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.
FlagT4 is a rationally designed live attenuated antigenically marked Classical Swine Fever Virus (CSFV) which harbors the capacity to differentiate infected from vaccinated animals (DIVA.) During FY 2012 FlagT4 was evaluated as a potential vaccine having a good minimal protective doses, lacking toxicity, but being genetically unstable, reverting to virulent phenotype when its genetic stability was analyzed using a reversion to virulence protocol. Changes in the genome of the modified FlagT4 virus were identified.
Conducted the following activities: (i) evaluated the contribution of each of the mutations discovered in the reversion to virulence, and (ii) designed and developed a derivative of FlagT4 containing genetic modification in the genome that would further stabilize the virus genome making mutations less feasible to be acquired during the serial passages on pigs. We were able to (i) determine the genetic changes responsible for the reversion to virulence of FlagT4; and (ii) carried out extensive genetic work on the genome of FlagT4 virus in order to genetically stabilize it. Four independent sets of site directed mutagenesis were performed altering genomic areas encoding for E1, E2, non structural proteins and 3’ UTR. This modified FlagT4 infectious clone will be further used to produce a new virus that will be assessed as a potential vaccine in swine.
No technologies were transferred in FY 2013. No publications were produced in FY 2013.