Dengue virus serotypic replacement of NS3 protease or helicase domain causes chimeric viral attenuation but can be recovered by a compensated mutation at helicase domain or NS2B, respectively.
Supernatants (~10 ml) were collected from BHK-21 or C6/36 cell cultures in T-75 flasks. Viral particles were precipitated by centrifugation at 15,000 g for 45 minutes in 40 % PEG8000 solution containing 10 mM Tris (pH8.0), 120 mM NaCl and 1 mM EDTA. After removing the supernatants, the remaining 0.5 ml solutions at the bottom of the tubes, containing the precipitates, were repeatedly mixed with 1.5 ml Trizol-LS (Ambion) and 0.4 ml chloroform. After the centrifugation of these mixed solutions at 12,000 g for 5 minutes, the upper layer, separated from Trizol/chloroform (bottom layer) was used for ethanol precipitation to collect viral RNA. The ethanol-precipitated pellet was dissolved by Tris (10 mM, pH8.0)-buffered DEPC-water and was purified with RNA clean & concentratorTM kit (Zymo Research). Library preparation and RNAseq sequencing were performed at Zymo Research Inc. (Irvine, CA), where each RNA sample (260 ng) was purified using Zymo-Seq RiboFree Total RNA Library kit (Zymo Research). Ribosomal RNA was removed from RNA samples, which were then reverse-transcribed to cDNAs. The produced cDNAs were ligated with P7 adaptor sequence at 3 end, followed by second strand synthesis and P5 adaptor ligation to the opposite sites of the double stranded DNAs. After purification by DNA size (300-600bp) with beads in the kit, index PCR was performed; initial denaturation at 95 C for 10 min; 16 cycles of denaturation at 95 C for 30 sec, annealing at 60 C for 30 sec, and extension at 72 C for 60 sec; and final extension at 72 C for 7 min. Successful library construction was confirmed with Agilents D1000 ScreenTape Assay on TapeStation. Libraries were sequenced on an Illumina Novaseq to a sequence depth of >30 million read pairs (150 bp paired-end sequencing).