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Inside the Memory Machine

Memory prostheses could help the sick and injured by stimulating the hippocampus, but they also raise ethical questions.

Lack of Evidence Plagues Neuroeducation Programs

Some neuroscience-inspired programs used in schools aren’t backed by solid scientific evidence.

The Man Who Rewrote the Tree of Life

Carl Woese pioneered the use of gene sequencing, discovered a third domain of life, and upended an entire field.

Aboveground nuclear bomb tests, like this French test over Fangataufa Atoll, created a pulse of radioactive carbon in the atmosphere.

Cold War Bomb Testing Is Solving Biology’s Biggest Mysteries

Our bodies are prolific artists, creating new cells throughout the body. Some cells, like those found in skin, hair, and the lining of the gut, are produced and discarded on a regular basis, like doodles on scrap paper. Other cells, like those in the adult brain and nervous system, have been viewed as more like the Mona Lisa. Once crafted, they remained forever. Or so scientists thought. Kirsty Spalding was one of the scientists who doubted that assessment. Spalding, once a postdoc at the Karolinska Institute in Stockholm, Sweden and now a professor there, knew there were tantalizing hints that the adult hippocampus—a seahorse-shaped region deep in the brain that is important for memory and learning—could regenerate neurons. But without knowing exactly when each neuron was created, scientists couldn’t say with any certainty that this was true. Spalding and her postdoc advisor Jonas Frisén had a hunch that a pulse of radioactive carbon created by above-ground nuclear tests during the Cold War could help solve the riddle. “A geopolitical phenomenon—this Cold War bomb testing—has, in a way, put a date stamp on everything and everybody,” Spalding says. The bomb pulse has been declining since the 1963 above-ground test ban treaty, creating a sort of clock they could exploit. By determining how many radioactive carbon atoms a cell contained, Spalding and Frisén hoped they could calculate its birthdate. To test theory, they needed neurons—a lot of them. Spalding’s curiosity eventually leading her to a slaughterhouse on the outskirts of Stockholm. Standing outside the low, gray industrial building, she watched as horses went in one side and, about 15 minutes later, a worker appeared on the other end, holding a head, neurons and all. “It was precisely as revolting as it sounds,” she says. Spalding would then spend hours chipping away to extract the necessary cells, a grisly procedure that was just the first in a decade-long stretch of hurdles she had to surmount. “Had we known how difficult it was going to be, we never would have stuck with it,” says physicist Bruce Buchholz, one of Spalding’s co-authors and an expert on bomb pulse dating at the Lawrence Livermore National Laboratory outside San Francisco. But Spalding persevered, and her hard work eventually paid off. Last June, she published a paper in which she conclusively stated that adult human did indeed build new neurons in their brains. The human hippocampus, Spalding and Frisén discovered, was continually creating small numbers of neurons. They and their co-authors had solved one of neuroscience’s longstanding mysteries. In the years leading up to that, Spalding and Frisén pioneered a new field of research, using the Cold War bomb pulse to answer a number of questions about human physiology, including neuron formation and lipid cycling. “It’s an amazingly powerful tool, whether you want to look at a fat cell or a brain cell,” Spalding says. Their work has been so fruitful that it could provide them with a lifetime worth of projects. But she and her collaborators can’t waste any time. By 2050, Frisén and Spalding estimate, the bomb pulse will have completely dissipated. <h2> Bombs Away </h2> The premise of bomb pulse dating is fairly straightforward. Most aboveground nuclear bomb testing happened between 1955 and 1963, and those detonations released untold numbers of neutrons into the atmosphere. These slammed into nitrogen atoms, causing their nuclei to eject a proton. What was once a seven-proton nitrogen became a six-proton carbon. But unlike most carbon atoms, which have six protons six and neutrons, this radioactive carbon, known as 14 C, retained nitrogen’s two extra neutrons, a difference in atomic mass that is small, but measurable. Normally, only a tiny fraction of the world’s carbon is 14 C, so little that scientists measure it in parts per trillion. The bomb pulse doubled this amount. While 14 C concentrations are still low even after the bomb pulse, the difference is obvious to scientists who know what they’re looking for. To measure the small amounts of 14 C, scientists use a technique called mass spectrometry, which sorts atoms by weight. When looking for carbon isotopes, the instrument strips carbon atoms of some of their electrons and launches them into a magnetic field, which alters each atom’s trajectory. Inertia causes heavier atoms, like 14 C, to take a wider path than lighter ones. By measuring how many carbon atoms travel along the various paths, scientists can determine how much 14 C is in a sample. By determining the age of the DNA, researchers can determine when, exactly, a cell was created. Atmospheric 14 C spiked in 1955 and rapidly dropped off after the Limited Test Ban Treaty was signed in 1963, which banned all aboveground detonations. Without nuclear explosions producing new 14 C, existing 14 C began to mix with other carbon sinks, diluting its concentration in the air. Radioactive 14 C is incorporated into all living things: by plants that use carbon dioxide for photosynthesis, by the animals that eat the plants, by the animals that eat other animals that have eaten plants. To our bodies, one type of carbon is as good as any other, so 14 C is used when building our cells, our proteins, and our DNA. A cell’s DNA reflects the amount of 14 C in the atmosphere at the time it was made. (For plants, that’s shortly after they fixed CO 2 containing 14 C; for animals, it’s when they ate a plant or animal containing 14 C.) Scientists measure the amount of 14 C in DNA because, while other molecules are frequently refreshed throughout a cell’s life, DNA remains constant. By determining the age of the DNA, researchers can determine when, exactly, a cell was created. <h2> The Great Debate </h2> Neuroscience dogma had long dictated that the adult human brain did not create any new neurons. The only time neuron numbers could increase was thought to be during fetal development and early childhood. Once the peak number was reached—usually around age four—it was all downhill. But by the late 1960s and early 1970s, rodent studies led some experts like Fred Gage, a neuroscientist at the Salk Institute in La Jolla, California, to question this notion. “We found stem cells in the hippocampus of adult mice and rats that could create new neurons,” Gage says. It was groundbreaking work, but at the time not everyone was convinced of its importance. “In order for this to have significance, we needed to know whether this occurred in humans.” To identify dividing cells in mice and rats, Gage had been using a molecule known as bromodeoxyuridine, or BrdU. BrdU is a synthetic nucleoside that can be incorporated into newly synthesized DNA in place of the standard nucleoside thymidine, the T in ATCG. Instead of thymidine’s standard methyl group, BrdU has a bromine atom, a small molecular difference which researchers can detect. If a cell divides shortly after BrdU is injected into the body, the DNA of the new cells will contain BrdU. DNA in new cells in a rat hippocampus is labeled here in green. At that time, in the mid-1990s, BrdU was mainly used in humans as a cancer diagnostic agent to determine which parts of a tumor were rapidly dividing. Gage saw this as an opportunity to test his hypothesis, so he obtained the brains of recently deceased cancer patients who had been treated with BrdU, checking if there were neurons in the hippocampus that contained the chemical. There were. “This was the first evidence that there were dividing stem cells in an adult brain, and that these cells could give rise to new neurons in the hippocampus,” Gage says. He <a href="http://www.nature.com/nm/journal/v4/n11/full/nm1198_1313.html"> published his data </a> in a 1997 paper in Nature Neuroscience . Although Gage felt he had settled the debate, not all scientists agreed. Some disputed his results because they came from cancer patients, whose bodies contained cells that weren’t behaving normally. “The reception was pretty rocky in the early days,” Gage says. “It was hard to accept that this could happen in mice and rats, not to mention people.” Across the Atlantic, in his Stockholm lab, Frisén read Gage’s paper with interest. He, too, had believed that humans could grow new neurons, but never had the evidence to back up his hunch. BrdU was potentially toxic and carcinogenic—no research safety committee was going to allow its use on healthy subjects. A rat hippocampus as seen through an ultra-widefield high-speed multiphoton laser microscope Frisén initially turned to radiocarbon dating, which archaeologists use to determine the age of ancient artifacts. At best, however, the technique could only date something to within one hundred years—not nearly sensitive enough for what he needed. In his literature search, however, he stumbled across a variation on the technique that also used 14 C. No one had used it to answer major questions in biology, though. “It was a high-risk project,” Frisén says. “I talked to all of the postdocs in my lab, and Kirsty was the only person willing to take on this research.” <h2> Hopes and Isotopes </h2> One of the first significant biological applications of bomb pulse dating came in forensic science back in the early 1990s. Detectives in Vienna had found two wealthy, elderly sisters dead in an apartment in 1992, and they called on University of Vienna physicist Walter Kutschera to help to solve the mystery. To disperse the women’s substantial estates and life insurance payouts, police needed to know which had died first or if they died at the same time. The detectives had heard about bomb pulse dating, and Kutchera was a resident expert. Could he help, the police asked? Kutschera agreed. “We focused on the cells that renewed quickly, like skin and hair. If you measure something that is renewed very regularly, you can determine when that person died,” he says. “This work spurred us on.” Working with forensic experts, Kutschera found that the newest cells in one of the sisters dated from 1988. Sister B, however, had cells that were born in 1989. The detectives ultimately concluded that Sister A had died at home while Sister B continued to live in the same apartment as her sister’s dead body. One or two years later, Sister B also died. Since Sister B was the last alive, the detectives could disperse the sisters’ estates according to Sister B’s wishes. Other forensic teams soon began taking advantage of bomb pulse dating. Detectives could determine date of birth and death for John and Jane Does, helping them put a name to a body. It would occasionally solve the case; other times it would give distraught relatives an answer to what had happened to their missing loved one. The forensics technique was also used to identify victims the deadly Indian Ocean tsunami in 2004. A large number of Swedish tourists had been in the tsunami’s path, and although officials knew who had gone missing, they were having trouble matching names with the bodies. The Swedish government asked Frisén and Spalding if they could use their expertise in bomb pulse dating to make the identifications. The experience emphasized for Frisén and Spalding how useful the technique could be. “This work actually spurred us on to keep going with some of our biological research,” Frisén says. <h2> Fat Chances </h2> Spalding’s work on the tsunami victims bolstered her confidence that 14 C dating could, in fact, accurately date not just individuals, but specific populations of cells in the body. Fortunately, she had already teamed up with Buchholz, the bomb pulse expert at Lawrence Livermore, to fine tune her technique. She then cast her net wide, dating intestinal, skeletal, brain, and blood cells, publishing the first results in July 2005 in Cell . Some cells, such as those in the cerebellum, were just three years younger than the person itself. Blood cells, on the other hand, were very young, confirming existing data about their rapid turnover. In the middle were intestinal cells, which Spalding found had an average lifespan of about 11 years. These results were crucial, Buchholz notes, because it showed the method would work in humans. “Before this study, it seemed bomb pulse dating was more of a curiosity question. People would mostly just say ‘Oh, yeah. We see it in people.’ It wasn’t used as a tool to do biology until Kirsty and Jonas just took this idea and ran with it,” Buchholz says. To Spalding, those first experiments were satisfying, but merely a step toward her goal. Once she proved the tool worked, she set out to use it. Her first target was fat. “It has very important endocrine functions,” says biologist Philipp Scherer from the University of Texas Southwestern Medical Center in Dallas. “Adipose tissue communicates with other organs using the hormones it creates. As you go from lean to obese, the fingerprint of these hormones changes. The bad ones go up and the good ones go down, so it’s important to understand the basic physiology of these cells.” “Before this study, it seemed bomb pulse dating was more of a curiosity question.” Spalding wanted to know whether fat cells were replaced more or less quickly in obese individuals compared with their leaner counterparts. This, she felt, might help explain not only why it’s so hard for many people to lose weight and keep it off, but also some of the health complications that occur in people with excess body fat. Spalding found that the age of cells in both obese and lean people were the same: around 10 years. Her calculations showed that, each year, around 8% of an adult’s fat cells die and are replaced by new cells, a rate that didn’t vary according to body mass index. But the story was different in children: obese kids added new fat cells much more quickly than their old cells died. Spalding also dated the fats inside adipose cells. She found that throughout a fat cell’s lifetime, lipids were replaced six times, on average. The individuals with the oldest lipids—and thus the slowest turnover rate—were more likely to be insulin resistant. The finding could pave the way for treatments that target the rate of lipid turnover to improve the health of obese individuals, even if it didn’t result in weight loss. “This technique is elegant and useful,” Scherer says. “Targeting fat cell turnover is where the field will have to go in order to get ahold of the obesity and Type 2 diabetes death spiral.” <h2> Results at Last </h2> After years of trial and error, tweaks, and fine tuning, Spalding was finally ready to test her and Frisén’s theory that human brains generated new neurons. The barriers, though, were still significant. Spalding first had to separate hippocampal neurons from the non-neuronal support cells that surrounded them. This took more than a year. She also had to isolate enough of their DNA—typically, she found only one 14 C atom for every twelve to fourteen cells. Most detection methods required a full milligram, but Spalding realized she could only isolate a few micrograms of material. She spent a year and a half optimizing her DNA extraction technique, yet it still wasn’t enough. Frustrated, Spalding turned to another brain region that had been hotly debated in neurogenesis circles—the olfactory bulb. For many mammals, smell is the dominant sense, and scientists had discovered newly formed cells in this region in mature animals. In humans, the olfactory bulb is bigger than the hippocampus, and it contains more cells and more DNA. Unfortunately, her results didn’t mirror findings in animals. Humans, <a href="http://www.cell.com/neuron/retrieve/pii/S0896627312003418"> she published in Neuron </a> , did not create new cells in the olfactory bulb. That wasn’t the end, though. Not long after Spalding published the Neuron study, she received word that a group of scientists at Uppsala University in Sweden had developed a new technique, a more sensitive one that could measure her tiny hippocampal samples. Here was new hope, though Spalding was still skeptical. “With each step, I thought I had it, I thought ‘This is great! I’m finally there,’ ” she says, “and then there would be another problem.” This time, however, was the exception. The determined scientist had finally cleared her last hurdle. The results were conclusive. Using 55 brains of individuals between the ages of 19 and 92, Spalding separated the hippocampal neurons and isolated their DNA. Buchholz then took the DNA and assayed their 14 C ratio. Their results showed that the average hippocampal neuron was only 20-30 years old, even in people who were more than 90 years old. Each day, the researchers estimated, the human brain creates 700 new hippocampal neurons. To the research community, the results were clear: neurogenesis occurs in humans, even long into adulthood. “We found a lot more hippocampal neurogenesis than anyone really expected,” Frisén says. After years of work and apparent dead ends, Spalding and Frisén finally had their answer. For some, that would be the end of it. But with the bomb pulse expected to dissipate by 2050, their real work is only just beginning. In the meantime, though, they can revel in overturning one of neuroscience’s long-held tenets. “It presents a whole new picture on our view of the brain,” Gage says of Spalding and Frisén’s research. “It’s not this static structure that just declines with age. Now we see the brain very differently.” Photo credits: <a href="http://www.flickr.com/photos/7969902@N07/511287693"> Pierre J./Flickr (CC BY-NC-SA 2.0) </a> , NIH, and National Center for Microscopy and Imaging Research Arner, P., S. Bernard, M. Salehpour, G. Possnert, J. Leibl, P. Steier, B.A. Buchholz, M. Eriksson, E. Arner, H. Hauner, T. Skurk, M. Ryden, K.N. Frayn, K.L. Spalding. 2011. "Dynamics of human adipose lipid turnover in health and metabolic disease." Nature 478(7367):110-113. DOI: <a href="http://dx.doi.org/10.1038/nature10426"> 10.1038/nature10426 </a> Bergmann, Olaf, Jakob Liebl, Samuel Bernard, Kanar Alkass, Maggie S.Y. Yeung, Peter Steier, Walter Kutschera, Lars Johnson, Mikael Landén, Henrik Druid, Kirsty L. Spalding, Jonas Frisén. 2012. "The Age of Olfactory Bulb Neurons in Humans." Neuron 74(4):634-639. DOI: <a href="http://dx.doi.org/10.1016/j.neuron.2012.03.030"> 10.1016/j.neuron.2012.03.030 </a> Eriksson, Peter S. , Ekaterina Perfilieva, Thomas Björk-Eriksson, Ann-Marie Alborn, Claes Nordborg, Daniel A. Peterson, and Fred H. Gage. 1997. "Neurogenesis in the adult human hippocampus." Nature Medicine 4:1313 - 1317. DOI: <a href="http://dx.doi.org/10.1038/3305"> 10.1038/3305 </a> Spalding K.L., E. Arner, P.O. Westermark, S. Bernard, B.A. Buchholz, O. Bergmann, L. Blomqvist, J. Hoffstedt, E. Näslund, T. Britton, H. Concha, M. Hassan, M. Ryden, J. Frisén, and P. Arner. 2008. "Dynamics of fat cell turnover in humans." Nature 453:783-787. DOI: <a href="http://dx.doi.org/10.1038/nature06902"> 10.1038/nature06902 </a>

Cold War nuclear bomb testing stamped a date on every cell, giving scientists the opportunity of a lifetime.

After Months of Slinking Across the Border, Chikungunya Transmitted in the U.S.-maho-beach-saint-martin.jpg

The island of Saint Martin—popular with tourists—has been hit hard by an outbreak of a mosquito-borne illness. Photo credit: Wilson Hui/Flickr (CC BY 2.0)

After Months of Slinking Across the Border, Chikungunya Transmitted in the U.S.

Mosquito-borne illness chikungunya has been lurking just outside the U.S. for months. Now it&#x27;s here.

Mosquitoes are the vectors for a number of diseases, including West Nile, that can cause chronic symptoms.

The Illness That Never Ends

Mark Henning is a self-described redneck who runs at full throttle. “You know those people who make YouTube videos of themselves doing stupid stuff? That’s me,” he says. So when Henning first began developing flu-like symptoms in the summer of 2013, the 47-year-old Nebraska native tried to brush it off. He continued working three jobs and helping friends with construction projects on the weekends. But instead of enjoying a pizza and beer afterwards, Henning went home. His doctor told him it was probably just the flu. After more than a month, Henning finally took off work one Thursday and Friday in early September, hoping that a few days to rest would cure him. Yet on Monday, he only managed a half day at work before returning home. He went to bed for a nap, but instead of falling asleep, he fell into a coma. Henning woke up in the hospital several days later. He had a 104.5° F fever and, finally, a diagnosis: West Nile encephalitis. “The doctors thought I was going to end up brain dead,” he says. “Down the hall, someone else with West Nile died while I was there.” Henning was in the hospital for a week and off work for two months. After that, his doctors told him that the worst was over. However, Henning’s health problems persisted. His gallbladder ruptured and had to be removed. He developed severe acid reflux for the first time in his life. His short-term memory slipped away from him. And perhaps most frustratingly, his doctors have no idea whether his symptoms might be linked to the West Nile encephalitis that nearly killed him more than two years ago. The idea that seemingly short-lived viral infections could cause long-term symptoms is fairly new to scientists. They long believed that once your body cleared an infection, its symptoms should disappear, too. But that isn’t always the case, says Kristy Murray, an epidemiologist and veterinarian at Baylor College of Medicine’s School of Tropical Medicine. “Some people are developing new problems over time,” Murray says, problems that appear related to their initial infections. More and more, researchers are coming to realize that Henning’s case is far from unique and that his experience isn’t merely paranoia or hypochondria. Some of the world’s most notorious viruses like Ebola, West Nile, chikungunya, and the newcomer, Zika, may cause health problems that linger for months or years, and scientists are only now beginning to grapple with the problem. <h2> A Mysteriously Chronic Malady </h2> When the New York City Department of Health asked epidemiologists from the CDC to investigate a cluster of encephalitis cases in 1999, Murray was one of the first people on the scene. A member of the CDC’s elite Epidemic Intelligence Service (EIS), her job was to track down the source of the outbreak. Murray’s work, along with a team of epidemiologists and virologists, uncovered West Nile virus as the culprit. Further investigations revealed that those encephalitis cases represented only a tiny fraction of people infected by the West Nile virus. Most people had no symptoms at all, and only 10% developed mild flu-like symptoms. Then, in 2002 and 2003, West Nile began a rapid spread westward across the United States, traveling from the Atlantic to the Pacific in near record time. By that time, Murray had finished her two-year stint in the EIS and had relocated to Houston, where she continued investigating West Nile. In 2002, there were more than 100 cases in the area, giving Murray the opportunity to study the disease in more depth. “Most people had no symptoms, but I was blown away by how ill some were. They were on respiratory support, on ventilators—they had severe paralysis. I was wondering if these people ended up recovering,” she says. To track the effects of West Nile over time, Murray began assembling a cohort of individuals across the spectrum of symptoms: those with the most severe forms of disease like meningitis and encephalitis, those with moderate flu-like symptoms who had tested positive at their doctor’s office, and those who had no symptoms at all and whose infections were identified only through blood bank screening. This initial group of 172 people gave Murray some of the <a href="https://www.researchgate.net/profile/Kristy_Murray/publication/7108772_Risk_factors_for_encephalitis_and_death_from_West_Nile_virus_infection/links/02e7e5225fe9a63e27000000.pdf"> most basic information about West Nile infections </a> that no one had gathered before. She found that those most likely to develop severe infections were older adults with hypertension and that even those with the most severe infections seemed to recover within a few weeks. “Most people had no symptoms, but I was blown away by how ill some were.” Eight years later, Murray once again assessed her group of patients only to discover that many of them had remained ill, a result she wasn’t expecting. Some of the most common complaints were fatigue, weakness, depression, difficulty walking (also known as ataxia), memory loss, paralysis, and blurred vision—symptoms that other West Nile survivors like Henning knew all too well. The symptoms were also frustratingly non-specific, as both Henning and those in the study cohort attested. Their physicians were often stymied by ongoing symptoms with no clear cause. Many of those with fatigue and depression <a href="http://wwwnc.cdc.gov/eid/article/13/3/06-0602_article"> also met the case definition </a> for chronic fatigue syndrome, also known as myalgic encephalitis or systemic exertion intolerance disease. Murray wasn’t prepared for this level of ongoing illness. “After eight years, 70% hadn’t returned to their baseline health status,” she says. The West Nile virus can infect the nervous system and kill neurons, especially in an area of the spinal cord called the anterior horn, which contains the motor neurons that travel to skeletal muscle. If too many of these neurons are damaged by West Nile infection, it could lead to lingering neurological symptoms, says Daniel Pastula, an arbovirus expert at the University of Colorado Denver. “If enough of these cells are killed, there aren’t enough in reserve to do their job,” he says. West Nile virus can also infect the kidneys, and nearly 40% of the Houston West Nile group had developed <a href="http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0040374"> some form of chronic kidney disease </a> . Until Murray analyzed the data, however, and shared her findings with the study group, almost no one had associated their current kidney disease with their past history of West Nile. Murray could only conclude one thing: the effects of the West Nile virus were lasting for a really long time in otherwise healthy individuals. The root cause remained a mystery. She didn’t know whether the symptoms were the result of the body’s immune response gone haywire or whether some people still harbored remnants of the initial infection that triggered ongoing symptoms. <h2> Another Outbreak </h2> While Murray toiled over her West Nile data, Marc Lecuit was struggling half a world away in his Paris lab to understand a different mosquito-borne disease. On the French protectorate of La Reunion in the Indian Ocean, an outbreak of chikungunya had swept through in 2005 and 2006, sickening more than one-third of the island’s inhabitants. Chikungunya rarely killed, instead causing joint pain so severe infected people often couldn’t sit or stand for several weeks. ( Chikungunya is derived from a Makonde word meaning “that which bends over.”) Similar to West Nile, the virus was known to scientists but had never before caused an outbreak of this magnitude. By 2007, the virus had left La Reunion and moved on, most famously arriving in the Americas in late 2013. But Lecuit, a virologist and epidemiologist at the Pasteur Institute, was hearing from physicians on La Reunion that some of their early chikungunya patients were experiencing joint pain for months, even years, afterward. They asked if Lecuit knew anything about this. He didn’t. Chikungunya virus particles as seen via transmission electron micrography (TEM). Until that point, most of Lecuit’s work had studied whether pregnant women could transmit chikungunya to their fetuses. Indeed they could, and the disease could lead to severe health problems, even death, in the newborn. But he didn’t know much about long-term symptoms in otherwise healthy adults, nor did anyone else, he realized. Lecuit soon arrived at the same question Murray had: Was this a chronic immune response or a chronic infection? “We didn’t really know what was causing this long-term pain,” he says. “There was no hard data proving this.” During the initial infection, the chikungunya virus makes billions of copies of itself in fibroblast cells, which make up the connective tissue commonly found in joints. The virus hijacks the fibroblasts’ cellular machinery, turning them into viral factories. This conversion ultimately kills the cells, which are disposed of by immune cells called macrophages. However, with so many cells and viruses to mop up, the macrophages may leave some debris in and around a patient’s joints for some time. Lecuit thinks the remaining bits of virus could give rise to the chronic condition. “The virus was where the symptoms now are.” People with more severe chikungunya infections are more likely to develop chronic joint pain, which provides some support for Lecuit’s hypothesis given that more severe symptoms are typically caused by a higher viral load. If the body can’t break down pieces of virus, it could lead to a prolonged, localized inflammation. And neither Lecuit nor other scientists have yet to discover any chemical markers in the blood to show that this inflammation is happening throughout the body. Instead, it appears limited to the initial site of chikungunya infection. That might not be the case for what’s happening with West Nile, however. Murray’s research shows that people experiencing prolonged symptoms after West Nile infection do have signs of a persistent, body-wide inflammatory response. And in a few of these individuals, Murray found <a href="http://jid.oxfordjournals.org/content/201/1/2.short"> very low levels of West Nile virus remaining in their urine </a> , which raised the possibility that a West Nile infection had become chronic. Fibroblast cells are found in connective tissue throughout the body. Lecuit isn’t convinced, noting that no other arboviruses had been found to cause long-term infection. To him, the idea that these viruses might set off an uncontrolled immune response was far more likely. Murray says that she hasn’t yet been able to conclusively show that virus is present in every person with long-term symptoms. If any virus is still present, it appears to be causing problems indirectly, by stimulating an ongoing hyperactive immune response, rather than causing symptoms directly. Stephen Maltby, a virologist and immunologist at the University of Newcastle in Australia, says that definitively linking a virus to longstanding symptoms is more difficult than people realize. Finding an association—that people infected by a virus are more likely to develop certain symptoms—is relatively straightforward, but causation is something else entirely. Consider the links between the Zika virus and microcephaly, a birth defect involving small head size, and Guillain-Barre Syndrome (GBS), an autoimmune reaction to an infection that leads to paralysis. While both arise during the infection as opposed to months or years after, they illustrate the difficulty in establishing causation. In countries like Brazil that have been hard hit by Zika, the medical community acknowledges that there have been spikes in babies born with microcephaly and people with signs of GBS. Certainly, signs are there—scientists found fragments of the Zika virus in the fluid that bathes the brains of babies with microcephaly. And a <a href="http://www.thelancet.com/journals/lancet/article/PIIS0140-6736(16)00562-6/abstract"> new study in The Lancet </a> of a Zika outbreak in French Polynesia provided some of the strongest evidence to date that infection could lead to GBS. But definitive proof has been much slower to arrive. “There’s a high risk that these kinds of spikes are really just noise, although everyone worries it’s the start of a trend,” Maltby says. “Until I saw some of this most recent data, I thought it might have been just a coincidence. Now I’m more convinced that there really might be something going on.” <h2> Following the Data Trail </h2> Murray has faced no small amount of skepticism from her fellow scientists that West Nile could create chronic problems. Other viruses, like herpesvirus and hepatitis C, are known for their ability to remain in the body for decades, but nothing in the genomes of West Nile or related viruses hinted at that ability. Beate Sander, a scientist at Public Health Ontario who has studied the long-term effects of West Nile infection, says that these phenomena probably aren’t newly evolved traits, but rather that scientists had never looked for them before. Part of the problem is that many studies on chronic symptoms haven’t been well organized or have suffered from low enrollment. Sander and her colleagues found inconsistencies and methodological problems in the way that 67 studies of chronic West Nile symptoms collected and analyzed their data. The result, Sander said, isn’t that these symptoms don’t exist, but that no one can say with any certainty how common they are or who they are most likely to infect. “If there are only a few cases dispersed over time and space, it’s impossible to gather the kind of data you need,” Sander says. “We didn’t have enough follow-up time until now.” As Sander suggests, that’s beginning to change. So long as viruses like West Nile, chikungunya, and Zika remained in regions where they originally evolved and where, presumably, many people were immune, they caused constant, low-level infections. They were a public health concern, but not a crisis, and that made it difficult for scientists like Murray and Lecuit to monitor chronic symptoms. “To track these kinds of outcomes, you have to see a lot of people get a viral disease,” says Pastula, the arbovirus expert. Yet once the viruses traveled outside that zone and came into contact with people who didn’t have immunity, they caused explosive outbreaks and produced a large number of patients. “If there are only a few cases dispersed over time and space, it’s impossible to gather the kind of data you need.” Sander found that many patients’ ongoing symptoms didn’t necessarily match the ones they experienced when they first developed West Nile, which makes it more difficult for physicians to link these later complaints to their previous infection. As Henning points out, “No one can tell me whether what’s happening to me is a result of West Nile or whether it’s just because I’m getting older.” “The virus doesn’t actually have to be in the system at the time” of the symptoms, Maltby says, since the initial infection could trigger an overactive immune response that lingers long after the body has cleared the virus. “That makes it really hard to prove causation when there’s no virus there anymore,” he says. The wide variety of symptoms, combined with their vagueness, is likely one reason that these effects hadn’t been noticed until recently. Unless someone is specifically looking for them, even in reasonably healthy, well-off Western populations, they’re easy to miss, Sander says. Previously, these viruses were confined to impoverished areas with less healthcare and more disease, especially afflictions like dengue and malaria with symptoms that mimicked these illnesses. “These symptoms have a lot of different causes, which means there’s lots of noise and it’s hard to understand what’s happening clinically,” Pastula says. Even if scientists are able to link Henning’s lingering symptoms to West Nile, it doesn’t necessarily mean that any curative treatments are close at hand. The same is true for Ebola and chikungunya and likely Zika. All too frequently, doctors can only attempt to treat the symptoms. But for Henning, being able to know what’s causing his malaise will go a long way to giving him peace of mind. For now, he tries to stay active, conducting a phone interview while cooking chicken for a charity luncheon at his local firehouse, for example. He still works long hours and hangs out with his buddies as much as his illnesses will allow. But he’s different now, and says he’ll never be able to go back to the person he was before. “The doctors thought I was going to die,” and that changes you, Henning says. “I’d really just like to know what’s going on, even if no one can do anything about it.” Photo credits: <a href="https://www.flickr.com/photos/25689440@N06/2777393311/"> Dan Bergstrom/Flickr (CC BY-NC) </a> , Cynthia Goldsmith/NIH, James J. Faust and David G. Capco/Arizona State University/NIH

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