Poliovirus-nonsusceptible Vero cell line for the World Health Organization global action plan

Figures

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  Figure 1. Generation of PVR1/PVR2 DKO Vero cell lines. (a) Diagram of PVR. The signal peptide and the N-terminus of the first domain (D1) of the three Ig-like domains, which is responsible for viral recognition, is encoded by exon 1 of the human PVR gene. Amino acid sequences are aligned with AGM (Chlorocebus sabaeus) or Vero PVRs (see also Supplementary Table 1). (b) Nucleotide sequences of the initial ATG codon and downstream region in Vero-PVR1 and Vero-PVR2. Genomic DNA of Vero cells were extracted and the coding region located on exon 1 of PVR1 and PVR2 were sequenced. DNA sequences of Vero-PVR1 and Vero-PVR2 are shown in lowercase letters; the predicted amino acid sequences are shown in capital letters. Vero-PVR2 was found to have a single-nucleotide variation (SNV) (†) in the open reading frame (ORF), probably due to heterozygosity of the gene. (c) DNA sequencing chromatogram of a Vero-PVR2 region. Sanger sequencing of Vero-PVR2 showed an SNV in the ORF of exon 1 (see also panel b).

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  Figure 2. Generation of PVR1/PVR2 double-knockout (DKO) cell lines. (a) Diagram for construction of PVR1/PVR2 double-deficient cell lines. In the first-round experiment, co-transfection of parental Vero cells with two plasmids expressing the sgRNA crV4 (targeting to PVR1) or crV3 (targeting to PVR2) gave only PVR1 single-knockout (SKO) cell lines as shown (Supplementary Fig. 2). One of the purified cell clones named Vero ΔPVR1-1 was subjected to a second-round experiment, in which the cells were transfected with the crV3-expressing plasmid, and two PVR1/2 DKO cell lines named Vero ΔPVR1/2–1 and Vero ΔPVR1/2–2, respectively, were obtained (see also Supplementary Fig. 2). In another experiment, transfection of Vero cells with the plasmid expressing crV2 (targeting to PVR1) gave a PVR1 SKO cell line named Vero ΔPVR1-2, and transfection of Vero ΔPVR1-2 cells with the crV3-expressing plasmid gave four PVR1/2 DKO cell lines: Vero ΔPVR1/2–3, Vero ΔPVR1/2–4, Vero ΔPVR1/2–5, and Vero ΔPVR1/2–6 (Supplementary Fig. 2). For construction of PVR2 SKO cells, Vero JCRB9013 cells were transfected with the crV3-expressing plasmid, and two mutant cell lines, Vero ΔPVR2-1 and Vero ΔPVR2-2, were obtained (see…

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  Figure 3. Cell morphology and growth curve of Vero and PVR knockout cell lines. (a) Cell morphology of the Vero cell line, two independent PVR1 SKO cell lines (ΔPVR1-1 and ΔPVR1-2), and two independent PVR2 SKO cell lines (ΔPVR2-1 and ΔPVR2-2; see also Supplementary Fig. 2). Scale bar: 200 µm. (b) Cells were seeded into 24-well plates at 1 × 104 cells/well and cultured for seven days. Live cells were counted each day and the proliferation curves are shown (means ± Standard Deviation, n = 3).

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  Figure 4. PV replication and viral RNA synthesis in the parental and PVR-disrupted Vero cell lines. (a–d) The indicated cell lines were infected with PV Sabin 1 and 3 strains at MOI of 0.01 (a–d) and 10 (e–h), and virus titers (a,b,e,g) and viral RNA (c,d,f,h) in the culture fluids were quantified at the indicated times post-infection (mean ± standard deviation, n = 3). See also Supplementary Fig. 3 for other Vero PVR-disrupted cell clones established in this study.