Fig 1.
Generation of clonal Aag2-derived cell lines originating from single cells.
(A) Brightfield microscopy image of heterogeneous Aag2 cell population consisting of multicellular ‘clusters’ (examples indicated by hashed lines throughout) and large rounded floating cells (arrows) interspersed across a loose monolayer. (B) FACS gating strategy illustrating selection of live single cells from DAPI-stained Aag2 cell suspension. (C) Resultant Aag2-derived clonal cell line morphologies following limited expansion; (i) similar appearance to parental Aag2 cells, (ii) highly clustered cells with no monolayer formation (some rounded floating cells present), (iii) only large rounded floating cells observable (individual cells and large multi-cell floating aggregates). Only those fourteen clones selected for further study are shown. Images were taken immediately following three-week expansion from single cells into confluent 24-well plate culture. # The Aag2-AF5 cell line was selected for CRISPR gene editing [60,61] (see main text). (D) Total number of clonal cell lines of each morphology generated. (E) Reversion of ‘clustered’ and ‘rounded’ clonal cell lines back to parental Aag2-like morphology following extended culture. Scale bar is 200 μm.
Fig 2.
Aag2-AF10 and Aag2-AF12 cell lines harbour barely detectable levels of phasi charoen-like virus.
(A) Short PCR amplicons spanning the three PCLV genome segments (L, M, S) amplified from RNA or genomic DNA isolated from parental Aag2 cells, either with or without a reverse transcription step (RT). Purified nucleic acids were treated with RNase or DNase prior to PCR. MDCK cell RNA, the cellular Rps7 gene/mRNA and the RNA virus NDV, which was spiked into cells immediately prior to RNA extraction, serve as controls. (B) Detection of the PCLV S segment and CFAV by RT-PCR in Aag2-derived clonal cell lines. Cellular Rps7 mRNA serves as a loading control. (C) Detection of the PCLV L, M and S genome (-ssRNA) and antigenome (+ssRNA) segments in select Aag2-derived clonal cell lines by sense-specific RT-PCR. Rps7 mRNA serves as a loading control. (D) PCLV L segment RT-qPCR ΔΔCt (normalised to Rps7 mRNA) for select Aag2-derived clonal cell lines expressed relative to parental Aag2 cell line at (i) early passages (Aag2-AF10, passage 2; Aag2-AF12, passage 3) and (ii) later (‘medium’) passages (Aag2-AF10, passage 8; Aag2-AF12, passage 12). (E) RT-qPCR quantification of viral RNA copies for PCLV L segment (i) and CFAV (ii) at late passages in Aag2 (parental), Aag2-AF5, Aag2-AF10 and Aag2-AF10 cells. Starting passages (“~P15”) were passage 15 (parental Aag2), 12 (Aag2-AF5), 14 (Aag2-AF10) and 13 (Aag2-AF12). Data points represent three independently passaged lines. Error bars represent standard deviation. ** P < 0.01; *** P < 0.001; ns, not significant (one-tailed Student’s t test). UD, undetected. # Aag2-AF5 cell line used for CRISPR gene editing [60,61].
Fig 3.
All Aag2-derived clonal cell lines have a functional RNAi pathway.
C6/36 cells, the parental Aag2 cell line and its derived clonal cell lines were transiently transfected with plasmids constitutively expressing firefly luciferase and Renilla luciferase (transfection control) in the presence of dsRNA directed against GFP (dsGFP) or firefly luciferase (dsLuc). Mean Renilla-normalised firefly luciferase (FFluc) expression is expressed relative to the dsGFP negative control. * P < 0.05; ** P < 0.01; *** P < 0.001; ns, not significant (one-tailed Student’s t test). Error bars represent standard deviation. # Aag2-AF5 cell line used for CRISPR gene editing [60,61] is highlighted in orange. PCLV-low clones Aag2-AF10 and Aag2-AF12 are highlighted in green; parental Aag2 cells and C6/36 cells (negative control) are shown in purple.
Fig 4.
Susceptibility of Aag2-derived clonal cell lines to infection with arboviruses.
(A, C, E, G) Single-step growth kinetics of DENV-2 (A), ZIKV (C), SINV (E) and VSV (G) in the parental Aag2 cell line (MOI 2). Grey shading highlights time points tested in Aag2-derived clonal cell lines. (B, D, F, H) Replication of DENV-2 (B) and ZIKV (D) at 1, 2 and 3 days post-infection, replication of SINV (F) at 6, 12 and 24 hours post-infection (hpi), and replication of VSV (H) at 6, 9 and 12 hpi in the parental Aag2 cell line and its derived clonal cell lines (all MOI 2). Grey shading indicates 0.5 Log10 above and 0.5 Log10 below peak extracellular titres detected in parental Aag2 cell line. Error bars represent standard deviation. # Aag2-AF5 cell line used for CRISPR gene editing [60,61] is highlighted in orange. PCLV-low clones Aag2-AF10 and Aag2-AF12 are highlighted in green; parental Aag2 cells and C6/36 cells (negative control) are shown in purple.
Fig 5.
Transfection efficiency of clone Aag2-AF5 relative to parental Aag2 cells.
(A) Cells were imaged at 10X magnification 48 h after transient transfection with a constitutively active GFP expression vector (pIEx-EGFP). (B) Quantification of transfection efficiency; differences are non-significant. (C) Cells were transiently transfected with a constitutively active firefly luciferase reporter plasmid (pIEx-luc) and luciferase activity was measured four days later. * P < 0.05 (two-tailed Student’s t test). RLU, relative light units. All error bars represent standard deviation. # Aag2-AF5 cell line used for CRISPR gene editing [60,61].
Fig 6.
Antimicrobial peptide induction in clone Aag2-AF5 compared to parental Aag2 cells.
Cells were stimulated with heat-inactivated E. coli (A), L. monocytogenes (B) or S. aureus (C) for 24 h and induction of DefD (i), CecB (ii) or CecD (iii) was measured by RT-qPCR. Gene induction is relative to the respective unstimulated cell line. * P < 0.05; ** P < 0.01; ns, not significant (two-tailed Student’s t test). Error bars represent standard error of the mean. # Aag2-AF5 cell line used for CRISPR gene editing [60,61].