The Human Virome's Permanent Mark

 

By Aaron Krol 

June 5, 2015 | In Stephen Elledge’s lab at the Harvard Longwood Campus in Boston, a unique collection of viruses lies dormant in a refrigerated slurry. They are bacteriophages ― viruses that prey on bacteria ― and harmless to us, but each of the nearly 100,000 varieties in the mixture does share something in common with a virus that infects humans. Specifically, each one has been engineered to create a single peptide, a fragment of a protein, that would normally be found in a human pathogen ― from the rhinoviruses behind the common cold, to notorious killers like HIV and the West Nile virus. The phages wear these peptides on their outer coats like identifying tags.

Together, the 100,000 tags in this viral goulash span every protein of every human-infecting virus in the vast UniProt database, where scientists around the world document protein structures. In effect, the slurry is a living library of viral peptides, which Elledge and his colleagues at the Howard Hughes Medical Institute of Brigham & Women’s Hospital have created to study the human virome, the vast and mysterious set of viruses that live in our bodies.

In a paper published this week in Science, Elledge and his coauthors, led by graduate students Tomasz Kula and George Xu, unveiled a new method that uses this phage mixture to test blood samples for over 200 species and 1,000 strains of virus at a time. The team speculates that their technique, named VirScan (VEERscan), could one day become a near-universal test for viral infections using just one drop of a patient’s blood, replacing one-off tests for specific types of virus.

“You’re looking across all viruses at once, without having to suspect ahead of time that maybe there’s a particular infection,” says Kula. “This opens up a lot of questions that simply couldn’t be asked before, because it wouldn’t be practical to look at every single virus individually.”

Is There Antibody in There? 

The virome doesn’t get as much love as its charismatic older brother, the microbiome. Studies of the bacteria that live inside us have caught the public imagination, showing that we contain a teeming diversity of critters whose populations affect everything from our diets to our immune systems. Thanks to cheap DNA sequencing, you can send samples of your microbiome to a lab and have a quick census taken; services like American Gut will even give you a colorful chart showing you which bacteria have been found and in what numbers. (Strictly the virome is part of the microbiome, which includes all the viruses, protozoa, and fungi living in one environment ― but bacteria are the stars of the show.)

Scientists can and do use DNA sequencing to study viruses too, but for several reasons, viral DNA is hard to work with. “Viruses don’t have a single gene that can be assayed like bacteria do,” says Kristine Wylie, a member of the McDonnell Genome Institute of Washington University who studies the human virome. With bacteria, researchers often start genetic tests by amplifying a gene called 16S rRNA, which all bacteria share some version of, but viruses don’t have one target that can be amplified. “Viruses have DNA genomes, RNA genomes, single-stranded, double-stranded ― it’s a very complex set of genomes that are all lumped together.”

Without a shortcut like 16S rRNA, virome studies have to comb through a lot of genetic material, most of which belongs to the viruses’ human hosts or other microorganisms. This signal-to-noise problem is made worse by the fact that viruses can be very shy. You might think a blood sample would turn up any viruses circulating in the body, but at different points in their lifecycles, viruses might be hiding quietly in the liver, or be present at such low levels that sequencing won’t pick them up. “One of the challenges is that you’re only going to capture the viruses that are circulating in the blood in large enough numbers,” says Kula.

This is the kind of problem the Elledge lab specializes in solving. Kula and his colleagues work on technologies that can run large volumes of complex lab procedures at once, from cloning DNA fragments to designing “promoters” that enhance the activity of specific genes. With VirScan, the team has taken this high-throughput approach to testing the human virome. Their solution piggybacks on the human body’s own system for recognizing viruses: antibodies, the proteins our immune cells produce to bind with the “epitopes” on viruses’ outer coats.

 

Unlike viruses, antibodies always circulate freely through the blood, making them easy to scoop up in blood samples. Tests used in hospitals to diagnose viral infections often look for the antibodies the immune system creates in response to those infections. These tests, however, are highly targeted: one test for one virus.

VirScan, on the other hand, can look at all the antibodies in a patient’s blood, by translating them into the language of DNA. The translators are those 100,000 varieties of bacteriophage with their special peptides.

It’s a simple process: the scientists mix a batch of phages with a patient’s blood sample, and let the circulating antibodies in the blood do their work, latching onto any phages whose epitopes they recognize. Once the antibodies have had time to find their matching epitopes, the scientists pull them out with magnetic beads and wash away any phages that haven’t been hooked.

Finally, DNA sequencing reveals which epitopes are still in the sample, captured by the patient’s own antibodies. Since these epitopes come from specific viruses documented in UniProt, each one can be matched to a viral infection that the patient’s body is fighting or has fought in the past. In this way, VirScan reveals much more than direct sequencing of a blood sample ― including shy viruses or even remnants of infections from years before.

“To me, the big thing that is exciting about this is the ability to look broadly,” says Wylie, who was not involved in the VirScan research. “That’s just not something that we’ve been able to do with antibodies before. A lot of work went into this, trying to be as comprehensive as they could.”

Stand Up and Be Counted  

It’s a little hard to tell how accurate VirScan is. So far, the team at the Howard Hughes Medical Institute has run VirScan on nearly 600 patients, including groups from the U.S., Peru, South Africa, and Thailand ― but because the technique looks for over 200 species of virus, the only way to check every result would be to run 200 targeted tests on each patient.

Early analysis, however, looks promising. A good reality check is simply to ask whether VirScan’s results are plausible: if the test reported that only 5% of patients had influenza antibodies, or that 90% had antibodies to the ebolavirus, that would be a red flag.

From this perspective, the results seem good. Very common infections, like rhinoviruses and herpesviruses, come up regularly in VirScan, while more exotic viruses are found rarely if at all. “Sure enough, for many common viral infections we’re detecting them at pretty high levels,” says Kula. “And for some viruses like CMV [one of the herpesviruses], which is known to infect about half the population, that is in fact what we saw.”

In a few cases, the team was able to get much more exact measures of accuracy, by recruiting patients who had already been tested for specific viruses ― including HIV and the hepatitis C virus. With these patients, VirScan agreed with other forms of testing more than 90% of the time, a very good record for a brand-new technology. Importantly, in 97 tests VirScan gave only one false positive result, claiming to find hepatitis C in a patient who reported being hepatitis-negative. Even in that case, the team believes their method may have picked up a past infection the patient was unaware of.

Nonetheless, there are a few results that don’t square with what we know about the human virome. For example, if VirScan were perfectly accurate, it should have detected poliovirus in almost every patient ― not because polio is a common illness, but because most people around the world are vaccinated against it, producing polio antibodies. Instead, poliovirus showed up in barely a third of samples. Similarly, antibodies to the chickenpox virus were found in less than a quarter of patients tested.

“What we pick up is just a snapshot of what antibodies you have in your blood at the time when we draw it,” says Kula. That makes VirScan sensitive to differences in how the immune system reacts to different viruses. Small viruses with few epitopes can sneak by undetected; closely related strains of virus can get confused; and infections from many years earlier can fade below VirScan’s level of sensitivity, as the antibodies gradually disappear. “Over time, you do maintain antibodies from infections in the past, but they do wane,” Kula says.

“With any technology, particularly one that’s broad, it’s going to work better for some things than others,” says Wylie. “[VirScan] is broad, and from what they’ve shown it works well, but that doesn’t mean it’s going to work perfectly for every virus… There’s always going to be limits of detection.”

When it comes to scanning the entire virome, VirScan is best thought of as a complement to ordinary sequencing studies. VirScan can pick up past infections; sequencing can tell you which viruses are present in the moment. VirScan can look quickly across all known human viruses; results from sequencing might take longer to interpret, but they can uncover viruses no one has seen before.

“I think there would be lots of applications of this technology to go along with other research,” Wylie adds. “I’m pretty excited to see how people might use it.”

Very Special Epitopes 

Maybe the most intriguing thing about VirScan is what it can tell us about our own immune systems.

Unlike past antibody tests, VirScan isn’t limited to a few viral epitopes that are known to trigger an immune response. The peptide library carried by the Elledge lab’s bacteriophages covers a huge variety of viral proteins. Looking at those peptides reveals not only which viruses a patient has fought, but also which specific peptides the immune system chose as its targets.

Even in their first round of 600 tests, Elledge’s team sees surprising patterns in the results. Presented with a wealth of epitopes from each viral species, our immune systems tend to choose from just a few ― often clustered close together in the viral genome.

“A lot of people who have been infected by a virus will actually generate antibodies against the same exact region,” says Kula. “This definitely surprised us… Why there is this similarity could be very informative.” In the paper, Kula and his colleagues suggest that some epitopes could be more exposed on viruses’ coats than others, or that our immune cells might create antibodies in non-random ways that bias them to certain peptide sequences. At this stage, we really don’t know.

What’s clear is that VirScan could be brought into a number of research areas where virome-wide sequencing, or targeted viral tests, have not given us the full picture. Wylie, for instance, is curious what antibodies would be found in a large study of children, a major focus of her own research on the virome. “You’re actually developing your immune repertoire at that time,” she says. “It would be interesting to see that history of what children had been exposed to, and what might be missing from the common pathogens in a child… We have to realize we’re a bit changed after these exposures, and this is one method of looking broadly at that.”

Kula, meanwhile, hopes that VirScan can shine a light on mysterious diseases like chronic fatigue syndrome and Kawasaki disease. Some experts suspect these illnesses are caused or exacerbated by viruses, but those suspicions are hard to confirm when no one knows which virus to look for. With VirScan, this is no obstacle.

Not that every VirScan study needs to be so exotic. “Understanding what viruses generally healthy people are carrying around is important,” says Wylie. “And understanding what viruses healthy people are carrying that could potentially be pathogens, should they become immunocompromised or have some other issue that causes the virus to reactivate ― all of those things are important.”

Like the microbiome, our relationship with the virome is lifelong but little understood. Until they make us sick, our viruses are invisible and seem inactive; but their battles with our immune systems, and the ripple effects on new infections and other microorganisms, don’t fully fade away with our fevers.

Instead, every virus we meet leaves its permanent mark ― if only you know where to look.