The vast virome

Studying complex diseases is like trying to solve a massive jigsaw puzzle with a blank box cover and who knows how many missing pieces. Scientists now realize that human genes form the borders of many disorders. But it turns out that the picture can’t be filled in without considering microbes, especially the bacteria and viruses that make the human body home.

Four years ago, evolutionary geneticist Vicente Pérez-Brocal found himself trying to complete the Crohn’s disease puzzle. As a member of a research group headed by Andrés Moya at the University of Valencia in Spain, Pérez-Brocal was tasked with determining if viruses fit into the picture.

Crohn’s disease is an inflammatory bowel disease thought to result when the immune system goes into overdrive, causing chronic inflammation that can damage the intestines and raise the risk of colon cancer. Genes and personal habits, such as diet and smoking, play a role, but there are still gaps. Scientists think infectious organisms could be involved, but still debate where those pieces belong.

Bacteria are obvious gap-fillers; they outnumber human cells 10-to-1 and influence nearly every biological process in the body. Some promote disease, others protect against it (SN: 6/18/11, p. 26). The microbiome — what scientists refer to as the collection of bacteria, fungi and other single-celled organisms that live in and on the body — has been a hot research topic for more than a decade. But bacteria aren’t the only microbes with which we humans share space.

The most abundant inhabitants of what many researchers are calling “the human ecosystem” are the viruses. So Pérez-Brocal reasoned they were worth a closer look.

Viruses are deceptively simple organisms consisting of genetic material packed in a protein shell. They are tiny and can’t replicate on their own, relying on human or other cells to reproduce.

And yet, scientists estimate that 10 quintillion virus particles populate the planet. That’s a one followed by 31 zeros. They outnumber bacteria 10-to-1 in most ecosystems. And they’re ubiquitous in and on humans.

Pérez-Brocal and others are learning that viruses, once seen only as foreign invaders that make people sick, are an integral part of human biology. Some cause major diseases, including influenza, AIDS and some cancers. Others, conversely, may promote health. Some may even help us gauge how well the human immune system works.

The study of people’s resident viruses, known collectively as the human virome, is “a whole new frontier in the understanding of humans,” and could become important for the future of medicine, says Forest Rohwer, an environmental microbiologist at San Diego State University.

Rohwer’s research indicates that viruses are part of the human defense system. Mucus studded with bacteria-infecting viruses called bacteriophage, or phage, may help protect host cells from invasive microbes, he and his colleagues reported June 25 in the Proceedings of the National Academy of Sciences (SN Online: 5/20/13). Within the mucus barrier that lines airways and intestines and coats the mouth and other orifices, the host and phage conspire to control the movement of bacteria. Anchored to sugars produced by host cells, phage infect and blow up invading bacteria that try to cross the mucus barrier.

As scientists take a census of the virome, they’ve begun to reveal these kinds of unexpected partnerships, but the work lags far behind that of the rest of the microbiome.

“We know a lot about the bacteria that inhabit humans,” says David Pride, an infectious disease doctor at the University of California, San Diego. In comparison, “we know absolutely nothing about the viruses.”

ID tags

Not that scientists haven’t been interested in viruses. Until recently there was just no good way to identify them, an important first step toward understanding the biology of health and disease. As a consequence, virome research is in its infancy.

Researchers have gotten a head start on cataloging bacterial denizens of the body because all bacterial cells contain a version of the 16S ribosomal RNA gene. That gene encodes an RNA component of protein-building machines called ribosomes. The 16S rRNA gene clearly flags bacteria because humans and other eukaryotes build their ribosomes with a different set of RNAs. Each bacterial species has a slightly different version of the gene, which researchers use to identify who’s who among microbial citizens of the human body.

Virus hunters aren’t so lucky. There is no analogous virus-identification tag. Instead, to look for viruses, researchers must sequence hundreds of thousands of bits of DNA from a sample — skin swabs, saliva, feces or mucus, for example. Scientists have gotten really good at generating these DNA sequences; the trick is figuring out what they are.

Some of these DNA bits come from human cells, some from bacteria and other microbes that occupy the body, such as archaea and fungi. Some bits may come from viruses, but it is hard to tell for sure, says Pérez-Brocal, because scientists have a limited set of characterized viruses to use as a guide for spotting new ones.

In their search for a viral cause of Crohn’s disease, Pérez-Brocal’s team examined the DNA in stool samples from eight healthy people and 10 people with the condition. After tossing out DNA that clearly came from humans and bacteria, the team was left with one pool of DNA that matched sequences from a database of viral DNA and another pool of unknown origin.

The researchers found less viral diversity in the Crohn’s disease patients. Only retroviruses appeared to be more abundant in people with Crohn’s disease than in healthy people, the team reported June 13 in Clinical and Translational Gastroenterology.

Retroviruses, including HIV, the virus that causes AIDS, insert copies of themselves into the host’s genome. Sometimes that insertion disrupts host genes, leading to cancer or other diseases.

To be fair, Pérez-Brocal and his colleagues have nothing more than guilt-by-association to link retroviruses and Crohn’s disease. They don’t yet know if a retroviral infection sparks the disease or if having an inflamed bowel makes it easier for retroviruses to gain a foothold. “We cannot say if this is cause or consequence,” Pérez-Brocal says. “At this point we’re just describing what we observe.”

Early virome studies indicate that there’s still much more to observe.

Every time Frederic Bushman samples a new person’s virome, he says, he finds new viruses. A microbiologist at the University of Pennsylvania Perelman School of Medicine in Philadelphia, Bushman has shown that no two people’s gut viruses are exactly alike (SN Online: 7/14/10). But once a person has picked up a community of bacteria-infecting phage, it tends to stick around. Fully 80 percent of the viruses present when the researchers first started tracking one man’s virome were still there more than two years later.

That’s not to say things stagnated, viromewise. The viruses themselves mutated rapidly; some changed up to 4 percent of their DNA over the course of the experiment, Bushman and his colleagues reported July 23 in the Proceedings of the National Academy of Sciences. That amount of mutation is similar to the degree of DNA difference between two viral species, he says, and could account for why people’s gut viruses are so individual.

Pride and his colleagues at Stanford, the University of California, San Diego and elsewhere tracked viruses from the mouths of four volunteers to get a sense of their viral history. The researchers used tags known as CRISPRs to observe the changing viral landscape. CRISPRs are bits of phage DNA that bacteria have chopped up and incorporated into their own chromosomes. When bacteria encounter a new phage, they check its DNA profile against this internal dossier and destroy those that have caused trouble before.

Those CRISPR tags help researchers determine which phage that the bacteria —  and therefore the human host — have encountered in the past.

In Pride’s study, volunteers spit into tubes four times over 11 months. In each person, a small number of tags — between 3 and 18 percent — were present in all the saliva samples, Pride’s team reported in the September 2012 Environmental Microbiology. But in every sample between 25 and 75 percent of the CRISPRs were new, indicating that bacteria are constantly facing fresh phage assaults.

Paradoxically, bacteriophage may play a crucial part in strengthening bacteria’s attacks on their human hosts. Phage may deposit genes for resisting antibiotics or for making toxins into the bacteria they infect, potentially producing virulent infections that can withstand antibiotic treatment.

Some bacteria use bacteriophage as a weapon against other bacteria, says Howard Hughes Medical Institute investigator Lora Hooper, a microbiologist and immunologist at the University of Texas Southwestern Medical Center in Dallas. Under certain conditions, a common intestinal bacterium called Enterococcus faecalis unleashes a Frankenstein bacteriophage composed of two different phages, she and her colleagues reported in the Proceedings of the National Academy of Sciences in October 2012. In experiments with mice, Hooper’s group showed that the composite phage gives E. faecalis strains an advantage over the competition when settling into the intestines.

Maybe researchers can use bacteriophage to shape the human microbiome in healthier ways. Using phage to control bacteria is a resurgence of an old idea. In the 1920s, doctors in the former Soviet Union and other Eastern European countries began using phage to treat specific bacterial infections. Unlike antibiotics, which kill bacteria indiscriminately, phage target only certain microbes for destruction.

Reviving this strategy will depend on finding or designing bacteriophage that will take out specific “bad bugs,” while somehow avoiding the bacteria’s ability to ward off attackers with CRISPRs. Researchers admit this type of microbial manipulation is still far in the future.

Immune system sentry

That doesn’t mean viruses, and viral surveys, can’t be useful in the near term.

Some viruses may act as bellwethers for the health of the immune system.

Stephen Quake, a geneticist and Howard Hughes Medical Institute investigator at Stanford University, and his colleagues were studying recipients of heart or lung transplants to learn why some people reject the organs. They collected blood from 96 transplant patients and examined bits of DNA floating in the samples. “We realized some of the DNA wasn’t human,” Quake says.

Of the nonhuman component of the patients’ blood, 73 percent came from viruses. The majority — 68 percent — of the viruses they found were anelloviruses, mysterious germs that don’t cause specific illness but have been linked to fevers in toddlers. Some of the transplant recipients had high levels of the viruses in their blood. It may sound counterintuitive, but “that’s good news if you have an organ transplant,” Quake says.

It means that immune-suppressing drugs are doing their job of weakening the immune system to prevent organ rejection, a very real danger for people who get hearts or lungs from unrelated strangers. People with lower levels of anelloviruses in their blood were more likely to reject their transplanted organs, Quake and his colleagues reported in the Nov. 21 Cell. Because the very immune reactions that keep the viruses in check can also turn against the foreign organ, measuring transplant patients’ load of anelloviruses may help predict who is likely to face organ rejection.

Even beyond transplants, doctors may be able to monitor anelloviruses to learn about the health of their patients’ immune systems. For example, people with HIV develop escalating levels of anelloviruses in their blood as their immune systems weaken, says Quake’s coauthor, Kiran Khush, a cardiac transplant surgeon at Stanford University.

“This is another big, red sign that these things [viruses] should be getting more attention,” Quake says.

Friend or foe?

In organ rejection, the anelloviruses are not the cause; they’re sentinels. But other maladies may have viral instigators. Figuring out which viruses are the culprits is a difficult task, says Kristine Wylie, a virologist at Washington University School of Medicine in St. Louis.

Wylie and her colleagues took blood samples and nasal swabs from infants and toddlers, some of whom had unexplained fevers. They wanted to see if DNA technology could quickly identify why the kids were sick.

The researchers found 25 different major categories of viruses, including many associated with illness, they reported in PLOS ONE in June 2012. Children with fevers tended to carry a heavier viral burden, both in the number and type of viruses. But even healthy kids had plenty of viruses in their noses and in their blood.

“Healthy subjects are just loaded with viruses,” Wylie says. Even viruses known to cause diseases such as the common cold were found in healthy kids. That makes it difficult to determine whether a particular virus is really making someone sick.

Some viruses previously thought innocent may cause harm. Rohwer was part of a team that found in 2005 that plant viruses, particularly the pepper mild mottle virus and other pathogens that affect fruit, grain and vegetable crops, are some of the most abundant viruses in human feces. Since plant viruses don’t infect human cells, researchers assumed that they were harmlessly passing through the digestive system. But in a 2010 study, the pepper virus was associated with fever, abdominal pain and itching in some people. The virus may accidently set off the immune system’s viral sensors and lead to inappropriate inflammation, or the symptoms may be an indirect result of eating spicy food, the researchers speculate.

To figure out which viruses are friends, foes or neutral passengers on the human body, scientists first need to identify them. Researchers still aren’t very good at recognizing new viruses, says Brian Jones, a molecular biologist at the University of Brighton in England. Hence the large pool of unknown samples in Pérez-Brocal’s and other researchers’ virome studies. But even if scientists improve their identification skills, it may take a long time to figure out what the viruses are doing in the body.

Based on what researchers have learned so far about the virome, Jones is convinced that viruses and other microbes “should be viewed as a part of us rather than something that lives in or on us.” They are part of the puzzle, the intricate ecosystem composed of human and microbial cells, all pushing and pulling at one another and subject to local conditions, such as diet and environment.

If he’s right, then knowing our viruses might help us know ourselves.