2012 Annual Report
1a.Objectives (from AD-416):
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.
2. Develop intervention strategies to control ASF virus by identifying virus-host determinants of virulence and transmission and by developing technologies to enable the development of ASF vaccines that are efficacious against the most prevalent ASF strains.
1b.Approach (from AD-416):
To develop strategies to control CSF virus and further vaccine and diagnostic platforms, improvements and further assessment will be conducted on ARS, PIADC previously developed live attenuated candidate marker vaccine strain (FlagT4v); inclusive of minimal protective dose response, toxicity, antibody response and genetic stability assessments. Studies will focus on protective innate immune mechanisms induced in swine after vaccination with live attenuated CSFV vaccine strains. Evaluation of native and modified forms of CSFV envelope proteins to analyze their capacity to induce rapid protective immune responses against infection with CSFV as a preliminary step in development of fast acting subunit marker vaccines.
Development of intervention strategies to control ASF virus will be done by identifying virus-host determinants of virulence and transmission and by developing technologies to enable the development of ASF vaccines that are efficacious against the most prevalent ASF strains. In vivo experimental models to study ASFV pathogenesis and host immune response in swine will be developed. Studies will focus on minimal lethal dose percentages, and early pathogenesis model with a virulent ASFV. The critical pathogenic and immunological mechanisms leading to protection against ASF will be identified. Technologies will be developed to enable the production of live attenuated vaccines for ASF.
FlagT4 is a rationally designed live attenuated Classical Swine Fever Virus (CSFV) strain. FlagT4 contains a double genetic determinant which produces attenuation and a positive and a negative antigenic marker which allow the differentiation between animals immunized (DIVA) with FlagT4 from those infected with a CSF wild type virus. During FY 2012 FlagT4 was evaluated as a potential vaccine, which included three different protocols performed in naïve pigs: (i) Determination of its Minimal Protective Doses (MPD); (ii) Determination of its toxicity, and (iii) Determination of genetic its stability. MPD of FlagT4 resulted to be 104 TCID, compatible with industrial requirements. FlagT4 was shown to lack toxicity. FlagT4 virus was found to revert to virulence when its genetic stability was analyzed using a reversion to virulence protocol. 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. An evaluation is being conducted to analyze the contribution of each of the mutations discovered in the reversion to virulence of FlagT4 virus after the passages. 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.
Advances were made in the development of a reliable DIVA test based in the lack of immune dominant highly conserved CSFV epitope recognized by monoclonal antibody 303. A non competitive ELISA was developed where 90% of the sera from animals immunized with FlagT4 could be serologically differentiated from animals infected with wild type CSFV strain.
The evaluation of the use of purified CSFV structural proteins as subunit experimental vaccines was initiated in FY 2012. The novelty of the project lies in the molecular modification of proteins Erns and E2 in order to increase their immunogenicity. 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. Immunization trials will be performed in FY 2013 with different protein doses and evaluated at different time post immunization.
In addition, the differences in the innate immune response of pigs infected with attenuated and virulent strains of CSFV were analyzed. Tonsils of animals infected with FlagT4, virulent Brescia virus or FlagT4 followed by over-infection with Brescia were analyzed to identify cells producing different host pro-inflammatory chemical mediators (PICM). Out of more than 40 PICM tested, we identified 8 that were significantly more expressed in FlagT4 infected as being produced in cultures treated with Imiquimod R837, a substance we showed to efficiently impede CSFV replication in swine macrophage cultures. Currently, we are analyzing the effect of these 8 PICM in virus replication first in swine marophage cultures and, if successful, in swine.
The discovery of the function of a viral protein (P7) in the virus replication cycle. As part of continuing efforts to develop better vaccines against Classical Swine Fever Virus (CSFV), the functional role of viral protein p7 was analyzed. ARS researchers at Greenport, New York have demonstrated that p7 belongs to a group of proteins called viroporines. Viroporines are viral proteins that have the role of producing pores in the interal membranes of the cell where the virus replicates. We demonstrated that p7 is essential for virus replication and growth in cell culture. A recombinat CSFV was created using mutations in the p7 area and showed that some of these mutations produced attenuation of virus virulence. This information can be useful in designing countermeasures not only against CSF but also other diseases caused by pestiviruses.
Gavrilov, B.K., Rogers, K., Fernandez Sainz, I.J., Holinka-Patterson, L.G., Borca, M.V., Risatti, G.R. 2011. Effects of glycosylation on antigenicity and immunogenicity of classical swine fever virus envelope proteins. Virology. 420:135-145.
Gladue, D.P., Holinka-Patterson, L.G., Largo, E., Fernandez Sainz, I.J., O'Donnell, V., Baker-Branstetter, R., Lu, Z., Ambroggio, X., Risatti, G.R., Nieva, J.L., Borca, M.V. 2012. Classical Swine Fever Virus p7 protein is a viroporin involved in virulence in swine. Journal of Virology. 86(12):6778-6791.