Persistent Rhesus Enteric Calicivirus Infection in Recombinant CHO Cells Expressing the Coxsackie and Adenovirus Receptor

Figures

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  Figure 1. ReCV infection in rCHO cell lines remains non-lytic through serial passages. (A). ReCV infection can be seen in LLC-MK2 cells with characteristic CPEs of cell rounding and detachment. Cultures are shown 48 h post infection (0.1 MOI). (B). No morphological changes are observable between uninfected and infected rCHO cell cultures. Cell monolayers at passage 9 are shown.

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  Figure 2. Cell viability and metabolic activity of mock- and ReCV-FT7-infected CHO-CAR+ cells. (A). Trypan blue exclusion assay results are shown, where the number of blues cells was counted in 5 view fields of mock- and ReCV-FT7-infected monolayers at passage 12 after 3 days of cell splitting. (B) Metabolic activity was measured at passage 12 after 3 days of cell splitting in 96-well white-walled tissue culture plates seeded with an equal number of cellsThe CellTiter-Glo 2.0 assay quantifying the amount of ATP was used according to the manufacturer’s protocol. The luminescence signal was recorded on a Cytation 5 multimode reader. Background-subtracted raw RLU values are shown. At least 3 wells were analyzed in 2 replicates. Means and standard deviations (SD) are graphed. Statistical significance was evaluated using a paired t-test; p ≤ 0.05 was considered statistically significant. ns: not significant.

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  Figure 3. ReCV infection in rCHO cell lines is persistent, and infectious virus is efficiently released into the medium. rCHO-CAR+ cells were infected with ReCV-FT7, Tulane virus, and ReCV-FT285. Cultures were passaged every 3 days and examined for CPE. Total virus titers and cell-free virus titers were determined by titration in LLC-MK2 cells. Means and standard deviations (SDs) are graphed. Statistical significance was evaluated using the one-way ANOVA test with multiple comparisons. * p ≤ 0.05; ** p ≤ 0.01; and *** p ≤ 0.0001.

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  Figure 4. ReCV-infected rCHO cells contain viral protein and replication intermediate dsRNA. (A). Mock and infected rCHO-CAR+ cells were stained with mouse serum with serotype-specific VN activity against ReCV-FT7. (B). Mock and infected rCHO-CAR+ cells were stained with the dsRNA specific monoclonal antibody J2. Monolayers treated with RNase III were also included. Images represent cultures at passage 6 of the persistently infected cell line.