title: RTG-2 - Marine non-pathogenic VHSV (1p49) - 3 hpi - replicate 3
design: Rainbow trout cells (RTG-2) were inoculated in vitro with either a freshwater VHSV isolate (J167) that is pathogenic to rainbow trout or a marine VHSV isolate (1p49) that is non-pathogenic to this fish species. Samples were collected at 0, 3, 6, and 12 hours post-inoculation and transcriptomic responses analysed using RNA-seq on an Illumina HiSeq 2500, with the aim to identify the molecular mechanisms responsible for VHSV pathogenicity in rainbow trout. Methods used: Low passages (p4-p7) of a freshwater VHSV isolate obtained from a VHS outbreak in farmed rainbow trout, belonging to genogroup Ia (J167) and a marine VHSV, isolated from sub-clinically infected herring Clupea harengus belonging to the genogroup II (1p49), which was shown to be non-pathogenic to rainbow trout were titered and inoculated in RTG-2 cells (CCL55, ATCC, LGS Standards), a rainbow trout gonad derived cell line. For the viral infections, approximately 10^4 RTG-2 cells were plated in 12-well plates containing maintenance medium ((MM) L-15 supplemented with 1 mM L-glutamine, 10% fetal bovine serum, Gibco (Paisley, GB) and 1% penicillin-streptomycin (Sigma)). Cell monolayers were inoculated with J167 and 1p49 at 0.01 MOI and incubated at 15°C. Inoculated cells were then harvested in triplicate at 3, 6 and 12 h post inoculation (hpi). For each sampling point, cells were washed with phosphate buffered saline (PBS) (Sigma) and detached by addition of 300 µL of RLT lysis buffer (Qiagen, Manchester, UK) for subsequent RNA extraction. Total RNA was extracted from each sample using an EZ1 RNA Cell Mini Kit v2.0 and EZ1 extraction robot (Qiagen, Manchester, UK) and samples were treated with RNase-DNase I to remove any DNA contamination during the RNA extraction procedures, following manufacturer's instructions. RNA was quantified using a Quantus™ fluorometer (Promega, UK). Illumina sequencing libraries were prepared from 100ng high quality RNA using poly-A isolation as part of the TruSeq Stranded mRNA Sample Preparation protocol (Illumina Inc.).