Fig. 2

Increased susceptibility to co-infection is only observed when virus is injected first. Zebrafish larvae were sequentially injected in the bloodstream with Sindbis virus (SINV) and Shigella at 72 and 96 hpf and larvae injected with SINV or Shigella alone were used as a control as depicted in Figure 1. Survival, bacterial burden, and viral replication were evaluated over time in SINV + Shigella (A,C,E) and in Shigella + SINV (B,D,E) sequentially infected larvae settings. (A,C,E) Data pooled from three independent experiments; (B,D,E) data from one experiment; see also Figures S2A,B in Supplementary Material. Survival curves of zebrafish larvae injected with SINV + Shigella (A) (blue curve) or with Shigella + SINV (B) (cyan curve) and incubated at 28°C. For both sequential co-infection settings, fish injected with Shigella (red curves) or with SINV (green curves) alone at the appropriate time point, and non-injected fish (black curves) were used as controls. n = 72 (A) or 24 (B) fish for each condition. (C,D) Bacterial burden quantification by enumerating live bacteria in homogenates from individual larvae sequentially co-infected with SINV + Shigella (C) (blue symbols) or Shigella + SINV (D) (cyan symbols) or with Shigella alone (red symbols) measured by plating onto LB immediately after Shigella injection and 24 h post Shigella injection. n = 15 (C) or 5 (D) larvae for each condition. (E,F). Viral replication measured by RT-qPCR from individual infected larvae in SINV-Shigella (E) (blue curve) or Shigella-SINV (F) (cyan curve) sequentially co-infected fish, or SINV (green curves). n = 15 (E) or 5 (F) larvae for each condition. (G) Representative images of virus (SINV-GFP) and bacteria (Shigella-DsRed) dissemination, determined by live imaging using a fluorescence stereomicroscope, of zebrafish larvae infected with SINV-GFP alone at 72 hpf, or with Shigella DsRed alone at 96 hpf, or sequentially co-infected with SINV-GFP first and Shigella-DsRed 1 day later. Non-infected larvae (CTRL) are also shown. The same infected larvae were live imaged 4 and 24 h post Shigella injection. Overlay of GFP and DsRed fluorescence is shown, except in SINV panels, where only GFP fluorescence was recorded.