CHARACTERIZATION OF GENETIC DETERMINANTS OF VIRULENCE IN AFRICAN SWINE FEVER VIRUS
Foreign Animal Disease Research
2013 Annual Report
1a.Objectives (from AD-416):
The objectives of this research project are (1) to assess the role of different regions of African Swine Fever Virus (ASFV) genomes in virus virulence in swine and (2) to understand the mechanisms of virus infection and persistence.
1b.Approach (from AD-416):
To fulfill objective 1, a set of genetically modified ASFV deletion mutants will be constructed from virulent wild-type viruses. Virulence of mutant viruses will be determined in swine relative to virulence of parental wild-type viruses.
To fulfill objective 2, infections with wild-type and mutant ASFV will be established in vivo and in relevant cells of swine origin in vitro. Persistently and acutely infected cells and recovered viruses will be analyzed by a combination of techniques including microscopy, fluorescence confocal microscopy, qPCR, sequencing and sequencing analysis. Isolated viruses will be characterized in vitro and in vivo relative to wild-type viruses.
Identification and characterization of host and viral mediating disease will be an important goal in this project. Overall experimental manipulation of identified host-virus interactions may be used for developing novel tools for controlling virus infection in vivo.
Previously, ARS, PIADC and UConn have designed strategies and produced recombinant African Swine Fever Viruses (ASFV) based on virulent isolate ASFV Georgia 2007. During FY 2013, efforts were directed to design and produce a set of deletion mutants named delta 9GL, delta CD2, delta MGF360-530, delta TK, delta EP152R, delta UK, and delta NL. Recombination plasmids carrying a reporter gene (beta GUS) flanked by genomic areas adjacent to the targeted gene were designed and produced to obtain mutant viruses by homologous recombination. These DNA constructs were used together with parental wild type virus, Georgia 2007, in transfection-infection of primary swine macrophages with the purpose of obtaining recombinant viruses. Recombinant viruses were recovered from blue colored plaques and puri¿ed to homogeneity by plaque assays. Recombination was verified by PCR followed by sequencing. Recovered mutant viruses are being characterized for growth in primary swine macrophages and for virulence in swine. It is expected that this virus will have an attenuated phenotype in swine.