Microcephaly is a neurodevelopmental disorder

Microcephaly is a neurodevelopmental disorder where the head is smaller than the typical size during fetal development and at birth, with the circumference less than 2 SDs below the mean (Centers for Disease Control and Prevention, 2016b). Babies with microcephaly can have a wide array of problems such as developmental delays, seizures, vision and hearing loss, and feeding difficulty. To date, little is known about the mechanism underlying ZIKV-associated microcephaly. Since a normal SB 203580 hydrochloride develops from neural stem cells (NSCs) and their differentiated neural cells, microcephaly is most likely associated with the abnormal function of these cells. Yet, many questions remain to be addressed, such as how human fetal brain NSCs or their progeny are susceptible to ZIKV infection, whether different strains of ZIKV infect NSCs with equal efficiency, if such infection affects functions of NSCs important in human brain development, and whether NSCs from different human origins respond to ZIKV equally. Recent evidence shows that ZIKV directly infects NSCs of the fetus and impairs growth in mice (Li et al., 2016; Wu et al., 2016). It has also been shown that ZIKV infects neural progenitors from human skin-cell-induced pluripotent stem cells (Garcez et al., 2016; Tang et al., 2016), but these studies used the murine neuro-adapted prototype strain (MR766) belonging to the African lineage. A more recent study showed that a 2015 Puerto Rico strain of ZIKV, PRVABC59, infects and kills primary human fetal neural progenitors (Hanners et al., 2016). However, none of these studies investigated the effect of ZIKV on neural stem cell functions, particularly their differentiation into neurons and glial cells, which is critical for brain development.

Results

Discussion
Here, we established an in vitro system using primary human fetal brain-derived NSCs from three different donors to characterize the effect of ZIKV infection. We particularly focused on an Asian-lineage ZIKV strain, Mex1-7, that was involved in the first outbreak in North America in late 2015 in Chiapas State (Guerbois et al., 2016). Interestingly, we found that the Mex1-7 strain had a substantial lower infectivity (1.5%) in primary hNSCs when compared with other strains (10% by FSS and 8% by DakAr) tested in this study, as well as compared with the 1947 African lineage MR766 strain (20%–80% infection rate) and ArB41644 strain (40% infection rate) reported previously (Garcez et al., 2016; Tang et al., 2016; Simonin et al., 2016). The high infectivity of MR766 may be explained by the fact that it has been passaged for over 150 times in suckling mouse brains and developed a neuro-adapted phenotype, a concern addressed previously (Miner and Diamond, 2016). An alternative explanation is that African ZIKV strains may have a higher infectivity rate than Asian ZIKV strains. In this study, the FSS and DakAr strains used were passaged 3 and 7 times, respectively, only in mosquito and mammalian cell culture and not in neural cells. On the other hand, the source of NSCs may also contribute to the discrepant infectivity of ZIKV. For example, our observation of less than 10% cell infection rates are more in line with recent reports (Hanners et al., 2016). Both this, and the fact that our study used primary cultured human fetal brain NSCs may account for differences compared with Tang et al. (2016) and Garcez et al. (2016) who used neural progenitor cells derived from human skin-cell-induced pluripotent stem cells. However, all of these results indicate that the type of neural cells, the viral lineage/strain from different sources, and the viral propagation methods need to be considered when interpreting ZIKV infectivity and functional consequences in human cells.
Microcephaly may result from abnormal proliferation or differentiation of NSCs during early development (Homem et al., 2015). We thus established an in vitro system to characterize the effect of Mex1-7 on survival, proliferation, and differentiation of three primary human fetal NSC strains: G010, K048, and K054. These three cell strains were derived from three human fetal donors at gestational weeks 9 (K048) and 13 (K054 and G010), around the end of the first trimester of pregnancy and within the time frame with high risk of viral-mediated abnormal neural development, including ZIKV-induced microcephaly (Cauchemez et al., 2016). Further, Mex1-7 infections decreased the cell population in all three cell strains without significant changes in Cas3 or BrdU during the proliferation stage. The lack of change of Cas3 could indicate that, during the proliferative stage, cell-cycle progression is halted but cells are not undergoing apoptosis. Since only a small percentage of hNSCs are infected (1.5%), a bystander effect may be occurring to inhibit proliferation. Alternatively, a necrotic mechanism of cell death could account for the decreased population. The population decrease is in general accordance with the previous studies using either human embryonic stem cells or induced pluripotent stem cells (Dang et al., 2016; Garcez et al., 2016; Qian et al., 2016; Tang et al., 2016). In addition SB 203580 hydrochloride to these in vitro studies, two groups have also shown that ZIKV infection in pregnant mice leads to inhibition of neural progenitor proliferation in mouse embryos by cell-cycle arrest (Wu et al., 2016; Li et al., 2016). Further studies are warranted to dissect the mechanisms underlying ZIKV inhibition of NSC proliferation both in vitro and in animal models in vivo.