-cell epitopes. The identified epitopes may be important for the formulation of immunogens (i.e. peptide vaccines) to provide broad cross-protection against diverse PRRSV strains. PARTICIPANTS: Osorio, FA, Pattnaik, AK, Ciobanu, D TARGET AUDIENCES: 1)Scientific peers at the PRRSV research community ( NC-229 2012 meeting and 2012 CRWAD) 2) Practitioners and industry stakeholders attending the PRRSV meetings 3) Commodity groups (National Pork Board), 4) International PRRSV researchers and international research consortia that have invited the NE members of NC229 to contribute expertise as external reviewers or as invited speakers. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
1) During the period reported, this project has produced a total of 5 publications in refereed journals. 2) Some of the novel information produced by this project has provided the entire foundation for successful competitive funding renewal achieved in the most recent round of USDA-NIFA awards (FY2012, PI AK Pattnaik). This renewal funding amounts to more than $ 480,000. 4) Based exclusively on investigations conducted for this project, three graduate students completed their doctoral degrees working in an important area of animal agriculture: domestic swine health. These three brand-new doctorates are now conducting independent post-doctoral research in 3 premier universities of the Nation: UC-Berkeley, U of Minnesota Medical School, and Virginia Tech.
Publications
- Anderson TK, Laegreid WW, Cerutti F, Osorio FA, Nelson EA, Christopher-Hennings J, Goldberg TL.(2012)Ranking viruses: measures of positional importance within networks define core viruses for rational polyvalent vaccine development. Bioinformatics. 2012 Apr 11.
- Jourdan SS, Osorio FA, Hiscox JA. Biophysical characterization of the nucleocapsid protein from a highly pathogenic porcine reproductive and respiratory syndrome virus strain. Biochem Biophys Res Commun. 2012 Mar 9;419(2):137-41.
- Jourdan SS, Osorio F, Hiscox JA.(2012) An interactome map of the nucleocapsid protein from a highly pathogenic North American porcine reproductive and respiratory syndrome virus strain generated using SILAC-based quantitative proteomics. Proteomics. 2012 Apr;12(7):1015-23.
- Subramaniam S, Beura LK, Kwon B, Pattnaik AK, Osorio FA.2012.Amino acid residues in the non-structural protein 1 of porcine reproductive and respiratory syndrome virus involved in down-regulation of TNF-α expression in vitro and attenuation in vivo.Virology. 2012 Oct 25;432(2):241-9
- Parida R, Choi IS, Peterson DA, Pattnaik AK, Laegreid W, Zuckermann FA, Osorio FA.Location of T-cell epitopes in nonstructural 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 10/01/10 to 09/30/11
Outputs
OUTPUTS: Objective 1 Elucidate the mechanisms of host pathogen To further our understanding of the role of the viral nsp1 in PRRSV life cycle, using nsp1beta, a proteolytically processed functional product of nsp1 as bait, we have identified the cellular poly (C)-binding proteins 1 and 2 (PCBP1 and PCBP2) as two of its interaction partners. The interactions of PCBP1 and PCBP2 with nsp1beta were confirmed by both co-immunoprecipitation in infected cells and/or in plasmid transfected cells and also by in vitro binding assays. During PRRSV infection of MARC-145 cells, the cytoplasmic PCBP1 and PCBP2 partially co-localize to the viral replication-transcription complexes. Furthermore, recombinant purified PCBP1 and PCBP2 were found to bind the viral 5' untranslated region (UTR). SiRNA-mediated silencing of PCBP1 and PCBP2 in cells resulted in significantly reduced PRRSV genome replication and transcription without adverse effect on initial polyprotein synthesis. Objective 3 Develop approaches for detection, prevention and control of PRRSV We had previously identified several immunodominant B-cell linear epitopes in the proteins of a type-II PRRSV strain FL12 (infectious cDNA clone-derived PRRSV pathogenic strain), of which, the epitope number 201 (EP-201) located at the carboxyl terminal region of the M protein was selected as a marker candidate. Comparison of the amino acid sequence of ~100 M proteins collected from the NCBI database revealed that this epitope is highly conserved across the type-II PRRSV strains. Moreover, a monoclonal antibody specific to EP-201 recognized 92% (n=81) type-II PRRSV isolates, confirming the conservation of this epitope. A triple mutant (TM) carrying 3 amino acid substitutions in the epitope 201 region of PRRSV FL12 was generated by site-directed mutagenesis. The TM was no longer recognized by anti-201 MAb. More importantly, the TM was stable in vivo and did not elicit antibodies to peptide 201. Collectively, the TM can be used as a DIVA marker vaccine strain. Current emphasis is directed at optimization of the peptide 201 ELISA companion assay. PARTICIPANTS: NE investigators: Osorio, Pattnaik, Ciobanu NE post-docs: Kwon(BJ) NE graduate students: Beura, Submaraniam External collaborators: Laegreid,W. (UIUC) Yoon, KJ (ISU) Christopher Hennings, J (SDSU) Nelson, E (SDSU) TARGET AUDIENCES: 1)Scientific peers at the PRRSV research commmunity ( 2010 International PRRS Symposium Meeting, organized by NC-229 and USDA-CAP2 PRRS group), 2) Practitioners and industry stakeholders attending the PRRSV meetings 3) Commodity groups (National Pork Boards), 4) International PRRSV researchers and international research consortiums that have invited the NE members of NC229 to contribute expertise as external reviewers or as invited speakers. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
1) At least three PRRSV-related refereed papers involving our laboratories have been published in refereed journals during the period covered in this report. 2) US and European Patent Title: Methods And Compositions For Vaccination Of Animals With PRRSV Antigens With Improved Immunogenicity. Inventors: Ansari, I, Osorio FA, and Pattnaik, AK Serial No. 12/064, Issued: October 27, 2009. During 2011 this patent is being explored by a veterinary biologics company 3) Provisional claim for invention: "A reverse genetics method to develop a PRRSV attenuated live vaccine strain with DIVA differential capacity that would permit distinguishing naturally infected from non-infected, just vaccinated animals" (Osorio, FA, Pattnaik, AK, Kwon, BJ, and Vu H.) filed on March 4, 2011, EFS ID: 9585692 Application No. 61449138
Publications
- Beura LK, Dinh PX, Osorio FA, Pattnaik AK.(2011) Cellular Poly(C) Binding Proteins 1 and 2 Interact with Porcine Reproductive and Respiratory Syndrome Virus Non-Structural Protein 1{beta} and Support Viral Replication. J Virol. 2011 Oct 5. In press
- Vu HL, Kwon B, Yoon KJ, Laegreid WW, Pattnaik AK, Osorio FA. (2011) Immune evasion of porcine reproductive and respiratory syndrome virus through glycan shielding involves both glycoprotein 5 as well as glycoprotein 3. J Virol. 2011 Jun;85(11):5555-64.
- Das PB, Vu HL, Dinh PX, Cooney JL, Kwon B, Osorio FA, Pattnaik AK.(2011)Glycosylation of minor envelope glycoproteins of porcine reproductive and respiratory syndrome virus in infectious virus recovery, receptor interaction, and immune response. Virology. 2011 Feb 20;410(2):385-94. Epub 2010 Dec 30.
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Progress 10/01/09 to 09/30/10
Outputs
OUTPUTS: Project 1.1 Glycoproteins of Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) in Infection and Immunity (Pattnaik A, Osorio F) Results to report on :Interaction of Minor PRRSV GPs with the Main PRRSV Receptor Overall, the main conclusion of this part of the project is that the GP4 protein is critical for mediating inter-glycoprotein interactions and, along with GP2a, serves as the viral attachment protein that is responsible for mediating interactions with CD163 for virus entry into susceptible host cell. This is an important concept for vaccinology of PRRSV. This output has been reported orally at the 2009 PRRSV International Symposium and published in Journal of Virology during 2010. Project 1.2 Porcine Reproductive and Respiratory Syndrome Virus Infection: Effects on Innate and Acquired Immune Responses (Osorio F, Pattnaik A) Results to report on : Anti-IFN effect of PRRSV NSP1Beta. In this tsudy we have identifed two important T cell epitopes in PRRSV NSP9 and two in the NSP10. We explored in detail the mechanisms involved in NSP1beta effect. We propose that this NSP1 -mediated subversion of the host innate immune response plays an important role in PRRSV pathogenesis.This output has been published in Journal of Virology during the year 2010 and presented orally at the 2009 PRRSV International Symposium. PARTICIPANTS: Nothing significant to report during this reporting period. TARGET AUDIENCES: 2009 and 2010 PRRSV International Symposiums PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
Work conducted at Nebraska under this project affects the multi-station collaborative network of NC-229 as follows: 1) Six PRRSV-related refereed papers involving our laboratories have been published during the period covered in this report. 2) ) US and European Patent Title: Methods And Compositions For Vaccination Of Animals With PRRSV Antigens With Improved Immunogenicity. Inventors: Ansari, I, Osorio FA, and Pattnaik, AK Serial No. 12/064, Issued: October 27, 2009. This patent is being explored now by a veterinary biologics company at this time.
Publications
- Doster AR, Subramaniam S, Yhee JY, Kwon BJ, Yu CH, Kwon SO, Osorio FA, Sur JH. (2010). Distribution and characterization of IL-10-secreting cells in lymphoid tissues of PCV2-infected pigs. Jour Vet Sci. 2010;11(3):177-183
- Subramaniam, S., Kwon, B. J., Beura, L. K., Kuszynski C. A., Pattnaik, A. K., and Osorio, F. A. (2010). Porcine reproductive and respiratory syndrome virus non-structural protein 1 suppresses tumor necrosis factor-alpha promoter activation by inhibiting NF-kB and Sp1. Virology, 406: 270-279 (E-pub ahead of print, Aug. 11, 2010
- Lawson S, Lunney J, Zuckermann F, Osorio F, Nelson E, Welbon C, Clement T, Fang Y, Wong S, Kulas K, 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. 2010 Jul 19;28(32):5356-64
- Das, P. B., Dinh, P. X., Ansari, I. H., de Lima, M., Osorio, F. A., and Pattnaik, A. K. (2010). The Minor Envelope Glycoproteins GP2a and GP4 of Porcine Reproductive and Respiratory Syndrome Virus Interact with the Receptor, CD163. J. Virology, 84: 1731-1740 (In Press, E-Publication ahead of Print, November 25, 2009)
- Beura, L. K., Sarkar, S. N., Kwon, B. J., Subramaniam, S., Jones, C., Pattnaik, A. K., and Osorio, F. A. (2010). Porcine Reproductive and Respiratory Syndrome Virus nonstructural protein nsp1 modulates host immune response by antagonizing IRF3 activation. J. Virology, 84: 1574-1584 (In Press, E-Publication ahead of Print, November 18, 2009)
- Subramaniam S, Sur JH, Kwon BJ, Pattnaik AK,Osorio FA (2010/11) A virulent strain of porcine reproductive and respiratory syndrome virus does not up-regulate interleukin-10 levels in vitro or in vivo. Virus Research, In Press
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