Superinfection exclusion creates spatially distinct influenza virus populations

Abstract

Interactions between viruses during coinfections can influence viral fitness and population diversity, as seen in the generation of reassortant pandemic influenza A virus (IAV) strains. However, opportunities for interactions between closely related viruses are limited by a process known as superinfection exclusion (SIE), which blocks coinfection shortly after primary infection. Using IAVs, we asked whether SIE, an effect which occurs at the level of individual cells, could limit interactions between populations of viruses as they spread across multiple cells within a host. To address this, we first measured the kinetics of SIE in individual cells by infecting them sequentially with 2 isogenic IAVs, each encoding a different fluorophore. By varying the interval between addition of the 2 IAVs, we showed that early in infection SIE does not prevent coinfection, but that after this initial lag phase the potential for coinfection decreases exponentially. We then asked how the kinetics of SIE onset controlled coinfections as IAVs spread asynchronously across monolayers of cells. We observed that viruses at individual coinfected foci continued to coinfect cells as they spread, because all new infections were of cells that had not yet established SIE. In contrast, viruses spreading towards each other from separately infected foci could only establish minimal regions of coinfection before reaching cells where coinfection was blocked. This created a pattern of separate foci of infection, which was recapitulated in the lungs of infected mice, and which is likely to be applicable to many other viruses that induce SIE. We conclude that the kinetics of SIE onset segregate spreading viral infections into discrete regions, within which interactions between virus populations can occur freely, and between which they are blocked.

Fig 1. The ability of IAV to cause SIE depends on the interval between primary and secondary infections.

(A) Confocal micrographs of cells infected with ColorFlu reporter viruses, which cause cells to express eGFP (green) or mCherry (magenta). Co-expression of both fluorophores is shown as white. MDCK cells were grown on glass coverslips, infected at an MOI of 1 FFU/cell for each virus, and fixed at 8 h post infection (hpi). Images were obtained using a 64× objective. Scale bar = 2 μm. (B) Flow cytometry of cells infected with reporter viruses. MDCK cells were first infected with ColorFlu-eGFP, before secondary infection at the time points indicated with ColorFlu-mCherry, with both viruses at MOI 1 FFU/cell. Representative plots are shown. (C) Kinetics of onset of SIE, determined from flow cytometry analysis; means and SD are shown (n = 6). Differences in the percentage of coinfected cells, compared to simultaneous infection (time = 0 h), were tested for significance by one-way ANOVA. By 3 h, the difference was significant (p = 0.0074), and at every subsequent time point (p < 0.0001). (D) The number of reporter viruses per cell that were able to cause expression of their fluorophore, with varying intervals between infection with primary (green) and secondary (red) viruses. Viruses were quantified as GFUs and RFUs, calculated from the proportions of red, green, and coinfected cells. The mean and SD are shown (n = 6). (E) The relationship between the expression of the secondary virus and the interval between infections, as shown in (D), modelled as an initial period of no SIE followed by an exponential increase in SIE. SST = 0.74. Underlying data can be accessed at the following address: http://dx.doi.org/10.5525/gla.researchdata.1370. FFU, focus forming unit; GFU, green forming unit; IAV, influenza A virus; MDCK, Madin–Darby canine kidney; MOI, multiplicity of infection; RFU, red forming unit; SIE, superinfection exclusion; SST, total sum of squares.

Fig 2. SIE kinetics are sensitive to the amount of primary infecting virus.

(A) To assess the effects of altering the ratios of primary (ColorFlu-eGFP, green) and secondary (ColorFlu-mCherry, magenta) infecting viruses, SIE was measured as in Fig 1. Five different conditions are shown, with the ratios of primary and secondary viruses for each experiment indicated as bars. The initial FFU/cell for 1:1 ratio is 0.66 for ColorFlu-eGFP and 0.72 for ColorFlu-mCherry. These data were used to calculate how the expression of secondary virus (RFU per cell) changes with the interval between infections. This is shown when changing the ratios of (B) primary and (C) secondary infecting viruses. The RFU per cell was then fit to a model describing an initial constant phase of 2 h, followed by exponential decay plateauing at 0 (B, C: left-hand panels). The SST of the models fitted for 1:1, 2.5:1, and 5:1 are 0.43, 0.18, and 0.067, respectively. The SSTs for the models fitted for 1:1, 1:2.5, and 1:5 are 0.43, 1.01, and 1.10, respectively. The half-life of the decay phase, after the initial constant phase of 2 h, was then calculated (B, C: right-hand panels). Differences between these intervals and those observed with a 1:1 ratio were determined by Kruskal–Wallis test (*p < 0.05). For all data the mean and SD are shown (n = 3). Underlying data can be accessed at the following address: http://dx.doi.org/10.5525/gla.researchdata.1370. FFU, focus forming unit; RFU, red forming unit; SIE, superinfection exclusion; SST, total sum of squares.

Fig 3. SIE does not inhibit coinfection between IAVs from a single focus of infection.

(A) Experimental design for investigating the role of SIE in the spread of coinfected foci. (B) Proposed models for the spread of coinfected foci. (C) The spread of coinfected plaques, showing the same region at 3 different time points. Viruses were seeded onto monolayers of MDCK cells, overlayed with agarose, and imaged every 24 h. Images were taken on Celigo fluorescence microscope and a representative field of view is shown. Scale bar = 2 mm. (D) A binary threshold was applied to images of plaques to distinguish coinfected cells (white) from singly infected cells (magenta or green); representative images of plaques at 48 hpi are shown. Scale bars = 1 mm. (E) The percentage of total plaque area that was coinfected, calculated from binarised images at taken at each time point. Box and whisker plots show the percentages of infected areas from 71 individual fields of view at 3 time points in 1 experiment. Differences between the coinfected percentage at different time points were tested for significance by one-way ANOVA (**** p < 0.0001). Underlying data can be accessed at the following address: http://dx.doi.org/10.5525/gla.researchdata.1370. IAV, influenza A virus; MDCK, Madin–Darby canine kidney; SIE, superinfection exclusion.

Fig 4. SIE allows only a small region of coinfection when 2 established regions of IAV infection meet.

(A) Representative image of plaque interaction. Reporter viruses were seeded onto monolayers of MDCK cells, overlayed with agarose, and imaged every 24 h. Representative images, taken on Celigo fluorescent microscope, are shown. Scale bar = 2 mm. (B) A binary threshold was applied to images of plaques to distinguish cells expressing the eGFP, mCherry, or both fluorophores together. Images of a representative plaque are shown. Scale bar = 1 mm. (C) The percentage of coinfected areas in comparison to total plaque area was calculated from images at taken at 72 hpi. The mean and the percentage areas of 86 individual fields of view from 1 experiment are shown. (D) Lung sections from infected mice at 6 dpi. C57BL/6 mice were intranasally inoculated with mixtures of mCherry and eGFP expressing viruses (500 PFU of each virus). Lungs were harvested, sectioned, and imaged with an LSM 880 confocal microscope (Zeiss) using a 20× objective; scale bar = 1,500 μm. (E) Enlarged image of a lesion showing coinfection; scale bars = 100 μm. Binarised images of the lesion were produced as described in (B). Underlying data can be accessed at the following address: http://dx.doi.org/10.5525/gla.researchdata.1370. dpi, days post infection; IAV, influenza A virus; MDCK, Madin–Darby canine kidney; PFU, plaque-forming unit; SIE, superinfection exclusion.