Zika virus impairs growth in human neurospheres and brain organoids

Abstract

Since the emergence of Zika virus (ZIKV), reports of microcephaly have increased considerably in Brazil; however, causality between the viral epidemic and malformations in fetal brains needs further confirmation. We examined the effects of ZIKV infection in human neural stem cells growing as neurospheres and brain organoids. Using immunocytochemistry and electron microscopy, we showed that ZIKV targets human brain cells, reducing their viability and growth as neurospheres and brain organoids. These results suggest that ZIKV abrogates neurogenesis during human brain development. P rimary microcephaly is a severe brain malformation characterized by the reduction of the head circumference. Patients display a heterogeneous range of brain impairments that compromise 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, and syphilis) are the main congenital infections that compromise brain development in utero (3).An 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, direct evidence has emerged that ZIKV is able to infect and cause the death of neural stem cells (15).We used human induced pluripotent stem (iPS) cells cultured as neural stem cells (NSCs), neurospheres, and brain organoids to explore the consequences of ZIKV infection during neurogenesis and growth with three-dimensional culture models. Human iPS-derived NSCs were exposed to ZIKV [multiplicity of infection (MOI), 0.25 to 0.0025]. After 24 hours, ZIKV was detected in NSCs (Fig. 1, A to D); viral envelope protein was evident 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 quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) ( Fig. 1F), providing evidence of productive infection.To investigate the effects of ZIKV during neural differentiation, mock-and ZIKV-infected NSCs were cultured as neurospheres. After 3 days in vitro (DIV), mock-infected NSCs generated round neurospheres. However, ZIKVinfected NSCs generated neurospheres with morphological abnormalities and cell detachment ( Mock-infected neurospheres presented the expected ultrastructural morphology of the nucleus and mitochondria (Fig. 3A). Viral particles were present in ZIKV-infected neurospheres, similar to those observed in murine glial and neuronal cel...

Zika virus disease: a current review of the literature

“…This was possibly because the African lineage strain of ZIKV was associated with low viremia in humans [77], whereas the Asian lineage strain of ZIKV, involved in current outbreaks (Pacific Islands and in South America), is spreading along long chains of human-to-human transmissions. Second, the differences between the genetic makeup of the African and Asian lineages and adaptive changes in the Brazilian strain might explain the recent emergence of ZIKV-associated complications in humans [78][79][80]. Third, microcephaly is a rare disease and has many causes [81] other than ZIKV.…”

“…Recently, a few studies [79,80,[82][83][84][85] have evidenced the pathogenic potential of Brazilian strain of ZIKV during the development of fetus in animal models. These studies concluded that ZIKV first infects the placenta and then the brain of fetus, where it preferentially infects neural progenitor cells and decreases their viability and growth as neurospheres through down-regulation of genes that are involved in cell and organ development, and up-regulation of genes that are involved in immune responses.…”

Translational potential of human brain organoids

“…These studies have provided evidence that human neural stem cells are selectively vulnerable35, 48 and more susceptible to premature differentiation50 upon Zika virus infection, providing a molecular explanation for the observed microcephaly. A further study suggested activation of innate immune responses as a possible mechanism for Zika‐induced neuroprogenitor cell death 47.…”

“…Several research groups applied the brain organoid model to study Zika infection, a current epidemic that has been strongly linked to newborn microcephaly 35, 46, 47, 48, 49, 50. These studies have provided evidence that human neural stem cells are selectively vulnerable35, 48 and more susceptible to premature differentiation50 upon Zika virus infection, providing a molecular explanation for the observed microcephaly.…”

Editorial brain malformation surveillance in the Zika era

“…ZIKV invades fetal neural progenitor cells and causes brain abnormalities by at least two general mechanisms: (1) direct destruction of neuronal progenitor cells and developing neurons, and (2) disruption of the differentiation, development, and migration of neurons in the developing brain (Garcez et al, 2016; Qian et al, 2016; Sarno et al, 2016; Tang et al, 2016; Cugola et al, 2016; Li et al, 2016). Both the Brazilian ZIKV strain and the Asian ZIKV strain cause abnormal cortical development in mice (Cugola et al, 2016; Li et al, 2016) that would be consistent with multiple different types of cortical malformations in human infants exposed in utero to ZIKV among both those with congenital microcephaly and those with normal head circumferences at birth.…”