Progress 10/01/00 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? African swine fever (ASF) is a highly lethal hemorrhagic disease of domestic swine with mortality rates approaching 100%. The causative agent, African swine fever virus (ASFV), is a unique and genetically complex DNA virus. It is the sole member of a newly named virus family and the only known DNA arbovirus. Cycling of virus between soft ticks of the genus Ornithodoros and wild pig populations (wart hogs and bush pigs) in sub-Saharan Africa provides a natural reservoir of virus that poses a constant threat to domestic pig populations worldwide. There is no vaccine for ASF. ASF poses a serious threat to the swine industry because it is a highly lethal disease; all domestic swine are susceptible to infection; there is a large natural reservoir of virus in nature and, apart from slaughter of infected
herds, there is no effective disease control strategy. A vaccine or other novel control strategy for ASF would remove ASF as a threat to the swine industry worldwide. Further, it would have significant impact on the problem of human malnutrition in areas of sub-Saharan Africa where ASF makes it impossible to raise swine. Given the uniqueness and genetic complexity of ASFV, this research will have much broader impact on swine infectious disease than ASF alone; it has already contributed novel concepts to our understanding of viral virulence/host range, anti-viral immune responses and pathogen-swine host-vector interactions in general. Either a vaccine or other novel control methods is needed to reduce the threat posed by this highly significant viral disease. The long term goal of the ASF research group at the Plum Island Animal Disease Center (PIADC) is development of an effective control strategy for this viral disease. This research project focuses on vaccination as a potential
control strategy. Research involves defining the protective immune response to ASFV. Specifically to: 1) Evaluate genetically engineered ASFV with deletions of virulence/host range genes as vaccines for ASF; 2)Identify and characterize ASF viral proteins that induce a protective immune response in swine; 3) Define antigenic variability ASFV strains; 4) Define critical protective host immune responses; and 5) Develop new methods for effective vaccine delivery in swine. This research will allow evaluation of vaccination as a potential control measure for ASFV and will provide both the theoretical and practical basis for subsequent vaccine development. 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: Construct and characterize ASFV chimeric viruses
and expression vectors containing selected viral genes/gene regions to map protective antigens (PA). Year 2.5: Identify PA using swine immunization and challenge experiments with ASFV chimeras and/or by identifying PA responsible for antibody- mediated protective immunity. Year 4: Complete fine mapping of PA and evaluate the most promising as vaccine candidates using swine immunization and challenge. 3. Milestones: A. An ASFV protein both necessary and sufficient for mediating monocyte infection inhibition (M-II) was identified and confirmed. Chimeric ASF viruses containing the M-II protein of one virus inserted into the genome of a second heterologous virus were constructed to be used in vaccination/challenge experiments to evaluate the antigen as a protective antigen. B. Future research work in FY-05: ASF chimeric viruses containing the M- II protein of one virus inserted into the genome of a second heterologous virus will be used in vaccination /challenge experiments to directly
assess the ability of the protein to induce a protective immune response in swine. Transcriptional profiling with ASFV infected macrophages will be used to define the cellular mechanisms of virus growth inhibition due to M-II. FY-06: Work will continue to characterize the virus-cell interactions underlying mII. Additional ASFV PA will be identified. Chimeras of heterologous ASF viruses viruses that fail to cross protect in swine - will be used to map ASFV PA in swine immunization/challenge experiments. Fine mapping of PA to individual genes will be done by construction additional chimeric viruses and evaluating them for their ability to induce cross protection. FY-07: Work will continue characterizing additional ASFV PA. The most promising PA will be tested for their ability to induce a protective immune response in swine. 4. What were the most significant accomplishments this past year? A. Single Most Significant Accomplishment during FY 2004: The significance of the recently
identified ASFV protein capable of mediating monocyte infection-inhibition (M-II) as a protective antigen is unknown. ASF chimeric viruses containing the M-II protein of one virus inserted into the genome of a second heterologous virus were constructed to be used in vaccination /challenge experiments to directly assess the ability of the protein to induce a protective immune response. Experiments are ongoing and will be completed in FY-05. The M-II protein as a major protective antigen would permit development of subunit or vectored ASFV marker vaccines - ones which would be suitable for use in the developed world for emergency disease management. B. Other Significant Accomplishments(s): None. 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. Our work in ASFV protective immunity has: 1) Clearly demonstrated the importance of the
humoral immune response in protective immunity to ASFV infection; 2) Demonstrated the existence of neutralizing antibodies in the serum of recovered ASFV infected animals; 3) identified for the first time a specific viral protein (p72) involved in mediating virus neutralization and mapped the protein epitope responsible; 4) Demonstrated that ASFV neutralizing antibodies are not sufficient for antibody-mediated protection and that other antibody-mediated immune mechanisms are responsible for the protective effect; 5) Anti-ASFV antibodies mediating a novel anti-viral mechanism, monocyte infection- inhibition (M-II) , were identified and shown to function in a strain- specific manner, suggesting a role for them in protective immunity; 6) Identified additional viral genes with possible significance to a protective immune response including those encoding, putative virion and infected cell membrane proteins, highly variable viral proteins and putative secreted viral virulence factors; 7)
Demonstrated the feasibility of using genetically engineered live attenuated ASF viruses as vaccines. A live attenuated ASFV was constructed by deleting the 9GL gene (a gene required for efficient growth in swine macrophages) . Pig infected (immunized) with this genetically engineered mutant virus were completely protected from lethal challenge with homologous and several heterologous virus strains indicating that highly virulent African ASFV field isolates can be attenuated by gene deletion and that these viruses induce a protective immune response in swine; 8) Several virulent African strains of ASFV were attenuated by deleting virulence associated genes and then used in cross protection studies in pigs. Cross protection was observed for all South African viruses examined indicating that protection from heterologous viruses within a geographic region can be achieved. These data indicate that ASFV strain variation may not be as great as previously thought thus, vaccination with a
single virus strain may be a viable option for controlling ASFV within a geographic region; and 9) Immune sera to different ASFV isolates were raised and tested in an in vitro monocyte infection-inhibition (M-II) assay where homologous and region-specific heterologous protective effects were observed. Significant correlation between the specificity of M-II assay and cross protection from heterologous viruses was observed. 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? Viruses, genomic sequences, cloned viral genes, protein expression vectors, and both
polyclonal and monoclonal antibody reagents have been provided to research and diagnostic laboratories in the US, UK, Spain, South Africa, the Netherlands, Denmark and China.
Impacts
(N/A)
Publications
- Neilan, J.G., Zsak, L., Lu, Z., Burrage, T.G., Kutish, G.F., Rock, D.L. 2004. Neutralizing antibodies to african swine fever virus proteins p30, p54 and p72 are not sufficient for antibody-mediated protection. Virology. 319(2):337-342.
- Zsak, L., Sur, J.H., Burrage, T.G., Neilan, J.G., Rock, D.L. 2003. African swine fever virus (asfv) multigene family (mgf) 360 and mgf 530 genes affect virulence, pathogenesis and immuno response in swine [abstract]. International Congress On Apoptosis. p. 618.
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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? African swine fever (ASF) is a highly lethal hemorrhagic disease of domestic swine with mortality rates approaching 100%. The causative agent, African swine fever virus (ASFV), is a unique and genetically complex DNA virus. It is the sole member of a newly named virus family and the only known DNA arbovirus. Cycling of virus between soft ticks of the genus Ornithodoros and wild pig populations (wart hogs and bush pigs) in sub-Saharan Africa provides a natural reservoir of virus that poses a constant threat to domestic pig populations worldwide. There is no vaccine for ASF. Either a vaccine or other novel control methods is needed to reduce the threat posed by this highly significant viral disease. The long term goal of the ASF research group at the Plum Island Animal Disease Center (PIADC) is development of an effective control strategy for this viral disease. This research project focuses
on vaccination as a potential control strategy. Research involves defining the protective immune response to ASFV. Specifically to: 1) Evaluate genetically engineered ASFV with deletions of virulence/host range genes as vaccines for ASF; 2) Identify and characterize ASF viral proteins that induce a protective immune response in swine; 3) Define antigenic variability ASFV strains; 4) Define critical protective host immune responses; and 5) Develop new methods for effective vaccine delivery in swine. This research will allow evaluation of vaccination as a potential control measure for ASFV and will provide both the theoretical and practical basis for subsequent vaccine development. 2. How serious is the problem? Why does it matter? ASF poses a serious threat to the swine industry because it is a highly lethal disease; all domestic swine are susceptible to infection; there is a large natural reservoir of virus in nature and, apart from slaughter of infected herds, there is no effective
disease control strategy. A vaccine or other novel control strategy for ASF would remove ASF as a threat to the swine industry worldwide. Further, it would have significant impact on the problem of human malnutrition in areas of sub- Saharan Africa where ASF makes it impossible to raise swine. Given the uniqueness and genetic complexity of ASFV, this research will have much broader impact on swine infectious disease than ASF alone; it has already contributed novel concepts to our understanding of viral virulence/host range, anti-viral immune responses and pathogen-swine host-vector interactions in general. 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: An ASFV protein capable of inducing anti-viral antibodies which mediate a novel anti-viral mechanism, monocyte infection-inhibition (M-II) was identified and shown to be variable among divergent ASFV strains. The association between the presence of M-II antibodies and protective immunity suggest this protein will be a major protective antigen for use in a subunit or vectored ASFV marker vaccine - a vaccine which would be suitable for emergency use in the developed world. B. Other Significant Accomplishments(s): None. 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. Our recent work in ASFV protective immunity has: 1) Clearly demonstrated the importance of the humoral immune response in protective immunity to ASFV infection; 2) Demonstrated the existence of neutralizing antibodies
in the serum of recovered ASFV infected animals; 3) identified for the first time a specific viral protein (p72) involved in mediating virus neutralization and mapped the protein epitope responsible; 4) Demonstrated that ASFV neutralizing antibodies are not sufficient for antibody-mediated protection and that other antibody-mediated immune mechanisms are responsible for the protective effect; 5) Anti-ASFV antibodies mediating a novel anti-viral mechanism, monocyte infection- inhibition (M-II), were identified and shown to function in a strain- specific manner, suggesting a role for them in protective immunity; 6) Identified additional viral genes with possible significance to a protective immune response including those encoding, putative virion and infected cell membrane proteins, highly variable viral proteins and putative secreted viral virulence factors; 7) Demonstrated the feasibility of using genetically engineered live attenuated ASF viruses as vaccines. A live attenuated ASFV
was constructed by deleting the 9GL gene (a gene required for efficient growth in swine macrophages). Pig infected (immunized) with this genetically engineered mutant virus were completely protected from lethal challenge with homologous and several heterologous virus strains indicating that highly virulent African ASFV field isolates can be attenuated by gene deletion and that these viruses induce a protective immune response in swine; 8) Several virulent African strains of ASFV were attenuated by deleting virulence associated genes and then used in cross protection studies in pigs. Cross protection was observed for all South African viruses examined indicating that protection from heterologous viruses within a geographic region can be achieved. These data indicate that ASFV strain variation may not be as great as previously thought thus, vaccination with a single virus strain may be a viable option for controlling ASFV within a geographic region; and 9) Immune sera to different ASFV
isolates were raised and tested in an in vitro monocyte infection-inhibition (M-II) assay where homologous and region-specific heterologous protective effects were observed. Significant correlation between the specificity of M-II assay and cross protection from heterologous viruses was observed. Findings will permit identification of viral proteins mediating M-II. 6. What do you expect to accomplish, year by year, over the next 3 years? Future research work in FY-04 - Recently identified ASFV protein(s) mediating M-II will be characterized and evaluated as protective antigens (PA) in swine. Transcriptional profiling with ASF infected macrophages will be used to define mechanisms of virus growth inhibition due to M-II. FY-05 ' Work will continue to characterize the virus-cell interactions underlying M-II. Additional ASFV PA will be identified. Chimeras of heterologous ASF viruses - viruses that fail to cross protect in swine - will be used to map ASFV PA in swine
immunization/challenge experiments. Fine mapping of PA to individual genes will be done by construction additional chimeric viruses and evaluating them for their ability to induce cross protection. FY-06 ' Work will continue characterizing additional ASFV PA. The most promising PA will be tested for their ability to induce a protective immune response in swine. 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? Viruses, genomic sequences, cloned viral genes, protein expression vectors, and both polyclonal and monoclonal antibody reagents have been provided to research and diagnostic laboratories in the US, UK, Spain, South Africa, the Netherlands, Denmark and China.
Impacts
(N/A)
Publications
- Burrage, T.G., Neilan, J.G., Lu, Z., Rock, D.L., Zsak, L. Characterization of African Swine Fever Virus (ASFV) Multigene Family (MGF) 360 Proteins Which Affect Virus Replication and Generalization of Infection in Ornithodoros Porcinus Ticks. Conference of Research Workers in Animal Diseases. 2002. Abstract p. 180.
- Zsak, L., Sur, J.H., Burrage, T.G., Neilan, J.G., Rock, D.L. African Swine Fever Virus Multigene Family 360 and 530 Genes Affect Viral Virulence and Pathogenesis in Swine. International Conference on Poxviruses and Iridoviruses. 2002. Abstract p. 56.
- Burrage, T.G., Neilan, J.G., Lu, Z., Rock, D.L., Zsak, L. African Swine Fever Virus Multigene Family Genes Affect Virus Replication and Generalization of Infection in Ornithodoros Porcinus Ticks. International Conference on Poxviruses and Iridoviruses. 2002. Abstract p.62.
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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? African swine fever (ASF) is a highly lethal hemorrhagic disease of domestic swine with mortality rates approaching 100%. The causative agent, African swine fever virus (ASFV), is a unique and genetically complex DNA virus. It is the sole member of a newly named virus family and the only known DNA arbovirus. Cycling of virus between soft ticks of the genus Ornithodoros and wild pig populations (wart hogs and bush pigs) in sub-Saharan Africa provides a natural reservoir of virus that poses a constant threat to domestic pig populations worldwide. There is no vaccine for ASF. Either a vaccine or other novel control methods is needed to reduce the threat posed by this highly significant viral disease. The long term goal of the ASF research group at the Plum Island Animal Disease Center (PIADC) is development of an effective control strategy for this viral disease. This research project
focuses on vaccination as a potential control strategy. Research involves defining the protective immune response to ASFV. Specifically to: 1) Evaluate genetically engineered ASFV with deletions of virulence/host range genes as vaccines for ASF; 2) Identify and characterize ASF viral proteins that induce a protective immune response in swine; 3) Define antigenic variability ASFV strains; 4) Define critical protective host immune responses; and 5) Develop new methods for effective vaccine delivery in swine. This research will allow evaluation of vaccination as a potential control measure for ASFV and will provide both the theoretical and practical basis for subsequent vaccine development. 2. How serious is the problem? Why does it matter? ASF poses a serious threat to the swine industry because it is a highly lethal disease; all domestic swine are susceptible to infection; there is a large natural reservoir of virus in nature and, apart from slaughter of infected herds, there is no
effective disease control strategy. A vaccine or other novel control strategy for ASF would remove ASF as a threat to the swine industry worldwide. Further, it would have significant impact on the problem of human malnutrition in areas of sub- Saharan Africa where ASF makes it impossible to raise swine. Given the uniqueness and genetic complexity of ASFV, this research will have much broader impact on swine infectious disease than ASF alone; it has already contributed novel concepts to our understanding of viral virulence/host range, anti-viral immune responses and pathogen-swine host-vector interactions in general. 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: ASFV antigens responsible for inducing a protective immune response in pigs as well as the extent of viral strain variation in nature are currently unknown. Chimeric ASF viruses representing approximately 40% of the genome were constructed and characterized by Southern blot and PCR methods. Immune sera to different ASFV isolates were raised and tested in an in vitro monocyte infection-inhibition (M-II) assay where homologous and region-specific heterologous protective effects were observed. Significant correlation between the specificity of M-II assay and cross protection from heterologous viruses was observed. Impact: Findings will permit the construction of chimeric viruses capable of inducing a protective immune response against heterologous ASF viruses. B. Other Significant Accomplishments(s): None. 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? Our recent work in ASFV protective immunity has: 1) Clearly demonstrated the importance of the humoral immune response in protective immunity to ASFV infection; 2) Demonstrated the existence of neutralizing antibodies in the serum of recovered ASFV infected animals; 3) identified for the first time a specific viral protein (p72) involved in mediating virus neutralization and mapped the protein epitope responsible; 4) Demonstrated that ASFV neutralizing antibodies are not sufficient for antibody-mediated protection and that other antibody-mediated immune mechanisms are responsible for the protective effect; 5) Anti-ASFV antibodies mediating a novel anti-viral mechanism, monocyte infection- inhibition (M-II), were identified and shown to function in a strain- specific manner, suggesting a role for them in protective immunity; 6) Identified additional viral genes with possible significance to
a protective immune response including those encoding, putative virion and infected cell membrane proteins, highly variable viral proteins and putative secreted viral virulence factors; 7) Demonstrated the feasibility of using genetically engineered live attenuated ASF viruses as vaccines. A live attenuated ASFV was constructed by deleting the 9GL gene (a gene required for efficient growth in swine macrophages). Pig infected (immunized) with this genetically engineered mutant virus were completely protected from lethal challenge with homologous and several heterologous virus strains indicating that highly virulent African ASFV field isolates can be attenuated by gene deletion and that these viruses induce a protective immune response in swine; and 8) Several virulent African strains of ASFV were attenuated by deleting virulence associated genes and then used in cross protection studies in pigs. Cross protection was observed for all South African viruses examined indicating that
protection from heterologous viruses within a geographic region can be achieved. These data indicate that ASFV strain variation may not be as great as previously thought thus, vaccination with a single virus strain may be a viable option for controlling ASFV within a geographic region. 6. What do you expect to accomplish, year by year, over the next 3 years? Future research work in FY-2003-2006 will include: 1) ASFV proteins mediating M-II will be identified and evaluated as protective antigens (PA). Predicted viral membrane proteins (V-MP) with a high degree of variability among strains will be targeted. Immune sera will be evaluated in M-II assays to detect biological function. In a second approach ASF chimeric viruses will be used to identify proteins which mediate M-II. The most promising candidate proteins - those which mediate M-II will be tested for their ability to induce a protective immune response in swine following immunization. 2) ASFV PA will be identified.
Chimeras of heterologous ASF viruses - viruses that fail to cross protect in swine - will be used to map ASFV PA in swine immunization/challenge experiments. Fine mapping of PA to individual genes will be done by construction additional chimeric viruses and evaluating them for their ability to induce cross protection. 7. What technologies have been transferred and to whom? When is the technology likely to become available to the end user (industry, farmer other scientist)? What are the constraints, if known, to the adoption durability of the technology? Viruses, genomic sequences, cloned viral genes, protein expression vectors, and both polyclonal and monoclonal antibody reagents have been provided to research and diagnostic laboratories in the US, UK, Spain, South Africa, the Netherlands, Denmark and China.
Impacts
(N/A)
Publications
- Neilan, J.G., Zsak, L., Lu, Z., Kutish, G.F., Afonso, C.L. and Rock, D.L. A Novel Swine Virulence Determinant in the Left Variable Region of the African Swine Fever Virus Genome 130224. 2002. Journal of Virology. v. 76(7). p.3095-3104.
- Zsak, L. and Neilan, J.G. Regulation of Apoptosis in African Swine Fever Virus Infected Macrophages. 2002. The Scientific World. v. 2. p. 1-7.
- Burrage, T.G., Shedlosky, E.C., Zsak, A., Rock, D.L. and Zsak, L. African Swine Fever Virus (ASFV) multigene family (MGF) 360 genes affect virus replication and generalization of infection in Ornithodoros porcinus ticks. Conference of Research Workers in Animal Diseases. 2001. Abstract p. 126P.
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Progress 10/01/00 to 09/30/01
Outputs
1. What major problem or issue is being resolved and how are you resolving it?
African swine fever (ASF) is a highly lethal hemorrhagic disease of domestic swine with mortality rates approaching 100%. The causative agent, African swine fever virus (ASFV), is a unique and genetically complex DNA virus. It is the sole member of a newly named virus family and the only known DNA arbovirus. Cycling of virus between soft ticks of the genus Ornithodoros and wild pig populations (wart hogs and bush pigs) in sub-Saharan Africa provides a natural reservoir of virus that poses a constant threat to domestic pig populations worldwide. There is no vaccine for ASF. Either a vaccine or other novel control methods is needed to reduce the threat posed by this highly significant viral disease. The long term goal of the ASF research group at the Plum Island Animal Disease Center (PIADC) is development of an effective control strategy for this viral disease. This research project focuses on vaccination as a potential control strategy. Research involves defining the protective
immune response to ASFV. Specifically to: 1) evaluate genetically engineered ASFV with deletions of virulence/host range genes as vaccines for ASF; 2) identify and characterize ASF viral proteins that induce a protective immune response in swine; 3) define antigenic variability ASFV strains; 4) define critical protective host immune responses; and 5) develop new methods for effective vaccine delivery in swine. This research will allow evaluation of vaccination as a potential control measure for ASFV and will provide both the theoretical and practical basis for subsequent vaccine development.
2. How serious is the problem? Why does it matter?
ASF poses a serious threat to the swine industry because it is a highly lethal disease; all domestic swine are susceptible to infection; there is a large natural reservoir of virus in nature and, apart from slaughter of infected herds, there is no effective disease control strategy. A vaccine or other novel control strategy for ASF would remove ASF as a threat to the swine industry worldwide. Further, it would have significant impact on the problem of human malnutrition in areas of sub-Saharan Africa where ASF makes it impossible to raise swine. Given the uniqueness and genetic complexity of ASFV, this research will have much broader impact on swine infectious disease than ASF alone; it has already contributed novel concepts to our understanding of viral virulence/host range, anti-viral immune responses and pathogen-swine host-vector interactions in general.
3. How does it relate to the National Program(s) and National Component(s)?
The research addresses objectives and goals outlined in the Action Plan for National Program 103 Animal Health (100%) under the Program Components of (1) Mechanisms of Disease, and (2) Strategies to Control Infectious Disease. Further, it 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 2001: The obvious approach for ASF control is vaccination. Unfortunately, given present knowledge, it is unclear that a safe and effective ASFV vaccine can be developed. ASFV vaccine development is significantly hindered by large gaps in our knowledge of the virus and the complex virus-host interactions involved in protective immunity. An additional complicating factor is ASFV strain variation and variability. Significant biological variation appears to exist between strains of ASFV, and cross protective immunity has proven difficult to achieve. Several virulent African strains of ASFV were attenuated by deleting virulence associated genes and then used in cross protection studies in pigs. Cross protection was observed for all South African viruses examined indicating that protection from heterologous viruses within a geographic region can be achieved. These data indicate that ASFV strain variation may not be as great as
previously thought thus, vaccination with a single virus strain may be a viable option for controlling ASF within a given geographic region. B. Other Significant Accomplishments(s): Although still poorly understood, some important aspects of ASF protective immunity are emerging. Protective immunity does develop in pigs surviving viral infection. Humoral immunity is a significant component of the protective immune response to ASF and ASFV antibodies are sufficient to protect pigs from lethal ASFV infection. However, how these antibodies function and the viral proteins responsible for their induction are as yet unknown. Development of subunit vaccines for ASF requires knowledge of viral proteins that induce protective host responses. Anti-ASFV antibodies mediating a novel anti-viral mechanism, monocyte infection-inhibition (M-II), were identified and shown to function in a strain-specific manner, suggesting a role for them in protective immunity. Knowledge of this important anti-viral
mechanism will permit identification of ASFV proteins that induce these antibodies, facilitating subunit on vectored vaccine development. C. Significant Accomplishments/Activities that Support Special Target Populations: N/A
5. Describe the major accomplishments over the life of the project including their predicted or actual impact.
This "bridging" research project is a natural extension of the terminated research project 1940-32000-031-00D ("Protective Immunity to ASF Virus Infection: Identification of Immunogenic Viral Pathogens"). The progress which is reported resulted from the above mentioned project. Our recent work in ASFV protective immunity has: 1) clearly demonstrated the importance of the humoral immune response in protective immunity to ASFV infection; 2) demonstrated the existence of neutralizing antibodies in the serum of recovered ASFV infected animals; 3) identified for the first time a specific viral protein (p72) involved in mediating virus neutralization and mapped the protein epitope responsible; 4) demonstrated that ASFV neutralizing antibodies are not sufficient for antibody-mediated protection and that other antibody-mediated immune mechanisms are responsible for the protective effect; 5) identified additional viral genes with possible significance to a protective immune response
including those encoding, putative virion and infected cell membrane proteins, highly variable viral proteins and putative secreted viral virulence factors; 6) identified ASF viral proteins necessary for a protective immune response in swine. ASFV MGF 360 proteins were required for induction of a protective immune response in vaccinated swine. This is the first identification of an ASFV protein associated with protective immunity in swine. Defining discrete protective antigens of the virus is the initial step toward development of a subunit ASF vaccine; one that would be compatible with emergency disease control for use in the developed world; 7) demonstrated the feasibility of using genetically engineered live attenuated ASF viruses as vaccines. A live attenuated ASFV was constructed by deleting the 9GL gene (a gene required for efficient growth in swine macrophages). Pig infected (immunized) with this genetically engineered mutant virus were completely protected from lethal
challenge with homologous and several heterologous virus strains indicating that highly virulent African ASFV field isolates can be attenuated by gene deletion and that these viruses induce a protective immune response in swine; and 8) several highly pathogenic African ASFV isolates were attenuated by deleting virulence-associated genes from their genomes and successfully evaluated in pigs as vaccine candidates. The availability of multiple attenuated viruses will, for the first time, allow cross protection studies to be performed in pigs to determine how many antigenic types of ASFV a vaccine must protect against. Overall, these data indicate that vaccination is a viable approach for disease control and that development of a live-attenuated vaccine for ASF may be feasible.
6. What do you expect to accomplish, year by year, over the next 3 years?
Future research work in FY 2002-2004 will include 1) ASFV protein mediating monocyte infection-inhibition (M-II) will be identified and evaluated as protective antigens (PA). Predicted viral membrane proteins (V-MP) with a high degree of variability among strains will be targeted. Immune sera will be evaluated in M-II assays to detect biological function. In a second approach ASF chimeric viruses will be used to identify proteins which mediate M-II. The most promising candidates - those which mediate M II - will be tested for their ability to induce a protective immune response in swine following immunization. 2) ASFV protective antigens (PA) will be identified. Chimeras of heterologous ASF viruses - viruses that fail to cross protect in swine - will be used to map ASFV PA in swine immunization/ challenge experiments. Fine mapping of PA to individual genes will be done by construction of additional chimeric viruses and evaluating them for their ability to induce cross protection.
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 & durability of the technology product?
Genomic sequences, cloned viral genes, protein expression vectors, and both polyclonal and monoclonal antibody reagents have been provided to research and diagnostic laboratories in the US, UK, Spain, South Africa, the Netherlands, Denmark and China.
8. List your most important publications in the popular press (no abstracts) and presentations to non-scientific organizations and articles written about your work (NOTE: this does not replace your peer-reviewed publications which are listed below)
Impacts
(N/A)
Publications
- Zsak, L., Lu, Z., Burrage, T.G., Neilan, J.G., Kutish, G.F., Moore, D.M., Rock, D.L. Africa Swine Fever Virus Multigene Family 360 and 530 Genes are Novel Macrophage Host Range Determinants. Journal of Virology. 2001. v. 75. p. 3066-3076.
- Tulman, E.R., Rock, D.L. Novel Virulence and Host Range Genes of African Swine Fever Virus. Current Opinion in Microbiology. 2001. v. 4. p. 456-461.
- Sur, J.H., Burrage, T.G., Neilan, J.G., Rock, D.L., Zsak, L. African Swine Fever Virus Multigene Family 360 and 530 Genes Affect Virulence, Pathogenesis and Immune Response in Swine. American Society for Virology. 2001. Abstract p. 84.
- Zsak, L., Sur, J.-H., Burrage, T.G., Neilan, J.G., Rock, D.L. African Swine Fever Virus Multigene Families 360 and 530 Genes Promote Infected Macrophage Survival. Miami Nature Biotechnology Winter Symposium. 2001. Abstract p. 97.
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