Location: Foreign Animal Disease Research
Project Number: 3022-32000-063-035-S
Project Type: Non-Assistance Cooperative Agreement
Start Date: Sep 1, 2022
End Date: Aug 31, 2025
Objective:
There is a need to understand the mechanisms producing genetic diversity among field isolates of African Swine Fever Virus (ASFV). The main objective of this project is to understand the possible role of specific ASFV genes in the process of generation genetic diversity among field isolates of the virus. The results of this project will contribute to the understanding of virus evolution in the field and its potential impact in the emergence of isolates with altered patterns of virulence and antigenicity. This knowledge will contribute to meeting some of the milestones and deliverables associated with within the parental project.
This project will have three sub-objectives (i) Development of mutant viruses. (ii) Characterization of mutant viruses in vitro. (iii) Analysis of mutant viruses during infection in pigs.
Approach:
African Swine Fever Virus is a highly virulent virus affecting swine which has had a devastating impact on the global swine industry. ARS, PIADC and the Friedrich Loeffler Institute (FLI) will conduct experimental studies investigating the role of specific ASFV genes in the generation of genetic diversity. These activities will include the development, by ARS, of recombinant ASFV harboring deletions or modifications of virus genes already know to be involved in the process of virus replication. In vitro characterization of the recombinant virus and experimental study of them in pigs to be carried out at the cooperator’s facility. Recombinant viruses will be transferred from ARS to the cooperator’s facility at Riems island, Greifswald, Germany.
Initially, the project will focus in the role of the ASFV O174L gene in the generation of genome diversity in ASFV. Analysis of the genome sequences of several Polish ASFV isolates revealed specific nucleotide sequence variation within the O174L gene, which encodes the DNA polymerase beta-like protein.
(i) Recombinant ASF (rASFV) viruses will be produced using the ASFV Georgia 2010 strain as template where the O174L gene will be either completely or partially deleted, or the putative active site modified (rASFV-O174L). These recombinant viruses will be later used to characterize the functionality of the O174L gene in experiment in vitro and in vivo.
(ii) The rASFV-O174L will be evaluated in terms of genetic variability present in their virus progeny, under controlled condition, in susceptible cell cultures. Next Generation Sequence (NGS) and differential PCR will be used to analyze the spectrum, frequency and characteristics of mutated subpopulations obtained in comparison with the parental virus.
(iii) In addition, the rASFV-O174L will be evaluated during experimental infection in domestic pigs. The variability of the virus progeny will be, again, evaluated and compare with the that of the parental virus as described for the in vitro assessment. The level of virulence of the rASFV-O174L will be also assessed considering kinetics and severity of the clinical signs, lethality, as well as virus replication and organ distribution during the animal infection.
