Figure 1.

Characterization of C-deletion mutants. (a) Sequence alignment of flavivirus C protein. The alignment was performed using the CLC main workbench software (Qiagen). The structural features of ZIKV C protein are indicated. Below are the virus strains with the corresponding GenBank access numbers: ZIKV strain FSS13025 (KU955593), DENV-2 strain New Guinea C (AF038403), DENV-1 strain Westpac (U88535), DENV-3 strain H87 (M93130), DENV-4 strain H241 (AY947539), JEV strain SA14–14-2 (JN604986), WNV strain NY99 (AF196835), YFV strain 17D (JX949181), and TBEV strain A104 (KF151173). (b) Diagram of C-deletion mutants in the context of ZIKV genome. Dashed boxes indicate the deleted segments. Amino acid positions are indicated. (c) Immunofluorescent assay (IFA) of RNA-transfected Vero cells. At given time points post-transfection, Vero cells were fixed and stained with NS3 antibody for viral protein expression (green). Nuclei were counterstained by DAPI (blue). (d) IFA of infected Vero cells. Naive Vero cells were infected with the supernatant that was harvested from the RNAtransfected cells at 72 h post-transfection. At 24 h p.i., cells were analyzed by IFA for viral NS3 expression. (e) Summary of C-deletion mutants after transfecting viral RNA into Vero cells. Percentages of NS3-expressing cells (NS3⁺) were calculated as follow: (NS3⁺ cell number) / (DAPI⁺ cell number)×100%. Supernatants at 72 h p.t. were used to infect naive Vero monolayers in 8-well chamber slides. At 24 h p.i., cells were fixed and monitored for NS3 expression by IFA. NS3⁺ cells were counted and the virus titers (IFU/ml) were calculated. Limit of detection, 10 IFU/ml; N.D., not detectable. (f) Amounts of extracellular viral RNA post-transfection. At indicated time points, viral RNA levels in supernatant were quantified by RT-PCR. The maximum amount of extracellular viral RNA from replicon RNA-transfected cells was set as the cutoff.