2012 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.
During FY 2012, efforts were made to understand the role of Classical Swine Fever Virus (CSFV) p7 protein in virus virulence in swine. Using bioinformatics, it was determined that the nonstructural protein p7 is a small highly hydrophobic polypeptide. The protein contains two hydrophobic stretches of amino acids interrupted by a short charged segment that are predicted to form trans-membrane helices and a cytosolic loop, respectively.
Using reverse genetics, partial in-frame deletions were introduced into p7 protein. These mutations turned out to be deleterious for virus growth, demonstrating that CSFV p7 function is critical for virus production in cell cultures.
A panel of recombinant mutant CSFVs was then created. Sequential stretches of p7 amino acids were substituted by alanine residues through the entire span of the protein in the genetic backbone of CSFV strain Brescia. The constructed recombinant viruses allowed the identification of the regions within p7 that are critical for virus production in vitro.
In vivo, some of these viruses were partially or completely attenuated in swine relative to the highly virulent parental CSFV Brescia strain, indicating a significant role of p7 in CSFV virulence.
Structure-function analyses in model membranes emulating the endoplasmic reticulum lipid composition confirmed that CSFV p7 is a pore-forming protein, and that pore-forming activity resides in the C-terminal transmembrane helix. Therefore, p7 is a viroporin which is clearly involved in the process of CSFV virulence in swine.
This work lead to a peer-reviewed publication: Gladue, D. P., L. G. Holinka, E. Largo, I. Fernandez Sainz, C. Carrillo, V. O'Donnell, R. Baker-Branstetter, Z. Lu, X. Ambroggio, G. R. Risatti, J. L. Nieva, and M. V. Borca. (2012). Classical Swine Fever Virus p7 Protein Is a Viroporin Involved in Virulence in Swine. Journal of Virology 86:6778-91.
In addition, we designed strategies and started to produce recombinant African Swine Fever Virus (ASFV). The first target for deletion has been 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.