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 and 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. 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. Although ASF has never been present in North and Central America, several outbreaks occurred in the Caribbean (Cuba, Haiti, Dominican Republic) and in Brazil during the 1980's. The disease has finally been eradicated from continental Europe (still present in the Italian island of Sardinia) after a 30 year presence in Spain and Portugal. The continued presence of ASF in many African nations is a constant threat to the rest of the world. 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, viral persistence and pathogen-swine host-vector interactions in general. Work here is focused on: 1) Identifying and characterizing viral genes responsible for virulence, viral latency and host range in both the swine and tick host; 2) Identifying host cell genes associated with host susceptibility and/or resistance; 3) Developing vaccines and/or other novel approaches for disease control that exploit virulence and host range mechanisms; and 4) Developing genomic based methods to rapidly identify and phenotypically characterize viruses. Identification of viral virulence-associated genes will permit the rational design and genetic engineering of live attenuated viruses for use as vaccines. Additionally, an understanding of these genes and their functions may permit development of novel approaches for disease control that exploit aspects of viral host range.
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 ASFV mutants containing deletions of macrophage host range genes (MGF360/530 genes) and other potential virulence and host range genes. Year 2.5: Complete in vitro evaluation of ASF mutants viruses and begin evaluation of swine and tick virulence phenotypes. Year 4: 1) Identify macrophage genes and gene networks affected by MGF360/530 genes to define their mechanism of action in macrophage host range; and 2) Complete virulence and pathogenesis studies in swine and ticks with mutant ASF viruses. 3. Milestones: A. Macrophage Transcriptional responses of ASFV and ASFV MGF360/530 gene deletion mutants were compared. MGF360/530 genes were found to suppress a host type I interferon response.
B. Future research work in FY-05: MGF 360/530 genes required for growth in macrophages, swine virulence and tick host range will be functionally characterized. Viral gene transcription, protein expression, protein localization in infected cells, and protein involvement in aspects of virus-cell interaction will be examined. Cellular proteins which physically interact with specific MGF 360/530 proteins will be identified using yeast two-hybrid assays. These approaches will better define MGF 360/350 gene function in suppressing a type I interferon response. FY-06: Whole genome comparisons have identified both highly conserved and variable genes in terminal ASFV genomic regions, which are known to contain important virulence and host range genes. Through this analysis, we have identified additional candidate genes with putative virulence and/or host range functions. To examine gene function, single gene deletion mutants of a pathogenic ASFV isolate will be constructed. Mutant viruses
together with parental and/or revertant viruses will be examined for their ability to replicate in porcine macrophage cell cultures in vitro and for their virulence in pigs. Viruses will also be examined in the tick host for alterations in infectious dose, initial midgut infection, generalization of infection, tissue tropism, viral persistence and transmission. FY-07: Mutant viruses identified in FY-06 with altered virulence and host range phenotypes will be examined using DNA microarray analysis of both swine and tick expressed sequence tags to define cellular transcriptional networks affected by the viral gene. The identification of cellular pathways affected by the viral gene will suggest molecular mechanisms for these genes in viral virulence and host range. 4. What were the most significant accomplishments this past year? A. Single Most Significant Accomplishment during FY 2004: ASFV multigene family (MGF) 360 and 530 genes affect viral growth in macrophage cell cultures,
virulence in pigs and tick host range. The mechanism by which these novel genes affect virus-host interactions is unknown. To define MGF360/530 function, global macrophage transcriptional responses of ASFV and ASFV mutants with MGF genes deleted were compared. MGF 360/530 genes were found to suppress a host type I interferon response. A molecular understanding of mechanisms of viral pathogenesis/host range will likely permit development of novel viral disease control strategies that are more efficacious and of greater utility. B. Other Significant Accomplishments(s): None. C. Significant Accomplishments/Activities that Support Special Target Populations: None. D. Progress Report: This report serves to document research conducted under a Specific Cooperative Agreement between ARS and Iowa State University (1940-32000- 036-05S). Procedures and reagents necessary to evaluate induction of cell-mediated immunity to viral vaccines in pigs were developed. These assays are currently being
used for evaluation of induction of T cell mediated immunity by the recombinant virus-vectored vaccines expressing Nipha virus F and G proteins. 5. Describe the major accomplishments over the life of the project, including their predicted or actual impact. Characterization of the ASFV genome - The genome of a pathogenic African ASFV isolate, Malawi Lil-20/1, was sequenced and analyzed. Data provided primary structure of all encoded genes and overall genome organization. Sequence homologies with other known genes have had great predictive value for proposing and examining ASFV gene function and have led to the identification of significant swine virulence and host range genes. Additionally, these data have also facilitated the identification of putative immunogenic proteins - viral membrane and envelope proteins. Identification of viral genes function in virulence and host range is difficult and very time consuming, especially when the genes are novel - lacking homology with other
known genes. Putative ASFV virulence and host range genes were identified using a novel comparative genome approach. Analysis of complete genome sequences of seven pathogenic field isolates of ASFV identified several highly conserved genes in regions of the viral genome known to contain other pathobiologically significant genes. Impact Comparative genomic approaches - facilitated by highly throughput DNA sequencing may greatly simplify identification of novel genes with critical functions in the host. The strategy developed here for ASFV will have broad applicability for characterization of other threat agent pathogens. Genetic engineering of the ASFV genome - Techniques were developed to genetically engineer pathogenic isolates of ASFV. These techniques which allow the addition or deletion of specific genes have permitted both genetic and reverse genetic research approaches for determining viral gene function. Recombinant ASFV gene-deletion mutants have been used to identify
and characterize swine virulence and host range genes. This technology has proven critical for engineering live-attenuated ASFV vaccine viruses. Identification and characterization of ASFV swine virulence and host range genes Using genetic based strategies and ASFV gene-deletion mutants, a number of pathobiologically significant viral genes, many of which are novel, have been identified and characterized. Virulence-associated genes, NL-S and UK were identified in pathogenic European ASFV isolates. Deletion of either of these genes from the viral genome resulted in complete attenuation of the virus in swine. A third distinct virulence determinant which includes multiple members of the multigene family 530 genes AVAD, was identified in the left variable region of the genome. These genes which are required for viral virulence are novel, lacking similarity to any other known genes. ASFV genes involved with the regulation of apoptosis in virus infected cells (bcl-2 and iap homologs)
and genes with functions involving immune evasion in the swine host (CD2 and IkB homologs) were identified and characterized. CD2 and IkB genes had not been previously described in any virus. This is the first example of a pathogen using of a CD2-like molecule in immune evasion. ASFV genes which affect macrophage host range were also identified and characterized. The thymidine kinase gene (TK) and a second gene 9-GL are required for efficient replication of virus in infected cells; deletion of either gene resulted in a 99% to 99.9-. reduction in viral growth and virulence attenuation. 9-GL is a novel gene whose function in virus replication is unknown. A novel macrophage growth-associated determinant which includes multiple members of the multigene family 360 genes was identified in the left variable region of the ASFV genome. These genes are novel lacking homology to other known genes. ASFV and CSF infect and replicate in cells of the monocyte-macrophage lineage. Revealing
molecular mechanisms involved in macrophage responses to infection is important to devise novel disease control strategies. A macrophage-specific swine CDNA microarray of 8029 clones was constructed and used to analyze the transcriptional profiles of swine macrophages infected with ASFV and gene deleted mutant viruses. Infection with virus caused a transcriptional response characterized by changes in the levels of expression of multiple macrophage genes. Up-regulated genes included novel and previously known genes, some of these involved in leukocyte differentiation, protein trafficking, endosome acidification and modulation of the antiviral response by nuclear factor kappa beta. Comparison of wild type and genedeleted viruses revealed that ASFV genes from families 360 and 530 suppress early innate host responses induced by interferon alpha/beta. Mutant viruses containing deletion of 360 and 530 genes displayed enhanced expression of interferon induced early response genes and
chemokines involved in the inflammatory response. Impact: These finding contribute to our understanding of swine resistance/susceptibility to viral infection and mechanisms of pathogenesis and may lead to early recognition of viral infection, predication of clinical outcome and identification of macrophage response genes associated with disease resistance. A novel viral virulence determinant which includes multiple members of the MGF 530 genes has been identified in the left variable region of the ASFV genome using in vivo marker rescue. These genes will be an important target for engineering liveattenuated ASF vaccine viruses. An understanding of pathogen-host interactions is critical for development of traditional and/or novel disease control strategies. The yeast two-hybrid system, used for the study of protein-protein interactions, was adapted specifically for investigating ASFV - swine macrophage interactions. ASFV infected swine macrophage CDNA libraries were constructed in
a yeast activation domain GAL4 vector. Library Quality was assessed by nucleotide sequence and bio-informatic analysis. Two ASFV genes (5HL and 6FR) screened in this system, identified positive host cell interactor proteins. 5HL, an ASFV gene previously found to have a role in the modulation of apoptosis, baited host genes predicted to have roles in the regulation of programmed cell death pathways and cell cycle modulation. 6FR, a novel gene which has been implicated to have a critical role in ASFV infection, baited genes involved in host cell transcriptional regulation. Overall, the identification of significant virulence and host range genes provides a rational basis for development of genetically engineered attenuated or nonreplicating (in the swine host) ASFV viruses for use as vaccines. Further, an understanding of how these novel genes function in the swine host will contribute to our overall understanding of viral virulence/host range, viral persistence and
pathogen-vector-host interactions; information that may have broad impact on swine infectious disease in general. Characterized latent infection with ASFV - Determined ASFV establishes a true latent infection in peripheral blood monocytes/macrophages, bone marrow and tonsil all recovered pigs, one in which viral replication does not occur. Knowledge of the existence of ASFV carrier animals is of critical importance for developing disease control programs in disease endemic regions. Characterized ASFV-tick host interactions - Our identification of a large number of highly conserved ASFV genes with no apparent role in the swine host suggested these genes may be associated with aspects of infection in the tick host. Colonies of ornithodoros ticks for use in investigating virus-tick interactions were established at PIADC from wild ticks collected in South Africa. Detailed pathogenesis studies of ASFV infection in the tick host revealed important aspects of this virus-host interaction.
Recombinant ASF viruses containing deletions of putative tick host range genes were constructed and examined in the ticks. The viral TK gene has recently been shown to be necessary for viral replication in midgut epithelial cells. And, a second tick host range determinant has been mapped to the left variable region of the ASFV genome. Identification of ASF viral genes functioning in host range in Ornithodoros porcinus ticks. Viral genes necessary for infecting ticks were identified. Multigene family 360 genes are required for efficient virus replication and generalization of infection in O.porcinus ticks. Successful identification of tick host range genes will permit the design of live attenuated ASFV vaccine viruses that are incapable of infecting or being transmitted by ticks. The functional characterization of these genes, will also likely contribute to our understanding of pathogen- arthropod host interactions in general. Functional genomics of cytoplasmic DNA viruses - A
viral genomics approach was used to identify putative ASFV arthropod host range genes. Two cytoplasmic DNA viruses representing distinct virus families, Chlorella virus PBCV-1 (330 Kbp) and the Melanoplus Sanguinipes Entomopoxvirus (236 Kbp) and a Baculovirus pathogenic for Culex nigripalpus were completely sequenced and analysed. The Chlorella virus genome represents the largest viral genome sequenced to date. These data provided information on the primary structure of all encoded genes, their organization, potential regulatory sequences and their evolutionary relationship to one another. In both viruses a number of novel and unexpected genes were identified. Sequence homologies with these viruses have led to the identification of ASFV genes with putative functions in virus-arthropod host interactions. This work has led to ASFV being placed into its own virus family, the Asfarviridae, and it has provided the genetic underpinning for all future research aimed at understanding the
complex biology and evolution of large cytoplasmic DNA viruses. Development of ASFV diagnostics and reagents - A PCR-based diagnostic assay for ASFV DNA, with a sensitivity equal to that of animal inoculation, was developed for use with blood and tissue samples. The assay is being used for research and diagnostic applications in the US, Spain, UK, South Africa, and China. Cloned genes, protein expression vectors, and both polyclonal and monoclonal antibody reagents have been developed and provided to research and diagnostic laboratories in the US, UK, Spain, South Africa, the Netherlands, and China. The genomes of Fowlpox virus, Marek's disease virus, Turkey herpesvirus and Swine pox were completely sequenced and analyzed. An understanding of the genetic basis of viral virulence and host range will permit the engineering of novel vaccine viruses and expression vectors with enhanced efficacy and greater versatility. The capripox viruses are responsible for highly significant foreign
animal diseases (FAD) found in Africa and Central Asia and significantly they can be confused with a disease of sheep present in the United States caused by a parapoxvirus (orf virus). To improve diagnostic assays to distinguish between these viruses and to improve on existing vaccines for these diseases the genomes of lumpy skin disease-virus (151 kpb, 156 genes), sheeppox virus (146 kpb, 150 genes) and goatpox virus (151 kpb and 150 genes) were completely sequenced and analyzed. Virus specific gene targets were identified as were genes important for viral virulence and host range. Impact: Genomic sequences of these FAD viruses will allow for development of highly sensitive and specific diagnostic tests. Additionally, an understanding of the genetic basis of viral virulence and host range will permit the engineering of novel vaccine viruses and expression vectors with enhanced efficacy and greater versatility and will contribute novel concepts of pathogen-host interactions. This
is fundamental information that will have broad impact on future strategies for controlling animal infectious diseases in general. 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, the Netherlands, Denmark and China. 7. List your most important publications in the popular press and presentations to organizations and articles written about your work.
This work has been presented to numerous groups representing other Federal Departments and Agencies and Academic Institutions(1999, 2000, 2001, 2002). Work was featured in the Agricultural Research Magazine, "Cracking the Code of Marek's Disease" July 2001, p. 16-17.
Impacts
(N/A)
Publications
- Afonso, C.L., Piccone, M.E., Zaffuto, K., Neilan, J.G., Kutish, G.F., Lu, Z., Balinsky, C.A., Gibb, T.R., Zsak, L., Rock, D.L. 2004. African swine fever virus multigene family 360 and 530 genes affect host interferon response. Journal of Virology. 78(4):1858-1864.
- Burrage, T.G., Lu, Z., Neilan, J.G., Rock, D.L., Zsak, L. 2004. African swine fever virus multigene family 360 genes affect virus replication and generalization of infection in ornithodoros porcinus ticks. Journal of Virology. 78(5):2445-2453.
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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 and 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. Work here is focused on: 1) Identifying and characterizing viral genes responsible for virulence, viral latency and host range in both the swine and tick host; 2) Identifying host cell genes
associated with host susceptibility and/or resistance;3) Developing vaccines and/or other novel approaches for disease control that exploit virulence and host range mechanisms; and 4) Developing genomic based methods to rapidly identify and phenotypically characterize viruses. Identification of viral virulence-associated genes will permit the rational design and genetic engineering of live attenuated viruses for use as vaccines. Additionally, an understanding of these genes and their functions may permit development of novel approaches for disease control that exploit aspects of viral host range. 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. Although ASF has never been present in North and Central
America, several outbreaks occurred in the Caribbean (Cuba, Haiti, Dominican Republic) and in Brazil during the 1980's. The disease has finally been eradicated from continental Europe (still present in the Italian island of Sardinia) after a 30 year presence in Spain and Portugal. The continued presence of ASF in many African nations is a constant threat to the rest of the world. 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, viral persistence 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: ASFV multigene family (MGF) 360 and 530 genes previously identified as host range genes in Omithodoros porcinus porcinus ticks were found to function in successful establishment of infection in phagocytic digestive cells of the tick midgut. Identification and characterization of ASFV genes with tick host range functions will permit design of live attenuated host range restricted ASFV vaccines for use in endemic areas. B. Other Significant Accomplishments(s): Rapid identification and diagnosis of a FAD is critical for successfully controlling, managing and eradicating the disease following
introduction. Rapid field-based tests to detect ASFV are not available. A rapid real-time TaqMan PCR assay was developed and evaluated in contact exposed pigs. The test was sensitive and specific for ASFV, as sensitive as virus isolation, and detected animals prior to the onset of clinical disease. A rapid, real-time PCR assay for ASFV provides a new diagnostic tool that conceivably will redefine emergency disease management and control strategies for this highly significant FAD. C. Significant Accomplishments/Activities that Support Special Target Populations: None. D. Progress Report: This report serves to document research conducted under a Specific Cooperative Agreement between ARS and Iowa State University (1940-32000- 036-05S). Nipah virus F and G genes were cloned into the HAd5 vector and the Nipah F gene into the PRV vector. The recombinant virus-vectored vaccines were shown to express proteins in vitro. Procedures and reagents necessary to evaluate induction of
cell-mediated immunity to viral vaccines in pigs were developed. The procedure uses four-color flow cytometry to monitor up regulation of the interleukin 2 receptor (CD25) on the surface of CD4+, CD8+, and Gamma Delta T cells. The use of four color flow cytometry allows for the simultaneous measurement of all 4 cell surface markers. This is important in pigs because they have a high percentage of double positive T cell subsets (ie, CD4 and CD8 positive, CD8 and gamma delta positive). These assays are ready to use for evaluation of induction of T cell mediated immunity by the recombinant virus-vectored vaccines expressing Nipah virus proteins. 5. Describe the major accomplishments over the life of the project, including their predicted or actual impact. Characterization of the ASFV genome - The genome of a pathogenic African ASFV isolate, Malawi Lil-20/1, was sequenced and analyzed. Data provided primary structure of all encoded genes and overall genome organization. Sequence
homologies with other known genes have had great predictive value for proposing and examining ASFV gene function and have led to the identification of significant swine virulence and host range genes. Additionally, these data have also facilitated the identification of putative immunogenic proteins - viral membrane and envelope proteins. Identification of viral genes function in virulence and host range is difficult and very time consuming, especially when the genes are novel - lacking homology with other known genes. Putative ASFV virulence and host range genes were identified using a novel comparative genome approach. Analysis of complete genome sequences of seven pathogenic field isolates of ASFV identified several highly conserved genes in regions of the viral genome known to contain other pathobiologically significant genes. Impact - Comparative genomic approaches - facilitated by highly throughput DNA sequencing - may greatly simplify identification of novel genes with critical
functions in the host. The strategy developed here for ASFV will have broad applicability for characterization of other threat agent pathogens. Genetic engineering of the ASFV genome - Techniques were developed to genetically engineer pathogenic isolates of ASFV. These techniques which allow the addition or deletion of specific genes have permitted both genetic and reverse genetic research approaches for determining viral gene function. Recombinant ASFV gene-deletion mutants have been used to identify and characterize swine virulence and host range genes. This technology has proven critical for engineering live-attenuated ASFV vaccine viruses. Identification and characterization of ASFV swine virulence and host range genes - Using genetic based strategies and ASFV gene-deletion mutants, a number of pathobiologically significant viral genes, many of which are novel, have been identified and characterized. Virulence-associated genes, NL-S and UK were identified in pathogenic European
ASFV isolates. Deletion of either of these genes from the viral genome resulted in complete attenuation of the virus in swine. A third distinct virulence determinant which includes multiple members of the multigene family 530 genes AVAD, was identi-fied in the left variable region of the genome. These genes which are required for viral virulence are novel, lacking similarity to any other known genes. ASFV genes involved with the regulation of apoptosis in virus infected cells (bcl-2 and iap homologs) and genes with functions involving immune evasion in the swine host (CD2 and IkB homologs) were identified and characterized. CD2 and IkB genes had not been previously described in any virus. This is the first example of a pathogen using of a CD2-like molecule in immune evasion. ASFV genes which affect macrophage host range were also identified and characterized. The thymidine kinase gene (TK) and a second gene 9-GL are required for efficient replication of virus in infected cells;
deletion of either gene resulted in a 99% to 99.9% reduction in viral growth and virulence attenuation. 9-GL is a novel gene whose function in virus replication is unknown. A novel macrophage growth-associated determinant which includes multiple members of the multigene family 360 genes was identified in the left variable region of the ASFV genome. These genes are novel lacking homology to other known genes. ASFV and CSF infect and replicate in cells of the monocyte-macrophage lineage. Revealing molecular mechanisms involved in macrophage responses to infection is important to devise novel disease control strategies. A macrophage-specific swine cDNA microarray of 8029 clones was constructed and used to analyze the transcriptional profiles of swine macrophages infected with ASFV and gene deleted mutant viruses. Infection with virus caused a transcriptional response characterized by changes in the levels of expression of multiple macrophage genes. Up-regulated genes included novel and
previously known genes, some of these involved in leukocyte differentiation, protein trafficking, endosome acidification and modulation of the antiviral response by nuclear factor kappa beta. Comparison of wild type and gene-deleted viruses revealed that ASFV genes from families 360 and 530 suppress early innate host responses induced by interferon alpha/beta. Mutant viruses containing deletion of 360 and 530 genes displayed enhanced expression of interferon induced early response genes and chemokines involved in the inflammatory response. Impact: These finding contribute to our understanding of swine resistance/susceptibility to viral infection and mechanisms of pathogenesis and may lead to early recognition of viral infection, predication of clinical outcome and identification of macrophage response genes associated with disease resistance. A novel viral virulence determinant which includes multiple members of the MGF 530 genes has been identified in the left variable region of the
ASFV genome using in vivo marker rescue. These genes will be an important target for engineering liveattenuated ASF vaccine viruses. An understanding of pathogen-host interactions is critical for development of traditional and/or novel disease control strategies. The yeast two-hybrid system, used for the study of protein-protein interactions, was adapted specifically for investigating ASFV - swine macrophage interactions. ASFV infected swine macrophage cDNA libraries were constructed in a yeast activation domain GAL4 vector. Library Quality was assessed by nucleotide sequence and bio-informatic analysis. Two ASFV genes (5HL and 6FR) screened in this system, identified positive host cell interactor proteins. 5HL, an ASFV gene previously found to have a role in the modulation of apoptosis, baited host genes predicted to have roles in the regulation of programmed cell death pathways and cell cycle modulation. 6FR, a novel gene which has been implicated to have a critical role in ASFV
infection, baited genes involved in host cell transcriptional regulation. Overall, the identification of significant virulence and host range genes provides a rational basis for development of genetically engineered attenuated or nonreplicating (in the swine host) ASFV viruses for use as vaccines. Further, an understanding of how these novel genes function in the swine host will contribute to our overall understanding of viral virulence/host range, viral persistence and pathogen-vector-host interactions; information that may have broad impact on swine infectious disease in general. Characterized latent infection with ASFV - Determined ASFV establishes a true latent infection in peripheral blood monocytes/macrophages, bone marrow and tonsil all recovered pigs, one in which viral replication does not occur. Knowledge of the existence of ASFV carrier animals is of critical importance for developing disease control programs in disease endemic regions. Characterized ASFV-tick host
interactions - Our identification of a large number of highly conserved ASFV genes with no apparent role in the swine host suggested these genes may be associated with aspects of infection in the tick host. Colonies of Ornithodoros ticks for use in investigating virus-tick interactions were established at PIADC from wild ticks collected in South Africa. Detailed pathogenesis studies of ASFV infection in the tick host revealed important aspects of this virus-host interaction. Recombinant ASF viruses containing deletions of putative tick host range genes were constructed and examined in the ticks. The viral TK gene has recently been shown to be necessary for viral replication in midgut epithelial cells. And, a second tick host range determinant has been mapped to the left variable region of the ASFV genome. Identification of ASF viral genes functioning in host range in Ornithodoros porcinus ticks. Viral genes necessary for infecting ticks were identified. Multigene family 360 genes are
required for efficient virus replication and generalization of infection in O.porcinus ticks. Successful identification of tick host range genes will permit the design of live attenuated ASFV vaccine viruses that are incapable of infecting or being transmitted by ticks. The functional characterization of these genes, will also likely contribute to our understanding of pathogen- arthropod host interactions in general. Functional genomics of cytoplasmic DNA viruses - A viral genomics approach was used to identify putative ASFV arthropod host range genes. Two cytoplasmic DNA viruses representing distinct virus families, Chlorella virus PBCV-1 (330 Kbp) and the Melanoplus Sanguinipes Entomopoxvirus (236 Kbp) and a Baculovirus pathogenic for Culex nigripalpus were completely sequenced and analysed. The Chlorella virus genome represents the largest viral genome sequenced to date. These data provided information on the primary structure of all encoded genes, their organization, potential
regulatory sequences and their evolutionary relationship to one another. In both viruses a number of novel and unexpected genes were identified. Sequence homologies with these viruses have led to the identification of ASFV genes with putative functions in virus-arthropod host interactions. This work has led to ASFV being placed into its own virus family, the Asfarviridae, and it has provided the genetic underpinning for all future research aimed at understanding the complex biology and evolution of large cytoplasmic DNA viruses. Development of ASFV diagnostics and reagents - A PCR-based diagnostic assay for ASFV DNA, with a sensitivity equal to that of animal inoculation, was developed for use with blood and tissue samples. The assay is being used for research and diagnostic applications in the US, Spain, UK, South Africa, and China. Cloned genes, protein expression vectors, and both polyclonal and monoclonal antibody reagents have been developed and provided to research and
diagnostic laboratories in the US, UK, Spain, South Africa, the Netherlands, and China. The genomes of Fowlpox virus, Marek's disease virus, Turkey herpesvirus and Swine pox were completely sequenced and analyzed. An understanding of the genetic basis of viral virulence and host range will permit the engineering of novel vaccine viruses and expression vectors with enhanced efficacy and greater versatility. The capripox viruses are responsible for highly significant foreign animal diseases (FAD) found in Africa and Central Asia and significantly they can be confused with a disease of sheep present in the United States caused by a parapoxvirus (orf virus). To improve diagnostic assays to distinguish between these viruses and to improve on existing vaccines for these diseases the genomes of lumpy skin disease-virus (151 kpb, 156 genes), sheeppox virus (146 kpb, 150 genes) and goatpox virus (151 kpb and 150 genes) were completely sequenced and analyzed. Virus specific gene targets were
identified as were genes important for viral virulence and host range. Impact: Genomic sequences of these FAD viruses will allow for development of highly sensitive and specific diagnostic tests. Additionally, an understanding of the genetic basis of viral virulence and host range will permit the engineering of novel vaccine viruses and expression vectors with enhanced efficacy and greater versatility and will contribute novel concepts of pathogen-host interactions. This is fundamental information that will have broad impact on future strategies for controlling animal infectious diseases in general. 6. What do you expect to accomplish, year by year, over the next 3 years? Future research work in FY-04 - MGF 360/530 genes required for growth in macrophages, swine virulence and tick host range will be functionally characterized. Viral gene transcription, protein expression, protein localization in infected cells, and protein involvement in aspects of virus-cell interaction will be
examined. Cellular transcriptional networks affected by these genes will be identified with DNA microarray analysis. Cellular proteins which physically interact with specific MGF 360/530 proteins will be identified using yeast two-hybrid assays. Together, these approaches will better define MGF 360/350 gene function in infection and immunity. FY-05 - Whole genome comparisons have identified both highly conserved and variable genes in terminal ASFV genomic regions, which are known to contain important virulence and host range genes. Through this analysis, we have identified additional candidate genes with putative virulence and/or host range functions. To examine gene function, single gene deletion mutants of a pathogenic ASFV isolate will be constructed. Mutant viruses together with parental and/or revertant viruses will be examined for their ability to replicate in porcine macrophage cell cultures in vitro and for their virulence in pigs. Viruses will also be examined in the tick
host for alterations in infectious dose, initial midgut infection, generalization of infection, tissue tropism, viral persistence and transmission. FY-06 - Mutant viruses identified in FY-05 with altered virulence and host range phenotypes will be examined using DNA microarray analysis of both swine and tick expressed sequence tags to define cellular transcriptional networks affected by the viral gene. The identification of cellular pathways affected by the viral gene will suggest molecular mechanisms for these genes in viral virulence and host range. 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, the Netherlands, Denmark and China. 8. List your most important publications in the popular press and presentations to organizations and articles written about your work. (NOTE: This does not replace your peer-reviewed publications listed below). This work has been presented to numerous groups representing other Federal Departments and Agencies and Academic Institutions(1999, 2000, 2001, 2002). Work was featured in the Agricultural Research Magazine, "Cracking the Code of Marek's Disease" July 2001, p. 16-17.
Impacts
(N/A)
Publications
- Afonso, C.L., Tulman, E.R., Lu, Balinsky, C.A., Moser, B.A., Becnel, J.J., Rock, D.L., Kutish, G.F. Genome Sequence of a Baculovirus Pathogenic for Culex nigripalpus. Journal of Virology. 2001. v. 75. p. 11157-11165.
- Tulman, E.R., Afonso, C.L., Lu, Z., Zsak, L., Sur, J-H., Sandybaev, N.T., Kerembekova, U.Z., Zaitsev, V.L., Kutish, G.F., Rock, D.L. The Genomes of Sheeppox and Goatpox Virus. Journal of Virology. 2002. v. 76. p. 6054- 6061.
- Neilan, J.G., Kutish, G.F., Lu, Z., Zsak, A., Zsak, L., Rock, D.L. The African Swine Fever Virus ORF 6FR interacts with infected cell nuclear proteins. Conference of Research Workers in Animal Diseases. 2001. Abstract p. 204.
- Moraes, M.P., Lu, Z., Afonso, C.L., Flores, E.F., Weiblen, R., Rock, D.L., Kutish, G.F. The genome of bovine herpesvirus type 5: Comparison with bovine herpesvirus type 1. Conference of Research Workers in Animal Diseases. 2001. Abstract p. 113P.
- Afonso, C.L., Tulman, E.R., Lu, Z., Zsak, L., Osorio, F.A., Balinsky, C., Kutish, G.F., Rock, D.L. The Genome of Swinepox Virus. Journal of Virology. 2002. v. 76. p. 783-790.
|
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 and 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. Work here is focused on: 1) Identifying and characterizing viral genes responsible for virulence, viral latency and host range in both the swine and tick host; 2) Identifying host cell genes
associated with host susceptibility and/or resistance; 3) Developing vaccines and/or other novel approaches for disease control that exploit virulence and host range mechanisms; and 4) Developing genomic based methods to rapidly identify and phenotypically characterize viruses. Identification of viral virulence-associated genes will permit the rational design and genetic engineering of live attenuated viruses for use as vaccines. Additionally, an understanding of these genes and their functions may permit development of novel approaches for disease control that exploit aspects of viral host range. 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. Although ASF has never been present in North and Central
America, several outbreaks occurred in the Caribbean (Cuba, Haiti, Dominican Republic) and in Brazil during the 1980's. The disease has finally been eradicated from continental Europe (still present in the Italian island of Sardinia) after a 30 year presence in Spain and Portugal. The continued presence of ASF in many African nations is a constant threat to the rest of the world. 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, viral persistence 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: Identification of ASF viral genes functioning in host range in Ornithodoros porcinus ticks. Viral genes necessary for infecting ticks were identified. Multigene family 360 genes are required for efficient virus replication and generalization of infection in O. porcinus ticks. Impact - A molecular understanding of ASFV tick host range and the viral genes responsible for this is critical for design of live-attenuated host range restricted ASFV vaccines. B. Other Significant Accomplishments(s): ASFV and CSF infect and replicate in cells of the monocyte-macrophage lineage.
Revealing molecular mechanisms involved in macrophage responses to infection is important to devise novel disease control strategies. A macrophage- specific swine cDNA microarray of 8029 clones was constructed and used to analyze the transcriptional profiles of swine macrophages infected with ASFV and gene deleted mutant viruses. Infection with virus caused a transcriptional response characterized by changes in the levels of expression of multiple macrophage genes. Up-regulated genes included novel and previously known genes, some of these involved in leukocyte differentiation, protein trafficking, endosome acidification and modulation of the antiviral response by nuclear factor kappa beta. Comparison of wild type and gene-deleted viruses revealed that ASFV genes from families 360 and 530 suppress early innate host responses induced by interferon alpha/beta. Mutant viruses containing deletion of 360 and 530 genes displayed enhanced expression of interferon induced early response
genes and chemokines involved in the inflammatory response. Impact - These finding contribute to our understanding of swine resistance/susceptibility to viral infection and mechanisms of pathogenesis and may lead to early recognition of viral infection, predication of clinical outcome and identification of macrophage response genes associated with disease resistance. An understanding of pathogen-host interactions is critical for development of traditional and/or novel disease control strategies. The yeast two-hybrid system, used for the study of protein-protein interactions, was adapted specifically for investigating ASFV - swine macrophage interactions. ASFV infected swine macrophage cDNA libraries were constructed in a yeast activation domain GAL4 vector. Library Quality was assessed by nucleotide sequence and bio-informatic analysis. Two ASFV genes (5HL and 6FR) screened in this system, identified positive host cell interactor proteins. 5HL, an ASFV gene previously found to
have a role in the modulation of apoptosis, baited host genes predicted to have roles in the regulation of programmed cell death pathways and cell cycle modulation. 6FR, a novel gene which has been implicated to have a critical role in ASFV infection, baited genes involved in host cell transcriptional regulation. Impact - Yeast two-hybrid analysis provides a powerful tool to define viral gene function in virulence and host range. 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? Characterization of the ASFV genome - The genome of a pathogenic African ASFV isolate, Malawi Lil-20/1, was sequenced and analyzed. Data provided primary structure of all encoded genes and overall genome organization. Sequence homologies with other known genes have had great predictive value for proposing and examining ASFV gene function and have led to the
identification of significant swine virulence and host range genes. Additionally, these data have also facilitated the identification of putative immunogenic proteins - viral membrane and envelope proteins. Identification of viral genes function in virulence and host range is difficult and very time consuming, especially when the genes are novel - lacking homology with other known genes. Putative ASFV virulence and host range genes were identified using a novel comparative genome approach. Analysis of complete genome sequences of seven pathogenic field isolates of ASFV identified several highly conserved genes in regions of the viral genome known to contain other pathobiologically significant genes. Impact - Comparative genomic approaches - facilitated by highly throughput DNA sequencing - may greatly simplify identification of novel genes with critical functions in the host. The strategy developed here for ASFV will have broad applicability for characterization of other threat
agent pathogens. Genetic engineering of the ASFV genome - Techniques were developed to genetically engineer pathogenic isolates of ASFV. These techniques which allow the addition or deletion of specific genes have permitted both genetic and reverse genetic research approaches for determining viral gene function. Recombinant ASFV gene-deletion mutants have been used to identify and characterize swine virulence and host range genes. This technology has proven critical for engineering live-attenuated ASFV vaccine viruses. Identification and characterization of ASFV swine virulence and host range genes - Using genetic based strategies and ASFV gene-deletion mutants, a number of pathobiologically significant viral genes, many of which are novel, have been identified and characterized. Virulence-associated genes, NL-S and UK were identified in pathogenic European ASFV isolates. Deletion of either of these genes from the viral genome resulted in complete attenuation of the virus in
swine. A third distinct virulence determinant which includes multiple members of the multigene family 530 genes AVAD, was identified in the left variable region of the genome. These genes which are required for viral virulence are novel, lacking similarity to any other known genes. ASFV genes involved with the regulation of apoptosis in virus infected cells (bcl-2 and iap homologs) and genes with functions involving immune evasion in the swine host (CD2 and IkB homologs) were identified and characterized. CD2 and IkB genes had not been previously described in any virus. This is the first example of a pathogen using of a CD2-like molecule in immune evasion. ASFV genes which affect macrophage host range were also identified and characterized. The thymidine kinase gene (TK) and a second gene 9-GL are required for efficient replication of virus in infected cells; deletion of either gene resulted in a 99% to 99.9% reduction in viral growth and virulence attenuation. 9-GL is a novel
gene whose function in virus replication is unknown. A novel macrophage growth-associated determinant which includes multiple members of the multigene family 360 genes was identified in the left variable region of the ASFV genome. These genes are novel lacking homology to other known genes. A novel viral virulence determinant which includes multiple members of the MGF 530 genes has been identified in the left variable region of the ASFV genome using in vivo marker rescue. These genes will be an important target for engineering live-attenuated ASF vaccine viruses. Overall, the identification of significant virulence and host range genes provides a rational basis for development of genetically engineered attenuated or non-replicating (in the swine host) ASFV viruses for use as vaccines. Further, an understanding of how these novel genes function in the swine host will contribute to our overall understanding of viral virulence/host range, viral persistence and pathogen-vector-host
interactions; information that may have broad impact on swine infectious disease in general. Characterized latent infection with ASFV - Determined ASFV establishes a true latent infection in peripheral blood monocytes/macrophages, bone marrow and tonsil all recovered pigs, one in which viral replication does not occur. Knowledge of the existence of ASFV carrier animals is of critical importance for developing disease control programs in disease endemic regions. Characterized ASFV-tick host interactions - Our identification of a large number of highly conserved ASFV genes with no apparent role in the swine host suggested these genes may be associated with aspects of infection in the tick host. Colonies of Ornithodoros ticks for use in investigating virus-tick interactions were established at PIADC from wild ticks collected in South Africa. Detailed pathogenesis studies of ASFV infection in the tick host revealed important aspects of this virus-host interaction. Recombinant ASF
viruses containing deletions of putative tick host range genes were constructed and examined in the ticks. The viral TK gene has recently been shown to be necessary for viral replication in midgut epithelial cells. And, a second tick host range determinant has been mapped to the left variable region of the ASFV genome. Successful identification of tick host range genes will permit the design of live attenuated ASFV vaccine viruses that are incapable of infecting or being transmitted by ticks. The functional characterization of these genes, will also likely contribute to our understanding of pathogen-arthropod host interactions in general. Functional genomics of cytoplasmic DNA viruses - A viral genomics approach was used to identify putative ASFV arthropod host range genes. Two cytoplasmic DNA viruses representing distinct virus families, Chlorella virus PBCV-1 (330 Kbp) and the Melanoplus Sanguinipes Entomopoxvirus (236 Kbp) and a Baculovirus pathogenic for Culex nigripalpus were
completely sequenced and analysed. The Chlorella virus genome represents the largest viral genome sequenced to date. These data provided information on the primary structure of all encoded genes, their organization, potential regulatory sequences and their evolutionary relationship to one another. In both viruses a number of novel and unexpected genes were identified. Sequence homologies with these viruses have led to the identification of ASFV genes with putative functions in virus-arthropod host interactions. This work has led to ASFV being placed into its own virus family, the Asfarviridae, and it has provided the genetic underpinning for all future research aimed at understanding the complex biology and evolution of large cytoplasmic DNA viruses. Development of ASFV diagnostics and reagents - A PCR-based diagnostic assay for ASFV DNA, with a sensitivity equal to that of animal inoculation, was developed for use with blood and tissue samples. The assay is being used for research
and diagnostic applications in the US, Spain, UK, South Africa, and China. Cloned genes, protein expression vectors, and both polyclonal and monoclonal antibody reagents have been developed and provided to research and diagnostic laboratories in the US, UK, Spain, South Africa, the Netherlands, and China. 6. What do you expect to accomplish, year by year, over the next 3 years? Future research work in FY-2003-2006 will include the following: Specific MGF 360/530 genes required for growth in macrophages will be identified. The minimal set of MGF 360/530 genes necessary for macrophage host range (MHR) will be further characterized. Viral gene transcription, protein expression, protein localization in infected cells, and protein involvement in aspects of virus-cell interaction will be examined. Cellular transcriptional networks affected by these genes will be identified with DNA microarray analysis. Cellular proteins which physically interact with specific ASFV proteins will be
identified using yeast two-hybrid assays. Together, these approaches will define viral gene function in MHR. Whole genome comparisons have identified both highly conserved and variable genes in terminal ASFV genomic regions, which are known to contain important virulence and host range genes. Through this analysis, we have identified additional candidate genes with putative virulence and/or host range functions. To examine gene function, single gene deletion mutants of a pathogenic ASFV isolate will be constructed. Mutant viruses together with parental and/or revertant viruses will be examined for their ability to replicate in porcine macrophage cell cultures in vitro and for their virulence in pigs. Viruses will also be examined in the tick host for alterations in infectious dose, initial midgut infection, generalization of infection, tissue tropism, viral persistence and transmission. Mutant viruses will be examined using DNA microarray analysis of both swine and tick expressed
sequence tags to define cellular transcriptional networks affected by the viral gene. The identification of cellular pathways affected by the viral gene will suggest molecular mechanisms for these genes in viral virulence and host range. 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, the Netherlands, Denmark and China. 8. List your most important publications and presentations, and articles written about your work (NOTE: this does not replace your review publications which are listed below) This work has been presented to numerous groups representing other Federal Departments and
Agencies and Academic Institutions(1999, 2000, 2001, 2002). Work was featured in the Agricultural Research Magazine, "Cracking the Code of Marek's Disease" July 2001, p. 16-17.
Impacts
(N/A)
Publications
- Afonso, C.L., Tulman, E.R., Lu, Balinsky, C.A., Moser, B.A., Becnel, J.J., Rock, D.L., Kutish, G.F. Genome Sequence of a Baculovirus Pathogenic for Culex nigripalpus. Journal of Virology. 2001. v. 75. p. 11157-11165.
- Tulman, E.R., Afonso, C.L., Lu, Z., Zsak, L., Sur, J-H., Sandybaev, N.T., Kerembekova, U.Z., Zaitsev, V.L., Kutish, G.F., Rock, D.L. The Genomes of Sheeppox and Goatpox Virus. Journal of Virology. 2002. v. 76. p. 6054- 6061.
- Neilan, J.G., Kutish, G.F., Lu, Z., Zsak, A., Zsak, L., Rock, D.L. The African Swine Fever Virus ORF 6FR interacts with infected cell nuclear proteins. Conference of Research Workers in Animal Diseases. 2001. Abstract p. 204.
- Moraes, M.P., Lu, Z., Afonso, C.L., Flores, E.F., Weiblen, R., Rock, D.L., Kutish, G.F. The genome of bovine herpesvirus type 5: Comparison with bovine herpesvirus type 1. Conference of Research Workers in Animal Diseases. 2001. Abstract p. 113P.
- Afonso, C.L., Tulman, E.R., Lu, Z., Zsak, L., Osorio, F.A., Balinsky, C., Kutish, G.F., Rock, D.L. The Genome of Swinepox Virus. Journal of Virology. 2002. v. 76. p. 783-790.
|
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. Research is focused on: 1) identifying and characterizing viral genes responsible for virulence, viral latency and host range in both the swine and tick host; 2) identifying host cell genes associated with host susceptibility and/or resistance; 3) developing vaccines and/or other novel
approaches for disease control that exploit virulence and host range mechanisms; and 4) developing genomic based methods to rapidly identify and phenotypically characterize viruses. Identification of viral virulence-associated genes will permit the rational design and genetic engineering of live attenuated viruses for use as vaccines. Additionally, an understanding of these genes and their functions may permit development of novel approaches for disease control that exploit aspects of viral host range.
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. Although ASF has never been present in North and Central America, several outbreaks occurred in the Caribbean (Cuba, Haiti, Dominican Republic) and in Brazil during the 1980's. The disease has finally been eradicated from continental Europe (still present in the Italian island of Sardinia) after a 30 year presence in Spain and Portugal. The continued presence of ASF in many African nations is a constant threat to the rest of the world. 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, viral persistence 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) Microbial Genomics, (2) Mechanisms of Disease, and (3) 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: Identification of viral genes function in virulence and host range is difficult and very time consuming, especially when the genes are novel - lacking homology with other known genes. Putative ASFV virulence and host range genes were identified using a novel comparative genome approach. Analysis of complete genome sequences of seven pathogenic field isolates of ASFV identified several highly conserved genes in regions of the viral genome known to contain other pathobiologically significant genes. Comparative genomic approaches - facilitated by highly throughput DNA sequencing - may greatly simplify identification of novel genes with critical functions in the host and will have broad applicability for characterization of other threat agent pathogens. B. Other Significant Accomplishments(s): The macrophage is the critical target cell for both ASFV and Classical Swine Fever virus. Gaining an understanding how this cell responds
to viral infection and the role of specific viral proteins in modifying or altering the cellular response has proven to be extremely difficult. To address this issue, c-DNA expression libraries were constructed from swine macrophages and analyzed by DNA sequencing. Approximately 6,000 swine genes (ESTs) have been identified including those encoding immunoregulatory proteins, transcription factors, proteins involved in signal transduction, cell death pathways and cell cycle regulation. Gene libraries are of sufficient complexity to be used for DNA microarray analysis and yeast two-hybrid screening. This research compliments work performed in other cell types at the Meat Animal Research Center (MARC), Clay Center, Nebraska. Monitoring changes in global gene expression during viral infection and virus-cell protein interactions will provide a fundamental understanding of virus- swine cell interactions and in the process suggest new viral or cellular targets that may be exploitable
for controlling and/or preventing infection. The capripox viruses are responsible for highly significant foreign animal diseases (FAD) found in Africa and Central Asia and significantly they can be confused with a disease of sheep present in the United States caused by a parapoxvirus (orf virus). To improve diagnostic assays to distinguish between these viruses and to improve on existing vaccines for these diseases the genomes of lumpy skin disease virus (151 kpb, 156 genes), sheeppox virus (146 kpb, 150 genes) and goatpox virus (151 kpb and 150 genes) were completely sequenced and analyzed. Virus specific gene targets were identified as were genes important for viral virulence and host range. Genomic sequences of these FAD viruses will allow for development of highly sensitive and specific diagnostic tests. Additionally, an understanding of the genetic basis of viral virulence and host range will permit the engineering of novel vaccine viruses and expression vectors with enhanced
efficacy and greater versatility and will contribute novel concepts of pathogen-host interactions. This is fundamental information that will have broad impact on future strategies for controlling animal infectious diseases in general. 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 1940-32000-032-00D ("Virulence and Host Range Genes of African Swine Fever Virus"). The progress which is reported resulted from the above mentioned project. Characterization of the ASFV genome - The genome of a pathogenic African ASFV isolate, Malawi Lil-20/1, was sequenced and analyzed. Data provided primary structure of all encoded genes and overall genome organization. Sequence homologies with other known genes have had great predictive value for proposing and examining ASFV gene function and have led to the identification of significant swine virulence and host range genes. Additionally, these data have also facilitated the identification of putative immunogenic proteins - viral membrane and envelope proteins. Genetic engineering of the ASFV genome - Techniques were developed to genetically engineer pathogenic isolates of ASFV. These techniques which allow the addition or deletion of
specific genes have permitted both genetic and reverse genetic research approaches for determining viral gene function. Recombinant ASFV gene-deletion mutants have been used to identify and characterize swine virulence and host range genes. This technology has proven critical for engineering live-attenuated ASFV vaccine viruses. Identification and characterization of ASFV swine virulence and host range genes - Using genetic based strategies and ASFV gene-deletion mutants, a number of pathobiologically significant viral genes, many of which are novel, have been identified and characterized. Virulence-associated genes, NL-S and UK were identified in pathogenic European ASFV isolates. Deletion of either of these genes from the viral genome resulted in complete attenuation of the virus in swine. A third distinct virulence determinant which includes multiple members of the multigene family 530 genes AVAD, was identified in the left variable region of the genome. These genes which
are required for viral virulence are novel, lacking similarity to any other known genes. ASFV genes involved with the regulation of apoptosis in virus infected cells (bcl-2 and iap homologs) and genes with functions involving immune evasion in the swine host (CD2 and IkB homologs) were identified and characterized. CD2 and IkB genes had not been previously described in any virus. This is the first example of a pathogen using of a CD2-like molecule in immune evasion. ASFV genes which affect macrophage host range were also identified and characterized. The thymidine kinase gene (TK) and a second gene 9-GL are required for efficient replication of virus in infected cells; deletion of either gene resulted in a 99% to 99.9% reduction in viral growth and virulence attenuation. 9-GL is a novel gene whose function in virus replication is unknown. A novel macrophage growth-associated determinant which includes multiple members of the multigene family 360 genes was identified in the left
variable region of the ASFV genome. These genes are novel lacking homology to other known genes. A novel viral virulence determinant which includes multiple members of the MGF 530 genes has been identified in the left variable region of the ASFV genome using in vivo marker rescue. These genes will be an important target for engineering live-attenuated ASF vaccine viruses. The genomes of Fowlpox virus, Marek's disease virus, Turkey herpesvirus and Lumpy Skin Disease Virus were completely sequenced and analyzed. An understanding of the genetic basis of viral virulence and host range will permit the engineering of novel vaccine viruses and expression vectors with enhanced efficacy and greater versatility. Complete genome sequencing of five selected ASFV isolates was completed. Preliminary analysis indicates more diversity in ASF viruses than previously recognized and the presence of at least three viral genotypes. Comparative ASFV genomics provides basic genetic information necessary
for developing new genetics-based approaches for rapid strain identification and typing. Strategies developed for ASF will likely have broad applicability for identification and characterization of other genetically complex threat-agent pathogens. Overall, the identification of significant virulence and host range genes provides a rational basis for development of genetically engineered attenuated or non-replicating (in the swine host) ASFV viruses for use as vaccines. Further, an understanding of how these novel genes function in the swine host will contribute to our overall understanding of viral virulence/host range, viral persistence and pathogen-vector-host interactions; information that may have broad impact on swine infectious disease in general. Characterized latent infection with ASFV - Determined ASFV establishes a true latent infection in peripheral blood monocytes/macrophages, bone marrow and tonsil all recovered pigs, one in which viral replication does not occur.
Knowledge of the existence of ASFV carrier animals is of critical importance for developing disease control programs in disease endemic regions. Characterized ASFV-tick host interactions - Our identification of a large number of highly conserved ASFV genes with no apparent role in the swine host suggested these genes may be associated with aspects of infection in the tick host. Colonies of Ornithodoros ticks for use in investigating virus-tick interactions were established at PIADC from wild ticks collected in South Africa. Detailed pathogenesis studies of ASFV infection in the tick host revealed important aspects of this virus-host interaction. Recombinant ASF viruses containing deletions of putative tick host range genes were constructed and examined in the ticks. The viral TK gene has recently been shown to be necessary for viral replication in midgut epithelial cells. And, a second tick host range determinant has been mapped to the left variable region of the ASFV genome.
Successful identification of tick host range genes will permit the design of live attenuated ASFV vaccine viruses that are incapable of infecting or being transmitted by ticks. The functional characterization of these genes, will also likely contribute to our understanding of pathogen-arthropod host interactions in general. Functional genomics of cytoplasmic DNA viruses - A viral genomics approach was used to identify putative ASFV arthropod host range genes. Two cytoplasmic DNA viruses representing distinct virus families, Chlorella virus PBCV-1 (330 Kbp) and the Melanoplus Sanguinipes Entomopoxvirus (236 Kbp). The Chlorella virus genome represents the largest viral genome sequenced to date. These data provided information on the primary structure of all encoded genes, their organization, potential regulatory sequences and their evolutionary relationship to one another. In both viruses a number of novel and unexpected genes were identified. Sequence homologies with these
viruses have led to the identification of ASFV genes with putative functions in virus-arthropod host interactions. This work has led to ASFV being placed into its own virus family, the Asfarviridae, and it has provided the genetic underpinning for all future research aimed at understanding the complex biology and evolution of large cytoplasmic DNA viruses. Development of ASFV diagnostics and reagents - A PCR-based diagnostic assay for ASFV DNA, with a sensitivity equal to that of animal inoculation, was developed for use with blood and tissue samples. The assay is being used for research and diagnostic applications in the US, Spain, UK, South Africa, and China. Cloned genes, protein expression vectors, and both polyclonal and monoclonal antibody reagents have been developed and provided to research and diagnostic laboratories in the US, UK, Spain, South Africa, the Netherlands, and China.
6. What do you expect to accomplish, year by year, over the next 3 years?
Future research work in FY-2002-2004 will include the following: Specific MGF360/530 genes required for growth in macrophages will be identified. The minimal set of MGF360/530 genes necessary for macrophage host range (MHR) will be further characterized. Viral gene transcription, protein expression, protein localization in infected cells, and protein involvement in aspects of virus- cell interaction will be examined. Cellular transcriptional networks affected by these genes will be identified with DNA microarray analysis. Cellular proteins which physically interact with specific ASFV proteins will be identified using yeast two-hybrid assays. Together, these approaches will define viral gene function in MHR. Whole genome comparisons discussed above have identified both highly conserved and variable genes in terminal ASFV genomic regions, which are known to contain important virulence and host range genes. Through this analysis, we have identified additional candidate genes with
putative virulence and/or host range functions. To examine gene function, single gene deletion mutants of a pathogenic ASFV isolate will be constructed. Mutant viruses together with parental and/or revertant viruses will be examined for their ability to replicate in porcine macrophage cell cultures in vitro and for their virulence in pigs. Viruses will also be examined in the tick host for alterations in infectious dose, initial midgut infection, generalization of infection, tissue tropism, viral persistence and transmissions. Mutant viruses will be examined using DNA microarray analysis of both swine and tick expressed sequence tags to define cellular transcriptional networks affected by the viral gene. The identification of cellular pathways affected by the viral gene will suggest molecular mechanisms for these genes in viral virulence and host range.
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, 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)
This work has been presented to numerous groups representing other of Federal Departments and Agencies and Academic Institutions(1999, 2000, 2001). Work was featured in the Agricultural Research Magazine, "Cracking the Code of Marek's Disease" July 2001, p. 16-17.
Impacts
(N/A)
Publications
- Afonso, C.L., Tulman, E.R., Lu, Z., Zsak, L., Kutish, G.F., Rock, D.L. The genome of swinepox virus. Conference of Research Workers in Animal Diseases. 2000. Abstract No. 177.
- Afonso, C.L., Tulman, E.R., Lu, Z., Zsak, L., Rock, D.L., Kutish, G.F. The genome of Turkey Herpesvirus. Conference of Research Workers in Animal Diseases. 2000. Abstract No. 121P.
- Tulman, E.R., Afonso, C.L., Lu, Z., Zsak, L., Kutish, G.F., Rock, D.L. The genome of lumpy skin disease virus. Conference of Research Workers in Animal Diseases. 2000. Abstract No. 183.
- Tulman, E.R., Afonso, C.L., Lu, Z., Zsak, L., Kutish, G.F., Rock, D.L. The genome of lumpy skin disease virus. Journal of Virology. 2001. v. 75. p. 7122-7130.
- Afonso, C.L., Tulman, E.R., Lu, Z., Zsak, L., Rock, D.L., Kutish, G.F. The genome of Turkey Herpesvirus. Journal of Virology. 2001. v. 75. p. 971-978.
- Afonso, C.L., Tulman, E.R., Lu, Z., Balinsky, C.A., Moser, B.A., Becnel, J.J., Rock, D.L., Kutish, G.F. The genome of culex nigripalpus baculovirus. American Society for Virology. 2001. Abstract p. 65
- Zsak, L., Burrage, T.G., Zsak, A., Sur, J-H., Rock, D.L. African swine fever virus (ASFV) 9GL gene is required for efficient replication in midgut and for generalization of infection in ornithodoros porcinus ticks. Conference of Research Workers in Animal Diseases. 2000. Abstract No. 174.
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