Obtaining IBV-M41 S1 Gene Sequence

To obtain the fragment coding for the M41-S1 gene, RNA was isolated from a virus stock of IBV-M41 (Animal Health Service, The Netherlands) using the QIAamp viral RNA Mini Kit (Qiagen, Germany). Reverse transcription was performed using the Transcriptor First Strand cDNA Synthesis Kit (Roche, Switzerland) with random hexamers according to the manufacturer’s protocol. PCR was performed with Phusion Hot Start II High-Fidelity DNA Polymerase (Thermo Fisher Scientific) using the primers listed in Table 1. To exchange the S1 domain of IBV-H52 for that of M41 in the plasmid used for targeted recombination, the M41-S1 PCR product and the previously generated plasmid containing the H52-S gene [16 (link)] were digested using restriction enzymes PacI and SnaBI (both New England Biolabs, USA), ligated and subsequently transformed in HB101 E. coli. Sequences were confirmed by automated nucleotide sequencing (Macrogen, The Netherlands). A step-wise ligation approach was used to obtain the H52 M41-S1 donor plasmid. To introduce mutations leading to individual N-to-A substitutions in N-glycosylation sites in the receptor binding domain (RBD) sequence, site-directed mutagenesis was performed using the primers listed in Table 1. The sequences of the plasmids containing the S1 gene with the introduced mutations were confirmed by automated nucleotide sequencing (Macrogen, The Netherlands).