Although being first isolated in 1947 from a sentinel rhesus monkey in forests of Uganda, until 2007 Zika Virus (ZIKV) outbreaks have been sporadic, being limited to tropical regions in Africa as well as Southeast Asia and only been associated with relative mild symptoms in about 20% of infected people. Autochthonous transmission of ZIKV outside these areas was only reported in 2007 in the Federal States of Micronesia (FSM), 2013 in French Polynesia, and in 2015 in South America, spreading to the Caribbean and North Americas within a few months. Concomitant with the advent of ZIKV in the Americas, ZIKV was implicated in the onset of neurological diseases in foetuses, neonates and adults, namely microcephaly and Guillan-Barre Syndrome (GBS); subsequently, studies in animal models (both mice and monkeys), brain organoids, neural stem cells (NSC) and human neural progenitor cells (hNPC) confirmed that ZIKV can induce abnormal brain development probably by inducing apoptosis of neural progenitor -but not mature neurons- in a Caspase dependent pathway either in ZIKV infected cells or via bystander apoptosis of uninfected neural crest cells via inducing the secretion of cytokines. As discussed before, the infection of the vaginal mucosa might promote the transmission of ZIKV to the placenta and/or of Hofbauer cells that might cross the placenta and thus infect the embryo during the early stages of neural development, thus explaining data retroactively obtained from the 2013 ZIKV outbreak that indicated an increased risk for microcephaly of infants born to mothers that were infected with ZIKV in the first two trimesters.
In addition to microcephaly however, several case reports from Brazil indicate that neonates born to ZIKV positive mothers display an array of eye malformations including blindness, intraretinal haemorrhages, chorioretinal atrophy, optic neuritis, lens sublaxation and bilateral iris colobomas, indicating that ZIKV might also affect eye development. Furthermore, in adults, ZIKV infection has been associated with conjunctivitis as well as uveitis in 10-15% of infected adults presenting themselves with ZIKV infection. Similar to EBOV, viral RNA can be detected in fluid samples taken from the anterior chamber of the eye, suggesting that ZIKV can either replicate in the eye or that ZIKV is at least present (but not necessarily replicating). ZIKV therefore joins other viruses that (potentially) can cause inflammation of the eye or blindness in neonates such as Human Cytomegalovirus (HCMV) which can be acquired both antenatal and intrapartum in addition to other viruses such as Adenovirus', Picornavirus', Herpes Simplex Virus 2 or Varicella Zoster Virus/Human Herpesvirus 3. So far however, an active eye infection as a result of ZIKV infection has not been reported to transmit to household contacts. More importantly however, the presence of viral RNA in the aqueous humor might indicate that following immunosuppression, ZIKV replication might resurface and thus might be horizontally transmitted via sexual contact or via mosquitoes and thus either sustain or restart a local. ZIKV outbreak.
In order to characterize the potential of ZIKV to cause uveitis, animal models were being used. As discussed before, ZIKV does not replicate well wt C57BL/6 mice partially because the viral NS5 protein does not block mouse derived STAT2 and thus antiviral signalling. Consequently, in mice, ZIKV pathogenesis in mice is studied either in immunocompromised mice such as AG129 or Ifnar-1 -/- mice; in theory however it might also be possible to use recombinant ZIKV generated using the recently published reverse genetic systems that contain the NS5 gene of the mouse adapted ZIKV MR766 (providing that the ZIKV MR766 NS5 inhibits mouse STAT2) . In accordance with previous results which also have been discussed in previous postings, wt mice treated with Ifnar-1 -/- antibody and infected with either ZIKV H/PF/2013 or ZIKV Paraiba 2015 (isolate from Brazil) do not develop any signs of disease although high viral titres can be detected in multiple organs, including the testes of male mice, which is in contrast with Ifnar-1 -/- mice that develop severe neuroinvasive infection with decreased survival. Viral RNA in both ZIKV H/PF/2013 and ZIKV Paraiba 2015 infected mice can be detected as early as 48 hrs p.i., increasing at day 6 p.i. (H/PF/2013) and day 7 p.i. (Paraiba 2015). Also, viral RNA could be detected in tears and in lacrimal glands of ZIKV Paraiba 2015 infected mice, suggesting the presence of either infectious viral particles and/or cell debris of ZIKV infected cells due to virus induced apoptosis of infected cells. The presence of infectious ZIKV in eyes at day 7 p.i. was confirmed by infecting immunocompromised AG129 mice eye homogenates of ZIKV Paraiba 2015 infected Ifnar-1 -/- mice, whereas both tears from day 7 p.i. nor eye homogenates from day 28 p.i. contained infectious virus, indicating that tears are not infectious and that infetious virus is not present in the eye after the acute phase of the infection. In general, eye homogenates derived from mice at day 7 p.i. , caused more severe symptoms in AG129 mice compared to ZIKV Paraiba 2015 infection, especially ocular pathology and conjunctivitis, despite similar viral titres in spleen, brain and eyes, suggesting that viral particles derived from the eye might be adapted (which at this point is speculative). One possibility to investigate if ZIKV derived from eye homogenates is adapted to cells in visual system might be to infect Ifnar-1 -/- or AG129 mice with virus grown in human retinal pigment cells (ARPE-19), retinal ganglion cells or adult retinal stem cells. Nonetheless, so far sequencing eye derived virus did not reveal substitutions except an increase single mutation in the viral NS2A gene (C to T at position 3895 or A to V), but the significance of this mutation has not been evaluated.
As discussed in a previous post, the in utero infection of foetal mice at E4.5 via the vaginal mucosa increases the severity of foetal and neonatal brain abnormalities compared to mice infected at E8.5, confirming previous observations during the ZIKV outbreak in French Polynesia that infants born to women who got infected with ZIKV during the first trimester display a higher percentage of microcephaly. Accordingly, the offspring of Ifnar-1 -/- crossbred with Ifnar-1 +/+ mice and infected in utero with ZIKV Paraiba 2015 at E6.5 but not E12.5 present themselves with intrauterine growth restriction (IUGR) with foetal demise and viral RNA in the brain without any (detectable) ocular defects. Viral RNA was only detected in 2 out 41 mice infected with either ZIKV H/PF/2013 or ZIKV Paraiba 2015 at day 8 post natal (or 1 week of age), indicating that ocular defects and persistence of viral RNA in the eye is indeed a rare occurrence following infection with ZIKV in utero, which is supported by epidemiological findings. In contrast, the infection of 1 week old wt mice with ZIKV Paraiba 2015 not only is lethal (in accordance with findings from the early 1950s using wt mice and the primary ZIKV MR766 isolate) but also leads to high viral RNA levels in the spleen, brain and the eye concomitant with prominent Caspase-3 dependent apoptosis of cells of the optic tract, the visual cortex and the lateral geniculate nucleus as well as other components of the visual cortex, although based on the data available it is not clear if apoptosis is also induced by "bystander apoptosis" of non-infected cells in addition to ZIKV virus infected cells. Immunodeficient AG129 infected postnatally also exhibit high viral titres at day 8 p.i. in the spleen, brain and eye irrespective if the parental or a mouse-adapted ZIKV Paraiba 2015 strain is used, suggesting that the IFN response in 1 week old wt mice is unable to inhibit viral replication irrespective of the ability of ZIKV to inhibit STAT2 signalling, which might be attributed to an immature immune system.
In contrast to 1 week old wt/AG129 mice, adult Ifnar-1 -/- mice infected with ZIKV Paraiba 2015 present themselves with extensive apoptosis of retinal cells in the neurosensory retina as measured using TUNEL staining that detects fragmented DNA and uveitis at day 6 p.i., accompanied by infiltration of inflammatory cells into both the anterior and posterior chambers of the eye in the absence of pan-retinal damage to the fundus. Although it is not clear how ZIKV Paraiba 2015 causes uveitis, it might be possible that the viral RNA triggers an inflammatory response that not causes localised apoptosis but also triggers the inflammation of the eye via Toll-like and/or RIG-1/MDA-5 mediated signalling pathways. In any case, viral infection is cleared by day 28 p.i.. Viral RNA can be detected in all regions of the eye between 6 and 8 days p.i. with the highest levels of viral RNA in the retinal epithelium/choroid complex, in particular bipolar and ganglion cell neurons as well as the optic nerve and cornea of ZIKV infected mice as detected by FISH and qRT-PCR, indicating that ZIKV can not infect the eye but also replicates in the eye.
In conclusion, similar to other viruses such as West Nile Virus, Hepatitis C Virus or EBOV, ZIKV can infect retinal tissue, causing ocular defects in adult as well as foetal/neonate mice. So far however further studies are needed to determine if ZIKV also replicates in retinal cells, which can be done using retinal cell lines. Also, it needs to be determined if ZIKV infection of the foetal brain downregulates the expression of genes that are required for the development of the visual system; experiments can be performed either by using foetal brain of in utero infected mice or alternatively brain organoids and/or neural stem cells. Data previously published suggest that genes encoding for proteins that are involved in the development of the visual system are differently expressed during brain development, which might explain the greater susceptibility of foetal mice infected at E6.5. Congenital ZIKV infection therefore might therefore not only target ocular tissue but also interfere with the development of the visual system which has been suggested by recent findings using SJL mice infected with ZIKV Brazil; it should be noted however that in congenital Ifnar-1 +/- foetuses derived from C57BL/6 Ifnar-1 -/- mice, no histological abnormalities are present unless they are rare and thus were not detected due to the sample size. Bystander apoptosis in non ZIKV infected cells can be induced by the secretion of cytokines by ZIKV cranial neural crest cells (CNCC). In contrast to other cell types, these cells undergo only limited apoptosis upon infection with ZIKV H/PF/2013 and secrete LIF, IL-60, PAI-1, VEGF, MCSF, TNF-α and IL-17 inducing apoptosis of co-cultured neurospheres whilst preventing viral induced apoptosis of infected CNCC in a paracrine manner. Further experiments however are needed to determine if bystander apoptosis is also affecting ocular precursor cells. In a sideline, 4 week old cerebral organoids express higher levels of VEGF compared to hNPC at week 0, so that it might be possible that ZIKV infection of 4 week old cerebral organoids even further increases the expression of VEGF in CNCC and thus prevents apoptosis of CNCC via a paracrine effect. Finally, if ZIKV replicates in retinal epithelial cells, then it might inhibit STAT3 signalling and thus lead to the degeneration of retinal epithelial cells.
|Figure: Gene groups that are upregualted in 8 week old cerebral organoids compared to 4 week old|
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