*2.2. Viruses*

RABV isolates used in this study comprised five recombinant virus clones and one non-recombinant lab virus. The recombinant field virus clones rRABV Dog and rRABV Fox have been described before [42]. SAD L16 is a recombinant virus clone of attenuated vaccine virus SAD B19 live vaccine virus [43]. The Evelyn Rokitnicki Abelseth (ERA) strain [44] is a progenitor virus strain of live vaccine virus SAD B19 [45] and was obtained from the FLI virus archive (FLI ID N 12829).

A cDNA full length clone (pRABV Rac) from a raccoon RABV isolate (Alabama, USA 1991; FLI archive ID N 13205) [46] was generated by full length RT-PCR amplification of the 12 kb cDNA genome with the primer pair 5-TCGATCCCGGGTCACGCTTAACAACAAAA-3/ 5-TAATACACCTGCCCATGCCGACCCACGCTTAACAAAAAAACAA-3. After PCR amplification of a 2.7 kb vector DNA fragment from pCMV HaHd ampR Br 322 ori with the primers 5-TCTGTTTGCTTGATGGTTTTTTTTGTCTTTGTTGTTTTTTTGTTAAGCGTGGGTCGGCATGGC ATCTCCAC-3 and 5-TTTTTGTAGATGATACTGTCTACTTCTTCTCTGATTTTGTTGTTAAGCGTGA CCCGGGACTCCGGGTTTCGTC-3, the fragments were combined by linear to linear recombination, and the resultant recombinant rRABV Rac virus was rescued and amplified according to previously described protocols [42]. The sequence of the full-length cDNA clone was deposited at GenBank (accession no. MN862283).

A CVS-11 (Challenge Virus Street-11; sequence accession no. LT839616) cDNA full length clone (pCVS-11) was generated by RT-PCR amplification of 7.5kb and 3.7 kb cDNA fragments from CVS-11 RNA with the primers pairs 5-AGTTTCAGACGTCTCAGTC-3/5-CTAGTAGGGATGATCTAGATC-3 and 5-GACTGAGACGTCTGAAACT-3/5 CATTGCAGATAGGATAGAG-3, respectively. The resultant fragments were assembled in combination with EcoRI-linearized 3.4 kb vector plasmid pCVS-11-termini by Hot Fusion reaction [47]. Plasmid pCVS-11-termini was generated by PCR amplification of a 2.7 kb DNA fragment from pCMV HaHd ampR Br 322 ori [42] with the primers 5-GACCCGGGACTCCGGGTTTC-3 and 5-GGGTCGGCATGGCATCTCCA-3, and Hot Fusion assembly with a synthetic DNA fragment containing the CVS-11 (accession no. LT839616) regions from nucleotide positions 1–580 (3-genome end) and 11759–11927 (5-genome end) separated by a non-viral EcoRI restriction site. The full-length cDNA clone was 99.99% identical to CVS-11 GenBank sequence no. LT839616 (1 nt mismatch in the N gene at nucleotide position 1263, which was already present in #LT839616 as a minor SNP (single nucleotide polymorphism), resulting in the amino acid exchange E398V). Recombinant rCVS-11 was rescued in Na42/13 neuroblastoma cells by co-transfection of pCVS-11, pCAGGS-based expression plasmids for RABV N, P, and L [48], and a pCAGGS-T7Pol vector comprising a codon-optimized bacteriophage T7 RNA polymerase gene. Three to six days after transfection, the supernatants were transferred to new Na42/13 cells. Two days after the transfer, infectious virus was identified by N and G protein-specific indirect immunofluorescence.

rRABV Dog, rRABV Fox, rRABV Rac, and rCVS-11 were amplified on Na42/13 cells. BSR T7/5 cells [49] were used for amplification of SAD L16 and ERA viruses. Infectious virus titers in cell culture supernatants were determined by end point dilution and titration on Na42/13 cells.
