Fig. 1: Generation and characterisation of the BinJ/JEVNSW/22-prME chimera.
a Schematic of the circular polymerase extension reaction (CPER) strategy to generate infectious DNA of chimeric BinJ/JEVNSW/22-prME. The prME genes of JEVNSW/22 (pink arrows) were inserted into the BinJV backbone (black arrows) (replacing the BinJV prME). OpIE2-CA, a modified Orgyia pseudotsugata multicapsid nucleopolyhedrosis virus immediate-early 2 promoter; HDVr-pA, hepatitis delta virus ribozyme–poly A, with the ribozyme autocleavage providing an authentic 3′ untranslated region (UTR). b IFA analysis of mock (left) and BinJ/JEVNSW/22-prME CPER (right) transfected C6/36 cells fixed 7 days post-transfection and immunolabeled with anti-flavivirus NS1 mAb 4G4 to confirm recovery of replicating virus (green signal). c Growth of chimera was analysed by infecting C6/36 monolayers in triplicate with BinJV, JEVNSW/22 or BinJ/JEVNSW/22-prME at an MOI of 0.1, before titrating on C6/36 monolayers in triplicate and determining viral titres by TCID50. Statistics were performed by one-way ANOVA whereby p = <0.0001 (****) or <0.005 (***). d IFA analysis by confocal microscopy of mock, West Nile virus, Kunjin subtype (WNVKUN, a mammalian cell infection control) and BinJ/JEVNSW/22-prME virus-infected C6/36 cells and mammalian cells at an MOI of 1. Cells were fixed and immunolabelled 5 days after infection. Mammalian cells: BSR (baby hamster kidney), Vero-76 cells (African green monkey kidney) and primary equine dermal fibroblasts. Virus replication was detected with anti-NS1 mAb 4G4 (green signal) and cell nuclei were stained with Hoechst 33342 (blue). Images taken at x40 magnification. Scale bar in (b) and (d) represents 100 μM.
