Optimized Propagation and Purification Protocols for Large-Scale Production of Rhinovirus C

Figures

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  Figure 1. Rhinovirus C15a (RV-C15a) binding and replication in HeLa-E8 suspension culture. (A) Suspension cultures of HeLa-E8 cells (5 × 107 cells) were inoculated with RV-C15a or RV-A34 (MOI of 10 PFUe/cell) and total viral yield was measured 24 h later by RT-qPCR. (B) Binding of RV-C15a and RV-A16 in the presence (medium A or PBS with Ca2+, Mg2+) or absence (medium B) of calcium. HeLa-E8 cells (106 cells) were inoculated with RV-C15a or RV-A16 (MOI of 10, 2 h), washed with PBS (3×) and cell-associated viral RNA was measured by RT-qPCR. Data are presented as mean ± standard deviation from three independent experiments. Statistical significance was assessed by unpaired, one-tailed t-test (A) or ordinary one-way ANOVA (B). *** p < 0.001; **** p < 0.0001.

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  Figure 2. RV-C15a binding and entry in suspension and adherent cultures of HeLa-E8 cells. (A) HeLa-E8 cells grown in suspension cultures (106 cells) and (B) adherent HeLa-E8 cells (~4 × 105 cells per well in a 12-well plate) were inoculated with RV-C15a (MOI of 10 PFUe/cell). After 2 h of incubation at either 4 °C or 34 °C, cells were treated with trypsin (5 min, 37 °C) or left untreated (control), washed with PBS (3×) and cell-associated viral RNA was quantified by RT-qPCR. Data are presented as mean ± standard deviation from three independent experiments. Statistical significance was assessed by unpaired, two-tailed t-test. *, p < 0.05; **, p < 0.01; ***, p < 0.001.

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  Figure 3. Optimizing RV-C15a yields in HeLa-E8 adherent cultures. We varied time after cell plating (A) and calcium concentration in media (B) and measured effects on RV-C15a binding at 2 hpi and progeny yields at 72 hpi by RT-qPCR. Data are presented as mean ± standard deviation from three independent experiments. Statistical significance was assessed by ordinary one-way ANOVA *, p < 0.05; **, p < 0.01.

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  Figure 4. Expression of CDHR3 in adherent HeLa-E8 cells. (A) CDHR3 surface expression was imaged in HeLa-E8 cells that were initially grown in suspension culture and then plated for 24, 48, or 72 h. (B) Mean surface expression was quantified from 3 to 4 representative areas per well in a 12-well plate. The error bars represent standard deviation of the mean. Statistical analysis was performed using a two-tailed unpaired t-test. *, p < 0.05; **, p < 0.01.

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  Figure 5. RV-C15a purification methods. (A) Cell lysates from three T75 flasks were pooled, mixed and clarified by low-speed centrifugation (10,000× g, 10 min), before dividing the material for purification using the three indicated methods. Total viral yields in the purified preparations were quantified by RT-qPCR. Data are presented as mean ± standard deviation from three independent experiments. (B) Virus yields after lipase treatment. Cell lysates from two T75 flasks were processed as in panel (A), treated or not with lipase and purified using ultracentrifugation. Data are presented as mean ± standard deviation from three independent experiments. Statistical significance was assessed by paired, two-tailed t-test. *, p < 0.05. (C) Monolayers of HeLa-E8 cells in 12-well plates were inoculated with lipase-treated or untreated (control) RV-C15a preparations (MOI of 10 PFUe/well). Viral RNA was quantified by RT-qPCR to measure cell-associated input virus at 2 hpi and virus progeny yields at 24 and 72 hpi. Data are presented as mean ± standard deviation from three independent experiments. Statistical significance was assessed by paired, two-tailed t-test. *, p < 0.05; **, p < 0.01. (D,E)…

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  Figure 6. Preferred methods for RV-C15a purification and concentration. The volume range for the virus precipitation method reflects the range used in our laboratory and is scalable to any number of flasks and lysate volumes. The volume range for ultracentrifugation applies to a single run and varies depending on rotor size.