Ez1 extraction robot

A European strain of KHV, K250, isolated from a KHV outbreak in the UK in 2007, was propagated in the common carp brain (CCB) derived cell line (ECACC 10072802) at 20 °C in EMEM media (Sigma, Gillingham, UK) supplemented with 2 mM Glutamine, 1% non-essential amino acids (Sigma, Gillingham, UK), 2% Foetal Bovine Serum (FBS), and 10 mM HEPES (Sigma, Gillingham, UK) [56 ].
The supernatant of cells showing cytopathic effects was harvested, clarified by centrifugation at 4000× g for 15 min, and stored at 4 °C.
To determine the viral dose, the copy number of the KHV orf90 gene was quantified in a Taqman qPCR test previously described [8 (link)]. Viral nucleocapsid was extracted from the clarified supernatant using the EZ1 Virus Mini Kit and the EZ1 extraction robot (Qiagen, Manchester, UK) following the manufacturer’s instructions. To generate a standard curve, a fragment of 1450 bp of the KHV orf90 gene containing the qPCR region was cloned into the pGem-T Easy plasmid vector (Promega, Southampton, UK) as described before [57 (link)]. The template (dsDNA) copy number was calculated using a QuantiFluor dsDNA kit in a Quantus fluorimeter (Promega, Southampton, UK), and a plasmid dilution series, from 10⁶ to 1 copy, was generated.

Total nucleic acid was extracted from 100 µL of the supernatant of EPC cells showing cytopathic effects (CPEs) using the EZ1 Virus mini kit and an EZ1 extraction robot (QIAGEN, Manchester, UK) following the manufacturer’s instructions. Reverse transcription (RT) was performed at 37 °C for 1 h in a total 20 μL reaction volume consisting of 200 U of M-MLV RT, M-MLV RT 5× reaction buffer (250 mM Tris-HCl, pH 8.3; 375 mM KCl; 15 mM MgCl₂; 50 mM DTT), 1 mM dNTP mix, 500 ng of random primers, 25 units RNasin^® Ribonuclease Inhibitor (Promega, Hampshire, UK) and 4 μL of viral nucleic acid extract.
Inoculated EPC cells showing CPEs were first subjected to a rhabdovirus ELISA test (SVCV Ag ELISA, TestLine, Brno, Czech Republic) and an SVCV-specific PCR and nested PCR using cDNA, as described by the OIE [10 ]. To check for other closely related spriviviruses or possible SVCV variants, a generic nested RT-PCR targeting the L gene of fish vesiculotype viruses was also tested [7 (link)].
To test for the presence of the agent of herpesviral hematopoietic necrosis (HVHN), cyprinid herpes virus-2 (CyHV-2)), a generic PCR and nested-PCR (CyHV-pol assay) targeting the herpesvirus DNA polymerase gene was conducted, as described before [11 (link)], using the extracted viral nucleocapsid of cells inoculated with two pool of samples.

Total RNA was extracted from each sample using an EZ1 RNA Cell Mini Kit v2.0 and EZ1 extraction robot (Qiagen, Manchester, UK). Following manufacturer’s instructions, RNase-DNase I (Qiagen Manchester, UK) was added to remove any DNA contamination during the extraction procedures. RNA was quantified using a Quantus™ fluorometer (Promega, Southampton, UK). Illumina indexed sequencing libraries were prepared from 100 ng high-quality RNA using poly-A isolation as part of the TruSeq Stranded mRNA Sample Preparation protocol (Illumina, Great Abington, UK). Tapestation analysis (Agilent, Worthing, UK) showed a library smear ranging in size from 200 bp to 900 bp with a peak at 415 bp without primer dimers. Samples were pooled in equimolar concentrations before denaturing, diluting, and 125 paired-end (PE) sequencing on an Illumina HiSeq2500 over two lanes of a high-output flowcell using v4 SBS (Sequencing by Synthesis) reagents (Illumina, Great Abington, UK).

RNA was extracted from each sample using an EZ1 RNA Cell Mini Kit v2.0 and EZ1 extraction robot (Qiagen, Manchester, UK). RNase-free DNase I treatment (Qiagen) was performed during the RNA extraction following manufacture instructions. RNA was diluted in 60 μL of elution buffer. 200 ηg of extracted RNA was reverse transcribed in a 20 μL reaction containing 0.25 mM each dNTP, 500 ng of random primers and 200 units M-MLV reverse transcriptase (Promega, Southampton, UK) at 37 °C for 1 h.
Taqman assays were then performed with 2 μL of cDNA containing 10 ng of input RNA, 500 nM of each primer and 250 nM of probe labelled with 6-FAM in 5′ and MGB in 3’, in a total volume of 20 μL by using the Taqman Universal PCR master Mix with AmpErase UNG (Applied Biosystem). qPCR and fluorescence detection were performed on a StepOne Real-Time PCR, software V2.3 (Applied Biosystem) at 50 °C for 2 min followed by 95 °C for 10 min then 40 cycles of 15 s at 95 °C and 1 min at 60 °C. Each sample was tested in duplicate. Molecular grade water was used as negative control for each master mix.