title: RNA seq of DENV1/3 EDI
design: Four chimeric DENV1/3 EDII, 4 EDIII and 4 DENV3/3 chimeric viruses were designed using our previously described tetrapartite DENV1 and 3 reverse genetics platforms (Messer et al., 2012; Messer et al., 2016; Young et al., 2020).  Briefly, 4 DNA fragments, which are flanked by class IIS restriction endonucleases, are excised from agarose gels and ligated together to reestablish an intact, DENV 1 wildtype and derivative genomes encoding chimeric E glycoproteins.  To generate chimeric E glycoproteins, DENV3 EDI, II and III sequences were synthesized in the DENV1 E glycoprotein backbone encoded in the A clone by BioBasic and assembled using Golden Gate cloning and molecular techniques. The DENV3/3 genotype chimeras encoded DENV3 G-III changes in the DENV3 G-IV A clone from BioBasic and was assembled as above. Full-length viral RNA was electroporated into Vero81 cells, a non-human primate cell line, or in C636 mosquito cells and incubated for 4 to 7 days at 37 or 32C, respectively. The supernatant was harvested, and cells were removed by centrifugation before freezing at -80C. Virus stocks were titered by fluorescent foci assay as previously described by our group (Young et al., 2020) and were subject to full-genome sequencing as previously described (Tse et al., 2022).