Recombinant wild-type and S510 and S598 variants of JHMV were generated as previously described [48, 49] . Briefly, overlapping extension polymerase chain reaction (PCR) was used to generate the S598 Q600Y and S510 W513R variants. Primers that overlapped the glutamine at residue 600 of the spike glycoprotein were (59 to 39) Q600Y fwd, ATGATCGCTGCTATATTTTTGCTAACATAT-TG; Q600Y rev, AATATGTTAGCAAAAATATAGCAGCGAT-CAT. Primers that overlapped the tryptophan at residue 513 were (59 to 39) W513R fwd, GTGAGTGTTCTCTTCGGAATGGGC-CCCATTTGCGCTCGGC; W513R rev, AGCGCAAATGG-GGCCCATTCCGAAGAGAACACTCAC. The outer primers for each targeted change were fwd, TGTTGATTGCGCCAG-CAGCTACATTAG; and rev, ACCTACGGATTGAACGCTAT-CATTGAC. Underlined nucleotides correspond to the nucleotides encoding the Gln to Tyr and Trp to Arg substitutions within S598 and S510, respectively. Recombinant viruses encoding the variant epitope(s) were selected, propagated and titered as previously described [49] . At least two independent isolates of each recombinant virus were analyzed.