Purelink mini total rna purification kit

For examining the inhibitory effect of tubacin on the synthesis of viral antisense RNA genomes, total RNAs of TE671 cells with JEV at an MOI of 0.1 were extracted using PureLink Mini Total RNA Purification Kit (ThermoFisher) 36 h post treatment tubacin, reverse transcripted into cDNA with antisense RNA-specific capture primer (5′-GCAGCAGAAGGAAAGACC GTGAT-3′), and followed by measuring antisense RNA genomes using SYBR Green Master Mix kit with JEV-specific primer pairs (5′-TCCACTTCCTCAACGCAATG-3′ at nucleotide 9724–9743 and 5′-CAGTCGTGCCAGCCATG-3 at nucleotide 9799–9783). The Real-time RT-PCR was performed by 7300 Realtime PCR system (Applied Biosystems, Foster City, CA, USA), and then the corresponding threshold cycle value (C_t) was measured. Relative levels of RNA genomes were normalized by the housekeeping gene GAPDH, described in a prior report [26 (link)].

For examining the synthesis of positive- and negative-sense RNA subgenomes, total RNAs of the transfected TE671 cells with JEV-EGFP replicon were extracted using PureLink Mini Total RNA Purification Kit (ThermoFisher, Waltham, MA, USA), reverse transcripted into cDNA with specific-capture primers, and followed by measuring positive- and negative-sense RNA subgenomes using SYBR Green-based real time PCR with JEV-specific primer pairs (Figure S3). Relative levels of RNA subgenomes were normalized by glyceraldehyde 3-phosphate dehydrogenase (GAPDH), described in a prior report [36 (link)]. To explore the expression of EGFP and JEV proteins, transfected cells were rinsed once with PBS, fixed with 4% formaldehyde in PBS at room temperature for 30 min, and permeabilized 0.1% Triton X-100, 10% BSA in PBS at room temperature for 1 hr. After blocking with 5% bovine serum albumin in PBS, permeabilized cells were stained using primary rabbit polyclonal antibodies, including anti-JEV E protein, anti-JEV NS3, and anti-JEV NS4B (GeneTex, Inc., Irvine, CA, USA), followed by the incubation with secondary AF546 goat anti-rabbit IgG (ThermoFisher, Waltham, MA, USA) in dark box for 2 h. After washing with PBS, stained cells were photographed using the immunofluorescence microscopy (Olympus, BX50, Tokyo, Japan).

A JEV-EGFP replicon, previously constructed in our laboratory^{10 (link)}, was used to explore whether CW-33A influenced viral protein translation and RNA genome replication stages of JEV life cycle. TE-671 cells were transfected with JEV-EGFP replicon and simultaneously treated with 10 μM of CW-33A or CW-33. After 36 hours post treatment, the images of replicon-derived EGFP reporter in JEV-EGFP replicon-transfected cells were taken by fluorescent and optical microscopies. The fluorescent intensity of EGFP reporter in treated transfected cells was quantified by Image J. For detecting the synthesis of JEV sense and antisense genomes, total RNAs of replicon-transfected TE-671 cells treated with CW-33A or CW-33 were purified using PureLink Mini Total RNA Purification Kit (ThermoFisher), and performed using SYBR Green-based real time PCR with JEV-specific primer pairs, as described in our prior report [9]. The corresponding threshold cycle value(C_T) for each sample was measured by 7300 Realtime PCR system (Applied Biosystems). Relative levels of JEV sense and antisense genomes were normalized by glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and then quantitated.