CVS-11 Nucleoprotein Gene Amplification

The complete nucleoprotein gene of challenge virus standard (CVS-11) was amplified using primers Lys001 (5’-ACGCTTAACGAMAAA-3’) and 304 (5’-TTGACAAAATCTTCTCAT-3’) as described previously [33 (link)]. Amplification products were purified using the Zymoclean Gel DNA Recovery Kit (Zymo Research) followed by cloning using the pGEM-T Easy vector system (Promega) according to the manufacturer’s instructions. Recombinant clones were characterized by sequencing using the BigDye Terminator V3.1 cycle sequencing kit (Thermo Fisher Scientific) and an ABI3500xL genetic analyser (Applied Biosystems) to determine orientation. A single recombinant clone containing the insert in the correct orientation with regard to the SP6 promoter was selected, and the insert was in vitro transcribed using the MegaScript kit (Ambion) according to the manufacturer’s instructions. In vitro transcribed RNA was purified using the RNA Clean and Concentrator-25 kit (Zymo Research) and quantified using the Qubit 3.0 fluorometer (Invitrogen). Contamination with plasmid DNA was ruled out with no-RT controls.