Gabriel E Definitely low risk of bias - Homogenous cell suspension - ZIKV-AS, ZIKV-AM, ZIKV-AF, Control Definitely low risk of bias - Homogenous cell suspension - ZIKV-AS, ZIKV-AM, ZIKV-AF, Control low risk of bias - Same media, solvent, plate Definitely high risk of bias - No evidence of blinding to study group involving organoids, author indicated that investigators were not blinded to allocation during experiments and outcome assessment Probably high risk of bias - Evidence of all organoids used was documented by table S2 **Cortical plate thickness measured only for ZIKV-AS, not ZIKV-AM or ZIKV-AF **Values not shown for TUNEL assay **Organoid diameter measured for ZIKV-AF only, not ZIKV-AS or ZIKV-AM of ventricular zone lumen exhibiting cells measured for ZIKV-AS and not ZIKV-AF Definitely low risk of bias - Viral culture, and titration described (MOI = 1) Definitely low risk of bias - Analysis of data by ImageJ software Definitely low risk of bias - All measured outcomes outlined are recorded in main text or supplementary material Garcez P Definitely low risk of bias - Homogenous cell suspension - ZIKV, DENV2, control Definitely low risk of bias - Homogenous cell suspension - ZIKV, DENV2, control low risk of bias - Same media, solvent, plate Definitely high risk of bias - No evidence of blinding to group involving organoids, author indicated that investigators were not blinded to allocation during experiments and outcome assessment Probably low risk of bias - Insufficient information provided about number of organoids (3 or 6 organoids used) Definitely low risk of bias - Viral culture, and titration described (MOI = 0.025 to 0.25) Definitely low risk of bias - Analysis of data by Harmony 5.1 software Definitely low risk of bias - All measured outcomes outlined are recorded in main text or supplementary material
Li C Definitely low risk of bias - Homogenous cell suspension - ZIKV, control Definitely low risk of bias - Homogenous cell suspension - ZIKV, control low risk of bias - Same media, solvent, plate Definitely high risk of bias - No evidence of blinding to study group involving organoids, author indicated that investigators were not blinded to allocation during experiments and outcome assessment Probably high risk of bias (not recorded) - Insufficient information provided about number of organoids (number of organoids not recorded) Definitely low risk of bias - Viral culture, and titration described (MOI = 1) Definitely low risk of bias - Analysis of data by Imaris or ImageJ software Definitely low risk of bias - All measured outcomes outlined are recorded in main text or supplementary material Li Y Definitely low risk of bias - Homogenous cell suspension - ZIKV, control Definitely low risk of bias - Homogenous cell suspension - ZIKV, control low risk of bias - Same media, solvent, plate Definitely high risk of bias - No evidence of blinding to study group involving organoids, author indicated that investigators were not blinded to allocation during experiments and outcome assessment Probably high risk of bias (not recorded) - Insufficient information provided about number of organoids (number of organoids not recorded) **Apoptosis by CASP3 not quantified **Full statistical method not disclosed for each experiment Definitely low risk of bias - Viral culture, and titration described (MOI = 1) Definitely low risk of bias - Analysis of data by ImageJ software Definitely low risk of bias - All measured outcomes outlined are recorded in main text or supplementary material Qian X Definitely low risk of bias - Homogenous cell suspension - ZIKV, control Definitely low risk of bias - Homogenous cell suspension - ZIKV, control low risk of bias - Same media, solvent, plate Definitely low risk of bias - Investigators blinded to culture conditions Probably high risk of bias - Insufficient information provided about number of organoids (3, 5, or 6 organoids used) **ZIKV-C strain limited to SOX2 and TBR2 staining **Statistical Definitely low risk of bias - Viral culture, and titration described (1x or 0.25x dilution) Definitely low risk of bias - Analysis of data by ImageJ software Definitely low risk of bias - All measured outcomes outlined are recorded in main text or supplementary material
method not reported CQ Probably high risk of bias - Homogenous cell suspension - ZIKV, no controls Probably high risk of bias - Homogenous cell suspension - ZIKV, no controls low risk of bias - Same media, solvent, plate Definitely low risk of bias - assays kept blind Probably high risk of bias (not recorded) - Insufficient information provided about number of organoids (number of organoids not recorded) Definitely low risk of bias - Viral culture, and titration described (MOI = 1 or 10) Definitely low risk of bias - Analysis of data by Harmony 5.1 software Definitely low risk of bias - All measured outcomes outlined are recorded in main text or supplementary material (only ZIKV reported) M Definitely low risk of bias - Homogenous cell suspension - ZIKV, control Definitely low risk of bias - Homogenous cell suspension - ZIKV, control low risk of bias - Same media, solvent, plate Definitely high risk of bias - No evidence of blinding to study group involving organoids, author indicated that investigators were not blinded to allocation during experiments and outcome assessment Definitely low risk of bias - Number of organoids provided for all experiments Definitely low risk of bias - culture, and titration described (MOI = 0.3 or 3.125) Definitely low risk of bias - Analysis of data by ImageJ or software Definitely low risk of bias - All measured outcomes outlined are recorded in main text or supplementary material Wells MF Definitely low risk of bias - Homogenous cell suspension - ZIKV-PR, control Definitely low risk of bias - Homogenous cell suspension - ZIKV-PR, control low risk of bias - Same solvent, plate Definitely high risk of bias - No evidence of blinding to study group involving organoids, author indicated that investigators were not blinded to allocation during experiments Probably low risk of bias - Insufficient information provided about number of organoids (4 or 6 organoids used) **Ki67 not quantified Definitely low risk of bias - Viral culture, and titration described (MOI = 0.1, 1, or 10) Definitely low risk of bias - Analysis of data by ImageJ software Definitely low risk of bias - All measured outcomes outlined are recorded in main text or supplementary material
and outcome assessment Xu M Definitely low risk of bias - Homogenous cell suspension - ZIKV, control Definitely low risk of bias - Homogenous cell suspension - ZIKV, control low risk of bias - Same solvent, plate Definitely high risk of bias - No evidence of blinding to study group involving organoids, author indicated that investigators were not blinded to allocation during experiments and outcome assessment Probably low risk of bias - Insufficient information provided about number of organoids (6 organoids used) Definitely low risk of bias - Viral culture, and titration described (MOI = 0.04 to 0.08) Definitely low risk of bias - Analysis of data in blinded fashion Definitely low risk of bias - All measured outcomes outlined are recorded in main text or supplementary material Yoon KJ Definitely low risk of bias - Homogenous cell suspension - of GPF and ZIKV-NS2A or DENV-NS2A via control Definitely low risk of bias - Homogenous cell suspension - of GPF and ZIKV-NS2A or DENV-NS2A via control low risk of bias - Same solvent, plate Definitely high risk of bias - No evidence of blinding to study group involving organoids, author indicated that investigators were not blinded to allocation during experiments and outcome assessment Definitely low risk of bias - Number of organoids provided for all experiments Definitely low risk of bias - of GFP and ZIKV-NS2A or in ventricular radial glia in forebrain organoids by Definitely low risk of bias - Analysis of data by Zen or ImageJ software Definitely low risk of bias - All measured outcomes outlined are recorded in main text or supplementary material
Zhou T Definitely low risk of bias - Homogenous cell suspension - ZIKV, control Definitely low risk of bias - Homogenous cell suspension - ZIKV, control low risk of bias - Same media, solvent, plate Definitely high risk of bias - No evidence of blinding to study group involving organoids, author indicated that investigators were not blinded to allocation during experiments and outcome assessment Definitely low risk of bias - 3 organoids provided for each experiments Definitely low risk of bias - Viral culture, and titration described (5x10^5 PFU/ml) Definitely low risk of bias - Analysis of data by software Definitely low risk of bias - All measured outcomes outlined are recorded in main text or supplementary material Table 4. Cellular effects of ZIKV according to lineage. Strains Viral replication after 72 hours Preferential infectivity Progenitor cells/radial glia cells (SOX2+, PAX6+, Nestin+, Phospho-vimentin+) Intermediate cells (TBR1+/TBR2+) Proliferating cells (Ki67+, phospho-H3) Mature neurons TUJ+) Apoptosis (TUNEL+, CASP3+) Viability (EdU+) Change in organoid size, area, volume, or thickness African (MR766) ↑ Progenitor cells > Mature neurons 19,25 ↓ ↓ 21 ↓ ↓ 21 ↑ ↓ 20 ↓
Latin America (PRVABC59, Brazilian, ↑ Progenitor cells > Mature neurons 19,21,26 ↓ ↓ ↓ 21 ↓ ↑ N/A ↓ Asian (FSS13025, H/PF/2013) ↑ 23 Progenitor > Mature neurons 21,25 ↓ 23 N/A N/A ↓ 23 ↑ 23,30 N/A ↓ 23