Location: Foreign Animal Disease Research2013 Annual Report
1a. Objectives (from AD-416):
Classical Swine Fever (CSF) is a highly contagious viral disease of swine. Controlling and eliminating the disease is dependant upon identification of CSF viral mechanisms involved in induction of disease generalization of infection, tissue tropism, host range, transmission, immunogenicity and strain virulence. Better understanding of these determinants will provide identification tools, vaccines and/or anti-virals. These determinants are linked to specific interactions between viral proteins with host cell proteins upon infection. To further characterize the molecular basis of CSFV and host-cell interactions ARS, PIADC and the University of Connecticut will identify swine macrophage proteins interacting with structural and non-structural CSFV proteins during infection. The effect(s) of these interactions on virulence, generalization of infection, tissue tropism, virus transmission, immunogenicity and induction of protection will be determined in swine.
1b. Approach (from AD-416):
1. A Yeast Two-Hybrid screening system will be used to identify cellular proteins, using a porcine macrophage expression cDNA library, currently available at ARS, PIADC, that interact with each of the Classical Swine Fever Virus proteins. 2. The University of Connecticut will conduct fine mapping of interacting host and viral proteins to identify specific binding residues or motifs mediating the interaction. 3. Mutant viruses harboring genetically modified binding motifs will be constructed and characterized in vitro and in vivo at ARS, PIADC. Particular emphasis will be placed on establishing the ability of mutant viruses to cause disease and to induce protection in swine, relative to parental virulent virus.
3. Progress Report:
Explored the role of a putative domain within structural CSFV protein E2 which appears to be, as predicted by bioinformatics analysis, a fusion peptide (FP). FPs are critical domains within viral structural proteins that mediates the interaction between virus and cell membranes during the process of entry/exit. CSFV E2 is a glycoprotein inserted into the outer envelope of CSFV virion which plays several roles during infection; from mediating attachment and entry to the host cell, to being the main target for the host immune response against CSFV. Introduced specially designed mutations that partially substitute some of the 15 amino acid residues composing the E2 putative FP. Substitutions within E2 were introduced in the context of a full-length DNA copy of CSFV strain Brescia (pBIC) to obtain mutant viruses lacking portions of the FP of E2. Some of these substitutions affected in a differential manner the production of E2 protein in infected cells as well as the yield of viral progeny. No technologies were transferred or publications produced in FY 2013.