Location: Foreign Animal Disease Research
Project Number: 8064-32000-063-008-I
Project Type: Interagency Reimbursable Agreement
Start Date: May 30, 2019
End Date: Sep 30, 2022
African Swine Fever Virus (ASFV) is the causative agent of African swine fever (ASF), a contagious viral disease in swine with increasing epidemiological importance worldwide. Currently the only promising vaccines have been live attenuated vaccines (LAV). Development of LAV have been largely based in the deletion of non-essential specific virus genes directly involved in virus virulence) in swine. Additional gene deletions in ASFV may be required to further attenuate current LAV increasing its safety as the current LAV have only been tested in experimental settings. Cross protection studies in ASFV have been very limited and have suggested that one vaccine strain will not protect against distant isolates of ASFV. Emerging viruses could require different or increased gene deletions. Therefore, determination of the essentiality of each gene in virus replication is of paramount importance in the identification of potential virus genetic determinant of virulence (VGDV) to be used in the development or improve safety of rationally designed LAVs. Specific objectives: 1. Design CRISPR/Cas9 plasmids and gRNA for gene deletions for all ORF in ASFV isolate Georgia. 2. Conduct isolation of ASFV viral DNA for full-length genome sequencing.
1. Design Clustered Regulatory Interspaced Short Palindrom Repeats ( CRISPR)/ ASSOCIATED WITH PROTEIN 9 (CAS9) Constructs: Because of the large number of constructs that will have to be synthesized targets will be split into two equal batches so that the first designed set can start synthesis before the second designed set. Below these will be called batch one and batch two. Synthesis of Batch One CRISPR/CAS9 Constructs: Test Batch One CRISPR/CAS9 Constructs for Delection Viablility: All constructs will be tested by infection/ transfection (I/T) in primary swine macrophages. Successful I/T will be monitored using fluorescence markers. The I/T will be passed one round to determine if virus was viable, which will be also monitored by fluorescence. Three consecutive independent rounds of replication will occur to characterize constructs as targeting non-essential virus genes. Synthesis of Batch Two CRISPR/CAS9 Constructs: Test Batch Two CRISPR/CAS9 Constructs for Deletion Viability. 2. Isolation of ASFV viral DNA for full-length genome sequencing: Test different commercial as well as in house formulated lysis buffers and DNA viral isolation protocols; determine percent of viral vs host NGS reads in different experimental conditions and; develop an optimize protocol for ASFV full length sequencing.