Plasmid construction.

MRV T1L rescue plasmids pT7-L1T1L, pT7-L2T1L, pT7-L3T1L, pT7-M1T1L, pT7-M2T1L, pT7-M3T1L, pBacT7-S1T1L, pT7-S2T1L, pT7-S3T1L, and pT7-S4T1L were prepared as described previously (12, 44). To create pT7-L1T1L-NLuc, which contains the NLuc gene (GenBank accession number {"type":"entrez-nucleotide","attrs":{"text":"KM359774","term_id":"692659521","term_text":"KM359774"}}KM359774), and the self-cleaving P2A peptide gene of porcine teschovirus-1 (GSGATNFSLLKQAGDVEENPGP), the NLuc-P2A gene cassette was amplified and inserted between nucleotides 124 and 125 of the L1 gene. To express NLuc-P2A as a fusion peptide with the L1 ORF N-terminal region, additional nucleotides (CC) were inserted before the NLuc-P2A cassette. Partial L1 gene nucleotide sequences spanning positions 19 to 124 were duplicated after the NLuc-P2A cassette to reconstitute the λ3 ORF.