Production of Recombinant African Swine Fever Viruses: Speeding Up the Process

Figure 1. Schematic diagram depicting the generation of recombinant African swine fever (ASFV) isolate lacking genes EP153R and EP402R using homologous recombination and single cell sorting. Homologous recombination between transfer plasmid p∆EP153R∆EP402R↔VP30mNeonGreen (A) and parental Benin∆DP148R (B) removed genes EP153R and EP402R (genome position: 67,051 to 68,775) from parental virus and replaced with a VP30mNeonGreen reporter gene cassette from the transfer plasmid to generate Benin∆EP153R∆EP402R∆DP148R (C). Gene maps were copied from SnapGene® Viewer software (GSL Biotech, San Diego, CA, USA; available at snapgene.com) and modified on Adobe® Illustrator® CS6 (Adobe Systems Inc., San Jose, CA, USA).

Figure 2. Purification of recombinant Benin∆EP153R∆EP402R∆DP148R via single cell isolation using FACS. The biparametric flow cytometry profiles depicted include two phases. Phase 1—pre-sorting: Panel 1—SSC-A vs FSC-A for total cells; Panel 2—SSC-A vs. SSC-H to obtain singlets; Panel 3—blue BP 530/30 nm-A vs. violet BP 450/40 nm-A to exclude autofluorescence cells, prominently in porcine bone marrow cells (PBMs); Panel 4—blue BP 530/30 nm-A vs. yellow-green (YG) BP 582/15nm-A to capture the mNeonGreen (mNG) positive cell subpopulation. Phase 2—post-sorting: Panel 5—total cells that were single cell sorted into 96-well plate containing purified PBMs; Panel 6—each dot represents an individual mNG positive cell that was sorted. The flow cytometry profiles are shown for (A) after the homologous recombination event in WSL-R cells; (B) second round of single cell sorting of recombinant ASFV-infected PBMs from a single mNG positive well, monitored under a fluorescent microscope; (C) third round of single cell isolation of a single mNG positive well, similar to (B), except the gating for mNG positive subpopulation was more stringent to capture the brighter selection of cells for single cell…

Figure 3. Representative PCR analyses of recombinant Benin∆EP153R∆EP402R∆DP148R showing the successful deletions of genes EP153R and EP402R. The extracted genomic viral DNA was subjected to PCR amplification and analysed by electrophoresis on 1.5% agarose gels. Panel depicts results from amplification of genes (A) DP148R (307 bp); (B) EP153R (297 bp); (C) EP402R (603 bp), (D) GUS (907 bp) and (E) mNeonGreen (634 bp). Lanes numbered 1 show analysis of samples from individual wells of purified Benin∆EP153R∆EP402R∆DP148R compared to (2) extraction negative control; (3) cell lysate control; (4) PCR negative control; (5) Benin∆DP148R control; (6) Benin∆EP402R control and (7) wildtype Benin 97/1 control.

Figure 4. Primary bone marrow cells were infected with the produced recombinant ASFV Benin∆EP153R∆EP402R∆DP148R at MOI of 1. (A) The images were taken 5 days post-infection, showing the bright field image of purified PBMs, the mNeonGreen expressed by Benin∆EP153R∆EP402R∆DP148R—infected cells (green) and a merged image overlaying total purified PBMs with the infected PBMs. Magnifications are indicated in the images (100×). Images were taken using the EXi Blue fluorescence microscopy camera and QCapture Pro Version 7 (QImaging, Tucson, AZ, USA). (B) The same recombinant ASFV-infected PBMs were analysed by flow cytometry, and the biparametric profiles leading to gating of mNeonGreen positive cells are depicted. Gating strategy to identify infected cells; Panel 1: SSC-A vs FSC-A for total cells; Panel 2: SSC-A vs. SSC-H to obtain singlets; Panel 3: blue BP 530/30 nm-A vs. violet BP 450/40 nm-A to exclude autofluorescence cells, finally blue BP 530/30 nm-A vs. Yellow-Green (YG) BP 582/15 nm-A (Panel 4) to capture the mNeonGreen (mNG) positive cell subpopulation.