PRJNA686429
Experimental evolution of Zika virus in Huh7.5.1 cells expressing deltaTIR TLR3
Type sample/origin:A clinical isolate of ZIKV from Brazil collected from a patient during the epidemics (PE243_KX197192) was amplified using Vero E6 cells.Supernatants were harvested and filtrated (0.45 micron) before storage at -80C. The serial passaging of viral populations was performed by inoculation of Huh7.5.1 cells expressing deltaTIR TLR3.At the indicated time, supernatants were harvested from infected cells. To avoid contamination of the libraries by cellular RNA released from dying cells, an additional amplification step was performed by passaging of the supernatant with the corresponding cells for 2 days, using MOI of 0.1. Supernatants (SN, 10ml) were concentrated using Vivaspin units with 100 MWA cut-of (Sartorius) by centrifugation at 3 000 g for 20 min at room temperature. Viral RNAs were extracted from the concentrated SN by phenol-chloroform extraction procedure.Preparation of sequencing librariesNext, RNAs were fragmented by sonication using Covaris M220 (peak incident 50 Watts; duty cycle 20%; 200 cycles per burst, time 200 seconds at 4C) using Covaris M220. Fragmented RNAs were concentrated with isopropanol precipitation, followed by analysis with Bioanalyzer (Agilent Technology) using Pico Total RNA Chip. The median size of the RNA fragments was around 150 nucleotides. We then generated tandem repeats of the fragment to reduce the error rate of next-generation sequencing (8). The steps of circularization of RNA fragments and retrotranscription were adapted from a previously described protocol (Acevedo et al. 2014). Briefly, circular RNAs were obtained by ligation using T4 RNA ligase (New England Biolabs), followed by phenol-chloroform extraction. The retrotranscription was performed using Superscript III (Life Technology). Then, libraries were prepared using the Ultra DNA Library Prep kit for Illumina (New England Biolabs). Additional steps of clean-up were performed using AMPure XP beads, ratio 1:0.8 (Agencourt) to remove free adaptors (done before the step of PCR enrichment of adaptor-ligated DNA) and to remove the free index adaptors (after the PCR enrichment of adaptor-ligated DNA). The quality of the libraries was assessed by Bioanalyzer (Agilent Technology) using High Sensitivity DNA Chip. The libraries were quantified using NEB Next Libraries Quantification kit for Illumina (New England Biolabs) and multiplexed at equimolarity.Deep sequencing methodsMultiplexed libraries were sequenced using HiSeq 2500XL (Illumina), using a 200-PE run at the EMBL Genecore Facility (Heidelberg, Germany).References:A. Acevedo, L. Brodsky, R. Andino, Mutational and fitness landscapes of an RNA virus revealed through population sequencing. Nature 505, 686-690 (2014).