Progress 10/01/04 to 09/30/09
Outputs
OUTPUTS: The previous 5-year project (2004-2009) has now been renewed for 5 additional years ( 2009-2014),under the same NC number( NC-229). The approval for Nebraska's new 5-year project is in progress. Therefore, we herein report the activities for the last year only , but also emphasizing on the main outputs obtained throughout the life of the project. Nebraska contributions to this project resulted in: 1)Data published in peer-reviewed scientific literature( see publications) 2) Data and interpretations published in industry newsletters and other publications targeted to the swine industry and allied providers ( see publications) 3)Presentations at meetings, workshops, and symposia attended by swine veterinarians and members of the swine industry. Examples include the annual International PRRS Symposium and meetings of the American Association of Swine Veterinarians, the International Pig Veterinary Society, the Iowa State Univ. Swine Health Conference, and the Conference of Research Workers in Animal Disease 4)Interaction with PRRSV researchers ( NC-229 members)from USA, Mexico, Canada, Belgium, Spain and China, respectively 5) Biological materials: Nebraska has contributed one PRRSV infectious clone used and published about by scientists worldwide.Nebraska has produced a monoclonal antibody anti-PRRSV M protein that is being marketed, by the Office of Technology Development of the university of Nebraska, amongst different veterinary biologics companies 6)Nebraska's participants in this multistate project are co-inventors in a University of Nebraska patent which in 2009 has been accepted in the US and Europe( Methods And Compositions For Vaccination Of Animals With PRRSV Antigens With Improved Immunogenicity ). 7) Nebraska participated in a successful Coordinated Agricultural Project that has been competitively funded with a total $ 1 million for four years ( 2009-2013). The awarded group involves four stations collaborating in NC-229: NE, IL, WI and SDSU PARTICIPANTS: The four researchers involved in the Nebraska chapter of this NC-229 project have collaborated in submission of a sucessfully funded Coordinated Agricultural project, together with other three stations: Illinois, Wisconsin, and South Dakota. During the life of this project training has been provided to 3 graduate students who received PhD degree, three other graduate students who received a Master degree. Research mentoring has been provided to 3 postdoctoral trainees and two visiting professors in sabbatical leaves from foreign universities. TARGET AUDIENCES: The data and interpretations of our group were published in industry newsletters and other publications targeted to the swine industry and allied providers. The presentations at meetings, workshops, and symposia attended by swine veterinarians and members of the swine industry. Examples include the annual International PRRS Symposium and meetings of the American Association of Swine Veterinarians, the International Pig Veterinary Society, the Iowa State Univ. Swine Health Conference, and the Allen D. Leman Swine Conference PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
Changes in Knowledge: Nebraska has history of extensive publications in the scientific literature on PRRS. During the last five years of this NC-229 project Nebraska contributed 20 refereed publications, many of them in journals of high impact factor.Examples for number of citations of a publiuctaio: 1) a 2004 publication that has been cited 64 times. 2) A 2006 publication that has been cited 57 times. Changes in Actions: Nebraska has contributed a PRRSV infectious clone that is being shared with PRRSV researchers worldwide. This reagent has become the gold standard and world-wide reference for PRRSV infectious clones, with consistent and reproducible pathaogenicity results in the hands of many different researchers time and time again. Changes in Conditions: Nebraska NC-229 researchers have produced new technology applicable to PRRSv vaccine that is now being marketed by the Office of Technology Development of the University of Nebraska. Such technology is currently being explored for license by one international company located overseas who has supported patenting costs for this product.
Publications
- De Lima, M. Ansari, I. H., Das, P. B., Ku, B., Martinez-Lobo, F. J., Pattnaik, A. K., and Osorio, F. A.(2009). GP3 is a Structural Component of the PRRSV Type II Virion. Virology, 390:31-36
- 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 nsp1b modulates host immune response by antagonizing IRF3 activation. J. Virology ( In press)
- 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 (In Press).
- Pattnaik AK, Osorio FA and Ansari IH. 2008. Patent Application for Methods and Compositions For Vaccination of Animals with PRRSV Antigens with Improved Immunogenicity (2008). Serial No. 12/064,877, Filing Date February 26, (2008) Ref: IP-987; Our Ref: 46589-74731).
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Progress 10/01/07 to 09/30/08
Outputs
OUTPUTS: Virulence of PRRSV. We know now that certain PRRSV non-structural proteins and two structural genes (ORF 5 and ORF2) are involved in virulence. This invites considering our novel information about GP2 in light of the previous reports stating that the major envelope of PRRSV (GP5) would not be the gGP that interacts with the cell receptor; this may then attract the attention of researchers to GP2 that we know now, through our research, is importantly associated to virulence. Along this this line, fine mapping of virulence will likely involve site directed mutagenesis and reverse genetics, using a pair of homologous wt /attenuated strains (JA142/ATP strain). The attenuated strain in this pair is the most commonly used vaccine in the US market. Immunopathogenesis and vaccinology of PRRSV. We have found that PRRSV evades the immune system by means of a glycan-shielding mechanism and that the de-glycosylation of the PRRSV GP5 enhances significantly the ability of the PRRSV strain to induce protective antibody response. These two concepts, added to the notion that PRRSV may have several serologic markers (immunodominant B-cell epitopes) that could be used for DIVA differentiation, make together a significant contribution to PRRSV vaccinology. A paper in press describes proof of the concept of slected epitopes serving as markers in a DIVA vaccine. Research tools to study PRRSV pathogenesis and immunity issues. We have been able to successfully establish a reverse genetics experimental system for PRRSV that serves as national and international reference. Several laboratories worldwide have requested and are successfully using our IC system. We know now that our IC is fully functional, being ours the PRRSV infectious clone system with best recorded evidence of stability and reproducible pathogenesis in vivo. PRRS strain diversity. By means of cross neutralization of PRRSV strains, using strain-specific neutralization sera; it may be possible to set the basis to cluster or subgroup the wide universe of strains of PRRSV in subtypes. These subtypes may have a direct correlation with cross protection. If true, this concept may help to define the minimal number of valences or specificities that should be present in a PRRSV vaccine for this product to be broadly protective. PARTICIPANTS: Between October 2007 and September 2008,the most significant impacts of work conducted at Nebraska's experimental station for this this multi-station NC-229 program are: 1) A cDNA infectious clone that was produced at Nebraska through funding received from USDANRICGP and from NPB was provided to different laboratories. Successful applications of this cDNA infectious clone in those labs are now being reported in the literature (Key et al. Arch Virol 152:1383-7; Yoo D.Proc. Int. PRRS Symp, December 1-2, 2006 poster No. 9, You et al.Virology, 378:34-47). 2) Six refereed papers based on our laboratories have been published during the period covered in this report Three of these manuscripts published during this period reflect interdisciplinary collaboration with international researchers , who in some cases had received funding provided by the U.S. NPB-PRRSV initiative directed to those overseas laboratories(You et al.Virology, 378:34-47, and Spencer et al Vaccine. 226:5653-9.) TARGET AUDIENCES: Between October 2007 and September 2008,the most significant impacts of work conducted at Nebraska's experimental station for this this multi-station NC-229 program are: 1) A cDNA infectious clone that was produced at Nebraska through funding received from USDANRICGP and from NPB was provided to different laboratories. Successful applications of this cDNA infectious clone in those labs are now being reported in the literature (Key et al. Arch Virol 152:1383-7; Yoo D.Proc. Int. PRRS Symp, December 1-2, 2006 poster No. 9, You et al.Virology, 378:34-47). 2) Six refereed papers based on our laboratories have been published during the period covered in this report Three of these manuscripts published during this period reflect interdisciplinary collaboration with international researchers , who in some cases had received funding provided by the U.S. NPB-PRRSV initiative directed to those overseas laboratories(You et al.Virology, 378:34-47, and Spencer et al Vaccine. 226:5653-9.) PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
The most significant impacts of work conducted at Nebraska's experimental station for this this multi-station NC-229 program are: 1) A cDNA infectious clone that was produced at Nebraska through funding received from USDANRICGP and from NPB was provided to different laboratories. Successful applications of this cDNA infectious clone in those labs are now being reported in the literature (Key et al. Arch Virol 152:1383-7; Yoo D.Proc. Int. PRRS Symp, December 1-2, 2006 poster No. 9, You et al.Virology, 378:34-47). 2) Six refereed papers based on our laboratories have been published during the period covered in this report Three of these manuscripts published during this period reflect interdisciplinary collaboration with international researchers, who in some cases had received funding provided by the U.S. NPB-PRRSV initiative directed to those overseas laboratories(You et al.Virology, 378:34-47, and Spencer et al Vaccine. 226:5653-9.)
Publications
- Flores-Mendoza, Silva-Campa E., Resendiz,M., Osorio, FA, and Hernandez J 2008. Porcine reproductive and respiratory syndrome virus (PRRSV) infects mature porcine dendritic cells and up-regulates IL-10 production, Clin Vaccine Immunol. 15(4):720-725
- You J H, Howella G, Pattnaik AK, Osorio FA , and Hiscox JA 2008. A model for the dynamic nuclear/nucleolar/cytoplasmic trafficking of the porcine reproductive and respiratory syndrome virus nucleocapsid protein based on live cell imaging. Virology, 378:34-47.
- DeLima M, Kwon B, Ansari IH, Pattnaik AK, Flores E F , and Osorio FA 2008. Development of a porcine reproductive and respiratory syndrome virus differentiable (DIVA) strain through deletion of specific immunodominant epitopes Vaccine 26: 3594-3600
- Kwon, B. J., Ansari, I. H., Pattnaik, A. K., and Osorio, F. A. 2008. Identification of virulence determinants of Porcine Reproductive and Respiratory Syndrome virus through construction of chimeric clones. Virology 380: 371-378
- Jar, A.M., Osorio, F.A., Lopez, O.J. 2008 Mouse x pig chimeric antibodies expressed in baculovirus retain the same properties of their parent antibodies. In press in Biotechnology Progress
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Progress 10/01/06 to 09/30/07
Outputs
New knowledge on PRRSV biology derived from this project: Theme: Virulence of PRRSV 1) We have learned that certain PRRSV non-structural proteins and two structural genes (ORF 5 and ORF2) are involved in virulence. This invites considering our novel information about GP2 in light of the previous reports stating that the major envelope of PRRSV(GP5) would not be the gp that interacts with the cell receptor, this may then attract the attention of researchers to GP2, that we know now, through our research, is importantly associated to virulence. Theme: Immuno-pathogenesis and vaccinology of PRRSV 2) We have found that PRRSV evades the immune system by means of a glycan-shielding mechanism and that the deglycosylation of the PRRSV GP5 enhances significantly the ability of the PRRSV strain to induce protective antibody response. These two concepts, added to the notion that PRRSV may have several serologic markers (immunodominant B-cell epitopes) that could be used for DIVA
differentiation, make together a significant contribution to PRRSV vaccinology . Theme: Research tools to study PRRSV pathogenesis and immunity issues 3) We have been able to successfully establish a reverse genetics experimental system for PRRSV that serves as national and international reference. Several laboratories worldwide have requested and are successfully using our IC system (24, 55). We know now that our IC is fully functional, being ours the PRRSV infectious clone system with best recorded evidence of stability and reproducible pathogenesis in vivo. Theme: PRRS strain diversity 4)By means of cross neutralization of PRRSv strains, using strain-specific neutralization sera, it may be possible to set the basis to cluster or subgroup the wide universe of strains of PRRSV in subtypes. These subtypes may have a direct correlation with cross-protection. If true, this concept may help to define the minimal number of valences or specificities that should be present in a PRRSV
vaccine for this product to be broadly protective
Impacts
Major conclusions of this project include the following new information on PRRSV Biology: Theme: Virulence of PRRSV 1) we know now that certain PRRSV non-structural proteins and two structural genes (ORF 5 and ORF2) are involved in virulence. This invites considering our novel information about GP2 in light of the previous reports stating that the major envelope of PRRSV (GP5) would not be the gp that interacts with the cell receptor; this may then attract the attention of researchers to GP2 that we know now, through our research, is importantly associated to virulence. Theme: Immunopathogenesis and vaccinology of PRRSV 2) we have found that PRRSV evades the immune system by means of a glycan-shielding mechanism and that the deglycosylation of the PRRSV GP5 enhances significantly the ability of the PRRSV strain to induce protective antibody response. These two concepts, added to the notion that PRRSV may have several serologic markers (immunodominant B-cell epitopes)
that could be used for DIVA differentiation, make together a significant contribution to PRRSV vaccinology. Theme: Research tools to study PRRSV pathogenesis and immunity issues 3) we have been able to successfully establish a reverse genetics experimental system for PRRSV that serves as national and international reference. Several laboratories worldwide have requested and are successfully using our IC system (24, 55). We know now that our IC is fully functional, being ours the PRRSV infectious clone system with best recorded evidence of stability and reproducible pathogenesis in vivo. We believe that all these points sustain the significance of renewing funding for this type of work in our labs. Theme: PRRS strain diversity 4) By means of cross neutralization of PRRSv strains, using strain-specific neutralization sera; it may be possible to set the basis to cluster or subgroup the wide universe of strains of PRRSv in subtypes. These subtypes may have a direct correlation with cross
protection. If true, this concept may help to define the minimal number of valences or specificities that should be present in a PRRSv vaccine for this product to be broadly protective
Publications
- De Lima M, Pattnaik A, Flores EF, and Osorio FA 2006. Serologic marker candidates identified amongst B-cell linear epitopes of Nsp2 and structural proteins of a North American strain of Porcine Reproductive and Respiratory Syndrome virus Virology 353: 410-421
- Kwon, B. J., Ansari, I. H., Osorio, F. A., and Pattnaik, A. K. 2006.Infectious clone-derived viruses from virulent and vaccine strains of porcine reproductive and respiratory syndrome virus mimic biological properties of their parental viruses in a pregnant sow model.Vaccine. 2006 Nov 30;24(49-50):7071-80.
- Lopez O J, Oliveira MF, Alvarez Garcia E, Kwon BJ, Doster A and Osorio FA 2007 Protection against porcine reproductive and respiratory syndrome virus (PRRSV) infection through passive transfer of PRRSV-neutralizing antibodies is dose dependent. Clin Vaccine Immunol. 2007 Mar;14(3):269-75
- Zuckermann, F., Alvarez Garcia E., Diaz Luque I, Christopher-Hennings, J, Doster A, Brito M, Osorio FA. 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 Jul 20;123(1-3):69-85.
- Spencer KA, Osorio FA, Hiscox JA. 2007 Recombinant viral proteins for use in diagnostic ELISAs to detect virus infection. Vaccine. 226;25(30):5653-9.
- Ramirez R, Moreno V, Diaz N, Osorio F, Ruiz A, Neira V, Quezada M. 2007 Evaluation of the pathogenesis and transmissibility of a Chilean isolate of porcine reproductive and respiratory syndrome virus (PRRSV) J. Vet. Med A 54: 1-8
- Expert Group (Reporter: Rock, D. 2007). Colloquium on Prospects for Development of an Effective PRRS Virus Vaccine College of Veterinary Medicine, University of Illinois, Urbana, IL. (AASV Newsletter August 13, 2007) URL: http://www.aasp.org/news/story.php?id=2527
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Progress 10/01/05 to 09/30/06
Outputs
Achieve biosecurity within herds by preventing the spread of virus within a herd and facilitating its elimination from endemically infected herds. Summarize research progress related to 1) the study of genetically resistant animals, 2) the development and use of antivirals, 3) the development and application of vaccines. Project: Mapping Genes of PRRSV involved in virulence. Some of the high points recently attained through our research can be summarized as follows. We know now that certain PRRSV NSPs and two structural genes are heavily involved in virulence: ORF5 and ORF2. It results unavoidably tempting to consider our novel information about GP2 in light of the notion that actually the major envelope of PRRSVGP5 is not the one that interacts with the cell receptor, (59), this may then switch the attention of researchers to this other glycoprotein, GP2, that we know now, through our research, that is associated to virulence. We have found that PRRSV evades the
immune system by means of a glycan-shielding mechanism and that the deglycosylation of the PRRSV gP5 enhances significantly the ability of the PRRSV strain to induce protective antibody response. These two concepts, added to the notion that PRRSV may have several serologic markers (immunodominant B-cell epitopes) that could be used to differentiate vaccination from infection, make all together the most significant contribution to PRRSV vaccinology so far. We have been able to successfully establish a reverse genetics experimental system for PRRSV that serves as national and international reference. Several laboratories worldwide have requested and are successfully using our IC system. We know now that our IC is fully functional, being ours the PRRSV infectious clone system with best recorded evidence of stability and reproducible pathogenesis in vivo.
Impacts
Output from CAP1: One MS Thesis based on CAP1-funded research has been published. One USDA NRI CGP project (new investigator award program) is being submitted (Nov 2006) based on data obtained exclusively though CAP1. One manuscript was submitted to a refereed journal based on data obtained through CAP1 support. Four laboratories from the US and abroad were freely provided with the cDNA infectious clone of wt PRRSV obtained at our institution. This cDNA clone was produced through funding received from a regular grant of USDANRICGP and from NPB. Several feed backs received consistently indicate a highly satisfactory performance of this reagent in all cases Besides some other collaborative publications, three refereed papers based on our laboratories have been published during the period covered in this report and two more are currently under review. One of the manuscripts sent involves collaboration of three NC-229 stations plus 2 more universities overseas.
Publications
- Ansari I H, Kwon B J , Osorio FA , and Pattnaik A K. 2006. Influence of N-Linked Glycosylation of Porcine Reproductive and Respiratory Syndrome Virus GP5 on Virus Infectivity, Antigenicity and Ability to Induce Neutralizing Antibodies. Journal of Virology, 80(8):3994-4004
- De Lima M, Pattnaik A, Flores EF, and Osorio FA. 2006. Serologic marker candidates identified amongst B-cell linear epitopes of Nsp2 and structural proteins of a North American strain of Porcine Reproductive and Respiratory Syndrome virus. Virology, 353:410-421
- Kwon, B. J., Ansari, I. H., Osorio, F. A., and Pattnaik, A. K. 2006. Construction and characterization of an infectious cDNA clone of a vaccine strain of porcine reproductive and respiratory syndrome virus. Vaccine, 24(49-50):7071-7080
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Progress 10/01/04 to 09/30/05
Outputs
Using reverse genetics, we generated a series of chimeric viruses containing specific genomic sequences of an attenuated PRRSV vaccine strain (Prime Pac) within the genomic context of a highly virulent infectious clone (FL-12). Eight viable chimeric viruses, encompassing the entire genome of PRRSV (Prime Pac), have been obtained. Five of these chimeras include all the non-structural open reading frames (ORFs): Most virulence determinants clustered in the structural genes of PRRSV. Some non-structural regions of the PRRSV genome (NSP3-8) exhibited a marked role in virulence. Meanwhile, other non-structural regions (NSP1-3, NSP10-12) showed an intermediate attenuation phenotype, while other non-structural (NSP9) or structural (ORF2) regions of the PRRSV genome could be ruled out as important determinants of virulence. We further dissected the structural regions for a finer mapping of individual ORFs of the PRRSV genome and generated 5 more chimeric viruses representing
the majority of each individual ORF, 3 through 7. Three putative N-linked glycosylation sites (N34, N44, and N51) are located on the GP5 ectodomain, where a major neutralization epitope also exists. To determine which of these putative glycosylation sites are used in PRRSV life cycle and the role of the glycan moieties in induction of neutralizing antibodies, we generated a panel of GP5 mutants containing single and multiple amino acid substitutions at these sites. In serum neutralization assays, the mutant viruses exhibited enhanced sensitivity to neutralization by wt PRRSV-specific antibodies. Furthermore, inoculation of pigs with the mutant viruses induced significantly higher levels of neutralizing antibodies against the mutant as well as the wt PRRSV, thus suggesting that the loss of glycan residues in the ectodomain of GP5 enhances both the sensitivity of these viruses to in vitro neutralization as well as the immunogenicity of the nearby neutralization epitope. These results
should have great significance for development of PRRSV vaccines of enhanced protective efficacy. This study is aimed at identifying PRRSV B-cell linear epitopes that would be consistently recognized by the humoral immune response of naturally infected animals. To this end, 213 overlapping 15-mer synthetic peptides covering the whole amino acid sequence of a non-structural protein (nsp2) and all the structural proteins of a North American strain of PRRSV (NVSL97-7895) were used in a peptide-based enzyme-linked immunosorbent assay. Interestingly, the Nsp2 was found to contain most linear epitopes when compared to the structural proteins. Analysis of the peptides spanning the amino acid sequence of all structural proteins of the NVSL97-7895 strain against convalescent sera (45dpi) revealed the presence of B-cell linear epitopes in all studied proteins. Despite a genetic diversity between different PRRSV genotypes (1), we found immunodominant epitopes in specific regions of the gp2, gp3,
gp5 and M protein which has been previously demonstrated to be recognized by immune sera raised against an European strain of PRRSV.
Impacts
The experiments dealing with reverse genetics using an infectious cDNA clone are significant to understand the virulence of PRRSv and its attenuation. Understanding the gene basis for the virulent phenotype of PRRSV is the basis for the development of new, safer, more rationally designed replicating vaccines. In addition, the identification of epitopes (small fragments) of PRRSV proteins that can be inactivated or eliminated from a live PRRSv may be the basis for the development of a marker vaccine. Along the same line, enhancement of the PRRSV- neutralizing antibody response by molecular modification of the PRRSV proteins is of high value for the development of more effective vaccines against PRRSv infections.
Publications
- Petry DB, Holl JB, Weber J , Doster AR, Osorio FA, and Johnson, RK. 2005. Biological responses to PRRSV in pigs of two genetic populations to PRRSV. J An Sci. 2005 July; 83(7):1494-502
- Macias-Duarte MJ, Yepiz-Plascencia G, Osorio FA, Pinelli-Saavedra A, Reyes-Leyva J, Hernandez J. 2005. Isolation and characterization of PRRS virus in Mexico. Vet Mex (In press)
- Ansari I H, Kwon B J , Osorio FA , and Pattnaik A K 2005. Influence of N-Linked Glycosylation of Porcine Reproductive and Respiratory Syndrome Virus GP5 on Virus Infectivity, Antigenicity, and Ability to Induce Neutralizing Antibodies, Journal of Virology, Accepted for publication
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Progress 10/01/03 to 09/30/04
Outputs
This work consisted of the Nebraska component of a successful multi-state NC- proposal t that reunited at least 12 major universities and research centers plus 60+ researchers. During the life of this project at Nebraska, four labs in two departments (VBMS and Animal Sciences) have worked in 1) Development an infectious clone for PRRSV, 2) Study of the protective effect of passive antibodies and 3) Genetic resistence to PRRSV infection. A major output of this project has also been the joint submission of an integrative research project to USDA-NRICGP which was funded at the level of 4.4 million dollars for 4 years in FY 2004, having Dr. M. Murtuagh (U. of MN) as PI. Three major lines of work have been started, through part of these competitive funds, in Nebraska: 1) Development of vesicular stomatitis vectors for delivery of important immunognes of PRRSV (Drs. Pattnaik, Osorio); 2) Sub-typing of PRRSV isolates by means of cross neutralization reactions (Dr.Osorio) and
3) Study of genetic resistance to PRRSV and its relationship to expression of certain genes of the host (Dr. R. Johnson and J. Weber). In addition, the national project has been renewed for five more years and will certainly be continued at Nebraska for the period 2004-2009.
Impacts
The experiments dealing with reverse genetics using an infectious cDNA clone are significant to understand virulence of PRRSv and its attenuation. Understanding the gene basis for the virulent phenotype of PRRSV is the basis for the development of new, safer, more rationally designed replicating vaccines. The results of passive protection of young pigs against PRRSV infection indicate that in this type of animals it is not possible to attain sterilizing immunity as easily as we previously observed in sows. This emphasizes that significant differences must exist between the PRRSV pathogeneses in adult and in young pigs. The level of protective antibodies that are required to be prepared in the immune system of the pig to overcome a PRRSv infection is significantly higher, suggesting that the level of PRRSV replication in the target cells of a young pig is significantly higher. Also important is the notion that absence of viremia in a pig does not correlate with lack of
infectivity. Even in absence of viremia an animal can shed and contagiate the infection to contact penmates. Genetics of PRRSv resistance/susceptibility It is important to identify traits that differentiate animals that respond differently to pathogens and to build the phenotypic and genotypic records to identify the genes involved. The different responses we obtained indicate an underlying genetic variation for infection with PRRSV.
Publications
- Bastos RG, Dellagostini OA, Barletta RG, Doster AR, Nelson E, Zuckermann, Osorio FA. 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, 22:467-474
- Truong HM, Lu Z, Kutish G, Galeota J, Osorio FA and Pattnaik AK. 2004. A Highly Pathogenic Porcine Reproductive and Respiratory Syndrome Virus Generated from an Infectious cDNA Clone Retains the In Vivo Markers of Virulence and Transmissibility Characteristics of the Parental Strain. Virology, 1(325)(2):308-19
- Lopez OJ and Osorio FA. 2004. The role of antibodies in PRRSV protective immunity, a short survey. Veterinary Immunology and Immunopathology, 102(3):155-63
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Progress 10/01/02 to 09/30/03
Outputs
One sub-project was conducted as part of Objective 3 of the NC-229 project: 1) Role of Antibodies in protective Immunity agaisnt PRRSV Lopez & Osorio (UNL), Platt (ISU), Nelson (SDSU) During the last year the area of concentration in this project has been primarily that of neutralizing epitope mapping and construction of chimeric mouse X swine neutralizing antibodies. We focused on the effect that virus bound to chimeric antibodies had on macrophage infection, mapping of neutralizing epitopes in different proteins of PRRSV, and inducing neutralizing antibodies in non-replicating immunogens in swine and mice. The results from these projects will provide important information that will be used to produce vaccines and hibridomas producing monoclonal antibodies with neutralizing activity against PRRSV. We are also working on the development of a test to determine the present of neutralizing antibodies in vaccinated pigs in an ELISA format. The data and reagents derived
from this research will be made available to all NC-229 participants. Another Sub-project was conducted as part of Objective 4 of the NC-229 project: 2) Use of Blocking ELISAs to Detect Antibodies anti_PRRSV in Non-Swine Species Osorio (UNL), Zimmermann (ISU), Nelson and Ferrin (SDSU). The overall objective of these experiments was to test the ability of existing immunoassays to detect antibodies to PRRSV in non-swine species. We first proved that commercial and experimental blocking ELISAs detect antibodies when these are actively induced in non-swine species. This result suggests that it would be possible to use immunoassays for detecting putative naturally-occurring PRRSV antibodies in non-swine species for diagnostic purposes. This has sgreat significance in the search for PRRSV natural reservoirs. Surprisingly, though, it was not possible in these experiments to experimentally infect Mallard ducks, reportedly the only non-swine-species capable of replicating PRRSV. This infection
was not possible either by replicating the exact conditions that had been successful in a single previous lab, or by using different routes and different strains of PRRS. Based on these results, we can conclude that the experimental inoculation of Mallard ducks with PRRSV is not a consistent occurrence. This perhaps reflects a need for additional experimental conditions that are so far unrecognized. Further work is necessary to clarify the role of Mallard ducks as reservoirs of PRRSV in nature. More conclusive evidence about the role of Mallard ducks and any other birds in PRRSV transmission could be obtained through the use of serology kits in serum samples collected from wild ducks and birds.
Impacts
The truly inter-institutional collaborative character of this regional research is documented by three articles accepted by to refereed journals which involve five different experimental stations that participate of NC-229: Nebraska, Mississippi, Iowa, South Dakota and Illinois. Part of this regional research is being supplemented by a private company interested in possible commercial applications ("Production of Mouse x Porcine Neutralizing Antibodies anti-Porcine Reproductive and Respiratory Syndrome Virus," $44,955, PIC USA (Sygen International) February 2002/February 2004). The most significant inter-institutional activity derived from this program has been our participation with the University of Minnesota (PI institution) and 12 other stations in an integrated National Research Initiative Competitive Grant for $4 million. The title of this inter-institutional grant proposal, entirely based on the NC-229 project is: "Integrated Control and Elimination of Porcine
Reproductive and Respiratory Syndrome Virus (PRRSV) in the US." The PI of this grant is Michael Murtaugh (U. of Minnesota), the funds requested are $4,000,000 and the proposed period of work is January 1, 2004 to December 31, 2007 (48 months). The grant involves 13 land grant universities and 4 resreach centers, plus etxernal industry collaborators.
Publications
- Wills RW, Doster AR, Galeota JA, Sur JH, Osorio FA. 2003 Duration of Infection and Proportion of Pigs Persistently Infected with Porcine Reproductive and Respiratory Syndrome Virus (PRRSV). Journal of Clinical Microbiology, 41(1):58-62
- Meier, WA Galeota J, OsorioFA , Husmann RJ, Schnitzlein W and Zuckermann FA. 2003. Gradual development of the Interferon-Gamma and Antibody Responses of Swine to Porcine Reproductive and Respiratory Syndrome Virus. Virology, 309(1):18-31
- Bastos RG, Dellagostini OA, Barletta RG, Doster AR, Nelson, E Zuckermann F and Osorio FA. 2003. 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, in press
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Progress 10/01/01 to 09/30/02
Outputs
Two main sub-projects were conducted as part of Objective 3 of the NC-229 project: 1) Demonstration of the Role of Antibodies in Preventing Clinical PRRSV disease and Providing Sterilizing Immunity (collaboration including Nebraska, Illinois and South Dakota experimental stations): (Narrative included as part of CRIS Report for NEB 14-105) 2) Identification of neutralizing and non-neutralizing epitopes in gp5 of PRRSV (collaboration including Nebraska and Iowa experimental stations): using peptide phage display method we have identified two epitopes present in the ectodomain of the PRRSV gP5. One of these epitopes (epitope B)was recognized both by the neutralizing monoclonal antibody ISU25-C1 and by swine neutralizing serum (NS) but not by swine non-neutralizing serum (NNS), indicating that it is a neutralizing epitope. Epitope B is sequential, conserved among isolates and is not immunodominant. Antibodies directed against it are detected in serum late after
infection. In contrast, the other epitope, which we named epitope A, is hypervariable and immunodominant. Antibodies against it appear early after infection with PRRSV. This epitope is recognized by swine NNA but is not recognized by neither neutralizing Mab ISU25-C1 nor swine NA, indicating that it is not involved in PRRSV neutralization. During infection with PRRSV, epitope A acts as a decoy eliciting most of the antibodies directed to GP5 and delaying the induction of neutralizing antibodies against epitope B for at least three weeks. A third sub-project was conducted as part of Objective 4 of the NC-229 project: Comparison of Three Commercial ELISA's for Detection of Antibodies to PRRSV (collaboration including Nebraska, Iowa and South Dakota experimental stations): The diagnostic kit that is most commonly used worldwide for the serologic detection of PRRSV antibodies is manufactured by Idexx Labs (ME, USA). This kit currently presents a characteristic high incidence of false
positive, which constitutes s serious problem when testing PRRSV-negative herds around the world. Currently indirect immunofluorescent serology (IFA) is used to confirm the serologic status of these singleton reactors. It was our interest to find some alternative ELISA test that could be used to confirm the serologic status of these singleton reactors. We evaluated, using an extensive collection of standard sera, two new commercially available PRRSV serologic kits: one manufactured in Canada and another in Spain. The conclusions were: 1) while the Idexx test shows a consistent level of acceptable sensitivity, the two new kits present poor sensitivity; 2) The poor sensitivity of the new tests make the character of a negative score negative obtained by these two tests uncertain; 3) This renders the tests unacceptable for the confirmation of the serological status of individual animals in large sero-negative populations. These new tests can not then replace the IFA test in the
verification of true serologic status of single Idexx reactors.
Impacts
The truly inter-institutional collaborative character of this regional research is documented by three articles accepted by to refereed journals which involve four different experimental stations that participate of NC-229: Nebraska, Iowa, South Dakota and Illinois. Part of this regional research is being supplemented by a private company interested in possible commercial applications (Production of Mouse x Porcine Neutralizing Antibodies anti-Porcine Reproductive and Respiratory Syndrome Virus, $44,955, PIC USA (Sygen International) February 2002/January 2003)
Publications
- Osorio FA, Galeota JA, Nelson E, Brodersen B, Doster A, Wills R, Zuckermann, F and Laegreid WW 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 Virology, In Press
- Bastos RG O, Dellagostin O, Barletta R, Doster A, Nelson E, Lopez O, Osorio FA. 2002. Construction and immunological evaluation of M. bovis BCG expressing GP5 and M protein of porcine reproductive respiratory syndrome virus, Vaccine, In Press
- Ostrowski M, Galeota JA, Jar AM, Platt KB, Osorio FA, Lopez OJ. 2002. Identification of neutralizing and non-neutralizing epitopes in the porcine reproductive and respiratory syndrome virus GP5 ectodomain. J Virol., 76:4241-50
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Progress 10/01/00 to 09/30/01
Outputs
Two main sub-projects were conducted as part of Objective 3 of the NC-229 project; 1) Demonstration of the Role of Antibodies in Preventing Clinical PRRSV disease and Providing Sterilizing Immunity (collaboration including Nebraska, Illinois and South Dakota experimental stations). (Narrative included as part of CRIS Report for NEB 14-105) 2) Identification of neutralizing and non-neutralizing epitopes in gp5 of PRRSV(collaboration including Nebraska and Iowa experimental stations): using peptide phage display method we have identified two epitopes present in the ectodomain of the PRRSV gP5. One of these epitopes (epitope B)was recognized both by the neutralizing monoclonal antibody ISU25-C1 and by swine neutralizing serum (NS), but not by swine non-neutralizing serum (NNS), indicating that it is a neutralizing epitope. Epitope B is sequential, conserved among isolates and is not immunodominant. Antibodies directed against it are detected in serum late after
infection. In contrast, the other epitope, which we named epitope A, is hypervariable and immunodominant. Antibodies against it appear early after infection with PRRSV. This epitope is recognized by swine NNA but is not recognized by neither neutralizing Mab ISU25-C1 nor swine NA, indicating that it is not involved in PRRSV neutralization. During infection with PRRSV, epitope A acts as a decoy eliciting most of the antibodies directed to GP5 and delaying the induction of neutralizing antibodies against epitope B for at least three weeks. A third sub-project was conducted as part of Objective 4 of the NC-229 project: Comparison of Three Commercial ELISA's for Detection of Antibodies to PRRSV (collaboration including Nebraska, Iowa and South Dakota experimental stations): The diagnostic kit that is most commonly used worldwide for the serologic detection of PRRSV antibodies is manufactured by Idexx Labs. ( ME, USA). This kit currently presents a characteristic high incidence of false
positive, which constitutes s serious problem when testing PRRSV-negative herds around the world. Currently indirect immunofluorescent serology (IFA) is used to confirm the serologic status of these singleton reactors. It was our interest to find some alternative ELISA test that could be used to confirm the serologic status of these singleton reactors. We evaluated, using an extensive collection of standard sera, two new commercially available PRRSV serologic kits: one manufactured in Canada and another in Spain. The conclusions were: 1) while the Idexx test shows a consistent level of acceptable sensitivity, the two new kits present poor sensitivity, 2) The poor sensitivity of the new tests make the character of a negative score negative obtained by these two tests uncertain.3) This renders the tests unacceptable for the confirmation of the serological status of individual animals in large sero-negative populations. These new tests can not then replace the IFA test in the
verification of true serologic status of single Idexx reactors.
Impacts
The truly inter-institutional collaborative character of this regional research is documented by two articles sent to refereed journals which involve three different experimental stations. Part of this regional research is being approached by a private company for commercial applications (see CRIS report for NEB 14-105)
Publications
- Sur JH, Doster AR, Galeota JA , and Osorio, FA. 2001. Evidence for the Localization of Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) Antigen and RNA in Ovarian Follicles in Gilts Vet Path 38:58-66
- Osorio FA, Galeota, JA, Nelson E, Brodersen, B, Doster, A., Wills, R, Zuckermann, F. and Laegreid WW. October 2001. Passive Transfer of Virus -Specific Neutralizing Antibodies Confers Protection against Reproductive Failure Induced by a Virulent Strain of Porcine Reproductive and Respiratory Syndrome Virus and Establishes Sterilizing Immunity, Submitted to J. of Virology
- Ostrowski, M., Galeota,J.A., Jar, A.M., Platt, K. B., Osorio, F.A. and Lopez, O.J. September 2001. Identification of Neutralizing and Non-neutralizing Epitopes in the Ectodomain of GP 5 pf Porcine Reproductive and Respiratory Syndrome Virus (PRRSV). Submitted to J. of Virology
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