Michaela Marziale

 Introduction. Zika virus (ZIKV) is a mosquito-borne virus that primarily infects human skin (dermal fibroblasts and epidermal keratinocytes), with the majority of infections being asymptomatic³. During 2015-2016 there was a ZIKV pandemic that lead to the World Health Organization (WHO) to declare a public health emergency of international concerns for microcephaly due to ZIKV infection². In order to better protect pregnant mothers and their children it is imperative that ZIKV infection and its lifecycle are better understood. Studies have shown that when ZIKV crosses the placental barrier it targets human neural progenitor cells (hNPCs) and enters these cells via an AXL receptor⁵. It has been proposed that after the virus has gained entry to the cell it hyper-activates Toll-like 3 receptors and this over-activation leads to impaired neurogenesis and activation of a pro-apoptotic pathway resulting in microcephaly^{1, 2, 6}. Methods. Embryonic stem cells were differentiated toward forebrain, midbrain, and hindbrain. Immunohistochemistry for TUJ1 and SOX1 was employed to specify neuronal and NPC populations in the cerebral organoids. This mimicked the neuronal migratory and differentiating pattern of the fetal brain. The cerebral organoids were then infected with ZIKV to observe the effect on fetal brain development in NPCs. Lastly, the authors used thiophenecarboxamidopropionate compound to competitively inhibit TLR3 on mouse neurospheres and human organoids. They then compared differential gene expression following TLR3 activation¹. Another experiment infected cells with ZIKV and quantified RNA using RT-PCR³. Results. It was found that ZIKV infected NPCs in organoid models^{1, 4}, and that the cells elongated morphology after infection suggested an unhealthy, apoptotic state. The neurospheres infected with ZIKV with and without TLR3 inhibitor showed a statistical difference, whereas there was no statistical difference between mock and ZIKV+inhibitor groups. Organoids inoculated with TLR3 inhibitor in the presence of ZIKV still showed some cell disruption which was shown by the non-smooth neuroepithelium, but the TLR3 competitive inhibitor reduced the severity of ZIKV mediated apoptosis and organoid shrinking seen in ZIKV only organoids. Pathway analysis of gene expression during TLR3 upregulation emphasized 41 genes involved in neuronal development¹. Conclusions. The data show an upregulation of TLR3 after ZIKV infection and that TLR3 inhibition reduces the ZIKV phenotype. This study suggests that ZIKV disrupts a TLR3 regulated network that controls neurogenesis and apoptosis. These findings along with others demonstrating the impact of TLR3 on neurogenesis are highly suggestive of the causal link between ZIKV and TLR3 hyperactivity^{1, 2, 3}.

1. Dang J, Tiwari S, K, Lichinchi G, et al. Zika Virus Depletes Neural Progenitors in Human Cerebral Organoids through Activation of the Innate Immune Receptor TLR3. Cell Stem Cell Vol 19, Issue 2, 4 August 2016, pp. 258-265.
2. Faizan M, I, Abdullah M, Ali S, Naqvi I, H, Ahmed A, Parveen S, Zika Virus-Induced Microcephaly and Its Possible Molecular Mechanism. Intervirology 2016;59:152-158
3. Hamel R, Dejarnac O, Wichit S, et al. Biology of Zika Virus Infection in Human Skin Cells. American Society for Microbiology: Journal of Virology Vol. 89:17. Doi: 10.1128/JVI.00354-15.
4. Tang H, Hammack C, Ogden S, et al. Cell Stem Cell Vol. 18: 587–590, 5 May 2016.  DOI:http://dx.doi.org/10.1016/j.stem.2016.02.016.
5. TJ Nowakowski, AA Pollen, Lullo Di, et al. Expression Analysis Highlights AXL as a Candidate Zika Virus Entry Receptor in Neural Stem Cells. Cell Stem Cell Vol 18: 519-6, 5 May 2016. doi: 10.1016/j.stem.2016.03.012.