Characterization of Genetic Determinants of Virulence in Csf and Asf
Foreign Animal Disease Research
2013 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.
Amendment I; objective 2: To genetically modify CSFV and ASFV respectively that will render viruses suitable for their use as modified vaccines.
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 parental virulent viruses. Virulence of mutant viruses will be determined in swine relative to virulence of parental viruses. We expect that introduced changes will render viruses phenotypically different from virulent parental viruses.
Relevant cellular signaling pathways altered by CSFV and ASFV infections will be revealed by further characterizing recombinant and parental viruses cycles in susceptible swine cells (i.e. primary swine macrophage cultures). The expression of relevant immunomodulatory genes will be assessed by qPCR. Identification of host cellular proteins interacting with viral proteins involved with virus virulence will be identified by means of the yeast two hybrid system. Colocalization of expressed virus proteins and interacting host proteins within infected macrophages will be performed by confocal microscopy.
To fulfill objective 2, infections with wild-type and highly attenuated mutant CSFV and ASFV will be established in vivo and animals will be challenged with wild type viruses to assess the protective efficacy of recombinant viruses in exposed swine. Identification and characterization of host and viral proteins 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.
Previously, we have designed strategies and produced recombinant African Swine Fever Viruses (ASFV) based on virulent isolate ASFV Georgia 2007. During FY 2013, we have identified, using comparative and analytic genomic techniques, several previously uncharacterized ASFV proteins. Those proteins were selected because they present homology to other viral proteins that have been shown to have important roles in virus pathogenesis or virulence. A particular protein, CLP, was identified because it harbors several motifs similar to poxvirus proteins that modulate complement cascade and that has been critical in virus virulence. We determined that CLP is critical for ASFV replication, since deletion of CLP gene is a lethal mutation. Recombinant ASFV having tagged the CLP protein, were developed to study the pattern of CLP expression. Yeast two hybrid methodology was used to identify swine host proteins interacting with CLP during infection. Furthermore, the areas of CLP being recognized by the host proteins were identified and ASF having mutated forms of CLP will be important to study the role of CLP in virus virulence.
No technologies have been transferred or publications produced during FY 2013.