FIG 5 .

In vitro and in vivo replication of MKWV and interferon-antagonism activity of MKWV NSs. (a) MKWV isolate MKW73 was used to inoculate cells derived from human (Huh7), tick (ISE6), or mosquito (C6/36). The supernatant of each cell line was collected every 2 days from immediately after inoculation (day 0) to 14 days postinoculation (dpi). (b) Phleboviruses were inoculated into cells derived from tick (ISE6) or sandfly (LL-5), and the supernatant of each cell line was harvested at 7 dpi. RNA extracted from the supernatant was subjected to quantitative RT-PCR to calculate the viral titer. Each data point indicates the mean from triplicate experiments, and error bars indicate standard deviations (SDs). (c) MKWV MKW73 and SFTSV isolate YG1 were used to inoculate 1-day-old mice (SM) and 3-week-old mice (YM). Tissues were collected at 3, 7, and/or 9 dpi, and viral titers were calculated as the 50% tissue culture infectious dose (TCID₅₀/gram). Error bars indicate SDs. (d and e) Luciferase activities under the control of the beta interferon promoter (d) and interferon-sensitive response element (ISRE) (e) were compared in NSs-expressing 293 cells stimulated by the expression of an activated form of RIG-I (d) and recombinant alpha interferon (e), respectively. All tests were done in triplicate, and the means ± SDs are shown.