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The apoptotic response induced by RGDV infection occurred via a caspase-dependent pathway in continuous cultured cells of R. dorsalis.

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posted on 2019-01-17, 19:04 authored by Qian Chen, Limin Zheng, Qianzhuo Mao, Jiejie Liu, Haitao Wang, Dongsheng Jia, Hongyan Chen, Wei Wu, Taiyun Wei

(A) RT-qPCR assay showing the relative gene expression for CASP2L, CASP8L or IAP was up-regulated by RGDV infection. Cultured cells were inoculated with purified RGDV (MOI of 1), then harvested at 48 hpi. Relative gene expression of CASP2L, CASP8L or IAP at 48 hpi was normalized to the mock-infected cells. His-Mg buffer-treated cells served as mock controls. (B) RT-qPCR assay showing the relative gene expression for RGDV P8 after treatment with the pancaspase inhibitor Z-VAD-FMK. Cultured cells were incubated for 4 h with 25 μM pancaspase inhibitor Z-VAD-FMK dissolved in DMSO. DMSO treatment served as the control. Cells were inoculated with purified virus (MOI of 0.1), achieving an early infection rate of about 20–30%, then assayed by RT-qPCR assay at 48 hpi. (C) The apoptotic response induced by RGDV infection facilitated viral accumulation in insect vector cells. Cultured cells in a monolayer were treated with dsCASP2L, dsCASP8L, dsIAP or dsGFP, then inoculated with RGDV (MOI of 0.1). At 72 hpi, cells were in situ labeled with TUNEL (green) and immunolabeled with virus-rhodamine (red), then examined by confocal microscopy. The TUNEL assay for dsRNAs-treated and His-Mg buffer-treated cells served as mock controls. Images are representative of three independent experiments. Bars, 40 μm. (D, E) The percentage of viral infection (D) or TUNEL-positive cells (E) in RGDV- or mock-infected cells treated with dsRNAs at 72 hpi. His-Mg buffer-treated cells served as mock controls. (F) RT-qPCR assay showing the relative gene expression for RGDV P8, CASP2L, CASP8L, and IAP after respective dsRNA treatment. The dsRNA-treated cells were inoculated with purified RGDV (MOI of 0.1), then assayed or harvested at 48 hpi. Means (±SD) from three independent biological replicates are shown. *P < 0.05, **P < 0.005. Data were analyzed with a two-tailed t-test in GraphPad Prism 7.

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