When the chickenpox virus reawakens in the brain

For most people, chickenpox ends in childhood. The blisters disappear, the fever subsides and life goes on. But the virus never disappears completely.

Instead, the varicella zoster virus – the virus behind both chickenpox and shingles – retreats to sensory nerve ganglia distributed throughout the nervous system, from the spinal cord to cranial nerves close to the brain. Here, it can remain hidden inside neurons for the rest of a person’s life.

Around 90% of the world’s population carries the virus throughout life. Up to one in three will experience the virus reactivating as shingles. In rare cases, the virus spreads further into the central nervous system and can cause encephalitis, meningitis or inflammation of the blood vessels in the brain which can lead to stroke.

This paradox has long fascinated researchers. How can almost the entire world’s population carry the virus for life but only a very few develop serious disease?

“When almost everyone carries the virus but only a very few become seriously ill, it tells us that there must be very precise protective mechanisms,” says Trine H. Mogensen, Professor of Infection Immunology, Department of Biomedicine, Aarhus University, and Senior Consultant at the Department of Infectious Diseases, Aarhus University Hospital, Denmark

New findings now suggest that a molecule inside neurons called RNA polymerase III may be part of the brain’s hidden antiviral alarm system.

The clue came from an unusual case involving identical twin sisters who repeatedly developed encephalitis following reactivation of varicella zoster virus.

“They were identical twins, meaning they are genetically largely identical, both of whom experienced reactivation of the chickenpox virus and inflammation of the brain. That pointed quite clearly to a genetic factor playing a role,” explains Trine H. Mogensen.

When the virus heads the wrong way

Varicella zoster virus is best known as the cause of chickenpox and shingles. In rare cases, however, it can spread into the central nervous system.

Normally, the virus lies dormant in sensory nerves along the spine. If it is reactivated, it typically travels out towards the skin, causing shingles with painful rashes. But in rare cases, the virus heads in the opposite direction.

“If the virus travels inwards/centrally along the nerve instead of outwards, it can end up in the spinal fluid or the brain,” says Trine H. Mogensen. “This is a serious disease that can cause brain damage, memory problems and cognitive impairment, and there is also a certain mortality.”

People who develop these complications without any obvious explanation have attracted particular attention.

Interest has grown as researchers have discovered that certain genetic defects in the host can dramatically increase the risk of specific infections without necessarily affecting the rest of the immune system.

Rare disease in a world of virus carriers

For most people, this virus never becomes more than a childhood memory or a case of shingles later in life. But when almost the entire population carries the virus, even very rare complications can matter.

“Reactivation in the brain occurs in perhaps one in 10,000 cases,” notes Trine H. Mogensen. “That sounds rare, but when virtually everyone carries the virus in a latent form, it still adds up to many patients.”

Treatment options are limited, and suitable experimental models are still lacking because the virus infects only human cells.

The lack of good experimental models means that the patients themselves have become an important source of new knowledge about the disease.

“Patients are teaching us something about biology and our immune system’s protection against viruses that we would otherwise never have discovered,” says Trine H. Mogensen. “When we find a genetic defect in a patient, we simultaneously gain a window into the mechanisms that normally protect us all.”

This strategy led precisely to the new study.

Genetic analysis of the identical twin sisters showed that both carried a mutation in POLR3F, which is part of RNA polymerase III.

“RNA polymerase III was originally described as a fundamental component of cell biology, but we are increasingly beginning to understand that it also functions as an antiviral system capable of detecting foreign viral DNA,” explains Trine H. Mogensen.

How do you study a virus that only lives in humans?

To investigate how neurons keep latent varicella zoster virus in check, the researchers had to recreate one of virology’s most challenging phenomena in the laboratory: viral latency and spontaneous reactivation in human nerve cells.

The virus establishes a lifelong latent infection in neurons.

“The chickenpox virus actually only infects human cells,” says Trine H. Mogensen. “That is why we cannot study the disease in conventional laboratory animals in the same way as influenza or coronavirus, and that is one of the reasons why we know less about this virus and how it causes disease.”

Instead, the researchers used a human neuroblastoma cell line called SH-SY5Y, which can be differentiated into mature neuron-like cells.

Using CRISPR/Cas9 gene editing, the team created cells with defects in POLR3F – the part of RNA polymerase III that was mutated in the twin patients.

Recreating the twins’ mutation in neurons

To investigate whether the mutation could explain the disease, the researchers engineered human neuron-like cells with corresponding genetic defects and infected them with varicella zoster virus.

Instead of completely removing the gene, the researchers created heterozygous mutant cells, which more closely resembled the patients’ genetic situation.

“This was important because the patients did not completely lack the protein,” explains Trine H. Mogensen. “They had mutations that only partly impaired the system.”

First, the team investigated acute infection. They infected both normal and genetically modified neuron-like cells with different strains of varicella zoster virus and measured viral activity over time using RT-qPCR.

But the most technically demanding part involved modelling viral latency and reactivation.

How the researchers woke the virus up again

The research group had already developed a model of latent chickenpox virus infection in sensory nerve cells, enabling them to mimic a process that normally unfolds over decades in the human nervous system.

To do this, the researchers infected differentiated neuron-like cells while treating them with aciclovir, which inhibits viral replication. Under these conditions, the virus entered a quiescent state with very low viral activity.

After a few days, the researchers withdrew the antiviral therapy.

They then monitored the cells for signs that the virus was beginning to wake up again. To do this, they used genetically modified viruses carrying fluorescent markers, which caused infected cells to glow red and green when specific viral genes became active.

Each fluorescent signal represented neurons in which the latent virus had resumed active replication.

Particularly interesting was the transition between latency and reactivation – the moment when the immune system either succeeds or fails in keeping the virus in check.

“It may well be precisely during this transition phase that the nervous system’s antiviral defences are most important,” explains Trine H. Mogensen.

Four times more viral awakenings

When the researchers compared normal neurons with cells carrying POLR3F defects, a clear pattern emerged: the virus became more difficult to keep under control.

Even during acute infection, the genetically altered neuron-like cells showed higher viral activity. But the biggest difference only became apparent when the researchers investigated reactivation.

After establishing a latent-like infection and withdrawing the antiviral treatment, the researchers monitored the cells for signs that the virus was beginning to wake up again.

In normal neurons, reactivation occurred occasionally.

In cells with POLR3F defects, it occurred far more frequently, with earlier reactivation and substantially greater viral replication and spread.

Overall, the mutated neuron-like cells showed around four times more spontaneous reactivation events, suggesting that even a partial impairment of the system can make it more difficult to keep the virus latent.

A hidden alarm system inside neurons

The findings suggest that RNA polymerase III acts as a kind of intracellular sentinel, helping neurons to detect viral activity and keep the virus suppressed before it breaks out into active infection.

“It was actually more pronounced than we had expected,” says Trine H. Mogensen. “This suggests that RNA polymerase III does not merely play a role during the initial infection but may be particularly important in keeping the virus suppressed throughout the latent phase.”

The identical twins did not have recurrent infections in general, nor did they show signs of a classic immunodeficiency. The problem arose specifically when varicella zoster virus was reactivated in the nervous system.

“That was precisely what made the patients so interesting,” adds Trine H. Mogensen. “They did not have a broadly weakened immune system. They appeared to have a very specific vulnerability to this one infection.”

The data therefore suggest that RNA polymerase III functions as part of a local antiviral surveillance system within neurons – a defence mechanism that may help keep viruses under control throughout life.

“We have traditionally thought of neurons as targets for infection,” says Trine H. Mogensen. “But they also appear to be part of the defence.”

Perhaps the virus never truly sleeps

The study also challenges the traditional view of latent viruses.

“We have long thought of latency as a kind of hibernation,” explains Trine H. Mogensen. “But our results fit better with a model in which the virus is constantly trying to break out, while the neuron is constantly trying to suppress it.”

“This is probably more of a constant tug-of-war between the virus and the host,” says Trine H. Mogensen. “Most of the time, the host wins, which is why we never notice anything.”

However, the researchers emphasise that the neuron model is still just a model.

“We must be careful not to overinterpret the results,” adds Trine H. Mogensen. “But when we see the same mechanism in both patients and the cell model, it gives us a strong indication that we are onto something biologically important.”

Now comes the hardest question

It is still unclear exactly how RNA polymerase III keeps the virus in check and why the effect appears to be particularly important during the transition between latency and reactivation.

“The next big question is to understand the mechanism itself,” says Trine H. Mogensen. “We know that the system plays a role and that it involves type I interferon, a key signalling molecule in the body’s antiviral defence. Now we need to find out exactly how neurons use it to keep the virus at bay.”

Interestingly, the effect was stronger with a clinical virus strain than with the laboratory-adapted strain the researchers used in parallel experiments. The researchers do not yet know why, but this finding suggests that naturally circulating viruses may interact differently with neuronal immune defences than laboratory variants.

More broadly, the findings suggest that lifelong viral latency may depend not only on immune cells patrolling the nervous system but also on antiviral defence mechanisms built directly into the neurons themselves.

Can the brain’s own defences be strengthened?

In the longer term, this new knowledge may have implications for treatment.

Today, antiviral drugs primarily attack the virus directly after reactivation. But if researchers learn to understand the mechanisms that naturally keep latent virus in check, it may become possible to strengthen the body’s own defence systems before disease develops.

“We actually only have one drug against the chickenpox virus, aciclovir,” notes Trine H. Mogensen. “It works best very early in the course of the disease, so there is a great need to understand the mechanisms better.”

This knowledge may also be important for families with inherited predispositions.

“We can actually offer genetic counselling, vaccination or preventive treatment to families in whom we find these genetic defects,” explains Trine H. Mogensen. “Some of the genes we have identified through this research have already been included in international recommendations of genes that should be tested in patients with severe herpesvirus infections, including both VZV and HSV infections affecting the central nervous system.”

This is where basic research suddenly becomes very concrete.

“It is very satisfying when we actually have something to offer patients,” she says. “Some families are genuinely relieved when we can tell them that other family members have not inherited the same genetic predisposition.”

Implications far beyond shingles

The implications are likely to extend beyond the chickenpox virus.

Many herpesviruses can remain hidden in the body for life, and researchers are increasingly beginning to view latency as an active process rather than a passive dormancy.

“I think we have previously underestimated how much is going on beneath the surface,” says Trine H. Mogensen. “There may be an ongoing battle between the virus and the host that we are only now beginning to understand.”

If this interpretation is correct, the insights from the study could be relevant to understanding how people live with chronic viral infections throughout their lives. They may also prove relevant to Alzheimer’s disease and other forms of dementia, since several studies have reported a reduced risk of dementia among people vaccinated against varicella zoster virus.

Why almost everyone keeps viruses at bay

However, the researchers stress that genetic explanations are only part of the picture.

“It is not that black and white,” says Trine H. Mogensen. “Genetics is one factor, but viral strains, hormones, physiology and environmental influences also affect the risk.”

The next step is therefore not just to understand RNA polymerase III better but to map the many other defence systems that are likely operating hidden within neurons.

“We are probably only seeing a small part of the picture,” notes Trine H. Mogensen. “There are likely many more mechanisms that help keep viruses under control throughout a person’s lifetime.”

Most people carry potentially dangerous herpesviruses in their bodies for decades without ever noticing them.

“The fascinating thing is that almost everyone lives with these viruses their whole life,” concludes Trine H. Mogensen. “And yet the vast majority never become seriously ill. This tells us that the body is incredibly good at maintaining a balance.”

The study therefore suggests that the peaceful coexistence between humans and herpesviruses may not be due to the viruses lying dormant but rather to nerve cells actively keeping them in check throughout life.