TITLE: Phylogenetic analyses of the JEV gene sequence
AUTHORS: Wenjing  Liu, Shihong Fu

[DESCRIPTION]
The E gene was amplified by semi-nested PCR. 

[STEPS]
SECTION: Nucleic acid extraction
1. Use Tianlong nucleic acid extraction instrument (automatic nucleic acid extraction instrument, model: np968. C, manufacturer: Suzhou Tianlong Biotechnology Co., Ltd.), and supporting nucleic acid extraction kit: Tianlong nucleic acid extraction kit (magnetic bead method EX-RNA/DNAvirus), manufacturer: Suzhou Tianlong Biotechnology Co., Ltd., for nucleic acid extraction. Perform the following operations in a biosafety cabinet. Add 200 μl of patient cerebrospinal fluid and serum specimens and mosquito grinding supernatant to each well in columns 1 and 7 of the 96-well plate provided with the nucleic acid extraction kit
SECTION: cDNA library preparation
2. 33 μl of RNA extract was placed in a 65 ° C water bath for 10 min.
SECTION: cDNA library preparation
3. Ice bath for 2 min
SECTION: cDNA library preparation
4. 32ul of RNA samples were pipetted into the first chain reaction tube in a Ready-To-Go kit kit (GE Healthcare, Little Chalfont, Buckinghamshire, UK) and allowed to stand at room temperature for 1 min. 
SECTION: cDNA library preparation
6.  37 ° C water bath for 1 h.
SECTION: cDNA library preparation
5. 1 ul of random primer (pdN6) (TaKaRa, Japan) was added to every sample.
SECTION: The E gene  amplification
7. The E gene sequence of JE virus was amplified by semi-nested PCR. The first round of amplification , using primers: JEV 3F: TTCATAGAAGGAGCCAGTGGA and JEV 3R: TCGTTTAAACTCGCGACTGA.he gene amplification system was 25 ul, including: 2 μl of cDNA template, 12.5 μl of GoTaq ® Green Master Mix-2 × (Promega, Madison, WI), 1 μl of upstream and downstream primers at 10 umol/L, and 8.5 μl of RNase Free Water. The reaction program was: 94 ° C, 8 min for 1 cycle; 94 ° C, 1 min, 55 ° C, 1 min, 72 ° C, 1 min for 35 cycles; 72 ° C, 10 min, keep in 4 ° C.    The second round of amplification sequence was 300 bp, using the first round of PCR product as a template with the forward primer JEV 3F and the reverse primer JEV 2R: TTTCCCGAAAAGTCCACATC.
SECTION: The E gene  amplification
8.  The second round of amplification sequence was 300 bp, using the first round of PCR product as a template with the forward primer JEV 3F and the reverse primer JEV 2R: TTTCCCGAAAAGTCCACATC,the amplification system and reaction program was same as the first round.
SECTION: C+PrM gene  amplification
9. The C+PrM gene sequence was also amplified by semi-nested PCR according to the same amplification system and  procedure as E gene. The primers used for the first round of PCR were JE-C+PrM-1F (CGTTCTTCAAGTTTACAGCATTAGC) and JE-C+PrM-1R (CCYRTGTTYCTGCCAAGCATCCAMCC)
SECTION: C+PrM gene  amplification
10. primers used for the second round were JE-C+PrM-1Fand JE-C+PrM-2R (CGYTTGGAATGYCTRGTCCG). The product of the first round of PCR was used as the template of the second round.The second round also use the same amplification system and  procedure as E gene.
SECTION: agarose gel electrophoresis
11. 5 µL of the amplification product was detected by 1% agarose gel electrophoresis .
SECTION: Nucleotide sequence determination
12. Nucleotide sequence determination was done by Sangon Biotech Co. Ltd. (Shanghai, China, Beijing Sequencing Department).
SECTION: Phylogenetic analyses
13. The viral gene nucleotide sequences were spliced and corrected using SeqMan II software (DNA Star, Madison, WI, USA). 
SECTION: Phylogenetic analyses
14. The JEV gene sequences used for phylogenetic analysis were downloaded from GenBank( Table S1) .
SECTION: Phylogenetic analyses
15. The ClustalW multiple sequence alignment was performed using BioEdit (Version 7.0, Hall 1999).
SECTION: Phylogenetic analyses
16. Neighbor-joining phylogenetic trees were drawn using MEGA6.0 with 1000 bootstrap replicates.