ZIKV infection of brain organoids: role of TLR-3 induced apoptosis due to changes in gene expression?

Circulating ZIKV strains can be separated into two clades, African and Asiatic with the former comprised of two groups (MR766 and the Nigerian cluster) and the latter into the Malaysian and Micronesian strain although the genetic diversity is rather low ( less than 12 % between the African and Asian clades).

ZIKV is transmitted by mosquitoes and can be readily isolated from Aedes mosquitoes in Africa (including Ae. Furcifer, Ae. luteocephalus, Ae. taylori, Ae. dalzieli, Ae. opok, Ae. vittatus, Ae. jamoti, Ae. flavicollis, Ae. grahami, Ae. taeniarostris, Ae. tarsalis, Ae. fowleri, Ae. metallicus, Ae. minutus, Ae. neoafricanus, Ae. Albopictus) as well as from Anopheles mosquitoes (An. coustani, An. gambiae), Mansonia (Ma. uniformis) and Culex (Cx. Perfuscus) whereas in Asia ZIKV is transmitted by Aedes aegypti. Mosquitoes are not however the primary host for ZIKV. Both in Africa and Asia, non-human primates have been proposed to be the reservoir for ZIKV with forest dwelling mosquitoes transmitting ZIKV to humans. Once established within the human population, ZIKV is transmitted by mosquitoes as well horizontally by sexual intercourse, vertical and parenteral.  

During the current outbreak of ZIKV in Brazil, ZIKV RNA was detected in amniotic fluid samples of women infected with ZIKV during pregnancy and ZIKV RNA has also been isolated from tissue (brain and CNS) of neonates born with microcephaly, suggesting that ZIKV infection of the mother might be a contributive factor in the observed increase of microcephaly cases in neonates. In addition to microcephaly, miscarriages have also been reported in ZIKV positive pregnant women, especially during the first trimester of pregnancy. Foetal deaths have been observed in women who were infected with ZIKV during the second and third trimester in addition to neonate death within 20 hrs following birth.

In addition to miscarriages, foetal and neonatal death, (congenital) ocular findings associated with microcephaly have been reported in 34.5% of infants examined that lead to eye damage.

In principle, teratogenic effects following viral infection of pregnant women is well documented for other viruses such as Herpes Virus simplex or Rubella Virus (RV). In the case of RV, the cytopathogenesis of infected foetal tissues suggests that RV infection of the foetus in utero induces extensive apoptosis and necrosis as well as mitotic defects of precursor cells, thus leading to abnormal organogenesis. These results are supported by results obtained in WI-38 human diploid fibroblasts showing that the viral 33A protein inhibits mitosis and the expression of the viral Capsid protein and all three structural proteins (but not the E1 or E2 protein separately) in RK13 cells induces apoptosis, is independent of the mitochondrial pathway probably as a result of the induction of the ER stress response.

In the case of ZIKV, apoptosis of precursor cells especially of neural progenitor cells, has been demonstrated for brain organoids as well as human progenitor stem cells (hNPC) as discussed previously. Foetal infection during the early stages however requires viral particles to cross the placental barrier. In the case of ZIKV, the expression of Interferon-l (IFN-l) in trophoblasts may limit ZIKV replication and thus the ability to infect embryonal or foetal cells. A recent paper however showed that at least in a small number of infection acquired microcephaly the maternal placenta allows the passage of infected Hofbauer cells since maternal histiocytes-immune cells of monocyte origin- are frequently found within the human placenta, have the ability to reach foetal vessels and and subsequently infect neuronal precursor cells, thus causing neuronal abnormalities associated with microcephaly and/or ocular abnormalities. Transplacental passage of infected histiocytes has been reported for other viral diseases, including seasonal (A/H1N1) influenza where the infection in early pregnancy caused second trimester foetal demise.
The notion that ZIKV can cause microcephaly is further supported by recent findings that in female Ifnar1 -/- as well as in wt mice treated with an inhibitor (MAR1-5A3)against Ifnar1, foetuses exhibit symptoms similar to those observed in infants born to ZIKV positive mothers that exhibit microcephaly. Viral RNA could be detected in the foetal brain of infected mice up to 16.5 days during embryonal development as well as in the placenta, suggesting that in Ifnar1 -/- mice ZIKV not only replicates in the placenta but also crosses the placenta and thus infects the embryo.

Research on ZIKV pathogenesis currently underway utilises cell lines, animal models and brain organoids, which are used to study the various aspects of the interaction of ZIKV with the host cell. ZIKV research thus benefits from both “traditional” (cell lines and animal models) as well as “modern” advances in cell biology. Whereas cell lines and animal models are used for a long time to study virus-host interactions, the use of hNPC and brain organoids is a relatively modern development. Brain organoids were developed to study neurodegenerative disorders and are stem cells and are human iPSC-derived neural progenitor cells (NPCs) that have differentiated into 3D organoid systems epitomize forebrain, midbrain, and hindbrain regions and thus represent a model of the developing brain.

The infection of mouse neurospheres as well as 10-day old human immature cerebral organoids with ZIKV MR766 exhibited a significant decrease in growth compared to mock infected samples as early as 24 hrs p.i. as well as viral replication, indicating that ZIKV indeed does decrease growth of brain organoids which has been shown to be due to the induction of apoptosis. The induction of apoptosis can be due to the downregulation of genes that inhibit apoptosis induced by a variety of processes including DNA damage, aberrant cell division or mitochondrial depolarisation as has been proposed ZIKV infection of hNPC or due to downregulation of genes regulating the aforementioned processes as a result of ZIKV induced antiviral signalling. Alternatively, ZIKV proteins and/or viral RNA (both dsRNA intermediates and ss genomic RNA) may activate the apoptotic response. 

Figure: Downregulation of gene groups in brain organoids compared to neural
stem cells

ZIKV infection of brain organoids, hNPC and A549 cells has been reported to induce caspase dependent apoptosis probably via the mitochondrial pathway which may be associated with the downregulation of genes associated with DNA replication and mitosis. Interestingly, human brain organoids exhibit a downregulation of in the expression of genes related to DNA replication, cell cycle progression, mitosis and apoptosis compared to human neuronal stem cells raising the possibility that brain organoids may be more sensitive to viral induced modulation of gene expression or to the induction of apoptosis by viral proteins and/or viral RNA; regrettably, as of now no data are available that compare the transcriptome of ZIKV infected neuronal stem cells with the transcriptome of ZIKV infected cell lines and brain organoids which may indicate whether ZIKV infection does indeed modulate the expression of genes similar to infected hNPC cells or not. 

In any case, the infection of cell lines and brain organoids with ZIKV as well as treatment with Poly (I:C) induces TLR-3 mediated antiviral signalling and subsequent apoptosis which can partially reversed by treatment of organoids with a TLR-3 inhibitor. 
These data indicate that ZIKV infection may trigger apoptosis  of foetal brain cells during the first trimester of foetal development and thus contributes to the malformation of the brain associated with microcephaly. In addition, Poly (I:C) induced TLR-3 signalling also downregulates the expression of two genes related to neurogenesis, Nestrin and Ephrin type-B receptor 2, both of which are also downregulated in ZIKV infected hNPC, suggesting that ZIKV indeed impairs neural development.

Interestingly, the expression of UNC93B1 is downregulated both in primary human trophoblasts (compared to JEG-3 cells) and in cerebral organoids (compared to hNPC). In humans, UNC9393B1 deficiency has been linked to predispose patients to HSV encephalitis that causes severe neurological damage. In mice, point mutations of UNC93B1 have been linked to prevent TLR-3 (as well as TLR-7 and -9) localisation to the endolysosomal compartment that contain the ligand and thus prevent activation of TLR mediated signaling pathways. If UNC93B1 deficiency contributes to ZIKV induced pathogenesis –not only in the developing foetus but also in adult patients- is not clear and needs to be investigated.

Figure: Downregulation of UNC93B1 in ZIKV infected hNPC and non-infected
brain organoids 

Both viral RNA and Poly (I:C) has been shown to induce apoptosis by activating both caspase-3 and caspase-8 dependent pathways through TLR-3 and ZIKV has been shown to induce apoptosis in both brain organoids and hNPC neuronal cells as well as in A549 cells. Interestingly, in ZIKV infected hNPC the expression of XIAP (X-linked inhibitor pf apoptosis) is upregulated, suggesting that ZIKV might be able to inhibit caspase-3 dependent apoptosis in hNPC.

Pending further studies, activation of TLR-3 by ZIKV RNA (dsRNA intermediate and/or genomic ssRNA) might induce the degradation of the TLR-3/RNA complex via the formation of autophagosomes and subsequent fusion of the autophagosome with the lysosome. Alternatively –or additionally- viral RNA located in early endosomes might be degraded following the fusion of the late endosome with the lysosome. Deficiency of UNC93B1 in brain organoids therefore would prevent the degradation of ZIKV RNA by autophagy and therefore promote viral replication.

Figure: TLR-3 is degraded in a UNC93B1 dependent manner upon binding to viral RNA

In conclusion, ZIKV might cross the placenta not by infecting placental cells but by migration of infected Hofbauer cells and subsequent infection of neuronal precursor cells followed by activation of TLR-3 dependent signalling pathways that induce both apoptosis and decrease the expression of genes related to neurogenesis. A recent study identified long noncoding RNAs (lncRNAs) that suppress the interferon response in human trophectoderm and primitive endoderm cells, suggesting that the expression of lncRNA in hNPC and brain organoids might contribute to ZIKV infection and ZIKV induced apoptosis. So far however this has not been investigated whereas the activation of TLR-3 has been shown to be involved in perinatal brain injury.

Further reading 

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