Virus populations dominated by DI particles give rise to a higher proportion of dually HA positive cells but a lower proportion of reassortant progeny viruses compared to virus populations with low DI content.

<p>Pan/99wt and Pan/99var viruses with matched passage histories were mixed in equal proportions (P0wt + P0var, P3wt + P3var and P4wt + P4var). Each mixture was used to inoculate MDCK cells at a range of MOIs, in triplicate. MOIs of P3 and P4 virus infections were calculated based on RNA equivalents relative to P0, such that comparable numbers of particles/cell were used in each case. A) Relationship between % HA positive cells and % dually HA positive cells. B) Relationship between % HA positive cells and % reassortment. C) Relationship between the proportion of HA positive cells that are dually HA positive and % reassortment. Results from two independent experiments are shown: data labeled as P0a were obtained in parallel with those for P3 and data labeled as P0b were obtained in parallel with those for P4.</p

Results observed with UV treated Pan/99wt and Pan/99var viruses match simulated co-infections in which each virus carries an average of 2.0 UV hits per genome.

<p>Experimental data points obtained with UV treated virus stocks, plotted with open circles, are overlaid on a black line indicating the model’s predicted relationships for % HA positive cells vs. % dually HA positive cells (A) and % HA positive cells vs. % reassortment (B). Computational viruses carried an average of 2.0 UV hits per genome when P_P settings found to best match the data obtained with untreated virus stocks were used (0.25, 0.5, 075, 0.75, 1, 1, 1, 1 for segments 1–8, respectively).</p

Impact on % infection, % co-infection, and % reassortment of introducing semi-infectious particles into the model.

<p>The probability that a given segment is present in each virus particle (P_P) was varied from 1.0 to 0.3 within the model. The color scale indicates the value assigned to P_P in each simulation. A) The relationship between % infected and % co-infected cells is not affected by changing P_P. B) The relationship between % HA positive cells and % dually HA positive cells varies with P_P, due to the potential for segments that are required for HA gene expression to be missing from the infecting virus particle(s). C) The relationship between % HA positive cells and expected % reassortment, averaged across all productively infected cells, changes markedly with P_P.</p

Proportion of polymerase segments intact in P3 and P4 viruses, relative to P0 viruses.

<p>Proportion of polymerase segments intact in P3 and P4 viruses, relative to P0 viruses.</p

Semi-infectious particles increase reassortment at a given MOI by increasing the proportion of infected cells that are co-infected.

<p>Results of computationally simulated co-infections are shown. In each simulation, multiplicity of infection in terms of total particles/ cell was held constant and is indicated by the color scale inset in panel (A). The same P_P value, plotted on the x-axis, was assigned to all eight segments and was varied from 0.3 to 1.0 in increments of 0.1. Twenty replicates were run, and the mean of these 20 runs is shown. In (A), the average % reassortment expected under each condition is plotted. Fully infectious progeny viruses generated from all infected cells are considered in this analysis. The results show that, at each MOI, the % of viruses that are reassortant increases with decreasing P_P. In (B), the average % reassortment for productively co-infected cells is plotted for each MOI condition. Here, only those fully infectious viruses generated in productively co-infected cells are considered. The results show that, regardless of MOI and P_P, the vast majority of fully infectious viruses emerging from co-infected cells have a reassortant genotype. In (C), the ratio of co-infected cells to singly infected cells is plotted on a log 10 scale. This plot shows that, at MOIs less than 3 particles/cell, this ratio increases with decreasing P_P. In other words, where P_P is lower, a greater proportion of cells are potential vessels for reassortment. A log 10 scale was used to allow visualization of all lines on one graph.</p

Increasing semi-infectious particle content by UV irradiation of virus stocks augments observed % reassortment.

<p>Pan/99wt and Pan/99var viruses were exposed to low dose UV light sufficient to reduce viral titers by ~10-fold (white circles) or were mock treated (black circles). Treated wt and var viruses were mixed in equal proportions and mock treated wt and var viruses were mixed in equal proportions. Each mixture was used to inoculate MDCK cells at a range of MOIs, in triplicate. Following a single cycle of infection, cell culture supernatants were stored and HA positive and dually HA positive cells were identified by flow cytometry. Clonal viral isolates (n = 18–21) derived from each cell culture supernatant were genotyped by high resolution melt analysis to allow calculation of % reassortment. A) Relationship between % HA positive cells and % dually HA positive cells. B) Relationship between % HA positive cells and % reassortment. Individual data points, each corresponding to one cell culture dish, are plotted. Data from mock treated samples (black circles) are also presented in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005204#ppat.1005204.g003" target="_blank">Fig 3</a> and are included here to allow direct comparison with data from UV treated samples.</p

Terminology used herein to describe different types of infected cells and virus particles.

<p>Terminology used herein to describe different types of infected cells and virus particles.</p

Varying P_P by segment yields good fit between modeled and observed relationships among HA positive cells, dually HA positive cells and reassortment.

<p>When P_P was allowed to vary among the segments (from 0.25 to 1.0 in increments of 0.25), several combinations of P_P values allowed a good fit between observational and modeled data. Shown here are the modeled results for the 1% of P_P combinations yielding the best fit (i.e. the top 28 of the 2800 settings tested). The 28 lines are colored from best (blue) to worst (red) fit. Experimental data points, plotted with black circles, are overlaid on these colored lines to allow comparison between experimental and modeled results for % HA positive cells vs. % dually HA positive cells (A) and % HA positive cells vs. % reassortment (B).</p

Impact on % reassortment of introducing defective interfering particles into the model.

<p>DI particles were introduced into the model by varying P_I values of PB2, PB1 and PA independently from 0.2–1.0 in increments of 0.2. Thus, 125 different settings for the prevalence of DI segments were tested. Computational virus populations A and B were assigned the same P_I values in each simulation. For each of the 125 settings, the potency with which the DI segments interfere with infectious virus production, DIX, was set to 0.01 (A), 0.1 (B) and 0.45 (C and D). In each panel, the color assigned to each of the 125 P_I settings reflects the product of P_I(PB2), P_I(PB1) and P_I(PA), with the highest product (0.75) in red and the lowest product (0.008) in blue. In addition, the line representing results obtained with P_I = 1.0 for all segments is shown in black for reference. (D) Experimental data obtained with standard virus stocks are plotted with black circles and overlaid on the modeled results for DIX = 0.45. This comparison indicates that, although the presence of mildly interfering DI segments enhances reassortment, this effect is not sufficient to account for the levels of reassortment observed with Pan/99wt and Pan/99var viruses.</p

Measurement of HA positive cells, dually HA positive cells and reassortment following co-infection of MDCK cells with Pan/99wt and Pan/99var viruses.

<p>Pan/99wt and Pan/99var viruses were mixed in equal proportions and used to inoculate MDCK cells at a range of MOIs. Infection at each MOI was performed in triplicate. Following a single cycle of infection, cell culture supernatants were stored and HA positive and dually HA positive cells were identified by flow cytometry. Clonal viral isolates (n = 18–21) derived from each cell culture supernatant were genotyped by high resolution melt analysis to allow calculation of % reassortment. Individual data points, each corresponding to one cell culture dish, are plotted.</p