Pbha vector

The strategy used to construct pBR-1,2PDO is presented (Additional file 1: Fig. S2) The pduP, pct, and yahK genes were synthesized by Bioneer Co. Ltd. (Korea). These sequences were cloned into the pBHA vector (Bioneer, Daejeon, South Korea), followed by nucleotide sequencing to confirm there were no errors introduced during cloning. A NheI-SpeI fragment containing the pct gene was inserted into the corresponding restriction sites downstream of the pduP sequence (pBHA-pdup). pBHA-pduP-pct-yahK was generated by sequential insertion of NheI-SpeI fragments containing pct (pBHA-pct) and yahK (pBHA-yahk) into the SpeI sites of pBHA-pduP. The lacZ promoter sequence (P_lacZ) was synthesized by Bioneer Co. Ltd. (Korea). These DNA fragments were cloned into the pGEM-T Easy vector, followed by nucleotide sequencing to confirm there were no errors introduced during cloning. pGEM-P_lacz-pduP-pct-yahK was obtained by inserting the NheI-SpeI fragment (containing pduP-pct-yahK from pBHA-pduP-pct-yahK) into the SpeI sites of pGEM-PlacZ. Finally, pBR322 was cleaved with ScaI, treated with alkaline phosphatase, ligated with a DNA fragment obtained by digestion of pGEM-PlacZ-pduP-pct-yahK with NotI, and then treated with the Klenow fragment to yield plasmid pBR-1,2PDO (pduP-pct-yahK). Electroporation was used to transform the final plasmid into K. pneumoniae [48 ].

The JEV GI/GIII intertypic virus was constructed with the K05GS envelope protein in the SA14-14-2 backbone. The prM/E proteins of the K05GS strain were codon-optimized and cloned into the pBHA vector with Bioneer to effectively express the target genes in humans. The cDNA of JEV SA14-14-2 was copied into the pACYC184 vector, and the E region of the infectious cDNA clone SA14-14-2 was changed with the in vitro-synthesized E region of Syn_GI. The plasmid that the prM/E region of the K05GS strain codon-optimized was Syn_GI, and it was subcloned into the pACYC184 vector by Bioneer. The E regions of K05GS and SA14-14-2 backbones were amplified with specific primers (Table S1), and each DNA fragment was cloned into the corresponding region with the In-Fusion Cloning System (Takara Bio, Seoul, Korea). After the cloning, plasmids were amplified from Escherichia coli HST08 cells.
To create the virus, we conducted transfection in BHK-T7 cells with Lipofectamine^® 2000 Reagent (Invitrogen™, Carlsbad, CA, USA). Cells were maintained in DMEM with 10% heat-inactivated FBS before transfection. After three days, the transfected cells were thawed thrice to obtain the viral supernatants. The complete recombinant infectious clone was termed SA14-GI env.

Control DNA sequences containing the target loci were generated by cloning them into a pBHA vector (Bioneer, Daejeon, Republic of Korea). DH5α competent cells (Thermo Fisher Scientific) were transformed with the resulting plasmids using a heat-shock procedure at 42 °C for 90 s, cultured on a selective agar plate with ampicillin (50 µg/mL), and incubated at 37 °C overnight. Plasmid DNA (pDNA) was extracted using an Exprep Plasmid SV Mini Kit (GeneAll, Seoul, Republic of Korea). The isolated pDNA was digested with the Bsal-HFv2 restriction enzyme (NEB, Ipswich, MA, USA) and purified using the Expin CleanUp SV Mini Kit (GeneAll). The final DNA fragments were verified by 1% agarose gel electrophoresis to confirm the successful generation of the control template DNA.

For the positive control, the UL75 (glycoprotein H) sequence of the CMV Merlin strain (GenBank accession number AY446894) was used. The UL75 nucleotide sequence was obtained from GenBank (http://www.ncbi.nlm.nih.gov/genbank/) and inserted into a pBHA vector (Bioneer Inc., Daejeon, Republic of Korea). The concentration was measured using NanoDrop 2000 (Thermo Fisher Scientific Inc., MA, USA), and the sequence was diluted for use as a positive control.

Nucleotide sequences containing an extended region of VP1 capsid protein G-H loop and a carboxyl-terminal region amino acid residues (Table 1) of O/Jincheon/SKR/2014 (NCBI accession no. KU991728), O/Andong/SKR/2010 (NCBI accession no. KC503937), and O1/Manisa/Turkey/69 (NCBI accession no. AY593823) for OVM and A/Pocheon/KOR/2010 (NCBI accession no. KC588943.1), A/Malay/97 (NCBI accession no. KJ933864.1), and A22/Iraq/24/64 (NCBI accession no. KY825717.1) for AVM were optimized for codon usage in E. coli by Genetyx-win version 4.0 software. In the OVM and AVM constructs, each epitope was flanked by inserting a linker (Gly-Pro-Gly-Pro-Gly) to improve the folding and function of the multiepitope protein. Restriction endonucleases EcoRI and XhoI were included at the constructs’ 5′ and 3′ prime terminals, respectively. Designed OVM and AVM constructs were synthesized and inserted into the pBHA vector (Bioneer, South Korea). Sequence-verified synthesized inserts were ligated into the pHis parallel1 (N-terminal His tag) prokaryotic expression vector using EcoRI and XhoI restriction enzyme sites by T4 DNA ligase (Takara Korea biomedical, Seoul, South Korea).