Attempts at the Development of a Recombinant African Swine Fever Virus Strain with Abrogated EP402R, 9GL, and A238L Gene Structure using the CRISPR/Cas9 System

Abstract

Introduction: African swine fever (ASF) is a pressing economic problem in a number of Eastern European countries. It has also depleted the Chinese sow population by 50%. Managing the disease relies on culling infected pigs or hunting wild boars as sanitary zone creation. The constraints on the development of an efficient vaccine are mainly the virus' mechanisms of host immune response evasion. The study aimed to adapt a field ASFV strain to established cell lines and to construct recombinant African swine fever virus (ASFV) strain.

Material and methods: The host immune response modulation genes A238L, EP402R, and 9GL were deleted using the clustered regularly interspaced short palindromic repeats/caspase 9 (CRISPR/Cas9) mutagenesis system. A representative virus isolate (Pol18/28298/Out111) from Poland was isolated in porcine primary pulmonary alveolar macrophage (PPAM) cells. Adaptation of the virus to a few established cell lines was attempted. The plasmids encoding CRISPR/Cas9 genes along with gRNA complementary to the target sequences were designed, synthesised, and transfected into ASFV-infected PPAM cells.

Results: The reconstituted virus showed similar kinetics of replication in comparison to the parent virus isolate.

Conclusion: Taking into account the usefulness of the developed CRISPR/Cas9 system it has been shown that modification of the A238L, EP402R, and 9GL genes might occur with low frequency, resulting in difficulties in separation of various virus populations.

Keywords: 9GL; A238L; African swine fever; CRISPR/Cas9; EP402R.

Fig. 1

Schematic representation of the selection of the suitable transfection kit and target cells

Fig. 2

Vero cells transfected with constructed CRISPR/Cas9 plasmid. In order to confirm successful transfection, a puromycin selection was applied, which led to an increased rate of cell death during the first three days post transfection, and later the cell culture started to grow in antibiotic supplemented medium (magnification 200 ×)

Fig. 3

Agarose gel (2%) showing the results of the conventional PCR to amplify whole sequences of targeted genes. Gene names at the top represent the amplified region. Numbers below gene names correspond to Table 5 numbers in brackets, 1000/500 – band size markers

Fig. 4

In vitro replication kinetics of the six generated deletion mutant (A – 9GL1Δ; B – 9GL2Δ; C – A238L1Δ; D – A238L2Δ; E – EP402R1Δ; F – EP402R2Δ) and parent ASFV/Pol18/28298/O111 isolates. PPAM cell cultures were infected (MOI 0.1) with both strains, and subsequently virus titres were estimated daily over 96 h post infection. Data represent means and standard deviations from three independent experiments. The sensitivity of virus detection was 1.8 HAD₅₀/mL