Figure 2. DVGs accumulate at a high rate in infected cells and are associated with the induction of the antiviral responses.

(A) SeV Cantell LD RNA and DVG RNA were extracted from SeV Cantell LD stocks or in vitro transcribed DVG RNA respectively. RNAs were treated with calf intestinal phosphatase, RNase A, RNase V1, and/or RNase A/V1. LLC-MK2 cells were transfected with 250 ng of control or treated RNAs and analyzed 4 h after transfection by RT-qPCR. (B) LLC-MK2 cells were transfected with the indicated doses of genomic RNA and DVG RNA. After 4 h, total RNA was extracted and expression of Ifnb and Il-6 was determined by RT-qPCR. (C) WT MEF cells were infected with SeV Cantell HD or SeV Cantell LD at the indicated mois. After 6 h, total RNA was extracted and the quantities of Ifnb mRNA, gSeV, and DVG were determined by RT-qPCR. (D) Total RNA was directly quantitated from SeV Cantell HD or LD solutions prepared to have the indicated infectious units. (E) WT MEF cells were infected with SeV Cantell HD at a moi of 3 TCID₅₀/cell and total RNA was analyzed by RT-qPCR at the indicated times post-infection. (F) Rate of gSeV and DVG replication calculated at 12 h post-infection. Gene expression is shown as copy number relative to the housekeeping genes Tuba1b and Rps11. Error bars indicate the standard deviation of triplicate measurements in a representative experiment (**p<0.01, ***p<0.001, ****p<0.001 by one-way ANOVA with Bonferroni post hoc test (A and C); ****p<0.001 by Unpaired, two tailed, t student test (F)).