Progress 01/22/01 to 01/21/06
Outputs
Progress Report 1. What major problem or issue is being resolved and how are you resolving it (summarize project aims and objectives)? How serious is the problem? Why does it matter? Classical Swine Fever Virus (CSFV), a highly infectious disease of swine, presents high rates of mortality and morbidity, depending upon the acting strain, in affected herds. Transmission of the virus is horizontal and vertical resulting in the presence of symptomatic carries that maintain and spread the virus in the infected herd. CSFV, as a foreign disease in the US, remains an important threat to the national pork industry. The disease is endemic in areas of Mexico, Central American and the Caribbean. These geographical areas of intense affluence of people and merchandise to US. Recent outbreaks in Western Europe, where the disease is sporadic, have had devastating economic consequences for the affected countries. Similar consequences should be expected if an outbreak of the diseases
occurs in the US. Improvement of the diagnostic methods and the development of new vaccines are needed to reduce the risk of the introduction of the disease and the management of the disease in the case that a disease outbreak occurs. The objectives of this research are focused on: (i) developing and evaluating rapid real time detection and diagnostic assays for Classical Swine Fever (CSF); and (ii) determining the genetic basis of mutanted CSFV virulence in order to develop and evaluate recombinant CSF viruses with virulence-associated genes as live attenuated antigenically marked vaccines. National Program 103, Animal Health (100%). This research addressed outcome 2, a safe and secure food and fiber system, and falls within both specific goals 2.1.2. and 2.1.4 of the ARS strategic plan. 2. List by year the currently approved milestones (indicators of research progress) FY2002: (1) Evaluate analytical sensitivity and specificity of CSF RT-PCR diagnostic test; and (2) Develop CSFV
infectious clones (IC) and construct chimeras of pathogenic and attenuated viruses. FY2003: (1) Evaluate CSFV RT-PCR in experimentally infected animals and optimize diagnostic sample; and (2) Characterize CSF chimeric viruses in vitro. FY2004: (1) Evaluating of diagnostic sensitivity and specificity of CSFV RT-PCR using field collected diagnostic samples; (2) Identify CSFV swine virulence and host range determinants using chimeric virus; and (3) Evaluate engineered live-attenuated CSFV viruses as vaccines. FY 2005: Evaluate different CSFV vaccine candidates containing positive and negative antigenic markers. FY 2006: (1) Development of new chimeric viruses based in the fine mapping of virulence-associated determinants and antigenic markers. (2) Discovery of new genetic determinanats associated with virulence. 4a List the single most significant research accomplishment during FY 2006. Identification of a Novel Virulence Associated Genetic Determinant in Glycoprotein E2 The problem
being addressed is the development of attenuated CSFV vaccines that are more efficacious and safer than existing vaccines. Comparative genomic analysis on the glycoprotein E2 gene among Pestiviruses (CSFV,Bovine Viral Diarrhea virus and Border Disease virus) showed critical differences in a particular area that is also recognized by a highly specific mAb (WH 303), the mAb WH 303 epitope (mAbWH303e). Systematic mutations affecting the WH303e toward the homologous sequence in other Pestivirus showed that the mAbWH303e is an important genetic determinant associated with virulence. Virus presenting a particular set of mutations in WH303e (Delta WH303e) was completely attenuated in swine. Importantly, this Delta WH303e virus lost reaction against mAb 303 making a negative antigenic marked virus to Delta WH303e virus. The attenuated phenotype of Delta WH303e virus opens the possibility of using it as a potential negative marker vaccine. Impact: this discovery will allow the development of
vaccine candidates targeting the E2 glycoprotein of CSFV. This research falls within component 1: Biodefense Research of the NP- 103 National Program. 4b List other significant research accomplishment(s), if any. Role of Glycosylation of Glycoprotein E2 in Virulence The problem being addressed is the development of attenuated CSFV vaccines that are more efficacious and safer than existing vaccines. Our studies of the role of glycosylation of structural proteins of CSFV demonstrated that glycosylation of E2 is critical in virus virulence in swine. Recombinant viruses (N1v) possessing mutations at specific putative glycosylation sites produced complete attenuation of a highly virulent virus. This is the first report demonstrating a role of glycosylation in virulence of Pestivirus during the infection in the natural host. Impact: The attenuated phenotype of N1?v opens the possibility of using it as a potential live attenuated vaccine. This research falls within component 1:
Biodefense Research of the NP- 103 National Program. 4d Progress report. 1) A series of mutant viruses containing systematic mutations at a highly conserved CSFV epitope (mAbWH303e) were created. The reactivity against the mAb recognizing the particular epitope was lost in some of those viruses becoming negatively antigenic marker virus. Those viruses were also completely attenuated in swine, thus they could be considered as potential negative marker vaccines. 2) Mutant viruses containing alterations in different predicted glycosylation sites of the structural glycoprotein E2 were constructed to study the role of glycosylation in the virulence/pathogenesis of CSFV in swine. Specific predicted glycosylation sites were detected as critical in altering virus virulence in swine. In addition with the CSFV research activity at PIADC, we have a Specific Cooperative Agreement with Dr. Risatti at the Department of Pathobiology in the University of Connecticut. Through this agreement we perform
research activities which closely complement those performed at PIADC. Progress report for the Specific Cooperative Agreement project number 58- 1940-32000-01S, between ARS and the University of Connecticut (58-1940-5- 0513), "CLASSICAL SWINE FEVER VIRUS: ROLE OF THE 3'UTR IN INFECTIVITY OF SWINE" The objective of this proposed research project is to assess the role of the Classical Swine Fever Virus 3UTR RNA conserved secondary structures in virus infectivity. To address the role of the conserved secondary structures of the 3UTR in CSFV cycle, we have begun a structural mapping of the 3 UTR. The entire 3 UTR was deleted from a clone encompassing the last 4000 nt of the CSFV genome. This clone will be used to rebuild the 3 UTR by adding oligonucleotides encompassing each of the conserved secondary structures predicted by computer modeling. The obtained clones will be then submitted to Plum Island Animal Disease Center for assembly of full- length clones. Viruses obtained from those
clones will be evaluated for genetic stability, ability to replicate in swine macrophages, growth kinetics, plaque morphology, and infectivity in swine. SUMMARY OF PROGRESS 1- Secondary structures of CSFV strain Brescia 3UTR were analyzed and compared with 3 UTR structures of CSFV virulent and attenuated strains. Three major structural features were predicted and oligonucleotides encompassing those areas were designed. 2- Synthetic oligonucleotides were directionally cloned into a plasmid carrying a partial region of non-structural protein NS5B. These clones are being sequenced to assess the integrity of the introduced changes. Upon sequence analysis clones will be sent to Plum Island for final assembling into the CSFV Brescia full-length cDNA clone. 5. Describe the major accomplishments to date and their predicted or actual impact. Rapid identification and diagnostic of the foreign disease is critical for successfully controlling and eradicating the disease following its
introduction. Rapid pen-side assays to detect CSFV were no available at the time this project started. A rapid real time fluorogenic hydrolysis probe TaqMan assay was developed and evaluate in pigs experimentally infected with a moderately virulent strain. This assay, highly specific and sensitive, outperform the current diagnostic standard test consisting of virus isolation). It allowed preclinical diagnosis of the disease up to 5 days prior to the appearance of the CSF clinical symptoms. This test was further evaluated under field conditions in Dominican Republic (where the disease is endemic) with results of sensitivity and specificity similar to those obtained during the analytical evaluation. Impact: this test is a new diagnostic tool that will redefine management and control strategies for this highly significant disease and will greatly reduce the chances of uncontrollable disease outbreaks. Engineering new marker live attenuated vaccines requires knowledge on the molecular
basis of the virulence in vivo. Using insertional mutagenesis we described a previously unknown genomic region in the E1 glycoprotein associated with virus virulence. Then, designing chimeric viruses between the virulent Brescia strain and the vaccine strain CS, we identified a previously non described area of the structural glycoprotein E2 involved in virulence in vivo. In both cases, viruses developed which contained either the mutation of E1 or E2 and were able to induce protection against the challenge 3 days post vaccination. By genetic manipulation, positive and negative markers were introduced in these attenuated mutants allowing the serological differentiation between he experimental vaccine strains and the wild type Brescia strain. Impact: A patent was filed (Novel classical swine fever virulence determinant and a novel classical swine fever vaccine, Patent application Number 11/316,755, USA). Now, two novel genetic determinants associated with virulence were discovered: (i)
A determinant associated with a CSFV conserved epitope in glycoprotein E2. (ii) Determinants associated with specific predicted glycosylation sites of the E2 glycoprotein. When mutated, both genetic determinants induce a complete attenuation of the virulent parental strain. Impact: A patent was filed (A novel virulence determinant within the E2 structural glycoprotein of classical swine fever virus, Patent application Number 11/443,132, USA). 6. What science and/or technologies have been transferred and to whom? When is the science and/or technology likely to become available to the end- user (industry, farmer, other scientists)? What are the constraints, if known, to the adoption and durability of the technology products? A novel virulence determinant within the E2 structural glycoprotein of classical swine fever virus, Patent application Number 11/443,132, USA. A patent has been filed with some of the Delta WH303e viruses demonstrating a novel mechanism to produce attenuation in
CSFV.
Impacts
(N/A)
Publications
- Risatti, G.R., Holinka, L.G., Lu, Z., Kutish, G.F., Tulman, E.R., French, R.A., Sur, J.H., Rock, D.L., Borca, M.V. 2005 Mutation of E1 Glycoprotein of Classical Swine Fever Virus Affects Viral Virulence in Swine. Journal of Virology 343: 116-127.
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Progress 10/01/04 to 09/30/05
Outputs
1. What major problem or issue is being resolved and how are you resolving it (summarize project aims and objectives)? How serious is the problem? What does it matter? Classical Swine Fever Virus (CSF), a highly infectious disease of swine, presents high rates of mortality and mobility, depending of the acting strain, in affected herds. Transmission of the virus is horizontal and vertical resulting in the presence of asymptomatic carriers that maintain and spread the virus in the infected herd. CSFV, as a foreign disease in the US, remains as an important threat for the national pork industry. The disease is endemic in areas of Mexico, Central America, and the Caribbean, all geographical areas with intense affluence of people and merchandise to US. Recent outbreaks in Western Europe, where the disease is sporadic, have had devastating economic consequences for the affected countries. Similar consequences should be expected if an outbreak of the diseases occurs in the
US. Improvement in the diagnostic methods and the development of new vaccines are needed to reduce the risk of the introduction of the disease and the management of the disease in the case that a disease outbreak occurs. The objectives of this research are focused to: (i) Develop and evaluate rapid real-time detection and diagnostic assays for Classical swine fever (CSF); and (ii) Determine the genetic basis of CSFV virulence in order to develop and evaluate recombinant CSF viruses with mutations of virulence- associated genes as live attenuated antigenically marked vaccines. This project addresses ARS Strategic Plan Goal #3 (Enhance Protection and Safety of the Nations Agriculture and Food Supply) under National Program #103, Animal Health and Well Being) and specifically addresses NPS-103 Mechanisms of Disease and Animal Immunology components; and Strategies to Control Infectious and Non-Infectious Disease. 2. List the milestones (indicators of progress) from your Project Plan.
Milestones: Year 1: (1) Evaluate analytical sensitivity and specificity of CSF RT-PCR diagnostic test; and (2) Develop CSFV infectious clones (IC) and construct chimeras of pathogenic and attenuated viruses. Year 2: (1) Evaluate CSFV RT-PCR in experimentally infected animals and optimize diagnostic sample; and (2) Characterize CSF chimeric viruses in vitro. Year 3: (1) Evaluating of diagnostic sensitivity and specificity of CSFV RT-PCR using field collected diagnostic samples; (2) Identify CSFV swine virulence and host range determinants using chimeric viruses; and (3) Evaluate engineered live-attenuated CSFV viruses as vaccines. Year 4: (1) Evaluate and introduce positive and negative antigenic markers in candidate vaccine strains. (2) Initiate optimization of vaccine candidates for early protection in swine. Year 5: (1) Complete optimization of vaccine candidates in swine. (2) Initiate fine mapping of virulence-associated genetic determinants to fine-tune attenuation of
vaccine virus in order to achieve early protection. (3) Develop a companion diagnostic test for differentiating vaccinated from infected animals. 3a List the milestones that were scheduled to be addressed in FY 2005. For each milestone, indicate the status: fully met, substantially met, or not met. If not met, why. 1. Complete optimization of vaccine candidates in swine. Milestone Fully Met 2. Initiate fine mapping of virulence-associated genetic determinants to fine-tune attenuation of vaccine virus in order to achieve early protection. Milestone Substantially Met 3. Develop a companion diagnostic test for differentiating vaccinated from infected animals. Milestone Substantially Met 3b List the milestones that you expect to address over the next 3 years (FY 2006, 2007, and 2008). What do you expect to accomplish, year by year, over the next 3 years under each milestone? FY-06: (1) A fine mapping of the originally identified virulence- associated genetic determinants will be
done. It is expected these newly designed mutants will be more efficient in both inducing early protection and seroligical response to the antigenic markers. (2) The diagnostic tests required for the detection of the serological responses to the antigenic markers included in the mutant virueses will be developed. Special emphasis will be focused in the development of mutant viruses where, trough-out the fine mapping of the originally identified virulence- associated genetic determinants, more restricted and characterized versions of those determinants will be used. It is expected these newly designed mutants will be more efficient in both inducing early protection and serological response to the antigenic markers. Additionally, new approaches will be used in order to detect and map the presence of unknown virulence-associated genetic determinants. Special attention will have the analysis of the role of specific genomic areas showing distinctive characteristics when compared with
other Pestiviruses. Also, the role of glycosilation of the structural proteins in the pathogenesis of the CSFV infection in pigs will be evaluated. Also, the diagnostic tests required for the detection of the serological responses to the antigenic markers included in the mutant viruses will be improved and standardized. FY-07: Work outlined in FY-06 will continue into FY-07. It is expected that information about the existence of new virulence-associated genetic determinants will be used, by themselves or in combination with the already characterized ones, in the developing of new vaccine candidates. Additionally, a main effort will be directed towards the identification and understanding of the innate and acquired immune mechanisms that mediated the status of early protection in pigs vaccinated with the mutant viruses. It is expected that the obtained information will be useful in the implementation of novel strategies and the development of new tools for inducing early protection
against the challenge with virulent CSFV. FY-08: Work outlined in FY-07 will continue into FY-08. This project, which was establish January 22, 2001 will terminate January 21, 2006. A bridge project will be established to allow for continuation of the project until the OSQR review is completed. 4a What was the single most significant accomplishment this past year? The development of recombinant vaccine-candidate viruses combining two different attenuation-related genetic determinants along with positive and negative antigenic markers. A sequence of these viruses were developed where a molecular dissection of both genetic and antigenic determinant were performed obtaining detailed information about the mapping of both, the attenuation associated determinant and the antigenic marker. The information obtained allowed the design of mutants viruses harboring positive and negative antigenic markers and inducing protection at 3 days post-vaccination. 4d Progress report. (1) Two different
viruses containing mutations in structural glyproteins E1 and E2 were developed containing a positive and negative antigenic marker respectively. Both viruses induced fully protection at 3 day post vaccination when challenged with the virulent Brescia virus. (2) A sequence of mutant viruses, containing different versions of the previously described attenuating mutations on glycoproteins E1 and E2 and both antigenic markers, positive and negative, were constructed. In general, this combination produced a moderate disability of the double mutated viruses in replicating in vivo, resulting as poor inducers of early protection. Recent results demonstrated that further genetic manipulation of the original mutations resulted in doubled mutant viruses inducing protection at 3rd days post vaccination. 19340-32000-038-02S: This report serves to document research conducted under a specific cooperative agreement between ARS and the University of Connecticut, Storrs. DNA material has been
produced by PIADC and transferred to UConn. Currently, specific primers to produce mutated 3' UTRs are being used to amplify the 3' end of the virus. 5. Describe the major accomplishments over the life of the project, including their predicted or actual impact. Rapid identification and diagnostic of a foreign disease is critical for successfully controlling and eradicating the disease following its introduction. Rapid pen-side assays to detect CSFV were no available at the time this project started. A rapid real time fluorogenic hydrolysis probe TaqMan assay was developed and evaluated in pigs experimentally infected with a moderately virulent strain. This assay, highly specific and sensitive (outperforming the current diagnostic standard test, virus isolation) allowed the preclinical diagnosis of the disease up to 5 days prior the appearance of CSDFV clinical symptoms. This test was further evaluated under field conditions in Dominican Republic (where the disease is endemic) with
results of sensitivity and specificity similar to those obtaining during the analytical evaluation. Impact: this test is a new diagnostic tool that will redefine disease management and control strategies for this highly significant disease. Engineering new markered live-attenuated CSFV vaccines requires knowledge on the molecular basis of the virus virulence in vivo. Previously, using insertional mutagenesis we described a previously unknown genomic region in the E1 glycoprotein associated to virus virulence. Now, designing chimeric viruses between the virulent Brescia strain and the vaccine strain CS, we identify a previously non described area of the structural glycoprotein E2 involved in virulence in vivo. In both cases, viruses developed containing either the mutation on E1 or E2 were able to induce protection against the challenge as early as 3 days post vaccination. By genetic manipulation, positive and negative markers were introduced in these attenuated mutants allowing the
serological differentiation between the experimental vaccine strains and the wild- type Brescia strain. Impact: the discovery and mapping of virulence- associated genetic determinants represent the critical first step in the development of rational design improved live-attenuated marker vaccines. 6. What science and/or technologies have been transferred and to whom? When is the science and/or technology likely to become available to the end- user (industry, farmer, other scientists)? What are the constraints, if known, to the adoption and durability of the technology products? The CSFV real-time RT-PCR test was transferred to APHIS.
Impacts
(N/A)
Publications
- Risatti, G.R., Kutish, G.F., Lu, Z., Holinka, L.G., French, R.A., Tulman, E., Rock, D.L., Borca, M.V. 2004. The E2 Glycoprotein of Classical Swine Fever Virus is a Virulence Determinant in Swine. Conference of Research Worksers in Animal Disese Meeting. P. 196.
- Risatti, G.R., Borca, M.V., Kutish, G.F., Lu, Z., Holinka, L.G., French, R. , Tulman, E., Rock, D.L. 2005 The E2 Glycoprotein of Classical Swine Fever Virus Affects Viral Virulence in Swine. Journal of Virology.79: 3787-3796.
- Risatti, G.R., Holinka, L.G., Lu, Z., Kutish, G.F., Callahan, J.D., Nelson, W.N., Tio, B.E., Borca, M.V. 2004. Diagnostic Evaluation of a Portable Real-Time Reverse Transcriptase PCR Assay for the Detection of Classical Swine Fever. Conference of Research Worksers in Animal Disese Meeting. P 194.
- Risatti, G.R., Holinka, L.G., Lu, Z., Kutish, G.F., Callahan, J.D., Nelson, W.M., Brea, T.E., Borca, M.V. 2005. Diagnostic Evaluation of a Portable Real-Time Reverse transcriptase PCR Assay for the Detection of Classical Swine Fever. Journal of Clinical Microbiology. 43 (1): 468-471.
- Risatti, G.R., Kutish, G.F., Lu, Z., Holinka, L.G., French, R.A., Sur, J.H. , Rock, D.L., Borca, M.V. 2004. A 19mer Peptide Insertion in the E1 Glycoprotein of Classical Swine Fever Virus Affects Viral Virulence in Swine. Conference of Research Worksers in Animal Disese Meeting. P.195
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Progress 10/01/03 to 09/30/04
Outputs
1. What major problem or issue is being resolved and how are you resolving it (summarize project aims and objectives)? How serious is the problem? What does it matter? Classical Swine Fever (CSF) is a highly infectious disease of swine. Morbidity and mortality rates in infected herds can be high. Infected sows can also transmit the virus to their offspring, creating asymptomatic carriers of the virus that shed virus in their feces, thereby contaminating the environment and posing risk to susceptible populations. As a foreign animal disease (FAD), an outbreak of CSF would significantly affect the U.S. pork industry and export markets. Improved CSF diagnostic tests and a marker live-attenuated CSF vaccine are needed to further reduce the risk posed by this FAD. Recent CSF outbreaks in Europe and the Caribbean (1997-2001) have had devastating economic consequences for the countries involved. A similar scenario would be expected should the disease be introduced into the
United States. Improvements in diagnostics and vaccines are needed to reduce the risk of disease introduction and to better manager a disease outbreak should it occur. The goal of this research is directed at: 1) Developing molecular epidemiological capabilities for CSF; 2) Developing rapid field diagnostic tests for CSF; 3) Developing laboratory diagnostic tests that will differentiate field from marker vaccine strains of CSF; 4) Defining the pathogenesis of moderately virulent CSF viruses in pigs; 5) Identifying CSF virulence and host range determinants; and 6)Developing a marker live attenuated CSF vaccine. 2. List the milestones (indicators of progress) from your Project Plan. National Program 103, Animal Health (100%) This research, addresses Outcome 2, a safe and secure food and fiber system, and falls within both specific goals 2.1.2 and 2.1.4 of the ARS strategic plan. Milestones: Year 1: 1) Evaluate analytical sensitivity and specificity of CSF RT-PCR diagnostic test.; and 2)
Develop CSFV infectious clones (IC) and construct chimeras of pathogenic and attenuated viruses. Year 2: 1) Evaluate CSFV RT-PCR in experimentally infected animals and optimize diagnostic sample; and 2) Characterize CSFV chimeric viruses in vitro. Year 3: 1) Evaluation of diagnostic sensitivity and specificity of CSFV RT-PCR using field collected diagnostic samples; 2)Identify CSFV swine virulence and host range determinants using chimeric viruses; and 3) Evaluate engineered live-attenuated CSF viruses as vaccines. 3. Milestones: A. Diagnostic evaluation of the CSFV RT-PCR test was completed using field collected samples from a disease endemic region. CSFV viruses with engineered mutations in virulence and host range genes/determinants and containing both positive and negative serological markers were shown to induce early protective immune responses in pigs. B. Future research work in FY-05: Functional genomic strategies will be used to fine map recently identified CSFV virulence
and host range determinants and to define additional regions of the genome suitable for use as negative markers. Engineered mutant viruses will be evaluated as markered live attenuated vaccine candidates (LAV) with emphasis on identifying those capable of inducing early protection. LAV containing multiple attenuating mutations and selective markers will be constructed and evaluated for protective efficacy. FY-06: Work outlined in FY-05 will continue into FY-06. In addition, efforts will be made to define the molecular mechanisms of CSFV virulence and host range genes/determinant in disease using discovery based genetic approaches and animal models. FY-07: Work outlined in FY-05 will continue into FY-06. In addition, efforts will be made to define the molecular mechanisms of CSFV virulence and host range genes/ determinant in disease using discovery based genetic approaches and animal models. 4. What were the most significant accomplishments this past year? A. Single Most
Significant Accomplishment During FY 2004: Rational engineering of markered live-attenuated(MLA) CSF vaccines requires knowledge of the genetic basis of viral virulence and host range. Reverse genetic and functional genomic strategies were used to map viral virulence determinants. A CSFV structural protein was identified as a major virulence determinant in swine. Viruses with engineered mutations in this protein and containing both positive and negative serological markers were constructed and shown to induce protective immune responses in animals. Research represents the first step toward rationally designed MLA CSF vaccines which will have improved safety, efficacy and utility. B. Other Significant Accomplishments(s): Diagnostic evaluation of the CSFV real-time PCR test developed in FY-02 was completed using field collected samples from a disease endemic region. Diagnostic sensitivity and specificity were high and the assay outperformed the current standard test, virus isolation.
The test provides a powerful new tool for emergency disease management and control. C. Significant Accomplishments/Activities that Support Special Target Populations: None. 5. Describe the major accomplishments over the life of the project, including their predicted or actual impact. Rapid identification and diagnosis of a FAD is critical for successfully controlling and eradicating the disease following introduction. Rapid field-based assays to detect CSFV are not yet available. A rapid real- time fluorogenic hydrolysis probe TaqMan assay was developed and evaluated in pigs experimentally infected with a moderately virulent CSF isolate, Haiti-96. The assay was@ highly sensitive and specific, detecting infected animals up to 5 days prior to the appearance of CSF clinical symptoms. Diagnostic sensitivity and specificity were high and the assay outperformed the current diagnostic standard test, virus isolation. Impact: This rapid, real-time RT-PCR assay for CSFV provides a new
diagnostic tool that will redefine disease management and control strategies for this highly significant FAD. Engineering new markered live-attenuated CSF vaccines requires knowledge of viral virulence and host range determinants. Currently, little is known about CSFV in this regard. Insertional mutagenesis was used to define CSFV virulence and host range determinants. A CSFV genomic region associated with viral virulence was identified. Mutant viruses with changes in this region were: 1) completely attenuated in pigs; and 2) capable of inducing a protective immune response in pigs. Impact: The mapping of genetic determinants associated with viral virulence and host range represents the first step toward rational design of improved markered live-attenuated CSF vaccines. National Program 103, Animal Health (100%) This research, addresses Outcome 2, a safe and secure food and fiber system, and falls within both specific goals 2.1.2 and 2.1.4 of the ARS strategic plan. 6. What
science and/or technologies have been transferred and to whom? When is the science and/or technology likely to become available to the end- user (industry, farmer, other scientists)? What are the constraints, if known, to the adoption and durability of the technology products? CSFV RT-PCR test transferred to APHIS.
Impacts
(N/A)
Publications
- Risatti, G.R., Kutish, G.F., Lu, Z., Holinka, L.G., French, R.A., Borca, M. V., Rock, D.L. 2004. The e2 glycoprotein of classical swine fever virus affects viral virulence in swine [abstract]. Positive Strand RNA Virus International Conference Proceedings. P. 109.
- Borca, M.V., Risatti, G.R., Kutish, G.F., Lu, Z., Holinka, L.G., French, R. A., Sur, J., Rock, D.L. 2004. A 19mer peptide insertion in the e1 glycoprotein of classical swine fever virus affects viral virulence in swine [abstract]. Positive Strand RNA Virus International Conference Proceedings. p. 109.
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Progress 10/01/02 to 09/30/03
Outputs
1. What major problem or issue is being resolved and how are you resolving it? Classical Swine Fever (CSF) is a highly infectious disease of swine. Morbidity and mortality rates in infected herds can be high. Infected sows can also transmit the virus to their offspring, creating asymptomatic carriers of the virus that shed virus in their feces, thereby contaminating the environment and posing risk to susceptible populations. As a foreign animal disease (FAD), an outbreak of CSF would significantly affect the U.S. pork industry and export markets. Improved CSF diagnostic tests and a marker live-attenuated CSF vaccine are needed to further reduce the risk posed by this FAD. The goal of this research is directed at: 1) Developing molecular epidemiological capabilities for CSF; 2) Developing rapid field diagnostic tests for CSF; 3) Developing laboratory diagnostic tests that will differentiate field from marker vaccine strains of CSF; 4) Defining the pathogenesis of
moderately virulent CSF viruses in pigs; 5) Identifying CSF virulence and host range determinants; and 6) Developing a marker live attenuated CSF vaccine. 2. How serious is the problem? Why does it matter? Recent CSF outbreaks in Europe and the Caribbean (1997-2001) have had devastating economic consequences for the countries involved. A similar scenario would be expected should the disease be introduced into the United States. Improvements in diagnostics and vaccines are needed to reduce the risk of disease introduction and to better manager a disease outbreak should it occur. 3. How does it relate to the National Program(s) and National Program Component(s) to which it has been assigned? National Program 103, Animal Health (100%) This research, addresses Outcome 2, a safe and secure food and fiber system, and falls within both specific goals 2.1.2 and 2.1.4 of the ARS strategic plan. 4. What were the most significant accomplishments this past year? A. Single Most Significant
Accomplishment during FY 2003: Rational engineering of markered live-attenuated CSF vaccines requires knowledge of viral virulence and host range determinants. Reverse genetic strategies were used to identify relevant determinants. Two distinct regions of the CSFV genome associated with viral virulence were identified. Mutant viruses were attenuated in pigs and capable of inducing a protective immune response. Identification of CSFV virulence determinants will permit the engineering of rationally designed markered live-attenuated CSF vaccines with improved safety, efficacy and versatility. B. Other Significant Accomplishments(s): The CSFV real-time PCR test developed in FY-02 was evaluated using field collected samples from a disease endemic region. The test exhibited high sensitivity and specificity, preclinical detection and it was found to be more sensitive than the current standard test, virus isolation. The test sets a new diagnostic standard for CSF and will provide a
powerful tool for emergency disease management and control strategies. C. Significant Accomplishments/Activities that Support Special Target Populations: None. 5. Describe the major accomplishments over the life of the project, including their predicted or actual impact. Rapid identification and diagnosis of a FAD is critical for successfully controlling and eradicating the disease following introduction. Rapid field-based assays to detect CSFV are not yet available. A rapid real- time fluorogenic hydrolysis probe TaqMan assay was developed and evaluated in pigs experimentally infected with a moderately virulent CSF isolate, Haiti-96. The assay was highly sensitive and specific, detecting infected animals up to 5 days prior to the appearance of CSF clinical symptoms. Impact: This rapid, real-time RT-PCR assay for CSFV provides a new diagnostic tool that will redefine disease management and control strategies for this highly significant FAD. This is a new project initiated in FY 2001.
Engineering new markered live-attenuated CSF vaccines requires knowledge of viral virulence and host range determinants. Currently, little is known about CSFV in this regard. Insertional mutagenesis was used to define CSFV virulence and host range determinants. A CSFV genomic region associated with viral virulence was identified. Mutant viruses with changes in this region were: 1) completely attenuated in pigs; and 2) capable of inducing a protective immune response in pigs. Impact: The mapping of genetic determinants associated with viral virulence and host range represents the first step toward rational design of improved markered live-attenuated CSF vaccines. 6. What do you expect to accomplish, year by year, over the next 3 years? Future research work in FY-04 ' Evaluation of the diagnostic sensitivity and specificity of the CSF real-time RT-PCR assay will be completed using field samples. Recently identified CSFV virulence and host range determinants will be fine mapped using
functional genomic strategies. Engineered mutant viruses will be evaluated as markered live attenuated vaccine (CAV) candidates. FY-05 ' LAV containing multiple attenuating mutations and positive and negative selective markers will be evaluated for protective efficacy. Molecular mechanisms of viral attenuation due to alteration of specific viral virulence and host range determinants will be defined using genetic approaches and animal models. FY-06 ' Work outlined in FY-05 will likely continue into FY-06. 7. What science and/or technologies have been transferred and to whom? When is the science and/or technology likely to become available to the end- user (industry, farmer, other scientists)? What are the constraints, if known, to the adoption and durability of the technology products? Rapid detection method transferred to APHIS.
Impacts
(N/A)
Publications
- Risatti, R.G., Callahan, J.D., Nelson, W.M., Boca, M.V. Rapid detection of Classical swine fever virus by a portable real-time reverse transcriptase PCR assay. Journal of Clinical Microbiology. 2003. v. 41(1). p. 500-505.
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Progress 10/01/01 to 09/30/02
Outputs
1. What major problem or issue is being resolved and how are you resolving it? Classical Swine Fever (CSF) is a highly infectious disease of swine. Morbidity and mortality rates in infected herds can be high. Infected sows can also transmit the virus to their offspring, creating asymptomatic carriers of the virus that shed virus in their feces, thereby contaminating the environment and posing risk to susceptible populations. As a foreign animal disease (FAD), an outbreak of CSF would significantly affect the U.S. pork industry and export markets. Improved CSF diagnostic tests and a marker live-attenuated CSF vaccine are needed to further reduce the risk posed by this FAD. The goal of this research is directed at: 1) Developing molecular epidemiological capabilities for CSF; 2) Developing rapid field diagnostic tests for CSF; 3) Developing laboratory diagnostic tests that will differentiate field from marker vaccine strains of CSF; 4) Defining the pathogenesis of
moderately virulent CSF viruses in pigs; 5) Identifying CSF virulence and host range determinants; and 6) Developing a marker live attenuated CSF vaccine. 2. How serious is the problem? Why does it matter? Recent CSF outbreaks in Europe and the Caribbean (1997-2001) have had devastating economic consequences for the countries involved. A similar scenario would be expected should the disease be introduced into the United States. Improvements in diagnostics and vaccines are needed to reduce the risk of disease introduction and to better manager a disease outbreak should it occur. 3. How does it relate to the national Program(s) and National Program Component(s) to which it has been assigned? National Program 103, Animal Health (100%) This research, addresses Outcome 2, a safe and secure food and fiber system, and falls within both specific goals 2.1.2 and 2.1.4 of the ARS strategic plan. 4. What was your most significant accomplishment this past year? A. Single Most Significant
Accomplishment during FY 2002: Rapid identification and diagnosis of a FAD is critical for successfully controlling and eradicating the disease following introduction. Rapid field-based assays to detect CSFV are not yet available. A rapid real- time fluorogenic hydrolysis probe TaqMan assay was developed and evaluated in pigs experimentally infected with a moderately virulent CSF isolate, Haiti-96. The assay was highly sensitive and specific, detecting infected animals up to 5 days prior to the appearance of CSF clinical symptoms. Impact: This rapid, real-time RT-PCR assay for CSFV provides a new diagnostic tool that will redefine disease management and control strategies for this highly significant FAD. B. Other Significant Accomplishments(s): Engineering new markered live- attenuated CSF vaccines requires knowledge of viral virulence and host range determinants. Currently, little is known about CSFV in this regard. Insertional mutagenesis was used to define CSFV virulence
and host range determinants. A CSFV genomic region associated with viral virulence was identified. Mutant viruses with changes in this region were: 1) completely attenuated in pigs; and 2) capable of inducing a protective immune response in pigs. Impact: The mapping of genetic determinants associated with viral virulence and host range represents the first step toward rational design of improved markered live-attenuated CSF vaccines. C. Significant Accomplishments/Activities that Support Special Target Populations: None. 5. Describe your major accomplishments over the life of the project, including their predicted or actual impact? This is a new project was initiated in FY-01. 6. What do you expect to accomplish, year by year, over the next 3 years? Future research work in FY-2003-2006: The diagnostic sensitivity and specificity of the CSF real-time RT-PCR assay will be determined. Validation of the test will be performed under field and diagnostic laboratory conditions in areas
of the world where CSF occurs. Functional genomic strategies will be used to identify additional CSF virulence and host range determinants. Information will be used to engineer and evaluate markered live attenuated vaccine candidates.
Impacts
(N/A)
Publications
- Risatti, G.R., Callahan, J.D., Borca, M.V., Sur, J.H., Clark, T., Nelson, W.M., Rock, D.L. Rapid detection of Classical Swine Fever Virus using a mobile Real Time PCR assay. Conference of Research Workers in Animal Diseases. 2001. Abstract p. 205.
- Risatti, G.R., Callahan, J.D., Nelson, W.M., Borca, M.V. Rapid detection of Classical swine fever virus using a portable real time RT-PCR assay. Pestivirus Symposium. 2002. Abstract p. 16.
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