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BRAIN Publication-derived

Zika Virus Infects Human Cortical Neural Progenitors and

Source Cugola et al. · University of São Paulo (USP), São Paulo, Brazil · 10.1038/nature18296

👤 Cugola FR, Fernandes IR, Russo FB, Freitas BC, Dias JL, Guimarães KP, Benazzato C, Almeida N, Pignatari GC, Romero S, Polber CM, Cunha I, Oliveira CL, Bhatt SM, Bhatt AH, Beltrão-Braga PC, Muotri AR ⏱ 35 days 📋 11 phases 🧫 Human iPSC

Abstract

Cugola et al. demonstrated that the Brazilian Zika virus strain causes severe growth attenuation and neural progenitor depletion in human cerebral organoids, providing definitive mechanistic evidence linking ZIKV to congenital microcephaly. Published in Nature during the 2016 Zika epidemic, this study showed the Brazilian strain was more neurotoxic than the African reference strain.

Cell source
Human iPSC
Application
Zika Virus Pathogenesis Research

Protocol overview

81 steps across 11 phases

Viral Isolation, Culture and Amplification Day 0–8
  1. 1 Reconstitute lyophilized ZIKV isolate
  2. 2 Prepare and maintain C6/36 mosquito cell culture
  3. 3 Inoculate C6/36 cells with viral samples
  4. 4 First subculture (T1) collection
  5. 5 Second subculture (T2) collection
  6. 6 Third subculture (T3) collection
Viral Titration Day 8–13
  1. 1 Prepare virus titration plates and medium
  2. 2 Perform serial dilutions of viral stocks
  3. 3 Seed porcine kidney epithelial (PS) cells
  4. 4 Overlay with carboxymethyl cellulose (CMC) medium
  5. 5 Incubate plates for plaque formation
  6. 6 Stain plaques and quantify viral titer
  7. 7 Store viral stocks
Generation of Human Neural Cells from Pluripotent Stem Cells Day 1–35
  1. 1 Maintain iPSC and hESC cultures on feeder-free plates
  2. 2 Initiate neural induction with dual SMAD inhibitors
  3. 3 Culture as Embryoid Bodies (EBs) in suspension
  4. 4 Plate EBs on matrigel and initiate rosette formation
  5. 5 Manually pick and dissociate neural rosettes
  6. 6 Expand neural progenitor cell (NPC) population
  7. 7 Differentiate NPCs into neurons
  8. 8 Generate neurospheres
  9. 9 Generate cerebral organoids
In Vitro Viral Infection of Neural Cells Day 1–4
  1. 1 Prepare viral inocula at defined multiplicities of infection (MOI)
  2. 2 Infect NPCs in monolayer cultures
  3. 3 Infect neurons in differentiated monolayer cultures
  4. 4 Infect neurospheres in suspension
  5. 5 Infect cerebral organoids
  6. 6 Maintain mock controls
  7. 7 Collect samples at defined timepoints
In Vivo Viral Infection of Pregnant Mice Day 10–13 of gestation through postnatal analysis
  1. 1 Prepare pregnant mice for infection
  2. 2 Prepare viral inocula for intravenous injection
  3. 3 Intravenous injection of pregnant mice
  4. 4 Observe pregnant dams daily post-infection
  5. 5 Collect and characterize newborn pups
  6. 6 Compute tomography (CT) imaging of pups
  7. 7 Harvest tissues from pups and dams
Nucleic Acid Extraction and Real-Time Quantitative PCR (qPCR) Day 1–3 (post-sample collection)
  1. 1 Extract RNA from cells, supernatants, or tissues
  2. 2 Quantify extracted RNA
  3. 3 Perform one-step RT-qPCR for viral RNA detection
  4. 4 Quantify viral RNA using standard curve
  5. 5 Analyze qPCR data
  6. 6 Perform qPCR for TAM receptor expression
  7. 7 Analyze gene expression using ΔΔCt or fold-change method
Transmission Electron Microscopy (TEM) Day 1–5
  1. 1 Prepare cell pellets for fixation
  2. 2 Primary fixation with glutaraldehyde
  3. 3 Post-fixation with osmium tetroxide
  4. 4 Dehydration with graded ethanol series
  5. 5 Transition with propylene oxide
  6. 6 Infiltration with embedding medium
  7. 7 Polymerization of embedding medium
  8. 8 Cut ultrathin sections
  9. 9 Stain ultrathin sections with heavy metals
  10. 10 Examine samples with transmission electron microscope
Immunofluorescence Staining and Confocal Microscopy Day 1–2
  1. 1 Fix cells or tissue sections with paraformaldehyde
  2. 2 Permeabilize fixed cells with Triton X-100
  3. 3 Block non-specific antibody binding
  4. 4 Incubate with primary antibodies
  5. 5 Wash cells to remove unbound primary antibody
  6. 6 Incubate with fluorescently labeled secondary antibodies
  7. 7 Wash and stain nuclei with DAPI
  8. 8 Mount slides with mounting medium
  9. 9 Acquire immunofluorescence images
  10. 10 Quantify immunofluorescence signal
Flow Cytometric Analysis of Cell Death Day 1–1 (sampling) + Day 2 (analysis)
  1. 1 Infect cells at defined MOI and timepoints
  2. 2 Harvest infected cells
  3. 3 Prepare cells for Annexin V/Propidium Iodide (PI) staining
  4. 4 Perform flow cytometric analysis
  5. 5 Quantify and analyze cell death data
Cell Death Pathway Analysis by qPCR Array Day 1–1 (sample collection and RNA extraction)
  1. 1 Collect brain tissue from infected and control mice
  2. 2 Extract total RNA from brain tissue
  3. 3 Perform RT² Profiler PCR Array for cell death pathways
  4. 4 Analyze gene expression data
  5. 5 Validate pathway results with additional qPCR
Histopathological Analysis and Light Microscopy Day 1–3
  1. 1 Prepare tissues for paraffin embedding
  2. 2 Perform graded alcohol dehydration
  3. 3 Perform xylene clarification (clearing)
  4. 4 Infiltrate tissues with paraffin wax
  5. 5 Embed tissues in paraffin blocks
  6. 6 Perform deparaffinization and rehydration
  7. 7 Perform hematoxylin-eosin (H&E) staining
  8. 8 Examine stained sections with light microscopy

Full SOP

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Attribution

This SOP was authored by Organthis based on the published method in Cugola et al.. The originating laboratory holds no rights in this SOP and has not endorsed it unless marked Verified.

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