2012 Annual Report
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.
This collaborative research agreement seeks to develop a live-attenuated Classical Swine Fever (CSF) virus which possesses DIVA (differentiation of infected animals from vaccinated animals) capabilities.
During FY 2012, FlagT4 virus was found to revert to virulence when its genetic stability was analyzed using a reversion to virulence protocol. This protocol include a five time serial passages of the vaccine virus in groups of naïve pigs. FlagT4 induced some CSFV symptoms in animals receiving the last serial passage. Changes in the genome of the virus during this serial passage were analyzed and specific mutations were identified. Specific studies are being currently conducted in order to evaluate the contribution of each of the mutations discovered in the reversion to virulence of FlagT4 virus after the passages. Additionally, genetic modification in the genome of FlagT4 virus are being currently incorporated in order to further stabilize the genomic structure of the virus making mutations less feasible to be acquired during the serial passages on pigs.
In addition we started the evaluation of the use of purified CSFV structural proteins as subunit experimental vaccines. The novelty of the project consists in the molecular modification of proteins E0 and E2 in order to increase their immunogenicity (addition of bacterial flagelline, or the expression of the monomers in a tretrameric arrangement). Genetic constructs for each of the product were assembled in a baculovirus recombinant plasmid. The corresponding recombinant baculoviruses were produced. Recombinant proteins were expressed as fusion products which were further purified by affinity chromatography.
The deliverables of this collaborative agreement compliment ARS in-house research project 1940-32000-056-00D, Countermeasures to Control Foreign Animal Diseases of Swine, objective 1, Develop intervention strategies to control and eradicate CSF virus by determining immune mechanisms mediating early protection and its application in blocking infection and preventing transmission and by discovering effective CSF vaccine and diagnostic platforms specifically designed for disease control and eradication.