E-MTAB-6614:Sample 14_p
Illumina HiSeq 1500 paired end sequencing; Structural and non-structural genes contribute to the genetic diversity of RNA viruses
Structural and non-structural genes contribute to the genetic diversity of RNA viruses
Sample 14_p
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.
Department of Microbiology & Immunology, University of Texas Medical Branch
E-MTAB-6614:Sample 14_p
Experimental Factor: strain
17D-204
Experimental Factor: genotype
NS2B-L109I substitution