Children of Zika

As the children born with Zika grow up, more developmental deficits are being noticed. New studies examine these changes from an epigenetic perspective and seek to improve detection of the virus.

But as soon as Zika caught global attention, it disappeared. The World Health Organization declared that Zika was no longer a global emergency and the active outbreaks ended. However, for all the families affected by the virus during that time, their journey is just beginning.

Generation Zika

Zika is a virus spread primarily by mosquitoes and sexual transmission. For most, an infection is quite mild causing minor or no symptoms. However, if an infection occurs during pregnancy, the virus can spread to the baby and result in severe birth defects including microcephaly (an abnormally small head) and brain malformations. Of the mothers with Zika during pregnancy, approximately 6% gave birth to babies with Zika-associated birth defects.

During the epidemic, thousands of babies were born with these birth defects. In Brazil alone, nearly 3000 babies were born with microcephaly. As these babies become toddlers and continue to grow, further developmental delays are beginning to form.

A recent CBS documentary featured the children of “Generation Zika” as they turn 3 years old. The film highlights the array of neurological and physical challenges the children are facing.¹ Many toddlers cannot walk or talk and suffer from vision impairment, muscle weakness, and seizures. The only treatment is intensive physical therapy. However, many families come from rural or low socioeconomic backgrounds. This makes paying for care and traveling to frequent appointments infeasible.

Despite the generosity of physical therapists and doctors who have volunteered their time and services, there is simply not enough available care to properly support all the affected children. Currently, the Brazilian government has not provided any financial or medical aid to the families and there are no plans to do so in the future. With the long-term health effects of Zika unclear, the pressing scientific question at hand is whether more Zika related neurological issues will appear later in life for affected children, especially for the 94% of babies who did not exhibit any birth defects.

Examining the Epigenetics

A recent study by Janssens et al. ² is attempting to address some of the questions that surround the development of babies born to mothers with Zika. Since the Zika virus can pass from a mother to a fetus during pregnancy and directly affect the fetus’s neurological development, researchers examined DNA methylation patterns within the genome of neural cells.

Embryonic stem cell-derived (ESC) brain organoids composed of multiple organized brain tissues were used as the best model for a developing brain. The complex organoida allowed for hetereocellular interactions to occur alongside any resultant regulation of gene expression and epigenetic patterning. ² The model organoids were then infected with the Zika virus.

Methylation levels were compared using whole-genome bisulfite sequencing to identify any significant methylome changes induced by Zika virus. To do this, researchers utilized the EZ DNA Methylation Gold Kit to perform the bisulfite conversion before constructing libraries.

Changes were identified in the DNA methylation patterns of the infected neurological cells. These changes were found at the genes that have been previously linked to brain disorders. The regions of methylation change were found in excess of 30-40% at regions near transcription start sites. This finding was very significant since the methylation changes near these regulatory regions dramatically influence gene expression, resulting in a variety of physiological symptoms depending on the genes with the epigenetic changes. Using the Direct-zol RNA Miniprep Kit, the group purified RNA and performed qRT-PCR to determine which genes were being expressed.

These regions were analyzed and linked to phenotypic gene expressions. Fifty-three genes close to these regions were linked to the microcephaly phenotype. Additional phenotypes were linked to genes associated with neurological and physical development disorders including intellectual disability, epilepsy, attention-deficit hyperactivity disorder, and craniofacial abnormalities.

The overarching question the study is trying to answer is whether Zika will cause more serious issues beyond microcephaly, even in individuals that did not show signs of infection at birth. Unfortunately, based on the results of this study, the affected children are very likely to develop mental health, cognitive, and behavior issues in the future due to their changes in DNA methylome.

Even children that were exposed, but did not show infection at birth, may have changes in their DNA methylome that could affect gene expression linked to neuropsychiatric disorders. Therefore, these disorders could develop later in life. There are still many unanswered questions about Zika and further research, particularly regarding the methylome of infected individuals is needed to better understand the true impact of the virus.  

Advances in Detection

While some researchers have focused on the outlook for the children of Zika, others have focused on optimizing the methods that are used to detect the virus in patients. Typically, diagnosing Zika has relied on detecting Zika RNA or antibodies in serum. But with reports of longer duration of virus shedding at higher concentrations, detection of Zika in urine has been an increasingly popular method. However, detecting Zika in urine possesses unique challenges due to the instability of RNA. Urine provides a good environment for RNase activity, up to 100x higher than in serum.

Due to this, a recent study by S.K. Tan et al. sought to address these issues with Zika stability in urine. The research team evaluated the effect of temperature, initial Zika levels, time between sample collection and extraction, and nucleic acid stabilizers like DNA/RNA Shield.

From their tests, researchers found that urine samples being evaluated for Zika can be stored at room temperature for up to 48 hours without significant impact on the levels of Zika RNA. Storing the urine at 4 °C in this window can help to further minimize degradation. However, if the samples will not be processed within this time frame, the researchers recommended using DNA/RNA Shield to stabilize the specimens before testing. The reagent detected all the Zika virus when compared to cold storage and improved quantitative recovery of the RNA.

In the aftermath of the Zika outbreak, there are many questions still left to answer as we work to improve treatment and detection of the virus. Health officials in several countries will be monitoring the Zika children for years to come to better understand the range of difficulties that they face and see if any problems arise for those children who were mildy affected or were asymptomatic at birth.

References:
1. Zika: Children of the outbreak. CBSN, 2019. CBSN Originals.
2. Janssens S et. al. Zika Virus Alters DNA Methylation of Neural Genes in an Organoid Model of the Developing Human Brain. ASM. 2018.