Figure 3.

In vitro recombination between SAM vaccine and WT alphavirus

(A) Schematic of experimental set-up. Vero cells were infected with WT alphaviruses and transduced with SAM vaccine. After 72 h, supernatants were serial passaged a total of four times (every 3–4 days) on HeLa and HeLa ΔG3BP cells, with CPE monitored by inverted bright-field microscope. (B) WT alphaviruses induced CPE in HeLa cells, but cannot replicate and induce CPE in HeLa ΔG3BP cells. CPE in HeLa ΔG3BP cells (ΔG3BP) indicated the likely presence of a chimeric alphavirus. In the first experiment in 24-well plates, 1/2 wells showed CPE for the combination GETV and SAM encoding mCherry (SAM-mCherry). In the second experiment in 96-well plates, 1/96 wells showed CPE for GETV and SAM-nLuc. (C) Schematic of the two chimeric alphavirus sequences (not to scale), with flanking sequences of the recombination sites. The predicted amino acid sequences are provided, although whether the SAM subgenomic promoter is active is unknown. (D) The replication kinetics of the SAM-mCherry-GETV chimera compared with TC-83 and GETV in Vero (Cercopithecus aethiops), HeLa (Homo sapiens), C6/36 (Aedes albopictus), and Chao Ball (Culex tarsalis) cells infected with 0.1 TCID₅₀/cell (n = 2). Virus titers determined by serial dilution assays (detection limit 3 log₁₀TCID₅₀/mL).