Strain-Dependent Consequences of Zika Virus Infection and Differential Impact on Neural Development

Abstract

Maternal infection with Zika virus (ZIKV) during pregnancy can result in neonatal abnormalities, including neurological dysfunction and microcephaly. Experimental models of congenital Zika syndrome identified neural progenitor cells as a target of viral infection. Neural progenitor cells are responsible for populating the developing central nervous system with neurons and glia. Neural progenitor dysfunction can lead to severe birth defects, namely, lissencephaly, microcephaly, and cognitive deficits. For this study, the consequences of ZIKV infection in human pluripotent stem cell-derived neural progenitor (hNP) cells and neurons were evaluated. ZIKV isolates from Asian and African lineages displayed lineage-specific replication kinetics, cytopathic effects, and impacts on hNP function and neuronal differentiation. The currently circulating ZIKV isolates exhibit a unique profile of virulence, cytopathic effect, and impaired cellular functions that likely contribute to the pathological mechanism of congenital Zika syndrome. The authors found that infection with Asian-lineage ZIKV isolates impaired the proliferation and migration of hNP cells, and neuron maturation. In contrast, the African-lineage infections resulted in abrupt and extensive cell death. This work furthers the understanding of ZIKV-induced brain pathology.

Keywords: Zika virus; neural progenitor cells; neurons.

Figure 1

Zika virus (ZIKV) isolate-specific growth and cytotoxicity in human pluripotent stem cell-derived neural progenitor (hNP) cells at six days post-infection. (A) hNP cells are maintained as a proliferating population in media with fibroblast growth factor (FGF) and leukemia inhibitory factor (LIF). Withdrawal of FGF and LIF from the media leads to differentiation and a population of immature neurons after 14 DIV, then post-mitotic neurons exclusively emerge after 28 DIV. (B–E) African-lineage ZIKV isolate (IbH) grew robustly and induced cell death in undifferentiated hNP cells and differentiating hNP cells, while Asian isolate (SPH) replicated more slowly with less extensive cell death. * demonstrates p < 0.05.

Figure 2

ZIKV isolate-specific growth and cytotoxicity in human neurons. (A,B) Viral replication and viability of hNP-derived nascent neurons (14 DIV) and (C,D) mature neurons (28 DIV) six days post-infection. * demonstrates p < 0.05.

Figure 3

Isolate-dependent ability of ZIKV to infect hNP cells and mature neurons. (A) ZIKV isolates IbH and SPH readily infect Vero cells within 48 h. (B) hNP cells are more susceptible to IbH infection than SPH infection, however neither isolate infected more than 13% of the population. (C) Mature neurons (28 DIV) were similarly more susceptible to ZIKV IbH infection than SPH infection. (D) Non-infected (0 MOI) did not demonstrate ZIKV E protein’s presence, but infected (SPH 10 MOI) cells after 48 h did contain ZIKV E protein in all three cell types.

Figure 4

ZIKV infection decreased hNP cell proliferation and migration. (A) Only infection with MOI 10 of African-lineage ZIKV demonstrated a significant impact on cell viability after 48 h, whereas infection with SPH did not decrease viability. (B) The proliferation of hNP cells after ZIKV infection was significantly decreased following infection with SPH as determined by quantification of Ki67 expression. (C) The migration of hNP cells was disrupted by both IbH and SPH infections. IbH infection severely inhibited migration at MOI 10 to such an extent that no acceptable data were collected (shown as ND). * demonstrates p < 0.05 significance between IbH and SPH and # demonstrates p < 0.05 significance difference from non-infected control.

Figure 5

ZIKV infection perturbs neurite outgrowth in human neural progenitor cell-derived neurons. (A) At 48 h post-infection, both African (IbH) and Asian (SPH) isolates had minimal impact on neuron viability. (B) Representative images of neurite outgrowth quantification. Hoechst stain labels nuclei of neurons and MAP2 identifies neurites. Both images contribute to the enumeration and quantification of neurite outgrowth characteristics. Scale bar = 50 micrometers (C–F). Infection with Asian-lineage ZIKV (SPH) did not reduce the number of valid neurons observed, yet infection with SPH (MOI 10) proved detrimental to neurite outgrowth by decreasing the quantity, length, and number of branch points per neuron. * demonstrates p < 0.05 significance between IbH and SPH and # demonstrates p < 0.05 significance difference from non-infected control.