Multiscale model of defective interfering particle replication for influenza A virus infection in animal cell culture

Figures

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  Fig 1. Addition of defective interfering particles (DIPs) can prevent virus-induced apoptosis and protect MDCKsus cells from standard virus (STV) infection. (A) Schematic depiction of the 12 different MOI and MODIP conditions used for infection experiments. (B) Viable cell concentration and (C) the fraction of apoptotic cells for infections with MOI 10−3 and MODIPs of 0, 10−3, 3 and 30. Results for all infection conditions are shown in S1 Fig.

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  Fig 2. Viral RNA dynamics in infected MDCKsus cells are strongly influenced by the applied concentrations of STVs and DIPs. Real-time RT-qPCR measurements of (A-F) viral mRNA and (G-L) vRNA levels for different infection conditions. The results for (left) FL segment 1, (middle) DI segment 1 and (right) FL segment 5 are shown. Results for the remaining infection conditions are presented in S5–S7 Figs.

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  Fig 3. Schematic depiction of the multiscale STV and DIP co-infection model. (Top) The population level of infection describes growth and apoptosis of uninfected cells, their infection by either STVs or DIPs, the co-infection of STV-only infected and DIP-only infected cells, virus-induced apoptosis of all infected cells and the lysis of apoptotic cells. STVs are released from STV-only infected and co-infected cells, DIPs are only released from co-infected cells and both are cleared via virus degradation. (Bottom) Virus entry, nuclear import, viral RNA and protein synthesis, nuclear export and progeny virion release in STV-only infected cells and co-infected cells is simulated using the same intracellular model. Figure adapted from [29].

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  Fig 4. Adaptations of viral RNA synthesis and cell growth mechanisms of the basic model improve the description of observed infection dynamics. (A+C+F) Dashed lines show model simulations of the basic model fitted to experimental data, (B+D+F) solid lines depict simulations of the extended model calibrated to the same measurements. (A+B) Dynamics of viral mRNA for segment 5, full-length segment 1 and defective interfering segment 1 using MOI 30 and MODIP 3. (C+D) Accumulation of viral DI mRNA for conditions L3 (MOI 10−3 and MODIP 3) and L30 (MOI 10−3 and MODIP 30). (E) Estimated values for the parameter kVSyn describing vRNA synthesis for different infection scenarios based on the applied MODIP-to-MOI ratio. The orange dotted line depicts the average kVSyn value for low MODIP infections (empty circles) and the blue dash-dotted line represents the dependency of kVSyn on the MODIP-to-MOI ratio for high MODIP conditions (full circles). The vertical black line separates infections only using STVs from infections with MODIP > 0. (F) Dynamics of the viable cell concentration for low MOI, high MODIP conditions L3 and L30. Experimental data for all other infection conditions are shown in S1 and…

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  Fig 5. The extended multiscale model captures infection dynamics on the intracellular and cell population level for all measured infection conditions. Curves depict simulations of the extended model fitted to (A-C) cell-specific viral mRNA, (D-F) cell-specific vRNA, (G-I) cell population and (J-L) extracellular virus titers measured in MDCKsus cell cultures infected with different amounts of influenza A/PR/8/34 (H1N1) and defective interfering particles (DI244). Results for MOI 10−3 and MODIP 0 (first column), 30 and 3 (second column), and 10−3 and 30 (third column) are shown. The figures presenting cell population dynamics (G-I) show fits to uninfected non-apoptotic, infected non-apoptotic, as well as the sum of uninfected and infected apoptotic cells. The extended model is based on Rüdiger et al. [27] and Laske et al. [23], but additionally considers primary transcription of viral mRNA, segment-specific viral mRNA production, a reduced vRNA synthesis for high MODIP conditions and a DIP-induced reduction of cell growth. Simulation results for all other infection conditions are shown in S3–S10 Figs.

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  Fig 6. STV infection suppression and DIP production are strongly affected by the infection conditions. (A+B) Infections of MDCKsus cells were simulated with the extended model using MOIs and MODIPs in the range of 10−8–102 and 10−4–102, respectively. The predicted concentrations of (A) infectious STVs and (B) total DIPs at 48 hpi determine the color of the heat map. The experimentally observed infection scenarios (+) are depicted. The solid red line indicates (A) an MODIP-to-MOI ratio of 104:1 and (B) the optimal multiplicity ratios for DIP production. For the switch between regular and reduced vRNA synthesis for low and high initial DIP concentrations a threshold value of FMODIP = 10−3 was used. Grey areas indicate that no production of either STVs or DIPs did occur. (C) The predicted fold-change for yields of infectious STVs and total DIPs at 24 hpi depending on a reduction or increase in the replication advantage of DI cRNAs over their FL counterpart is presented. The parameter FAdv was varied in the range of 0 to 1000% of its estimated value. The vertical red line indicates the replication advantage estimated during model calibration, i.e., FAdv = 0.32.