Ready to go kit

Genomic DNA was extracted for genotyping using PCR, as previously reported (Edwards et al., 1991 ). Total RNA was prepared with TRIZOL reagent (Invitrogen, Carlsbad, CA, USA) and TURBO DNA-free Kit (Applied Biosystems, Foster City, CA, USA). cDNA synthesis was performed with Ready-To-Go Kit (GE Healthcare Life Sciences, Stockholm, Sweden) and analyzed by reverse transcription PCR (RT-PCR). PCR primers were designed by Primer3². Quantitative real-time PCRs (qPCRs) with iQ SYBR Green Supermix (Bio-Rad, Techview, Singapore) were performed by using MyiQ thermocycler and iQ5 optical system (Bio-Rad). qPCR data were normalized to the internal control of PP2AA3 (PP2A) (Czechowski et al., 2005 (link)).

Total RNA was extracted from the grinding fluid of sandfly specimens and the supernatants of infected BHK-21 cells and C6/36 cells using the Viral RNA Mini Kit (QIAamp; Qiagen, Valencia, CA, USA), according to the manufacturer’s instructions. The extracted RNA was immediately incubated in a 65 °C water bath for 10 min and then quickly transferred to an ice bath for 2 min. RNA (32 µL) was added to the first-strand reaction tube of the Ready-To-Go kit (GE Healthcare, Little Chalfont, Buckinghamshire, UK). The reaction tube was incubated at room temperature for 1 min, and then 1 µL random primer (pd(N)6) (TaKaRa, Shiga, Japan) was added, before further incubation at 37 °C for 1 h. The total volume of the resulting cDNA library was 33 µL, which was used immediately or stored at −40 °C for further use [10 (link),14 (link),15 (link)].

Viral DNA was extracted from 200-μL aliquots of virus-infected BHK-21 cell culture supernatants using a QIAamp DNA BloodMini Kit (Qiagen). Viral RNA was extracted from 140-μL aliquots of virus-infected BHK-21 cell culture supernatant using a QIAamp Viral RNA Mini Kit (Qiagen) according to the manufacturer’s instructions. cDNA was made with a Ready-To-Go kit (GE Healthcare) using random hexanucleotide primers. Samples were then amplified as described previously [9 (link)]. Amplification products were sequenced at the National Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention. The Ion Sequencing Kit (Life Technologies) was used with the Personal Genome Machine (PGM) sequencer as described in the Ion Sequencing Kit User Guide [17 (link)] (part no. 4467391 rev. B, 04/2011).
RT-PCR was performed to fill in gaps between viral gene sequences obtained with Ion Torrent sequencing using contig-specific primers. Total viral RNA was extracted as described above, cDNA was generated by reverse transcription, and used as a template for complete genome amplification. Next, a set of specific primers was designed to amplify each segment of the viral genome and the amplification products were sequenced using the Sanger method (Table 2).

Total RNA was extracted from 140μl of BHK-21 or C6/36 cell supernatants using QIAamp Viral RNA Mini Kit (Qiagen) according to the manufacturer’s instructions. Next, the cDNA of the three TIBOV virus strains was synthesized using a Ready-To Go kit (GE Healthcare) using random hexanucleotide primers. The whole viral genome of the three TIBOV strains was then amplified using the 10 gene segment-specific amplification primers. Next, their nucleotide sequences in the gene amplification products were determined (Li et al., 2014 (link)). The viral genome sequences for the three virus isolates were deposited in the GenBank under the accession numbers (XZ0923: OR712119-OR712128(10 segments); HN11121: OR712129-OR712138(10 segments); HNQZ1927: OR712139-OR712146(8 segments, 2 segments were not sequenced successfully)).

Total RNA was extracted from cells using the RNeasy Mini Kit (Qiagen). Concentrations were measured with a Nanodrop spectrophotometer. Only samples with ratios between 1.8 and 2.0 were further analyzed. A total of 1 μg of total RNA was reverse-transcribed using the Ready-To-Go kit (GE Healthcare). Reverse-transcribed material (5 ng for each sample) was amplified using the TaqMan Universal PCR Master Mix (Applied Biosystems) and the appropriate primers to evaluate gene expression (probe ID available upon request) in the 7500 Real Time PCR System. The expression levels for all genes were normalized to the average levels of the housekeeping gene ACTB and referred to the relevant control samples.

RNA was extracted from all collected samples (serum, CSF, and mosquito grinding supernatant) using a Tianlong Nucleic Acid Automatic Extractor (model: Np968.c; Suzhou Tianlong Biotechnology Co., Ltd., Jiangsu, China) with the Tianlong Nucleic Acid Extraction Kit (EX-RNA/DNA virus, Suzhou Tianlong Biotechnology Co., Ltd, Suzhou, China). All operations were carried out in accordance with the manufacturer’s instructions [18 ]. The Ready-To-Go kit (GE Healthcare, Little Chalfont, Buckinghamshire, UK) and random primers (pdN6) (TaKaRa, Shiga, Japan) were used to prepare the cDNA library [13 (link),18 ].
qRT-PCR detection of JEV and WNV was performed on extracted RNA using the Stratagene real-time PCR instrument (model MX300; Thermo Fisher Scientific, Waltham, MA, USA) and the AgPath-IDTM One-step RT-PCR Kit (AM1005, Thermo Fisher Scientific, USA). Universal primers and probes for JEV (detect JEV genotype I, III and V) [18 ] and specific primers and probes for WNV [19 ] were used. Then the positive samples detected by qRT-PCR with JEV universal primers were amplified using primers specific for JEV E gene. The PCR products were subjected to nucleotide sequence determination and JE virus phylogenetic analyses were used to confirm the JEV genotype.