The small heads observed in microcephalic children reflect a physically smaller brain—specifically, the neocortex is thinner than in a normal brain. The neocortex, the largest part of the cerebral cortex of the brain, is composed of six distinct layers of neurons, which are established during embryonic development. First, glial cells originating from progenitor cells in the ventricular zone extend their processes throughout the cortex and anchor at the pia, the outer surface of the brain. These long fibers provide a scaffold on which neurons, produced from the same progenitor cells, migrate outwards to establish the six layers of the cortex.
Neuronal migration is impaired during Zika virus infection. Brain slice cultures from embryonic day 15 mice were infected with 105 PFU of Zika virus and at 4 dpi, were fixed and stained with antibody against vimentin to mark the radial glia progenitor (RGP) basal processes, which are the fibers upon which bipolar neurons migrate. ZIKV infection perturbed the RGP scaffold compared with control slices.
Glial fibers are visible as parallel tracks in the mouse embryonic brain slice cultures stained with an antibody to vimentin, a protein component of the fibers (image, left panel). When embryonic brain slice cultures were infected with Zika virus, the structure of the glial tracks was altered. Instead of parallel tracks, the fibers assumed a twisted morphology that would not allow neurons to travel from the ventricular zone to the developing neocortex (image, right panel). Disruption of glial fibers was observed after infection with Zika viruses isolated from 1947 to 2016.
These results suggest that Zika virus-mediated disruption of glial fibers during embryonic development contributes to microcephaly: if neurons cannot migrate to the pial surface, the neocortex will be thinner.
Rosenfeld AB, Doobin DJ, Warren AL, Racaniello VR, Vallee RB. 2017. Replication of early and recent Zika virus isolates throughout mouse brain development. Proc Natl Acad Sci U S A 114:12273–12278.
Monolayer and suspension cell cultures do not reproduce the cell type diversity and architecture typical of tissues and organs. One way to overcome this limitation is by the use of organotypic slice cultures, which can be produced from a variety of organs, including brain, liver, and kidney. These cultures are prepared by slicing embryonic or postnatal rodent organs into 100- to 400-micrometer slices. They are placed on substrates, such as porous or semiporous membranes, and bathed in cell culture medium. Such cultures remain viable for 1 to 2 weeks. The effect of Zika virus infection on neuronal migration has been examined in organotypic brain slice cultures derived from embryonic mice (Box 2.3).
Another type of three-dimensional cell system is the multicellular, self-organizing organoid that approximates the organization, function, and genetics of specific organs. Organoids are derived from either pluripotent stem cells (iPSCs or embryonic stem cells) or adult stem cells from different organs. Organoids that model many organs such as intestine, stomach, esophagus, and brain have been established, and many have been validated for the study of a variety of viral infections (Fig. 2.3). For example, for years propagation of human noro-viruses eluded virologists until the development of intestinal organoids.
The differentiation of stem cells into organoids depends on growth conditions and nutrients. For example, one type of brain organoid can be established from human pluripotent stem cells by embedding the cells in a gelatinous protein mixture that resembles the extracellular environment of many tissues. In the absence of further cues, the stem cells differentiate into structures typical of many diverse brain regions, including the cortex. In contrast, the production of intestinal organoids requires agonists of a particular signal transduction pathway. Current attempts to improve organoid cultures include the addition of immune cells, vasculature, and commensal microorganisms, to more accurately reflect the details of tissue and organ architectures.
Figure 2.3 Production of organoids from stem cells. The different germ layers shown (endoderm and ectoderm) may be derived from embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs) in vitro with specific differentiation protocols. After transfer into 3-dimensional systems these cells produce organoids that recapitulate the developmental steps characteristic of various organs.
Air-liquid interface cultures are used to model the respiratory tract, a major site of virus entry and infection. This organ presents a challenge because its structure differs from the pharynx to the alveoli. In the trachea and bronchi, the epithelium comprises a single layer of columnar cells which contact the basement membrane. In the alveoli the epithelium is made of a thin, single cell layer to facilitate air exchange. Air-liquid interface cultures may be produced from primary human bronchial cells or respiratory cell lines (Fig. 2.4).
Because viruses are obligatory intracellular parasites, they cannot reproduce outside a living cell. An exception comes from the demonstration in 1991 that infectious poliovirus could be produced in an extract of human cells incubated with viral RNA, a feat that has not been achieved for any other virus. Consequently, most analyses of viral replication have used cultured cells, embryonated eggs, or laboratory animals. For a discussion of whether to call these different systems in vivo or in vitro, see Box 2.4.
Evidence of Viral Reproduction in Cultured Cells
Before quantitative methods for measuring viruses were developed, evidence of viral propagation was obtained by visual inspection of infected cells. Some viruses kill the cells in which they reproduce, and they may eventually detach from the cell culture plate. As more cells are infected, the changes become visible and are called cytopathic effects.
Many types of cytopathic effect can be seen with a simple light or phase-contrast microscope at low power, without fixing or staining the cells. These changes include the rounding up and detachment of cells from the culture dish, cell lysis, swelling of nuclei, and sometimes the formation of a group of fused cells called a syncytium (Fig. 2.5). High-power microscopy is required for the observation of other cytopathic effects, such as the development of intracellular masses of virus particles or unassembled viral components in the nucleus and/or cytoplasm (inclusion bodies), formation of crystalline arrays of viral proteins, membrane blebbing, duplication of membranes, and fragmentation of organelles.
Figure 2.4 Production of airway-liquid interface cultures of bronchial epithelium. (A) Epithelial cells are seeded onto a permeable membrane and cell culture medium is supplied on both apical (top) and basal (bottom) sides. (B) When the cells are confluent, medium on the apical side is removed. Contact of the cells with air drives differentiation of cells towards types found in the airways, such as goblet cells, ciliated and nonciliated cells, and basal cells. Cultures may be produced that mimic tracheobronchial cells, with different cell types, or human alveolar cells with only two cell types (not shown).
