Location: Foreign Animal Disease Research
Project Number: 8064-32000-060-06-S
Project Type: Non-Assistance Cooperative Agreement
Start Date: Aug 1, 2015
End Date: Feb 28, 2019
This research project seeks to develop recombinant attenuated viruses of African Swine Fever Virus (ASFV) through genetic manipulation of paretal African Swine Fever Virus-Georgia virus (ASFV-G virus), a field virus of the genotype II currently circulating and causing disease in Western Asia/Eastern Europe. The obtained recombinant viruses will then be used to test their protective efficacy against ASFV challenge and to study the molecular basis of ASF disease in swine with particulalrly emphasis on the host immune response to the virus. Multiple gene deletions will be introduced into ASFV to allow for the additive effect of multiple gene deletions which each individually cause some degree of attenutation. Structural-functional studies comparing predicted ASFV type ORFs to protein databases containing information regarding protein motifs, protein signatures, and protein domains to infer putative protein functions will be conducted and their effects analyzed. Transcriptional profiles of macrophages (in vitro) and of swine (in vivo) upon infection with recombinant viruses and virulent parental virus will be generated for determining host-pathways involved in the response to infection. Information suggests that specific viral determinants are responsible for phenotypic differences in ASFV virulence, replication in swine macrophages and the differential induction of cytokine expression, however additional information is needed. This research project is aimed at defining the roles that viral proteins have in modulating ASFV virulence, attenuation, and induction of protection and to understand the host response to infection to elucidate mechanisms of disease manifestation. Specific objectives include: 1. Preliminary assessment of selected viral proteins was conducted by UConn and ARS who identifyied ASFV genes which are involved in virus virulence and could be targeted for ASFV attenuation. The parental strain ASFV-G was developed to yield a recombinant virus containing a single gene deletions of the K196R gene while a second virus was generated that harbored a single gene mutation of B119L gene. Further development will include the generation of a second generation of recombinant mutants viruses where double gene deletions or a combination of single and multiple gene deletions such as members of the multigene families (MGF) 360 and 505 will be developed from ASFV-G to yield a double/multiple deletion mutants with increased attenuation phenotype in swine being potentially safer if used as live attenuated vaccine. 2. An evaluation of the residual virulence of second generation ASFV-G viruses in swine. 3. Generation of the host transcriptional profiles in vitro and in vivo upon infection of macrophages or swine with single mutant viruses, double/multiple mutant viruses and parental virulent viruses. 4. Assessment of induction of prtective immunity in swine against homologous challenge using second generation ASFV-G viruses. 5. Assessment of induction of protective immuinity in swine against heterologous challenge using second generation ASFV-G viruses.
1. Second generation of recombinant ASFV mutants viruses will be generated by homologous recombination using single gene deletion mutants as targets for genetic modifications. For instance, attenuated recombinant virus delta 9GL (B119L gene) will be used as template to delete the K196R gene (thymidine kinase). 2. Commercial breed swine that are susceptible to ASF will be experimentally challenged with second generation ASFV-G viruses, single gene/s ASFV mutants and wild type virus to assess the degree of attenuation. Induction of disease, as well as quantification of viral loads in tonsils, blood, nasal cavity will be measured. 3. This task is aimed to characterize by means of functional genomics the response of swine macrophages and pigs to infection with second generation ASFV-G viruses, single gene/s ASFV mutants and wild type virus. RNA Seq and/or qPCR will be used to describe the transcriptional profiles of infected and non-infected cells/pigs. 4. Commercial breed swine will be exposed to attenuated recombinant viruses and then challenged with parental ASFV-G (homologous challenge) to assess protective efficacy. The presence of ASFV antibodies and INF-gamma production will be assessed in exposed-challenged animals for establishing correlates of protection. 5. Commercial breed swine will be exposed to attenuated recombinant viruses that conferred protection against the parental virus ASFV-G (homologous challenge) and then challenged with heterologous ASFV. Heterologous viruses defined as those currently circulating in East Africa, causing severe ASF outbreaks, and genetically differing from ASFV-G at least 30% at whole genome level. The presence of ASFV antibodies and INF-gamma production will be assessed in exposed-challenged animals for establishing correlates of protection.