Primary microcephaly is a
severe brain malformation characterized by the reduction of the head
circumference. Patients display a heterogeneous range of brain impairments,
compromising motor, visual, hearing and cognitive functions (1).
Microcephaly is associated
with decreased neuronal production as a consequence of proliferative defects
and death of cortical progenitor cells (2). During pregnancy, the primary
etiology of microcephaly varies from genetic mutations to external insults. The
so-called TORCHS factors (Toxoplasmosis, Rubella, Cytomegalovirus, Herpes
virus, Syphilis) are the main congenital infections that compromise brain
development in utero (3).
The increase in the rate of
microcephaly in Brazil has been associated with the recent outbreak of Zika
virus (ZIKV) (4, 5), a flavivirus that is transmitted by mosquitoes (6) and
sexually (7–9). So far, ZIKV has been described in the placenta and amniotic
fluid of microcephalic fetuses (10–13), and in the blood of microcephalic
newborns (11, 14). ZIKV had also been detected within the brain of a
microcephalic fetus (13, 14), and recently, there is direct evidence that ZIKV
is able to infect and cause death of neural stem cells (15).
Here, we used human induced
pluripotent stem (iPS) cells cultured as neural stem cells (NSC), neurospheres
and brain organoids to explore the consequences of ZIKV infection during
neurogenesis and growth with 3D culture models. Human iPS-derived NSCs were
exposed to ZIKV (MOI 0.25 to 0.0025). After 24 hours, ZIKV was detected in NSCs
(Fig. 1, A to D), when viral envelope protein was shown in 10.10% (MOI 0.025)
and 21.7% (MOI 0.25) of cells exposed to ZIKV (Fig. 1E). Viral RNA was also
detected in the supernatant of infected NSCs (MOI 0.0025) by qRT-PCR (Fig. 1F),
supporting productive infection.
Fig. 1
ZIKV infects human neural stem
cells.
Confocal microscopy images of
iPS-derived NSCs double stained for (A) ZIKV in the cytoplasm, and (B) SOX2 in
nuclei, one day after virus infection. (C) DAPI staining, (D) merged channels
show perinuclear localization of ZIKV. Bar = 100 μm. (E) Percentage of ZIKV
infected SOX2 positive cells (MOI 0.25 and 0.025). (F) RT-PCR analysis of ZIKV
RNA extracted from supernatants of mock and ZIKV-infected neurospheres (MOI
0.0025) after 3 DIV, showing amplification only in infected cells. RNA was
extracted, qPCR performed and virus production normalized to 12h post-infection
controls. Data presented as mean ± SEM, n=5, Student’s t test, *p < 0.05,
**p < 0.01.
To investigate the effects of
ZIKV during neural differentiation, mock- and ZIKV-infected NSCs were cultured
as neurospheres. After 3 days in vitro, mock NSCs generated round neurospheres.
However, ZIKV-infected NSCs generated neurospheres with morphological
abnormalities and cell detachment (Fig. 2B). After 6 days in vitro (DIV),
hundreds of neurospheres grew under mock conditions (Fig. 2, C and E).
Strikingly, in ZIKV-infected NSCs (MOI 2.5 to 0.025) only a few neurospheres
survived (Fig. 2, D and E).
Fig. 2
ZIKV alters morphology and
halts the growth of human neurospheres.
(A) Control neurosphere
displays spherical morphology after 3 DIV. (B) Infected neurosphere showed
morphological abnormalities and cell detachment after 3 DIV. (C) Culture
well-plate containing hundreds of mock neurospheres after 6 DIV. (D)
ZIKV-infected well-plate (MOI 2.5-0.025) containing few neurospheres after 6
DIV. Bar = 250 μm in (A) and (B), and 1 cm in (C) and (D). (E) Quantification
of the number of neurospheres in different MOI. Data presented as mean ± SEM,
n=3, Student’s t test, ***p < 0.01.
Mock neurospheres presented
expected ultrastructural morphology of nucleus and mitochondria (Fig. 3A).
ZIKV-infected neurospheres revealed the presence of viral particles, similarly
to those observed in murine glial and neuronal cells (16). ZIKV was bound to
the membranes and observed in mitochondria and vesicles of cells within
infected neurospheres (Fig. 3, B and F, arrows). Apoptotic nuclei, a hallmark
of cell death, were observed in all ZIKV-infected neurospheres analyzed (Fig.
3B). Of note, ZIKV-infected cells in neurospheres presented smooth membrane
structures (SMS) (Fig. 3, B and F), similarly to those previously described in
other cell types infected with dengue virus (17). These results suggest that
ZIKV induces cell death in human neural stem cells and thus impairs the
formation of neurospheres.
Fig. 3
ZIKV induces death in human
neurospheres.
Ultrastructure of mock- and
ZIKV-infected neurospheres after 6 days in vitro. (A) Mock-infected neurosphere
showing cell processes and organelles, (B) ZIKV-infected neurosphere shows
pyknotic nucleus, swollen mitochondria, smooth membrane structures and viral
envelopes (arrow). Arrows point viral envelopes on cell surface (C), inside
mitochondria (D), endoplasmic reticulum (E) and close to smooth membrane
structures (F). Bar = 1 μm in (A) and (B) and 0.2 μm in (C) to (F). m =
mitochondria; n = nucleus; sms = smooth membrane structures.
To further investigate the
impact of ZIKV infection during neurogenesis, human iPS-derived brain organoids
(18) were exposed with ZIKV, and followed for 11 days in vitro (Fig. 4). The
growth rate of 12 individual organoids (6 per condition) was measured during
this period (Fig. 4, A and D). As a result of ZIKV infection, the average
growth area of ZIKV-exposed organoids was reduced by 40% when compared to brain
organoids under mock conditions (0.624 mm2 ± 0.064 ZIKV-exposed organoids
versus 1.051 mm2 ± 0.1084 mock-infected organoids normalized, Fig. 4E).
Fig. 4
ZIKV reduces the growth rate
of human brain organoids.
35 days old brain organoids
were infected with (A) MOCK and (B) ZIKV for 11 days in vitro. ZIKV-infected
brain organoids show reduction in growth compared with MOCK. Arrows point to
detached cells. Organoid area was measured before and after 11 days exposure
with (C) MOCK and (D) ZIKV in vitro. Plotted quantification represent the
growth rate. (E) Quantification of the average of mock- and ZIKV-infected
organoid area 11 days after infection in vitro. Data presented as mean ± SEM,
n=6, Student’s t test, *p < 0.05.
In addition to MOCK infection,
we used dengue virus 2 (DENV2), a flavivirus with genetic similarities to ZIKV
(11, 19), as an additional control group. One day after viral exposure, DENV2
infected human NSCs with a similar rate as ZIKV (fig. S1, A and B). However,
after 3 days in vitro, there was no increase in caspase 3/7 mediated cell death
induced by DENV2 with the same 0.025 MOI adopted for ZIKV infection (fig. S1, C
and D). On the other hand, ZIKV induced caspase 3/7 mediated cell death in NSCs,
similarly to the results described by Tang and colleagues (15). After 6 days in
vitro, there is a significant difference in cell viability between ZIKV-exposed
NSCs compared to DENV2-exposed NSCs (fig. S1, E and F). In addition,
neurospheres exposed to DENV2 display a round morphology such as uninfected
neurospheres after 6 days in vitro (fig. S1G). Finally, there was no reduction
of growth in brain organoids exposed to DENV2 for 11 days compared to MOCK
(1.023 mm2 ± 0.1308 DENV2-infected organoids versus 1.011 mm2 ± 0.2471
mock-infected organoids normalized, fig. S1, H and I). These results suggest
that the deleterious consequences of ZIKV infection in human NSCs, neurospheres
and brain organoids are not a general feature of the flavivirus family. Neurospheres
and brain organoids are complementary models to study embryonic brain
development in vitro (20, 21). While neurospheres present the very early
characteristics of neurogenesis, brain organoids recapitulate the orchestrated
cellular and molecular early events comparable to the first trimester fetal
neocortex, including gene expression and cortical layering (18, 22). Our
results demonstrate that ZIKV induces cell death in human iPS-derived neural
stem cells, disrupts the formation of neurospheres and reduces the growth of
organoids (fig. S2), indicating that ZIKV infection in models that mimics the
first trimester of brain development may result in severe damage. Other studies
are necessary to further characterize the consequences of ZIKV infection during
different stages of fetal development.
Cell death impairing brain
enlargement, calcification and microcephaly is well described in congenital
infections with TORCHS (3, 23, 24). Our results, together with recent reports
showing brain calcification in microcephalic fetuses and newborns infected with
ZIKV (10, 14) reinforce the growing body of evidence connecting congenital ZIKV
outbreak to the increased number of reports of brain malformations in Brazil.
Supplementary Materials
