Brent Palmer's first inkling about long COVID started in the early days of the pandemic, before the term "long COVID" even existed.

Some of his friends had caught the virus while on a ski trip and returned home to Colorado with the mysterious, new illness. It was a frightening time — and an irresistible opportunity for Palmer, who studies the immune response to infectious diseases like HIV.

In particular, he was keen to see how their T cells, part of the immune system's arsenal for fighting infections, were handling the novel infection. So he started taking samples of their blood. And there was one subject in particular who intrigued him. She was still dealing with heart palpitations, chest pains and trouble breathing more than six weeks after her acute illness.

In the lab, Palmer noticed that she still had a very high percentage of these T cells directed at the virus compared to her husband who'd also fallen ill, but didn't have lingering symptoms. "They had very different responses," says Palmer, an associate professor of medicine at the University of Colorado.

Typically, T cells aimed at the virus reach very high levels during the acute infection and over time they drop off, but Palmer noticed that wasn't the case as he began studying more patients with these persistent symptoms.

"These individuals had frequencies that were 100-fold higher in some cases than the individuals that didn't have any persistent symptoms," he says.

In fact, Palmer says it was like their body was still fighting the virus. It led him to questions: "Despite the fact that they test negative in a nasal swab, is the virus still persisting in the lungs? Is it persisting in some other organ?"

Other scientists trying to uncover the roots of long COVID have asked some version of that same question for the last few years now. This theory of "viral persistence" posits that some people never fully eliminate the infection, that viral reservoirs are subsisting in the body and possibly triggering the symptoms of long COVID.

While a direct link isn't yet proven, this line of research has gained momentum as more evidence has emerged indicating that some people seem to harbor "viral reservoirs'' after their initial illness has passed. It's far from the only explanation under consideration, but some scientists see it as one of the most promising.

"There's enough evidence to place viral reservoirs as one of the leading drivers of long COVID," says microbiologist Amy Proal, who's president of PolyBio Research Foundation, which is spearheading research into viral persistence.

A reservoir... but where?

Much of the research related to viral persistence has come from two sources: evidence of the virus persisting in different parts of the body and signs that the immune system is still revved up in some people months after the acute illness.

For example, Palmer and his team ultimately found that patients with long COVID had higher frequency of T cells specific to the virus than those who didn't have persistent symptoms — and that was also associated with higher levels of systemic inflammation, worse lung function and longer duration of symptoms like cough and difficulty breathing. Other teams have made similar types of observations about the ongoing immune response in long COVID patients.

Then there are all the corners of the body where scientists have discovered that genetic material or proteins from the virus appear to linger. Patients with persistent symptoms have traces of the virus in their stool many months after being infected, while tissue samples from the gut have also shown evidence of viral persistence. In other reports, biopsies taken from different parts of the body have led to similar conclusions, that parts of the virus can be recovered from some patients many months later.

A painstaking autopsy study, published at the end of last year, has also strengthened the case for viral persistence.

By examining people who died following a case of COVID-19, Dr. Daniel Chertow was able to find evidence of the "virus in over 30 different cell types" and "in tissues throughout the body and all the major organs."

In one case, there was genetic material from the virus in a person who died more than 200 days after contracting COVID-19. In another, Chertow and his team were able to culture virus from tissue samples taken from the brain of someone who'd died within two weeks of their illness.

"It provides definitive evidence that the virus is capable of spreading all over the body and that it's capable of persisting in parts all over the body," says Chertow, who's an investigator at the NIH Clinical Center and the National Institute of Allergy and Infectious Diseases.

Chertow cautions that only so much can be extrapolated from the study because it wasn't examining long COVID patients specifically. The subjects also tended to be older and had underlying health problems — a very different population than those with long COVID who were healthy and had a mild case of COVID-19 before developing chronic symptoms.

"Ultimately, the role of viral persistence in causing or contributing to the clinical symptoms and signs that people with long COVID experience is yet to be determined," he says, "but our paper provides strong biologic plausibility that it may contribute."

Unanswered questions

All of this research has brought newfound attention to the concept of viral persistence in the aftermath of an acute illness, says Dr. Michael Peluso, an infectious disease specialist at the University of California, San Francisco who's studying long COVID.

He notes this was a "game changer" in our understanding of Ebola, after researchers realized that survivors of that disease could harbor viral reservoirs in parts of the body that were difficult for the immune system to reach. But Peluso stresses there are still key gaps in the research as it relates to long COVID. For one, the evidence of viral persistence has come from people who have persistent symptoms and others who don't.

"It does seem we can recover particles of this virus for much longer than we initially expected in at least some people," says Peluso. "It's not clear yet whether there is a direct relationship between these measurements and the person's clinical condition."

Mohamed Abdel-Mohsen — an associate professor at the Wistar Institute's Vaccine and Immunotherapy Center — echoes this assessment, saying there is now "overwhelming evidence" of a viral reservoir in some individuals, but to what "degree that contributes to the mechanism or the symptoms of long COVID" remains to be seen.

More than one cause. How are they related?

If there's any consensus though, it's that researchers don't anticipate viral persistence will serve as the only explanation for all of those who suffer from the condition.

"No one thinks that every long COVID patient has the exact same thing happening," says Proal.

And there are other possible mechanisms under consideration. Those include autoimmunity and other troubles with the immune system, tiny blood clots and inflammation of the lining of blood vessels, tissue damage, the reactivation of dormant viruses, and signaling problems with the brainstem and the vagus nerve, among others.

"Right now, we have these buckets," says Peluso. "Ultimately, we need to connect all of the dots and see how these different mechanisms might be interrelated and whether particular pathways relate to specific subsets of symptoms," he says.

An added challenge is that the definition of long COVID has varied considerably from study to study. And while researchers have identified different clusters of symptoms, the diagnosis can still serve as a sort of catch-all for myriad post COVID-19 problems, making it all the more difficult to study.

Even the relatively straightforward term "viral reservoir" can obscure a yet-to-be-solved mystery. What is actually persisting?

There are many definitions of a reservoir, but there's nothing yet to suggest that people with long COVID have "actively replicating infectious virus that can spread to others," says Peluso. "For me, what [reservoir] means is there are at least pieces of the virus, even if they're not infectious, that are altering people's immune responses after the infection and potentially contributing to their symptoms," he says.

Proal's foundation has recently helped launch a series of studies that use sophisticated imaging and tissue biopsies to put together a more precise picture of these reservoirs.

It's possible the "genetic backbone" of the virus is producing viral proteins in certain tissues — the most likely sites being the gut, lungs and nerves — which then spill into the bloodstream, says Proal. The reservoir could look different depending on where it's located, and could be replicating and making proteins at certain times, and inactive at others.

"There's all kinds of possibilities and nuances," she says. "Does it persist more in certain body sites over others? If it does persist in one body site, does it have a different mechanism for how it sticks around there?"

Not mutually exclusive

Viral persistence is an appealing direction for long COVID research because it could be compatible with other leading theories related to the condition's pathophysiology.

"These could all actually converge and come together in an interrelated way, so by no means are they necessarily mutually exclusive," says Dr. Linda Geng, co-director of Stanford University's long COVID clinic.

A viral reservoir could wreak havoc on the immune system in any number of ways. "A chronic reaction to something could lead to immune dysfunction and inflammation," says Abdel-Mohsen.

For example, Palmer notes that T cells make small proteins, called cytokines, that can drive inflammatory responses, which "over long periods of time are not good for your body."

Viral reservoirs might also spark the phenomenon of microclotting that has been observed in some patients, and neurological symptoms by way of the vagus nerve, which runs throughout the body and can convey inflammatory signals to the brain, says Proal. "That can activate brain cells that drive neuroinflammation and cognitive issues."

At the Wistar Institute, Abdel-Mohsen has found evidence that fungi, which should reside in the lungs and gut, are leaking into the bloodstream of some people with long COVID.

"This can cause chronic inflammation" that then changes immune cells so they produce metabolites with neurotoxic properties," he says. And it's possible that viral reservoirs hiding in the gut are helping trigger this "vicious cycle," beginning with local inflammation and damage to the gut barrier.

While all these theories need to be carefully investigated, Abdel-Mohsen says that, in many cases, "each one could be happening independently and leading to a problem, but they can also lead to each other."

"There's still a lot of questions remaining, and many of the studies are smaller studies," says Geng, who's leading a clinical trial to test whether a 15-day course of the antiviral Paxlovid helps long COVID patients.

The results are expected later this year. If it does work, that would suggest the medication had extinguished a lingering infection.

"There are still plenty of people who are suffering and we really need to be able to figure out answers for them," Geng says.