1a.Objectives (from AD-416)
Classical Swine Fever Virus (CSFV) is a highly contagious disease affecting swine world-wide. CSFV control methods include prophylactic vaccination or eradication in the event of an outbreak. Disease-free countries do not allow the use of currently available live attenuated virues (LAVs) as tools to control disease. Use of current LAV also does not allow for the differentiation of infected animals from vaccinated animals (DIVA) after application. ARS, PIADC has developed an experimental LAV marker vaccine strain (FlagT4) which serves as a positive marker and produces complete protection against CSFV strain Brescia.
The objectives of this project are 1. Produce and evaluate FlagT4 master seed stock and determine efficacy and minimal dose response. 2. Develop and optimize a rapid molecular test to differentiate between FlatT4 and wild type viruses and develop companion serological and genetic DIVA tests. 3. Identify cellular proteins that interact with CSFV proteins and construct genetically modified viruses lacking this interaction. These viruses will be evaluated for their ability to induce disease and induce protection in swine against CSFV.
1b.Approach (from AD-416)
Objective 1: FlagT4 master seed stock will be produced and evaluated for revision to virulence in swine. Safety data will be developed for use of the vaccine off-PIAC. Efficacy testing and minimal dose response challenge testing will be conducted. Determine the efficacy and minimal protective dose of FlagT4 master seed stock in challenge studies.
Objective 2: Develop and optimize a rapid molecular test (RT-PCR) to differentiate FlagT4 vaccinated from wild-type viruses. Develop serological and genetic companion tests compatible with FlagT4.
Objective 3: Identify cellular proteins that physically interact with CSFV proteins. Determine the residues in the viral proteins that are important for interaction with cellular proteins. Construct and characterize mutant viruses harboring genetically modified binding motifs. Viruses presenting complete attenuation will be assessed as potential vaccine strains and bioprophylactic.
1. A preliminary assessment of FlagT4 virus as a vaccine was conducted. FlagT4 virus has been developed at ARS, PIADC and consists in a rationally designed antigenically marker vaccine. In house preliminary experiments performed in the past demonstrated that at a dose of 105 tissue culture infectious dose (TCID) TCID50/animal, vaccinated animals were protected against the challenge since the second day post vaccination. The assessment included the determination of the minimal protective dose (MPD 50%), the presence of shedding virus and the test of virus toxicity. Samples from these experiments are still being processed in the laboratory to detect presence of virus. In general FlagT4v demonstrated to have its MPD50 between 102 and 103 (TCID50%). FlagT4 did not show any degree of toxicity when inoculated at high doses either at the site of inoculation or at the general level.