Nucleospin rna virus isolation kit

Viral RNA was extracted with the NucleoSpin RNA virus isolation kit (Macherey-Nagel) after the manufacturer’s instructions. 1 μl of viral RNA was applied to each reaction. Quantification of genomic SARS-CoV RNA was done using the SuperScript III one-step reverse transcriptase-PCR system (Invitrogen) with the Platinum Taq DNA polymerase according to the manufacturer’s recommendations and these primers (SARS-F: 5’-CCCGCGAAGAAGCTATTCG-3’, SARS-P: Fam-5’-ACGTTCGTGCGTGGATTGGCTTTG-3’-BHQ, SARS-R: 5’-AGTTGCATGACAGCCCTCTACA-3’). An established SARS-CoV standard curve was used in each run [69 (link)] to quantify RNA copies per ml.
To analyze IFN-β expression in Calu-3 and Rhinolophus alcyone cells (provided by authors Christian Drosten and Marcel A. Müller), mRNA was extracted with the NucleoSpin RNA isolation kit (Macherey-Nagel). Real-time PCR was done as described previously [70 (link)] using the following primers: Hu-IFNB1-F: 5’-AGGATTCTGCATTACCTGAAGG-3’, Hu-IFNB1-P: Fam-5’- TCCACTCTGACTATGGTCCAGGCA-3’-ZEN and Hu-IFNB1-R: 5’-GGCTAGGAGATCTTCAGTTTCG-3’ (Calu-3) and Rhi-IFNB1-F: 5’-AAATAATGGAGGAGGAAAACTTCAC-3’, Rhi-IFNB1-P: Fam-5’-CGAAACATGAGCACGCTGCACCTG-3’-BHQ and Rhi-IFNB1-R: 5’- CGCCTGATCCTTAGGTAGTAATTCT-3’ (Rhinolophus alcyone). To determine the induction of IFN-β relative to TBP (Calu-3) and β-actin (Rhinolophus alcyone) the 2^−ΔΔCt method was applied [55 (link)].

RNA was extracted from selected samples from P5 of the passaging experiment and from the original C6/36-derived BgV stock using the Macherey Nagel Nucleospin RNA Virus isolation kit, following the manufacturer’s protocol–without carrier RNA. This RNA was sequenced as described in the earlier section detailing the sequencing of BgV/WNV-prME on a NextSeg 500 (see above). The reads were assembled to the published sequence of BgV (KU308380.1) using Geneious 8.1.9 with default parameters.
RNA was extracted from all other BgV positive supernatants using the same kit following manufacturer’s instructions. The mutation sites were amplified by RT-PCR using SuperScript III One-Step RT-PCR System with Platinum Taq DNA Polymerase (Invitrogen) and BgV specific primers (see S1 Table). The resulting PCR products were resolved on a 1% agarose gel, excised and gel purified using Nucleospin gel and PCR clean up kit (Macherey Nagel), before they were sent for Sanger sequencing at the AGRF. The sequences were aligned to a BgV reference and each other in Geneious 8.1.9.

PCR products were sequenced using BigDye technology and processed by Sanger sequencing by the Australian Genome Research Facility (AGRF). The resulting chromatograms were analysed using Contig Express (Vector NTI, Informax, Gaithersburg, MD, USA).
Viral RNA was extracted from samples using the Macherey Nagel Nucleospin RNA Virus isolation kit, following the manufacturer’s protocol. First strand cDNA was transcribed using Superscript III (Invitrogen, Carlsbad, CA, USA) according to the manufactures instructions and amplified using Phusion (Finnzymes, Thermo Fischer Scientific, Queensland, Australia) and sequenced by AGRF. The resulting chromatograms were analysed using Contig Express (Vector NTI, Informax, Gaithersburg, MD, USA).