Rotavirus Spreads in a Spatially Controlled Manner

Figures

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  Figure 1. Rotavirus infection spreads in a spatially restricted manner. A confluent monolayer of MA104 cells is infected with WT rotavirus (RV) expressing UnaG at an MOI of 0.012, 0.003, or 0.0012. Virus infection is imaged using live-cell microscopy every 30 min for 16 h. (A) Representative brightfield (gray) and UnaG WT RV images (green). Scale bar = 100 μm. Arrows mark a primary infected cell through its lifetime as it dies and becomes a colony of infected cells. (B) The schematic of rotavirus infection and spread depicting primary infection, the cell death of primary infected cells and secondary infection forming a spatially restricted infected cellular colony surrounding the initial primary infected cells. (C) Quantification of the time to primary infection (time to detect UnaG). (D) Quantification of the time to cell death relative to the time to primary infection. (E) Quantification of the time to colony formation relative to the time of primary infected cell death. N = 15 fields of view from 3 independent experiments; and ns = not significant.

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  Figure 2. Rotaviruses are released and require trypsin activation for their spread: (A) A confluent monolayer of MA104 cells is infected with WT rotavirus (RV) expressing UnaG at an MOI of 0.003. Virus supernatants are collected at 4, 6, 8, 10, and 12 hpi. The production of de novo infectious rotavirus particles was analyzed by plaque assay. (B–H) A confluent monolayer of MA104 cells is infected with UnaG WT rotavirus (RV) at an MOI of 0.003. Trypsin is added at 1, 8, 10, 12, 14, 16, 18, or 20 hpi. Virus infection is imaged using live-cell microscopy every 30 min for 24 h. (B) Schematic showing the times of addition of trypsin following rotavirus infection. (C) Representative brightfield (gray) and UnaG WT RV images (green). Scale bar = 100 μm. (D) Quantification of the number of infected cells per field of view. (E) Quantification of the number of primary infected cells per field of view that leads to the formation of spatially restricted infected colonies (secondary infection of neighboring cells) at 16 hpi. (F) Quantification of the time to primary infection (time to detect UnaG). (G) Quantification of the time to cell death relative to the time to primary infection. (H) Quantification…

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  Figure 3. Trypsin neutralization impacts the formation of rotavirus-infected cellular colonies. A confluent monolayer of MA104 cells is infected with UnaG WT rotavirus (RV) at an MOI of 0.003. Trypsin is added at 1 hpi and is inactivated by the addition of FBS at 6, 8, 10, and 12 hpi. Virus infection is imaged using live-cell microscopy every 30 min for 16 h. (A) Schematic showing the time of addition of FBS following rotavirus infection. (B) Representative brightfield (gray) and UnaG WT RV images (green). Scale bar = 100 μm. (C) Quantification of the number of infected cells per field of view. (D) Quantification of the number of primary infected cells per field of view that leads to the formation of spatially restricted infected colonies (secondary infection of neighboring cells) at 16 hpi. N > 3, statistics are performed by two-way ANNOVA. Scale bar represents standard deviation. ** = p < 0.01.

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  Figure 4. Blocking P2Y1 signaling reduces primary rotavirus infection and virus production. A confluent monolayer of MA104 cells is infected with UnaG WT rotavirus (RV) at an MOI of 0.003. Cells are either mock-treated with media +DMSO or treated with 10 μM of BPTU at 0, 4, and 8 hpi. Virus infection is imaged using live-cell microscopy every 30 min for 24 h. (A) Representative brightfield (gray) and UnaG WT RV images (green). Scale bar = 100 μm. (B) Quantification of the number of infected cells per field of view at 24 hpi. (C) Quantification of the number of primary infected cells per field of view that leads to the formation of spatially restricted infected colonies (secondary infection of neighboring cells) at 16 hpi. (D) Quantification of the time to primary infection (time to detect UnaG). (E) Quantification of the time to cell death relative to the time to primary infection. (F) Quantification of time to colony formation relative to the time of primary infected cell death. (G) Supernatants from A are harvested at 12 hpi, and the production of de novo infectious rotavirus particles is analyzed by plaque assay. (H) Same as G except supernatants are harvested at 24 hpi. (A–F) N = 10–15…

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  Figure 5. High MOI increases the kinetics of primary rotavirus infection. A confluent monolayer of MA104 cells is infected with UnaG WT rotavirus (RV) at an MOI of 0.064, 0.64, 6.4, and 64. Virus infection is imaged using live-cell microscopy every 30 min for 24 h. (A) Representative brightfield (gray) and UnaG WT RV images (green). Scale bar = 100 μm. (B) Quantification of the number of infected cells per field of view. (C) Quantification of the time to primary infection (time to detect UnaG). N > 3. Statistics are performed by two-way ANNOVA. Scale bar represents standard deviation. * = p < 0.05, ** = p < 0.01, *** = p < 0.001, **** = p < 0.0001.