Schmallenberg virus, Akabane virus 93FMX strain (KVCC-VR0000064), and Aino virus KSA9910 (KVCC-VR0000064) were maintained in Vero cells. All cell lines were grown in Dulbecco’s modified Eagle’s medium (GibcoBRL, Gaithersburg, MD, U.S.A) supplemented with 5 % heat-inactivated fetal bovine serum (GibcoBRL) in a humidified 5 % CO2 atmosphere at 37 °C. Schmallenberg virus was kindly provided by Friedrich Loeffler Institute (FLI). The Akabane virus and Aino virus were acquired from the Korea veterinary Culture Collection (KVCC) .
Culicoides samples were pooled (30–40) and added to 2 ml containers with ceramic beads. Samples were ground for 30 s with 1 ml of cold phosphate-buffered saline (PBS). Ground samples were centrifuged (1 min, 4 °C), and supernatants were harvested. Total viral RNA was extracted from each control viruses, blood samples, serum samples, bovine brain samples, and pooled culicoides samples using the Maxwell®16 research instrument system (Promega, Medison, Wisconsin, USA) with Maxwell®16 viral total nucleic acid purification kit (Promega AS1150), according to the manufacturer’s instructions.
Specific primer and probe design
Oligonucleotide primers and probes were used to amplify the genes encoding the S segment of SBV (Accession No: HE649914), AKAV (Accession No: AF034942) and AINV (Accession No: AF034939). These conserved viral genome regions were chosen as the best candidates for the generation of specific primers and virus-specific probe sequences for SBV, AKAV and AINV. The primer and probe sequences, fluorophores and quenchers as follows: Primer Common F 5’ – TGACTGCAGAAGARTGGATGA-3’, Common R 5’ – GAATCCA GATTTGGCCCA -3’, SBV Probe 5’ – FAM-ACAGAAATAAAAGCTGCT-BHQ1-3’, AKAV Probe 5’ – HEX-ATCTAAGTTGGACGCA-BHQ1-3’, and, AINV Probe 5’ – Cy5-A TGCTGTCCGTGCA-BHQ2-3’.
Preparation of RNA controls
Plasmids containing target sequences of the mRT-qPCR for SBV, AKAV and AINV were produced using the pGEM®-T Easy Vector Systems (Promega), and RNA was transcribed using the MEGAscript® Kit (Ambion) according to manufacturer’s instructions. RNA quality and integrity was confirmed using Nanodrop 2000 (Thermo scientific), and stored at −20 °C.
One-step sRT-qPCR and one-step mRT-qPCR
The one-step sRT-qPCR reaction was used to detect each control RNAs or viral RNA using specific primer, probe and viral RNA. The one-step mRT-qPCR reaction contained all three control RNAs or viral RNAs, a mixture of all three primer pairs and probe in the same tube. The one-step sRT-qPCR and one-step mRT-qPCR were tested using AgPath-ID One-Step RT-PCR Kit (Applied Biosystems). The reaction contained 12.5 μl 2x RT-PCR buffer, 1 μl 25x RT-PCR enzyme mix, 5 μl RNA template, 5 μl primer-probe mix (Final concentration of 3.2 μM for each primer and 200nM for each probe) and RNase free water to final volume of 25 μl. All reaction were performed on a BioRad CFX96 with the following cycling parameters; 45 °C for 10 min, 95 °C for 10 min and then 45 cycles of 95 °C for 15 s 52 °C for 20s. The primer and probe concentrations for each assay were individually optimized using in-house protocol. Each control RNAs and viral RNAs were quantified using Nanodrop 2000 (Thermo scientific).
Sensitivity of one-step sRT-qPCR and one-step mRT-qPCR
The sensitivity of one-step sRT-qPCR and one-step mRT-qPCR was evaluated on serial ten-fold dilutions of control RNA or viral RNA. For spiking assay, serial ten-fold dilutions of the control RNAs were used to spike each of the matrix nucleic acid extracted from the ten samples (e.g. Bovine whole blood, serum and brain, Goat serum, and Culicoides). Also, serial ten-fold dilutions of the viruses were used to spike each of the ten samples (e.g. Bovine whole blood, serum, brain, Goat serum, Culicoides), from which RNA was extracted using Maxwell®16 viral total nucleic acid purification kit (Promega AS1150). The matrix nucleic acid and the samples used for the spiking assay were all tested negative against SBV, AKAV, and AINV, prior to use. Ten-fold serial dilutions of the control RNA or viral RNA were prepared from starting solutions containing 2.4 × 106 copies (10 5.6 TCID 50/ml) for SBV, 9.62 × 106 copies (10 5.5 TCID 50/ml) for AKAV, and 5.23 × 106 copies (10 5.2 TCID 50/ml) for AINV.
Inter assay and intra assay
Reproducibility of the Inter- and Intra-assay were examined in triplicate using ten-fold serial dilutions of the three control RNAs (starting solutions containing 2.4 × 106 copies for SBV, 9.62 × 106 copies for AKAV, and 5.23 × 106 copies for AINV).
Specificity of one-step sRT-qPCR and one-step mRT-qPCR
Bovine ephemeral fever virus (10 4.1 TCID 50/ml, KVCC-VR1300041), Chuzan virus (10 3.2 TCID 50/ml, KVCC-VR000109), Ibaraki virus (10 4.6 TCID 50/ml, KVCC-VR000109), West Nile fever virus NY99 stain (10 6.1 TCID 50/ml) West Nile fever virus B956 strain (10 6.7 TCID 50/ml), Japanese encephalomyelitis virus (Anyang strain, 10 7.1 TCID 50/ml, KVCC-VR1200026) and bluetongue virus (10 4.5 TCID 50/ml, RSArrr001). West Nile fever viruses were acquired from ATCC and bluetongue viruses were kindly provided by the Pirbright Institute. The other viruses were received from the Korean Veterinary Culture Collection (KVCC). Each probe was tested against three control RNAs and the viral RNA.
Bovine whole blood samples (n = 112) were collected at abattoir in Jeju island in 2013. Bovine and goat serum samples (n = 100) were collected with 20 samples being collected from national surveillance of Foot and Mouth disease by the Animal and plant Quarantine Agency (QIA) from each 5 farms located in various provinces. Bovine brain samples (n = 123) initially were submitted for etiological findings to the Animal and plant Quarantine Agency (QIA), Culicoides samples (157 pools) of various species were collected through the national vector surveillance program for arboviral infectious disease in collaboration with local entomological experts . Samples were maintained at −70 °C until used. The collected clinical samples were tested by one-step mRT-qPCR and real-time reverse-transcriptase PCR for AKAV and AINV, and real-time reverse-transcriptase PCR for SBV as described previously [1, 13].