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2018 Annual Report
Objectives
1. Develop intervention strategies to control and eradicate Classical Swine Fever (CSF), including determining immune mechanisms mediating early protection and its application in blocking infection and preventing transmission, and discovering effective CSF vaccine platforms specifically designed for disease control and eradication. Immune mechanisms mediating early protection and its application in blocking infection and preventing transmission will be developed. Studies designed to develop effective CSF vaccine platforms specifically designed for disease control and eradication will be completed.
Sub-Objective 1.i: Determine immune mechanisms mediating early protection and its application in blocking infection and preventing transmission.
Sub-Objective 1.ii: Discover effective CSF vaccine platforms specifically designed for disease control and eradication.
2. Develop intervention strategies to control African Swine Fever (ASF) including identify functional genomics of virus-host determinants of virulence and transmission, determining host mechanisms of ASF immune protection, determining host mechanisms of ASF disease tolerance in wild suids. Additional efforts include the identification of effective ASF vaccine platforms specifically designed for disease control and eradication.
Sub-Objective 2.i: Identify novel virus-host genetic determinants of virulence by systematic screening of almost all previously uncharacterized virus genes.
Sub-Objective 2.ii: Discover effective ASF vaccine platforms specifically designed for disease control and eradication.
Approach
The development of intervention strategies to control Classical Swine Fever will based on research of live attenuated vaccines (LAV). Research will be aimed at determining the virological and immunological factors present in animals that are protected at early times post vaccination, emphasizing on expression profiles of pro-inflammatory chemical mediators (PCMs) produced the first few days after vaccination. The potential therapeutic effect of any PCMs identified will then be assessed. An evaluation of the second generation marker live attenuated vaccine (LAV) FlagT4Gv vaccine will be conducted focusing on toxicity, immunogenicity, protective effect and genetic stability. Efforts will be devoted to develop and optimize serological DIVA (to differentiate infected from vaccinated animals) tests to accompany the FlagT4G strain. Additional vaccine candidates and companion DIVA tests will also be assessed.
To develop strategies to control African Swine Fever Virus (ASFV) studies will be conducted to provide information about the mechanisms of viral replication, virus-host interaction and virulence in the natural host. This information will be used to identify genes that determine the viral virulence that could targeted for deletion of mutation in order to yield attenuated viral strains with potential as vaccine candidates. Identification of candidate target genes will be determined through in silico analysis and/or interaction with host proteins. Full characterization of selected genes will include their interaction with host proteins, production of recombinant ASFV to assess the protein functionality in vitro and virulence during infection in swine. It is anticipated that this research will lead to the identification of genes which may be modified or deleted to created attenuated virus strains for use in vaccine development. In addition to testing attenuated strains containing single gene deletions, strains containing two or more gene deletions/modifications will be produced and assessed to evaluate their ability to protect against homologous and heterologous virulent strains. Efforts will also include the development of a stable cell line capable of supporting ASFV growth for use in commercial vaccine production. As contingency to the LAV approach, experimental subunit vaccines will be tested for their ability to protect against homologous virulent ASFV. The vaccine antigens will be delivered using, a modified vaccinia Ankara virus (MVA) vector co-expressing the ASFV recombinant proteins. These vectors will be assessed in their efficiency of expressing the ASFV recombinant proteins and their immunogenicity and efficacy in protecting swine against challenge.
Progress Report
During FY 2018, limited progress was made in the development of intervention strategies to control and eradicate Classical Swine Fever (CSF) and African Swine Fever (ASF). The lack of availability of animal facilities at Plum Island has severely affected our ability to advance in any aspect of our research activity requiring animal studies. Therefore, we could not to perform an evaluation of the marker live attenuated virus (LAV) FlagT4Gv vaccine strain in swine. We have concentrated most of our efforts in laboratory work not requiring animal experimentation. We have developed a novel approach to identify and characterize regions of CSFV major structural glycoprotein E2 that specifically interact with swine host proteins. This methodology allowed us to fine map E2 amino acids residues interacting with 20 different swine proteins during virus replication cycle. This information allowed us to developed 10 recombinant CSFV mutants that have abolished their ability to interact with a specific host protein. These recombinant viruses will be used to understand the effect of each of these virus-host interactions on the mechanism of virulence during the infection in swine. This effort is expected to produce information regarding the binding site in CSFV E2 of approximately 25 host proteins. In addition, we have continued our investigation of the biological significance of CSFV nonstructural protein p7. Previous work in our laboratory defines p7 as a critical protein for virus replication and virus virulence in swine, playing a role as viroporine, a virus factor producing pores in the host cell membrane. During the last year, we discovered that viroporin activity is mediated by producing two different types of pores, which can be blocked by independent mechanisms. In addition, we have discovered that the interaction pf p7 with a host protein CALM-G is involved in the process of calcium mediated cell activation. Interestingly, we also discover that p7, itself or through its association with CALM-G, is able to modulate the intracellular calcium concentration. These studies are unique in terms of characterization of the biological role of p7 and are seminal in understanding viroporin function in several other virus models such as hepatitis C, dengue, etc.
In the area of ASFV, we developed a series of attenuated vaccine strains ASFVdelta9GL/deltaUK/deltaNL, ASFVdelta9GL/deltaUK/deltaMGF, ASFVdelta9GL/deltaUK/delta205 based on our previously developed ASFVdelta9GL/deltaUK vaccine strain in order to strength its genetic stability and safeness. These new strains were obtained by specifically deleting additional virus genes in the ASFVdelta9GL/deltaUK genome. The protective effectiveness and safety profile of these new vaccine candidates will be tested once the animal facility is again available at the Plum Island Animal Disease Center (PIADC). In addition, we continued the identification of virus genes which are important in virus virulence. Ten different genes, previously selected by functional genetics were further analyzed for their ability to interact with host genes, by yeast two-hybrid methodology, and choose to further research. We developed recombinant ASFV harboring deletions or modified forms of these genes. The analysis of the virulent phenotype of these recombinant viruses will be performed once the animal facility is again available at PIADC. In addition, we were the first to introduce the use of a powerful tool, CRISPR-Cas9 methodology, in the development of recombinant ASFV viruses. Using the CRISPR-Cas9 methodology, we increased efficiency of producing the desired recombinatory event over the standard technique used in our laboratory by 1,000 times. We continued our efforts to establish a cell line able to support ASFV replication as a possible substrate for vaccine production. So far, none of the tested cell lines fully supported virus replication. We have established contact with different laboratories around the world possessing diverse swine-derived cell lines as a possible source of cell substrates to be tested in the near future. In addition, our effort to develop an ASFV experimental subunit vaccine using poxviruses as vaccine vector, which is a modify vaccine Ankara and raccoon pox viruses, are halted until the animal facilities are again available at PIADC.
Accomplishments
1. Vaccines for African Swine Fever. African Swine Fever (ASF) is a devastating and highly lethal disease of pigs for which there are no effective vaccines. ARS scientists at the Plum Island Animal Disease Center in Orient, New York were the first to utilize the CRISPR-Cas9 metholology in the development of potential vaccine candidates against ASF. This tool is critical to the study of the function of specific genes and in the development of experimental ASF vaccines. The use of this methodology is at least 1,000 times more efficient than standard techniques for producing the desired genetic changes in the virus. This recently published work will have significant impact on the rapid development of vaccines against this devastating disease.
Review Publications
Borca, M.V., Holinka-Patterson, L.G., Berggren, K., Gladue, D.P. 2018. CRISPR-Cas9, a tool to efficiently increase the development of recombinant African swine fever viruses. Scientific Reports. 8(3154). https://doi.org/10.1038/s41598-018-21575-8.
Largo, E., Gladue, D.P., Aguilella, V.M., Alcaraz, A., Borca, M.V., Nieva, J.L. 2018. Mutation-induced changes of transmembrane pore size revealed by combined ion-channel conductance and single vesicle permeabilization analyses. Biochimica et Biophysica Acta. 1860:1015-1021. https://doi.org/10.1016/j.bbamem.2018.01.012.
Borca, M.V., O'Donnell, V., Holinka-Patterson, L.G., Ramirez, E., Vuono, E., Berggren, K., Alfano, M., Carey, L.B., Richt, J.A., Risatti, G.R., Gladue, D.P. 2018. The L83L ORF of African swine fever virus strain Georgia encodes for a non-essential gene that interacts with host protein IL-1ß. Virus Research. 249:116-123. https://doi.org/10.1016/j.virusres.2018.03.017.
Alonso, C., Borca, M.V., Dixon, L., Revilla, Y., Rodriguez, F., Escribano, J.M. 2018. ICTV Virus taxonomy profile: Asfarviridae. Journal of General Virology. 99:613-614. https://doi.org/10.1099/jgv.0.001049.
