Fig. 7

Gene knockdown and the effect on LGTV replication and production in IDE8 cells. IDE8 cells were treated with dsRNA to silence selected transcripts and subsequently infected with LGTV at MOI 0.01. a Transcripts coding for Argonaute (Ago 30) and Dicer (Dcr 90) were amplified by RT-PCR using dsT7-Ago 30 or dsT7-Dcr 90 primers and visualised by agarose gel electrophoresis. A representative 1 % agarose gel from one of the three experiments is shown; upper lanes show Ago 30 and Dcr 90 PCR products, lower lanes show beta actin PCR products. b Gel-electrophoresis images were used to semi-quantify mRNA knockdown of Ago 30 and Dcr 90 with Image Lab software (BioRad) normalised to beta actin control. c Knockdown of mRNA of the genes listed in the x-axis was quantified using qRT-PCR with qRT-PCR primers (Additional file 1). Gene expression was normalised to beta actin and is shown relative to eGFP-dsRNA controls. d Viral RNA levels were determined by qRT-PCR using LGTV NS5 primers at 48 h p.i.. The data was normalised to beta actin and is presented for each of the genes listed in the x-axis, and for cells that were not treated with any dsRNA and then infected with LGTV (Virus), as fold change relative to eGFP dsRNA controls. e Infectious virus present in the supernatant was titrated by plaque assay at 48 h p.i. and the titres are presented for each of the genes listed in the x-axis, and for cells that were not treated with any dsRNA and then infected with LGTV (Virus), as fold change relative to titres in the eGFP-dsRNA control. The mean with standard error of three independent experiments is shown, including only those replicates in which the knockdown was validated. Statistical significance was calculated using two-way ANOVA Fisher’s LSD test (* p < 0.05, ** p < 0.001)