Fig. 1: Recovery of YYV and EILV infectious clones by CPER.
A Schematic of the YYV genome, overlapping fragments and linker fragment used for CPER assembly. B Schematics of alphavirus genomes showing virus and chimera construction. Construct 1 (C1): EILV (blue) infectious clone; construct 2 (C2), EILV/YYV-sP chimera – EILV backbone (blue), YYV structural proteins (green); construct 3 (C3), EILV/RRV-sP/YYV-3’UTR: EILV 5’ UTR and non-structural proteins (blue), RRV structural proteins (red), YYV 3’UTR (green), with addition of UC nucleotides prior to the poly(A) tail; Construct 4 (C4): final YYV infectious clone (YYVEILV; green) including the UC nucleotides. C IFA staining of C6/36 cells infected constructs of panel B. Mock and infectious clone/chimera infected and fixed cell monolayers were probed with anti-dsRNA (see also Fig. S4A), anti-YYV E2 (6C12), anti-EILV E2 (1E1) or cross-reactive anti-CHIKV capsid (5.5G9) mAbs (green). Nuclei were stained with Hoechst 33342 (blue). Images taken at 40× magnification. D Comparative growth kinetics of EILV, YYVEILV (YYV infectious clone including 5’ terminal 11 nt of EILV genome) and YYVTALV (YYV infectious clone including 5’ terminal 11 nt of TALV genome) in C6/36 cells, infected at an MOI of 0.1 (n = 3). Statistical analyses: two-way ANOVA, and comparisons between individual time-points with Tukey’s multiple comparisons test. *P ≤ 0.05. E Representative image of gradient-purified YYV. The blue band is concentrated virus, and the white band is cell debris from C6/36 cells. F SDS-PAGE analysis of gradient-purified infectious clones and chimeric viruses. G TEM of purified YYV imaged at ×100,000 magnification. H IFA analysis of insect and mammalian cells infected with YYV, YYV/RRV-sP, and RRVWT or West Nile virus (WNVWT) virus at an MOI of 1. YYV-infected cell lines probed with anti-YYV mAb 6C12, YYV/RRV-sP and RRV with cross-reactive anti-CHIKV mAb 5.5G9, and WNV with anti-flavivirus envelope mAb 4G2. Nuclei were stained with Hoechst 33342. Images taken at 63× magnification.
