How can Zika virus infection in the uterus lead to microcephaly in newborns?

A new study led by researchers at Baylor College of Medicine revealed how in utero Zika virus infection can lead to microcephaly in newborns.

The team discovered that the Zika virus protein NS4A disrupts brain growth by hijacking a pathway that regulates the generation of new neurons.

The findings point at the possibility of developing therapeutic strategies to prevent microcephaly linked to Zika virus infection. The study appears today in the journal Developmental Cell.

Patients with rare genetic mutations shed light on how Zika virus causes microcephaly

“The current study was initiated when a patient presented with a small brain size at birth and severe abnormalities in brain structures at the Baylor Hopkins Center for Mendelian Genomics (CMG), a center directed by Dr. Jim Lupski, professor of pediatrics, molecular and human genetics at Baylor College of Medicine and attending physician at Texas Children’s Hospital,” said Dr. Hugo J. Bellen, professor at Baylor, investigator at the Howard Hughes Medical Institute and Jan and Dan Duncan Neurological Research Institute at Texas Children’s Hospital.

This patient and others in a cohort at CMG had not been infected by Zika virus in utero.

They had a genetic defect that caused microcephaly.

CMG scientists determined that the ANKLE2 gene was associated with the condition. Interestingly, a few years back the Bellen lab had discovered in the fruit fly model that ANKLE2 gene was associated with neurodevelopmental disorders.

Knowing that Zika virus infection in utero can cause microcephaly in newborns, the team explored the possibility that Zika virus was mediating its effects in the brain via ANKLE2.

In a subsequent fruit fly study, the researchers demonstrated that overexpression of Zika protein NS4A causes microcephaly in the flies by inhibiting the function of ANKLE2, a cell cycle regulator that acts by suppressing the activity of VRK1 protein.

Since very little is known about the role of ANKLE2 or VRK1 in brain development, Bellen and his colleagues applied a multidisciplinary approach to tease apart the exact mechanism underlying ANKLE2-associated microcephaly.

The fruit fly helps clarify the mystery

The team found that fruit fly larvae with mutations in ANKLE2 gene had small brains with dramatically fewer neuroblasts—brain cell precursors – and could not survive into adulthood.

Experimental expression of the normal human version of ANKLE2 gene in mutant larvae restored all the defects, establishing the loss of Ankle2 function as the underlying cause.

“To understand why ANKLE2 mutants have fewer neuroblasts and significantly smaller brains, we probed deeper into asymmetric cell divisions, a fundamental process that produces and maintains neuroblasts, also called neural stem cells, in the developing brains of flies and humans,” said first author Dr. Nichole Link, postdoctoral associate in the Bellen lab.

Asymmetric cell division is an exquisitely regulated process by which neuroblasts produce two different cell types.

One is a copy of the neuroblast and the other is a cell programmed to become a different type of cell, such as a neuron or glia.

Proper asymmetric distribution and division of these cells is crucial to normal brain development, as they need to generate a correct number of neurons, produce diverse neuronal lineages and replenish the pool of neuroblasts for further rounds of division.

“When flies had reduced levels of Ankle2, key proteins, such as Par complex proteins and Miranda, were misplaced in the neuroblasts of Ankle2 larvae.

Moreover, live imaging analysis of these neuroblasts showed many obvious signs of defective or incomplete cell divisions.

These observations indicated that Ankle2 is a critical regulator of asymmetric cell divisions,” said Link.

Further analyses revealed more details about how Ankle2 regulates asymmetric neuroblast division. They found that Ankle2 protein interacts with VRK1 kinases, and that Ankle2 mutants alter this interaction in ways that disrupt asymmetric cell division.

The Zika connection

“Linking our findings to Zika virus-associated microcephaly, we found that expressing Zika virus protein NS4A in flies caused microcephaly by hijacking the Ankle2/VRK1 regulation of asymmetric neuroblast divisions.

This offers an explanation to why the severe microcephaly observed in patients with defects in the ANKLE2 and VRK1 genes is strikingly similar to that of infants with in utero Zika virus infection,” Link said.

“For decades, researchers have been unsuccessful in finding experimental evidence between defects in asymmetric cell divisions and microcephaly in vertebrate models.

The current work makes a giant leap in that direction and provides strong evidence that links a single evolutionarily conserved Ankle2/VRK1 pathway as a regulator of asymmetric division of neuroblasts and microcephaly,” Bellen said.

“Moreover, it shows that irrespective of the nature of the initial triggering event, whether it is a Zika virus infection or congenital mutations, the microcephaly converges on the disruption of Ankle2 and VRK1, making them promising drug targets.”

Another important takeaway from this work is that studying a rare disorder (which refers to those resulting from rare disease-causing variations in ANKLE2 or VRK1 genes) originally observed in a single patient can lead to valuable mechanistic insights and open up exciting therapeutic possibilities to solve common human genetic disorders and viral infections.

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Zika virus (ZIKV) has been known since 1947 [1], and was historically regarded as a geographically restricted virus causing minor symptoms in humans. In 2013, this view started to change when ZIKV occurrence in French Polynesia was associated to development of Guillain-Barre syndrome, a serious autoimmune condition affecting the peripheral nervous system.

Only two years later, the virus landed in the Americas, where it was responsible for a major outbreak becoming a serious public health concern.

It is predicted that over 800,000 people were infected by ZIKV in the American continent between 2015 and 2018 [2]. Following this epidemic, several studies endorsed a causal relationship between ZIKV infection and neurological disorders, both in congenitally-exposed newborns and in adult patients.

The development or severity of these conditions cannot be predicted based on genetic or environmental factors, which reinforces the importance of long-term clinical surveillance of individuals exposed to ZIKV. Here, we review the epidemiology of ZIKV-associated complications and discuss experimental and clinical evidence of potential therapeutic approaches.

ZIKV Infection Is Associated to Late Detrimental Effects to the Developing and Mature Nervous Systems

A spectrum of neuropathological conditions has been reported in newborn babies exposed to ZIKV in uterus, being microcephaly the most severe outcome. Infants who develop ZIKV-induced microcephaly show recurrent seizures [3], and severely compromised neuropsychomotor development is frequently reported [4]. Several alterations were described after brain tomography of babies congenitally exposed to ZIKV, including widespread calcifications, cerebral atrophy, brainstem and cerebellar hypoplasia, as well as ventriculomegaly [5] (Figure 1A).

Extensive research has focused on unraveling the mechanisms of congenital microcephaly induced by ZIKV [6,7,8].

However, increasing evidence from clinical and experimental studies suggest that even infants considered healthy at birth can develop severe long-term neurological complications as a consequence of exposure. According to US hospital records, 6% of children infected with ZIKV in uterus showed malformations detectable at birth.

Importantly, the percentage of infants with ZIKV-associated neurological complications rose to 14% when babies considered normal at birth were followed-up after leaving hospital [9]. Among the most common symptoms reported were personal-social developmental delays, swallowing difficulties, and a myriad of motor dysfunctions including compromised mobility, muscular hypertonia, dystonic movement, and impaired fine motor skills [9,10,11].

Post-natal onset microcephaly was also reported, and studies suggest that 60% of normocephalic babies exposed to ZIKV in uterus present seizures at some stage of development [11]. Several alterations were also described in brains of normocephalic ZIKV-exposed babies assessed by neuroimaging tools, including subcortical calcifications, increased frontal cerebrospinal fluid space and ventricle enlargement [12].

Infants exposed to ZIKV during the epidemic in the American continent are still under their third year of life, making it impossible to predict which of these symptoms may persist and if others may emerge. Animal models are useful tools to investigate the mechanisms of infection and its complications, and whereas most studies have focused on unraveling the mechanisms of ZIKV-induced microcephaly, a few others have provided insight into the mechanisms underlying late ZIKV-induced neurological damage.

Using a mouse model of ZIKV infection, our group was able to predict possible long-term alterations in children after a congenital infection. Since the stage of development of the mouse brain at early postnatal period resembles the events taking place in the human brain during the second and third trimester of pregnancy [13,14,15], we performed ZIKV infection shortly after birth (post-natal day 3).

Indeed, we found that infected pups presented neuropathological hallmarks that closely resembled those seen in brains of babies after ZIKV congenital infection. Over 90% of mice showed spontaneous seizures during childhood, and intensity of seizures decreased as animals grew into adulthood. Moreover, even though these episodes resolved when animals became adults, mice infected neonatally with ZIKV remained more susceptible to chemically-induced seizures. Besides, ZIKV-infected mice showed motor and cognitive disabilities in adulthood. Similar results were obtained in another study, in which authors demonstrated that early-life infection with an African ZIKV strain results in persistent memory impairments in mice [16].

These findings were further confirmed in non-human primates infected in uterus or shortly after birth. As adults, these neonatally-infected macaques developed an atypical emotional response [17] and neuropathological characterization of brains showed reduced white matter volume, ventriculomegaly, and decreased hippocampal growth in ZIKV-infected animals.

Neurological complications following ZIKV infection are not limited to cases of congenital infection [18].

Mounting clinical evidence show that ZIKV infection in adult patients may result in encephalitis [18,19,20], encephalomyelitis [18,20], acute myelitis [18,19], chronic inflammatory demyelinating polyneuropathy [21], and Guillain-Barre syndrome [19,22,23] (Figure 1B). Episodes of septic shock were also reported, and in rare cases these complications were shown to be fatal [21,24].

In addition, ZIKV-associated Guillain–Barre syndrome may, in some cases, lead to development of chronic pain [18]. One study found that 40% of patients still showed some kind of neurological alteration after 1 year of symptom onset, and after two years some patients had not yet fully recovered [25].

Collectively, these reports demonstrate that ZIKV infection is also detrimental to the mature nervous system and is associated with a spectrum of neurological syndromes that may culminate in long-term consequences and even mortality. Whether these neurological consequences are a direct effect of ZIKV replication or are secondary to the host immune response still need to be investigated; a question that could lead to important clues on how to prevent long-term consequences of infection. Different research groups have shown beneficial effects of both antiviral and anti-inflammatory strategies in preventing long-term behavioral consequences of infection.

These findings are reviewed in the following sections.

Another matter of concern involves the ability of ZIKV to persist in certain organs and tissues, since the late consequences of viral persistence remain unknown. ZIKV usually reaches detectable levels in the serum of patients within 3–10 days after onset of symptoms, shortly returning to undetectable levels [26].

Even after viral clearance from blood and up to 200 days after infection, detectable levels of ZIKV RNA were found in placenta of pregnant women [27]. ZIKV was also shown to persists in semen samples up to 93 days after the onset of symptoms, reinforcing the high risk of sexual transmission [28,29].

ZIKV RNA was found in brains of babies exposed in utero on average 163 days after infection, and one study described high viral loads after post-mortem analysis of the brain of a 5-month-old infected infant [5].

The ability of ZIKV to replicate for long periods in specific tissues has also been demonstrated in animal models. In adult non-human primates infected subcutaneously with ZIKV, virus remained in neuronal tissue, cerebrospinal fluid, and lymph-nodes, while in the serum levels were undetectable after only seven days post-infection [30,31].

Further evidence of persistent replication in neuronal tissue were found in immunocompetent mice infected during the neonatal period, where ZIKV negative RNA strand was detectable in the brain into adulthood (100 dpi), an indicative of active viral replication [32]. Altogether, these results provide evidence that ZIKV is capable of persisting in certain tissues long after resolution of symptoms and clearance of virus from blood.

Factors that may contribute to viral persistence remain unknown, as well as if new rounds of active viral replication and reoccurrence of symptoms can occur at later stages of life, including moments of compromised immune response or as consequence of other viral infections.

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More information: Nichole Link et al, Mutations in ANKLE2, a ZIKA Virus Target, Disrupt an Asymmetric Cell Division Pathway in Drosophila Neuroblasts to Cause Microcephaly, Developmental Cell (2019). DOI: 10.1016/j.devcel.2019.10.009

Journal information: Developmental Cell
Provided by Baylor College of Medicine