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Department of Microbiology & Immunology, University of Texas Medical Branch

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Namespace:Department of Microbiology & Immunology, University of Texas Medical Branch

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Protocols: Yellow Fever infectious clones (IC) were generated by site-directed mutagenesis (SDM) of either pANCR 17D (IC) or pANCR Asibi IC as a template. QuickChange XLII SDM kit (Strategene) along with mutagenic primer sets were used to generate infectious clones as per the manufacturer’s protocol. Stock cDNA YF IC were amplified in E. coli (MC1061) and plasmid harvested by large-scale extraction. Full-length YF RNA was generated by in vitro transcription of purified cDNA using SP6-Scribe standard RNA in vitro transctiption kit (Cellscript) according to the manufacturer’s protocol; the reaction was incubated at 37oC for 2.5 hours. The entire reaction was electroporated into 6.7x106 monkey kidney Vero cells. Cell supernatant was collected when infected cells showed 80% cytopathic effect (CPE). RNA from the YF ICs were extracted using QIAamp Viral RNA mini kit (Qiagen), following the manufacturer’s guidelines. NGS libraries were constructed using TruSeq RNA v2 kit as recommended by the manufacturer.