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United States Department of Agriculture

Agricultural Research Service

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Location: Foreign Animal Disease Research

2011 Annual Report

1a. Objectives (from AD-416)
The objectives of this project are: (1) to assess the role of different regions of Classical Swine Fever Virus (CSFV) and African Swine Fever Virus (ASFV) genomes in virus virulence in swine; and (2) to understand mechanisms of virus infection and persistence.

1b. Approach (from AD-416)
To fulfill objective 1, a set of genetically modified full-length cDNA copies of CSFV and a set of ASFV deletion mutants will be constructed from virulent wild-type viruses. Virulence of mutant viruses will be determined in swine relative to virulence of parental wild-type viruses. To fulfill objective 2, infections with wild-type and mutant CSFV and ASFV will be established in vivo and in relevant cells of swine origin in vitro. Persistently and acutely infected cells and recovered viruses will be analyzed by a combination of techniques including microscopy, fluorescence confocal microscopy, qPCR, sequencing and sequencing analysis. Isolated viruses will be characterized in vitro and in vivo relative to wild-type viruses. Identification and characterization of host and viral mediating disease will be an important goal in this project. Overall experimental manipulation of identified host-virus interactions may be used for developing novel tools for controlling virus infection in vivo.

3. Progress Report
We were able to identify the cellular protein IQGAP1 which plays a pivotal role as a regulator of cytoskeleton interaction within the CSFV Core protein. Sequence analysis identified residues within CSFV Core protein that maintain homology to regions within the matrix protein that mediate binding to IQGAP1. Alanine-substitution within Core regions I, II, III and IV identified residues that specifically mediate the Core-IQGAP1 interaction. Recombinant CSFV viruses harboring alanine substitutions at residues 207ATI209 (I), 210VVE212 (II), 213GVK215 (III), or 232GLYHN236 (IV) have defective growth in primary swine macrophage cultures. In vivo, substitutions of residues in areas I and III yielded viruses that were completely attenuated in swine. These data show that the interaction of Core with an integral component of cytoskeletal regulation plays a role in the CSFV cycle. In addition, we designed strategies and started to produce recombinant ASFV virus. The first target for deletion is the 9GL gene known to be involved in ASFV virulence. The virulent ASFV strain used in these studies is Georgia 2007, responsible for causing severe outbreaks of the disease in the Caucasus region and Russia since it was introduced accidentally into the Republic of Georgia in 2007. This approach will enable us to produce a recombinant virus that may confer protection against challenge with homologous wild-type Georgia 2007 virus. This strategy may have implication in the design of vaccines against that virus. This project was monitored through email and telephone exchange, as well as site visits to ARS, PIADC and the University of Connecticut (UConn). In addition, two UConn scientists were stationed at ARS, PIADC and conducted research on this project in FY 2011.

4. Accomplishments

Last Modified: 06/26/2017
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