Location: Foreign Animal Disease Research
Project Number: 3022-32000-063-023-S
Project Type: Non-Assistance Cooperative Agreement
Start Date: Jul 1, 2025
End Date: Jun 30, 2027
Objective:
African swine fever (ASF) remains a significant threat to domestic pigs.It is currently prevalent in many parts of the world with a huge economic burden. The development of effective vaccines has been based in the production of recombinant attenuated viruses, a very complicated and time consuming methodology. Thus, there is a major need for better engineering tools for ASFV to generate modified strains to understand its biology and the development of safer vaccine candidate strains more rapidly. Recently, the Cooperator developed a synthetic genomics reverse genetics system for ASFV genotype IX strains to enable rapid combinatorial genome-wide modifications. The objective of this project is to develop a reverse genetic systems for the ASFV Georgia2010 (genotype II) and the virulent field isolate Asia Hybrid I/II (genotype I/II), the prevailing strains currently affecting the pig industry in Asia, Europe and Hispaniola. These two tools will greatly improve the ARS ability to produce recombinant derivative viruses from these two isolates facilitating the study of virus gene function and the development of safer and more efficacious live attenuated vaccines.
Approach:
The approach involves cloning individual overlapping fragments or “parts” that comprise the African swine fever virus (ASFV) genome which can be modified individually and in parallel and then assembled into full-length genomes from the wild-type and modified parts in a mix-and-match manner using yeast’s homologous recombination pathway. Live viruses are reconstituted from these recombinant genomes using a novel CRISPR/Cas9-inhibited self-helper virus, in effect enabling the rapid generation of combinatorial mutant strains. The methodological steps to set the synthetic genomics reverse genetics system for ASFV genotype II and I/II will include (i). First, the overlapping parts of the virus genome will be cloned using transformation-associated recombination (TAR) in yeast. Then, (ii) these parts will be modified and then used to assemble full-length genomes. (iii) Host cell lines will be generated that express Cas9 together with guide RNAs that target essential genes to inhibit replication of the wild-type genotype II or I/II so that they can serve as the self-helper viruses. (iv) Live virus reconstitution will be performed in the host cells using the self-helper virus system previously developed by the Cooperator. (iv) Besides the development of the parental virus of both genotypes (I and I/II). Several recombinant chimeric viruses will be produced.