Semi-continuous Propagation of Influenza A Virus and Its Defective Interfering Particles: Analyzing the Dynamic Competition To Select Candidates for Antiviral Therapy

Figures

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  FIG 1. Semi-continuous propagation of influenza A virus and DIPs. (A) PR8 seed virus depleted in DI vRNAs was used for infection. Results of segment-specific RT-PCR for Seg 1 to 8 followed by agarose gel electrophoresis are shown. Signals corresponding to FL and DI vRNAs are indicated. The upper, middle, and lower thick bands of the DNA ladder indicate 3,000, 1,000, and 500 bp, respectively. (B) Experimental setup of the small-scale two-stage cultivation system in shake flasks (scheme adapted from Tapia et al. [68]). MDCK(sus) cells were grown in the CSS and VS. After an initial batch and semi-continuous phase (CSS and VS not coupled), the cells in the VS were infected with the seed virus (A) at an MOI of 0.1. The semi-continuous production mode was initiated 0.5 dpi, where cells were transferred from the CSS into the VS (V2) at regular time intervals, while fresh medium was added (V1 or V3) and virus harvest was taken for monitoring (V4). (C) Periodic oscillations of total and infectious virus titers during the production. The vRNA level of Seg 5 (indicating total virus particle concentration) was quantified by real-time RT-qPCR and infectious virus titer by TCID50 assay. (D)…

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  FIG 2. Diversity, distribution and de novo generation of deletion junctions during semi-continuous propagation of IAV. Deletion junctions were identified by Illumina-based NGS and subsequent analysis via the ViReMa algorithm (32). (A) Number of different deletion junctions located on the respective genome segment(s). (B) Fraction of all deletion junctions located on the respective genome segment(s). This fraction describes the ratio of the total number of detected deletion junctions for one segment to the total number of deletion junctions on all genome segments. (C) De novo formation of deletion junctions. The vRNA level of Seg 5 (indicating total virus particle concentration, as shown in Fig. 1C) was quantified by real-time RT-qPCR. Samples not analyzed by NGS are indicated by open circles. The illustration includes the results of one experiment.

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  FIG 3. Dependency of the length of DI vRNAs on their accumulation during semi-continuous propagation of IAV. Deletion junctions were identified by Illumina-based NGS and subsequently analyzed via the ViReMa algorithm (32). Fractions of individual deletion junctions were calculated based on the ratio of the number of NGS reads of one individual deletion junction to the number of NGS reads of all deletion junctions located on all eight segments. The means of DI vRNA length (calculated by fitting a normal distribution function) are indicated by dashed vertical lines, and the corresponding lengths are shown. Representative time points are illustrated. The illustration includes the results of one experiment.

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  FIG 4. Deletion junction sites of DI vRNAs during semi-continuous propagation of IAV. Deletion junctions were identified by Illumina-based NGS and subsequently analyzed via the ViReMa algorithm (32). DI vRNA 3′ and 5′ lengths indicate the number of retained nucleotides prior to and after the deletion junction, respectively, at the corresponding vRNA ends. The packaging signal is indicated as gray areas and is divided into the incorporation signal (dark gray area) and bundling signal (light gray area). Representative time points are illustrated. The color code from red to blue shown on the right denotes the fraction of the individual deletion junction, which was calculated based on the ratio of the number of NGS reads of one individual deletion junction to the number of NGS reads of all deletion junctions located on all eight segments. Additionally, the circle radii increase with higher fractions. The diagonal black line indicates equal DI vRNA 3′ and 5′ lengths. The illustration includes the results of one experiment.

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  FIG 5. Deletion junction sites of DI vRNAs on non-polymerase-encoding segments during semi-continuous propagation of IAV. Deletion junctions were identified by Illumina-based NGS and subsequently analyzed via the ViReMa algorithm (32). DI vRNA 3′ and 5′ lengths indicate the number of retained nucleotides prior to and after the deletion junction, respectively, at the corresponding vRNA ends. The packaging signal is indicated as gray areas and is divided into the incorporation signal (dark gray area) and bundling signal (light gray area). Representative time points are illustrated. The color code from red to blue shown on the right denotes the fraction of the individual deletion junction, which was calculated based on the ratio of the number of NGS reads of one individual deletion junction to the number of NGS reads of all deletion junctions located on all eight segments. For graphs where no breaking points are shown, no DI vRNAs were detected at the selected time points. Additionally, the circle radii increase with higher fractions. The diagonal black line indicates equal DI vRNA 3′ and 5′ lengths. The illustration includes the results of one experiment.

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  FIG 6. Propagation of DI vRNAs showing the highest gains or losses in their fractions during semi-continuous propagation of IAV. Deletion junctions were identified by Illumina-based NGS and subsequently analyzed via the ViReMa algorithm (32). Fractions of individual deletion junctions were calculated based on the ratio of the number of NGS reads of one individual deletion junction to the number of NGS reads of all deletion junctions located on all eight segments. (A) Fraction of individual DI vRNAs belonging to the group of top five gains, losses, and gains (de novo) of the fraction over cultivation time. Top gains (de novo) indicate newly formed deletion junctions with the highest fraction at the end of cultivation. (B) Deletion junction position of the top 15 gains, losses, and gains (de novo). DI vRNA 3′ and 5′ lengths indicate the number of retained nucleotides prior and after the deletion junction, respectively, at the corresponding vRNA ends. The packaging signal is indicated as gray areas and is divided into the incorporation signal (dark gray area) and bundling signal (light gray area). The illustration includes the results of one experiment.

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  FIG 7. Interfering efficacy of DIPs derived from DI vRNAs showing the highest gain, loss, or gain (de novo) in their fraction during semi-continuous propagation of IAV. Purely clonal DIPs containing a deletion in Seg 1 (derived from DI vRNAs showing the top gains and top loss) were generated using a modified reverse genetics methodology for reconstitution of purely clonal IAV DIPs (40). Next, DIPs were multiplied in genetically engineered MDCK-PB2(sus) cells (expressing PB2) in a shake flask at a multiplicity of DIP (MODIP). (A and B) Interference assay. MDCK(adh) cells were infected with STV only at an MOI of 0.01 (NC) or coinfected with the corresponding DIP, resulting in a DIP/STV ratio of 3,224 (number of DIPs, 4.25 × 108 virions; number of STVs, 1.32 × 105 virions, derived from the HA titer). For comparison, DI244, a prototypic, well-characterized DIP (23–25, 27), was used. (A) Infectious virus release, quantified by plaque assay. (B) Total virus particle release, measured by HA assay. The illustration includes the results of three independent experiments (n = 3). Error bars indicate the SD. One-way analysis of variance (ANOVA) followed by Tukey’s multiple-comparison test (*, P <…