Development Real-Time PCR Assays to Genetically Differentiate Vaccinated Pigs From Infected Pigs With the Eurasian Strain of African Swine Fever Virus

Lauro Velazquez-Salinas; Elizabeth Ramirez-Medina; Ayushi Rai; Sarah Pruitt; Elizabeth A Vuono; Nallely Espinoza; Douglas P Gladue; Manuel V Borca

doi:10.3389/fvets.2021.768869

Development Real-Time PCR Assays to Genetically Differentiate Vaccinated Pigs From Infected Pigs With the Eurasian Strain of African Swine Fever Virus

Front Vet Sci. 2021 Oct 27:8:768869. doi: 10.3389/fvets.2021.768869. eCollection 2021.

Authors

Lauro Velazquez-Salinas^{1

2}, Elizabeth Ramirez-Medina¹, Ayushi Rai^{1

3}, Sarah Pruitt¹, Elizabeth A Vuono^{1

4}, Nallely Espinoza¹, Douglas P Gladue¹, Manuel V Borca¹

Affiliations

¹ Agricultural Research Service, United States Department of Agriculture, Plum Island Animal Disease Center, Greenport, NY, United States.
² Department of Anatomy and Physiology, Kansas State University, Manhattan, KS, United States.
³ Oak Ridge Institute for Science and Education (ORISE), Oak Ridge, TN, United States.
⁴ Department of Pathobiology and Population Medicine, Mississippi State University, Mississippi, MS, United States.

Abstract

Currently, African swine fever virus (ASFV) represents one of the most important economic threats for the global pork industry. Recently, significant advances have been made in the development of potential vaccine candidates to protect pigs against this virus. We have previously developed attenuated vaccine candidates by deleting critical viral genes associated with virulence. Here, we present the development of the accompanying genetic tests to discriminate between infected and vaccinated animals (DIVA), a necessity during an ASFV vaccination campaign. We describe here the development of three independent real-time polymerase chain reaction (qPCR) assays that detect the presence of MGF-360-12L, UK, and I177L genes, which were previously deleted from the highly virulent Georgia strain of ASFV to produce the three recombinant live attenuated vaccine candidates. When compared with the diagnostic reference qPCR that detects the p72 gene, all assays demonstrated comparable levels of sensitivity, specificity, and efficiency of amplification to detect presence/absence of the ASFV Georgia 2007/1 strain (prototype virus of the Eurasian lineage) from a panel of blood samples from naïve, vaccinated, and infected pigs. Collectively, the results of this study demonstrate the potential of these real-time PCR assays to be used as genetic DIVA tests, supporting vaccination campaigns associated with the use of ASFV-ΔMGF, ASFV-G-Δ9GL/ΔUK, and ASFV-ΔI177L or cell culture adapted ASFV-ΔI177LΔLVR live attenuated vaccines in the field.

Keywords: ASF real time PCR; ASFV; genetic DIVA test; live attenuated vaccine; phylogenetics.