In the final push to end polio, global health planners are embarking on an unthinkably ambitious and potentially risky move. They’re switching 155 countries—a good portion of the world—from one polio vaccine to another.
This will require moving millions of doses of a new vaccine into place over the course of two weeks in late April, while sequestering the remaining stocks of the old one.
And that’s only one of the many maneuvers necessary to truly end polio, which in the 1980s caused more than 350,000 cases of paralysis a year. So far in 2016, there have been only nine cases in two countries: Afghanistan and Pakistan.
The vaccine switch is part of the final strategy to put a noose around the few remaining cases, by improving the match between the viruses that remain in the wild and the vaccine that suppresses them. If it goes as planned, it will improve children’s immunity to wild-type polio while removing their vulnerability to a variant of the disease that can be accidentally caused by the vaccine itself.
It looks like the goal is in sight. But polio has slipped from control before.
“This is the largest, the fastest, and [a] unique event that is taking place,” Dr. Michel Zaffran, the director of polio eradication at the World Health Organization, said in a phone call with reporters Thursday morning. “This is an unprecedented event that has never been done before in the world.”
After almost 30 years of trying, the move has the potential to finally stop any new cases of polio from occurring. But planners acknowledge that the move carries some risk: It could accidentally ignite an outbreak of the type of polio caused by the vaccine.
“We are anticipating there will be at least one event we will have to respond to,” said Dr. Steven Cochi, who serves as a senior liaison between the eradication campaign and the Centers for Disease Control and Prevention.
Shots or Drops?
To understand the complexity of this, it helps to remember a little history. The start of the effort to control polio, back in the 1950s, was a competition between two scientists: Jonas Salk, who developed an injectible vaccine using killed virus, and Albert Sabin, who formulated a vaccine taken by mouth that relies on living but weakened polio.
Salk’s vaccine ended up ruling in the industrialized world. But Sabin’s became the foundation of the international eradication campaign, not just because it can be administered even by people with no medical training, but because, as the virus gets into the gut and attaches there, it produces copies that pass out of the body in feces and create immunity in anyone else who picks it up.
That strength turned out to be a weakness, because as the live virus reproduces, it can mutate from its weakened form into a virulent disease-causing type, and cause polio in any of those nearby who would otherwise have been protected when the vaccine virus was shed. Last year, when there were only 74 cases of polio in the world, 27, more than a third, were caused by what is called “vaccine-derived” virus.
Polio virus comes in three “types,” or strains—known for simplicity as types 1, 2 and 3—that are different enough from each other that they all must be included in the vaccine. Type 2 is the most efficient at attaching to the gut, and partly because of that, it became the first strain to be eradicated; it has not been seen in the wild since 1999. But for the same reason, it is the strain most likely to cause vaccine-derived cases. So the new vaccine being rolled out on Sunday deletes the Type 2 weakened virus.
Why Change All at Once?
That substitution will only work if everyone in the world who is using oral polio vaccine, or OPV, switches at the same time; if one country continued to use the three-type vaccine, it could put others at risk. So beginning this weekend, thousands of volunteers and monitors will fan out, across the developing world and also in industrialized societies such as the Russian Federation which are still using OPV, to make sure the new vaccine is delivered on schedule and, crucially, kept cold as it goes.
To reduce the vulnerability inherent in the switch, as many countries as possible were supposed to give children one shot of the injectable vaccine, known as IPV, to make sure their immunity was as high as possible. But planners acknowledged Thursday that there is a shortage of IPV, and not all children may have received the protective dose.
Most people stop shedding the vaccine virus in two to four weeks; that, Cochi said, is considered the window of vulnerability post-switch in which an outbreak might spark. There are also rare cases in which people with immune-system disorders hang onto the virus and shed it for years; since they are not made sick by it, they are very hard to spot. (To find them, some countries screen sewage for the presence of polio.)
Will This Cause an Outbreak?
Hypothetically, a long-term shedder carrying mutated type 2 polio virus could ignite an outbreak at any time. But Zaffran said, with unusual frankness, that in the countries that would be most vulnerable, immune-deficient children often do not live long; and in the countries where good medical care sustains their lives, immunization rates are already high enough to make the possibility of an outbreak null.
Nevertheless, Cochi said that to keep any potential outbreaks from spreading, stockpiles of the old oral vaccine will be kept on hand in each country, and million of doses of a new, Type 2-only vaccine are ready for emergency deployment if needed.
Planners hope the giant vaccine switch is the beginning of the endgame of eradication. It is late—they thought they would get to the goal 16 years ago—and each delay has been costly. The next steps will also be expensive and complex: first rolling out IPV across the world, and then scouring laboratories for any forgotten frozen samples that might harbor the polio virus.
But in the end, if they are successful, polio will become the second human disease eradicated from the world.
If Zika virus comes to the United States, will the US blood supply be at risk?
Because the disease has demonstrated that it can pass via blood from mother to fetus, and via other bodily fluids between sexual partners, the question lurks in the back of most discussions of Zika’s likely arrival on the US mainland. And because there is not now a test for donated blood, keeping the virus out of the blood supply relies on people adhering to restrictions published by the Food and Drug Administration that ask travelers to defer donating for a period of time—an imprecise deterrent, but currently as good as it gets.
The concern for the blood supply is reasonable. In 2002, when West Nile virus was newly arrived in the United States, transfusions given to a teenage accident victim—who died of her injuries, and became an organ donor—caused that disease to pass to all four recipients of her organs. Dengue, another mosquito-borne illness that is burgeoning in Central and South America and has become established in south Florida, has also passed between blood donors and recipients, though there are only a few cases on record. And Zika virus was identified in 3 percent of donated blood in French Polynesia in late 2013 and early 2014, when the virus first landed in that area.
The concern has been sharpened by a new analysis, published Wednesday in the journal PLoS Currents Outbreaks, that plots the range of the mosquito species known to carry Zika against the numbers of travelers who arrive from the Zika zone. The researchers—from several US government agencies, North Carolina State University and University of Arizona, and Durham University in England—predicted that cities within the mosquito’s range are at highest risk of local transmission of Zika if they have international airports, or airports receiving connecting flights from those hubs. Other cities receiving large numbers of travelers from the Zika transmission zone were at moderate or lower risk if they fell near the edge of the mosquito’s range. So, for instance, Miami, Orlando, Jacksonville, Tallahassee, and New Orleans were at high risk of receiving the disease; New York, Atlanta and Houston were at moderate risk, and Dallas, Denver and Los Angeles at low risk.
What was jaw-dropping in the study, though, were the sheer numbers of people who arrive in US cities from the Zika transmission zone: up to 1 million per month in Miami and New York, 500,000 per month in Atlanta, Houston, New York and Dallas, and millions per month through the ground border crossings of San Diego, El Paso and Laredo.
To prevent Zika contaminating the blood supply, the FDA issued guidelines last month addressing blood donation and this month regarding donated cells and tissues. For blood donation, the agency recommended that blood agencies ask people to defer donation for four weeks after experiencing Zika symptoms, traveling in the Zika transmission zone, or having sex with a man who either had the symptoms or traveled in the Zika zone. For tissues such as ligaments and corneas, and cells (which include sperm and eggs), the agency extended the deferral to six months. The FDA has not placed any restrictions on donation of solid organs, arguing that because they are both life-saving and in short supply, the benefit outweighs the risk.
Dr. Matthew Kuehnert, who is director of the office of blood, organ and other tissue safety at the Centers for Disease Control and Prevention, and is serving as the lead for the blood safety team in the CDC’S Zika response, said knowing how far to go to protect the blood supply is challenging because data is so sparse.
“There is little that we know about transfusion transmission of Zika, although I think we should assume it can happen,” he said by phone. “From the data that has been collected on Zika, about 80 percent of people don’t know they are infected. There is a period of viremia”—when virus circulates in the blood—”but we don’t know how long that viremia is. It is thought to be 7-10 days, but as we start to collect more data we may find it is longer than that.”
“It is possible we could get a transfusion or transplant transmission case before we even know local transmission of Zika is occurring.”
A problem, Kuehnert pointed out, is that because symptoms are the signal of an infection, only people who show signs of Zika infection—mostly fever, headache, rash and red eyes—are being interviewed and tested to add to knowledge about the disease. People who do not experience symptoms are not visible to investigators. They also become viremic; but since they are not interviewed or tested, the duration of their viremia, when the virus in their blood could pass into a blood donation, is not being uncovered. And there are early signals that, even after it passes out of the blood, the virus can take shelter in other tissues and fluids. “Zika can be sexually transmitted long after viremia is thought to be gone, so there are likely protected sites where it can hide,” he said. “Thus there might be blips of viremia occurring after symptoms have resolved. So there is a lot of potential for transfusion transmission.”
Those considerations apply in areas where Zika is not yet locally established. Where it is—which in the United States is Puerto Rico (160 cases as of March 9), American Samoa (13 cases) and the Virgin Islands (1 case)—blood is assumed to be a risk, and workarounds are being urgently sought. Because there is no test for Zika in donated blood—an approved test is “weeks to months away,” Kuehnert said—the only alternative is to use what are called “pathogen reduction” treatments, which inactivate viruses. Currently, pathogen reduction can only be used on platelets and plasma; red blood cells can be altered by pathogen reduction, and authorities are urgently searching for better techniques..
In a sign of how quickly an epidemic can upset the balance of blood supplies, Puerto Rico is now receiving outsourced blood from the US mainland, via a joint effort of three blood-collection agencies—the American Red Cross, Blood Centers of America, and America’s Blood Centers—and the Department of Health and Human Services. The CDC estimates the current need for clean blood and blood products in Puerto Rico is 2,500 units of red blood cells, and an additional 1,000 units of other blood products, every week.
Despite the protections put in place by the FDA, public health authorities are braced for the possibility that transfusion-associated Zika could begin occurring in the United States. “This could happen at any time,” Kuehnert acknowledged.
He added: “It is possible we could get a transfusion or transplant transmission case before we even know local transmission of Zika is occurring,” because the illness that necessitates a transfusion—or the immunosuppressive drugs that transplants recipients take—make them more vulnerable to disease. “We are doing a lot of work to be prepared.”
The U.S. Centers for Disease Control and Prevention has responded to the discovery that Zika virus can be transmitted sexually, recommending that if pregnant women’s partners have traveled to areas where the virus is circulating, they either rigorously use condoms, or abstain from sex til the pregnancy has ended.
It is one of several pieces of news that are complicating the debate over how best to protect against Zika infection, which is exploding in South and Central America and is linked to an apparent epidemic of birth defects in Brazil. On Wednesday, the European Centre for Disease Prevention and Control said it had been notified of Zika being passed via blood transfusion. Also on Friday, the United Nations chief of human rights, and subsequently the secretary general, said that countries which restrict access to birth control and abortion—which includes many of the 31 countries where the disease is burgeoning—must repeal those laws in response to Zika. And Brazilian health authorities said they have detected virus in the saliva and urine of infected people, though they could not draw conclusions about whether it is transmitted by those body fluids.
The CDC’s recommendations are a response to the announcement Tuesday by Dallas County Health and Human Services Department that a man in Dallas transmitted the virus to his sexual partner after returning from traveling in Venezuela. (The department did not specify the gender of the man’s partner and said a pregnancy was not involved.) They also come after a woman in Hawaii, who was infected while living in South America in the early stages of pregnancy, gave birth to a baby with the Zika-related birth defect microcephaly. The CDC has recommended that pregnant women not travel to 30 countries and jurisdictions where the virus is circulating, in the Caribbean, Central and South America, and the Pacific Islands, as well as the US territory of Puerto Rico.
In a conference call with reporters Friday, CDC director Dr. Thomas Frieden cautioned that it has been 70 years since an infectious disease has posed a direct threat to fetuses—rubella, in that case. (See “Way Before Zika, Rubella Changed Minds on Abortion” at National Geographic News.)
“We are not aware of any prior mosquito-borne disease associated with such a potentially devastating birth outcome on a scale anything like what appears to be occurring with Zika,” he said. “Because this phenomenon is so new we are quite literally discovering more about it each and every day.”
The CDC’s new recommendations say:
Men who live in or have traveled to places where Zika virus is being transmitted, and whose partner is pregnant, should either abstain from sex as long as the pregnancy lasts, or should use condoms for vaginal, anal or oral sex.
Men who live in or travel to places where Zika is being transmitted, and have a sexual partner who is not currently pregnant, may want to consider either using condoms or abstaining from sex.
The CDC earlier recommended that pregnant women who live in areas where Zika is circulating should strictly avoid mosquito bites.
Asked about sexual transmission to women who are not pregnant or to same-sex partners, Frieden pointed out that Zika is most often a mild illness in adults, though it has been linked to the paralytic syndrome Guillain-Barre. “Our primary concern and priority here is the protection of pregnant women,” he said.
In a separate set of recommendations, the CDC updated its advice for pregnant women who may have been exposed to Zika, suggesting that women who have traveled where it is circulating be tested for the virus (from 2 to 12 weeks after returning to the United States) even if they do not show symptoms. The agency cautioned though that the tests can return false results because they cross-react to infection with other mosquito-borne viruses, and Frieden said the tests may be in short supply for a while. “We wish more tests were available; our laboratories are literally working around the clock to get tests kits out,” he said. “Not everyone who wants a test will be able to get it.”
The CDC has made Zika a reportable disease in the United States, meaning that state health authorities should report new diagnoses to the agency. So far, Frieden said, the CDC has been told of 51 cases in the United States, 50 imported and the single sexually associated case, and 21 in Puerto Rico. Of those 72 cases, seven are pregnant women, he said. (The CDC maintains a map, not yet updated with those numbers.)
The Centers for Disease Control and Prevention has responded to growing alarm over the Zika virus epidemic in Central and South America with quickly published guidelines covering health care and tests for pregnant women who may have been exposed to the virus.
The guidelines come on the heels of the CDC’s recommendation last Friday night that US women who are pregnant, or planning to become pregnant, avoid traveling to the 13 countries where transmission of Zika has occurred, and also to the US territory of Puerto Rico.
Zika, which is transmitted by mosquitoes, arrived in South America in 2014 and ignited a pandemic. Most of the adult cases, which number more than 1 million, have been mild. (It is generally accepted that four out of five people infected with Zika do not develop symptoms; so the true number of those infected is likely more than 5 million.) But in Brazil, there has been an epidemic of a birth defect called microcephaly—smaller than usual brains and heads in newborns— that is associated temporally, and by some lab tests, with Zika infection. So far in Brazil there have been more than 3,500 cases of microcephaly. Zika has come to the United States as well, with local transmission in Puerto Rico and an imported case in the county surrounding Houston, and on Friday, a baby born in Hawaii to a woman who lived in Brazil while she was pregnant was diagnosed with Zika microcephaly. Today, the Illinois Department of Public Health disclosed that it is monitoring two pregnant women who traveled to Zika transmission areas.
(Update, Jan. 20: According to Florida media, that state’s department of health has announced three cases in Florida, all travel-related.)
The CDC’s guidelines today offer advice for pregnant women who traveled to a location where Zika is circulating, whether or not the woman reports symptoms of Zika infection: sudden fever, a rash, conjunctivitis, and joint pain. Broadly, women with a travel history and symptoms should have blood drawn to be tested for Zika infection—the test can be performed only by the CDC and some health departments—and if positive, should have regular ultrasounds to track fetal development and should be seen by one of several specialists. Pregnant women who traveled to a Zika area but did not experience symptoms are recommended to undergo ultrasounds first, and to seek a test to confirm infection if there are abnormalities in the imaging.
Within the text of the recommendations, which were published as an early release from the CDC’s weekly journal Morbidity and Mortality Weekly Report, there are hints of how complex this emerging situation has become. There is no vaccine for Zika, so as prevention the agency can recommend only “wearing long-sleeved shirts and long pants, using U.S. Environmental Protection Agency-registered insect repellents, using permethrin-treated clothing and gear, and staying and sleeping in screened-in or air-conditioned rooms.” There is no specific treatment, so it can recommend only “rest, fluids, and use of analgesics and antipyretics. Fever should be treated with acetaminophen.” (The CDC specifically rules out aspirin, because the mosquito-borne diseases chikungunya and dengue are also circulating in the areas where Zika is, and dengue can lead to hemorrhagic fever—so drugs that can increase bleeding are not recommended.)
The limited options for confirming Zika in a fetus are especially difficult, since amniocentesis—which could yield a sample for testing—also carries a risk of miscarriage. The CDC says:
Zika virus RT-PCR testing can be performed on amniotic fluid. Currently, it is unknown how sensitive or specific this test is for congenital infection. Also, it is unknown if a positive result is predictive of a subsequent fetal abnormality, and if so, what proportion of infants born after infection will have abnormalities. Amniocentesis is associated with an overall 0.1% risk of pregnancy loss when performed at less than 24 weeks of gestation…. early amniocentesis (≤14 weeks of gestation) is not recommended. Health care providers should discuss the risks and benefits of amniocentesis with their patients.
The CDC has also published guidance for health care professionals here, and explanations of how to send samples for testing here.
Update, Jan. 22: The CDC has added Barbados, Bolivia, Ecuador, Guadeloupe, Saint Martin, Guyana, Cape Verde, and Samoa to its “don’t travel if pregnant” list.
As Yogi Berra (or Niels Bohr or Samuel Goldwyn) is supposed to have said, it’s difficult to make predictions, especially about the future. It’s especially dangerous to try to predict the behavior of infectious diseases, when small unpredictabilities in climate or trade or the behavior of governments can bring a problem that we thought was handled roaring back to life.
But as 2016 opens, it is fair to say that the disease public health experts are pinning their hopes on, the one that might truly be handled this year, is polio. There were fewer cases last year than ever in history: 70 wild-type cases, and 26 cases caused by mutation in the weakened virus that makes up one of the vaccines, compared to 341 wild-type infections and 51 vaccine-derived ones the year before. Moreover, those wild natural infections were in just two countries, Afghanistan and Pakistan, and the vaccine-derived cases were in five. The noose is tightening.
The most that health authorities can hope for this year is to end transmission of polio. The ultimate goal is eradication, which has happened only twice—for one human disease, smallpox, and one animal one, rinderpest. To declare a disease eradicated requires that the entire world go three years without a case being recorded. If there are no polio cases in 2016, eradication might be achieved by the end of 2018.
Which would make for nice round numbers, because the polio eradication campaign began in 1988. It is safe to say that no one expected it would take anywhere near this long; the smallpox eradication campaign, which inspired the polio effort, reached its goal in 15 years.
Smallpox was declared eradicated in 1980, so long ago that most people have no knowledge of how devastating a disease it was, or even what a case of the disease looked like. (There are survivors left, but they are aging; the last person infected in the wild, Ali Maow Maalin of Somalia, died in 2013.) In the same way, we’ve forgotten how difficult it is to conduct an eradication campaign. Smallpox was the first campaign that succeeded, but it was the fifth one that global authorities attempted. In its success, it demonstrated what any future campaign would need: not just a vaccine that civilians could administer, but an easy-to-access lab network, granular surveillance, political support, huge numbers of volunteers, and lots and lots of money.
In its own trudge to the finish, the polio eradication campaign has stumbled over many of those, from local corruption to extremist opposition to the still almost unbelievable interference of the CIA (which I covered here and here), along with the virus’s own protean ability to cross borders (to China) and oceans (to Brazil).
But now, at last, the end does look in sight. I asked Carol Pandak, director of the Polio Plus program at Rotary International — which since 1988 has lent millions of volunteers and more than a billion dollars to the eradication campaign — how she thinks the next 12 months will go.
“We are getting closer,” she told me. “We have only two endemic countries left. Of the three types of the virus, type 2 was certified eradicated in September, and there have been no type 3 cases globally for three years. And Pakistan and Afghanistan have goals to interrupt transmission internally in May 2016.”
The diminishment of wild polio paradoxically creates greater vulnerability to vaccine-derived polio, which happens when the weakened live virus used in the oral vaccine mutates back to the virulence of the wild type. The only means of defusing that threat is to deploy the killed-virus injectable vaccine, which is widely used in the West but until recently was considered too expensive and complex to deliver in the global south.
To begin the transition, Pandak said, countries that still use the oral vaccine have agreed to give one dose of the injectable as part of routine childhood immunizations for other diseases. That should strengthen children’s’ immune reactions to polio, so that the reversion to wild type — which occurs as the weakened virus replicates in the gut — does not take place.
In the smallpox campaign, when eradicators thought they were almost done, there was a freak weather event—the worst floods that Bangladesh had experienced in 50 years—that triggered an internal migration and redistributed the disease. Polio is just as vulnerable to last-minute disruptions, especially since the two remaining endemic countries are hotspots of unpredictability. Travelers from Pakistan actually carried polio into Afghanistan in August.
“In Pakistan, the army has committed to providing protection for vaccinators in conflict areas,” Pandak told me, “and another strategy that has been successful has been to set up border posts to immunize people as they are fleeing areas of conflict and military operations. I have seen Rotary volunteers staffing 24/7 kiosks in train stations and toll booths, so that we can get people wherever they happen to be.”
There is no question that hurdles remain. By the World Health Organization’s order, polio is still considered a “public health emergency of international concern,” which requires countries where the disease is extant to either ensure its citizens are vaccinated before leaving, or prevent their crossing the border. And polio still lives quiescently in lab freezers all over the world, and those will have to be searched and their contents eliminated lest a lab accident bring the disease alive again (a warning that was recently circulated for rinderpest as well). Plus, up til now, the injectable vaccine has been made by starting with a virus that is not only live but virulent, posing the risk that a lab accident that could release it; British scientists announced on New Year’s Eve that they may have found a way to weaken it while still yielding a potent vaccine.
When it goes, if it does, polio will gift the world not only with its absence, but also with the abundant health infrastructure that was set up to contain and eliminate it, and can be turned to other uses. When I talked to Pandak, she sounded excited at the possibility that countries and volunteers would be able to turn their attention away from a single disease and toward ensuring the overall health of children.
“We have been doing this for 30 years,” she said. “We’ll continue to fundraise, advocate and raise awareness to the last case. We are committed to seeing this to the end.”
Over the past fifty years, Marek’s disease—an illness of fowl—has become fouler. Marek’s is caused by a highly contagious virus, related to those that cause herpes in humans. It spreads through the dust of contaminated chicken coops, and caused both paralysis and cancer. In the 1970s, new vaccines brought the disease the under control. But Marek’s didn’t go gently into that good night. Within ten years, it started evolving into more virulent strains, which now trigger more severe cancers and afflict chickens at earlier ages.
Andrew Read from Pennsylvania State University thinks that the vaccines were responsible. The Marek’s vaccine is “imperfect” or “leaky.” That is, it protects chickens from developing disease, but doesn’t stop them from becoming infected or from spreading the virus. Inadvertently, this made it easier for the most virulent strains to survive. Such strains would normally kill their hosts so quickly that they’d die out. But in an immunised flock, they can persist because their lethal nature has been neutered. That’s not a problem for vaccinated individuals. But unvaccinated birds are now in serious trouble.
This problem, where vaccination fosters the evolution of more virulent disease, does not apply to most human vaccines. Those against mumps, measles, rubella, and smallpox are “perfect:” They protect against disease and stop people from transmitting the respective viruses. “You don’t get onward evolution,” says Read. “These vaccines are very successful, highly effective, and very safe. They have been a tremendous success story and will continue to be so.”
He is more concerned about the next generation of vaccines that are being developed against diseases like HIV and malaria. People don’t naturally develop life-long immunity to these conditions after being infected, as they would against, say, mumps or measles. This makes vaccine development a tricky business, and it means that the resulting vaccines will probably leak to some extent. “This isn’t an argument against developing those vaccines, but it is an argument for ensuring that we carefully check for transmission,” says Read.
“The candidate Ebola vaccines are also foremost in my mind,” he adds. “Some of the monkey trials suggest that they may be perfect, but we need to be very confident that they don’t leak. If they do, and some vaccinated individuals are capable of passing on Ebola, that might lead to the evolution of very dangerous pathogens.”
He is also concerned about animal vaccines, which are often leaky. These include vaccines against Newcastle disease in poultry, Brucella in livestock, and especially bird flu. When bird flu outbreaks hit American and European farms, the birds are culled. But in Southeast Asia, they’re often vaccinated, “and those vaccines are leaky,” says Read. “It creates an analogous situation to Marek’s.” The birds might survive more lethal forms of the virus, which they could then spread to each other—and potentially to people.
Read first proposed the “imperfect vaccine hypothesis” back in 2001, on purely theoretical grounds. It proved controversial, not least because he had neither experimental evidence nor case studies to support the idea. Then, a colleague told him that the hypothesis might explain the increasing virulence of Marek’s disease. “I wrote the name down, misspelled it, and couldn’t find anything in the literature!” Read says. He only heard about the condition again when he was asked to speak at a Marek’s conference. There, someone put him in touch with Marek’s expert Venugopal Nair from the Pirbright Institute.
The duo infected vaccinated and unvaccinated chicks with five different strains of Marek’s virus, of varying virulence. They found that when unvaccinated birds are infected with mild strains, they shed plenty of viruses into their surroundings. If they contract the most lethal strains, they die before this can happen, and their infections stop with them. In the vaccinated chicks, this pattern flips. The milder strains are suppressed but the lethal ones, which the birds can now withstand, flood into the environment at a thousand times their usual numbers.
Read and Nair also found that the “lethal” strains could spread from one vaccinated individual to another, and that unvaccinated chickens were at greatest risk of disease and death if they were housed with vaccinated ones.
All of this is consistent with the imperfect vaccine hypothesis. It doesn’t prove that imperfect vaccines drove the evolution of today’s extra-virulent strains, “and we may never know for sure why those evolved in the first place,” Read writes. Other factors, like the fact that modern chickens are genetically similar or raised in dense, crowded conditions, may have also played a role. Still, it’s at least clear that vaccines can keep virulent strains in circulation. “For the chicken industry, these results are actually an argument for getting the vaccine,” says Read. “Any chicken that doesn’t get it is at even greater risk than it would be in the 1950s.”
“This work may drive change in the way that vaccines are developed and tested, so that there is much greater emphasis on their ability to prevent infection and transmission, rather than only on their ability to prevent clinical disease,” says Joanne Devlin from the University of Melbourne. “I think that would be a positive step.”
Katherine Atkins from the London School of Hygiene and Tropical Medicine agrees. “While more theoretical work is now being conducted prior to vaccine roll-outs,” she says, researchers need to look beyond how vaccines curb epidemics. They must also consider “the long-term evolutionary consequences of new vaccine introduction.”
But Vincent Racaniello from Columbia University says, “We still do not have any proof that allowing a virus to replicate in a vaccinated individual will select for more virulent viruses.” The new results simply show that leaky vaccines allow virulent viruses to spread—not that they allow those viruses to evolvein the first place. The only way of doing that is to infect vaccinated chickens with mild strains and see if more virulent ones arise after many rounds of transmission.
Racaniello is also unconvinced that the effect would generalise to other vaccines. For example, the Salk polio vaccine—one of two that are used—is a little leaky. “People who are immunized can be infected with poliovirus and the virus can replicate in their guts, be shed, and transmitted to others,” says Racaniello. “This behaviour has been well documented in human populations, yet the virulence of poliovirus has not increased for the 50+ years during which this vaccine has been used.”
That is no reason to rest on our laurels, says Read. It’s important to at least check for the emergence of deadlier viruses if vaccines are imperfect—and perhaps to take preventative measures. For example, a leaky malaria vaccine could be paired with bed nets that would stop mosquitoes from spreading more virulent strains of malarial parasites to unvaccinated people. “If someone developed [such a vaccine] and it worked, we should go ahead and use it, but not think of it as a magic bullet,” says Read. “I’d say that anyone who is vaccinated against malaria should be under a bed net too.”
Reference: Read, Baigent, Powers, Kgosana, Blackwell, Smith, Kennedy, Walkden-Brown & Nair. 2015. Imperfect Vaccination Can Enhance the Transmission of Highly Virulent Pathogens. PLoS Biol http://dx.doi.org/10.1371/journal.pbio.1002198
In June 2013, starfish on the western coast of North America started wasting away. At first, their arms curled from the tips, and they tied themselves into pretzel-like knots. Their bodies deflated. White festering sores appeared on their flesh. As the lesions spread, their flesh rotted away and their arms fell off. Within days, healthy animals had disintegrated into mush.
This condition, known as sea star wasting syndrome (SSWS), was recorded as far back as the 1970s, but the scale of this recent event is unprecedented. It has hit at least 20 species all along the Pacific, from Alaska to California. In less than a year, huge, thriving populations have completely wasted away.
As the stars blinked out, scientists compiled a list of possible causes that included storms, rising temperatures, and pollutants. But an infection always seemed likely. The disease seemed to move from place to place with the character of a spreading epidemic. Most tellingly, starfish in aquariums started dying too. These animals were housed in controlled captive environments but they were immersed in water pumped in from the surrounding ocean—and that was enough to kill them. Filtering the water through sand didn’t help. The only measure that spared the stars was sterilising the water with ultraviolet light. Whatever was killing the animals was microscopic and biological.
Now, a team of scientists led by Ian Hewson from Cornell University have identified the most likely culprit behind the grisly outbreak—a new virus that they call sea star-associated densovirus, or SSaDV.
Densoviruses are best known for infecting insects and crustaceans, but last year, Hewson’s team discovered them in a group of Hawaiian sea urchins. “We didn’t associate those viruses with any disease,” he says. “I jokingly told a colleague: Boy, what if there was some kind of mass mortality?” When the SSWS outbreak hit, the team leapt at the chance to study it.
First, they blended tissues from wasting starfish and passed them through filters with extremely small pores—small enough to exclude bacteria but big enough to let viruses through. They inoculated healthy starfish with these extracts, and the animals started wasting within a couple of weeks. If they boiled the extracts first, the animals were unharmed. This confirmed that the disease was transmissible and was caused by something the size of a virus. By sequencing the killer extract, the team showed that it contained the genome of a new densovirus—SSaDV.
They then collected tissue samples from 465 wild starfish, belonging to three species. The symptomatic animals were more likely to carry SSaDV than their healthy peers, and in higher numbers. And the more viruses they had, the worse their symptoms were.
The association wasn’t perfect: some diseased starfish showed no signs of the virus, while some healthy ones did. But Hewson thinks that there are easy explanations for these patterns. Diseases animals don’t have the virus everywhere. “If you take a sea star and divide it into pieces, you can detect virus in 80 percent of them. So 20 percent will be a false negative,” he says. There’s also a lag between infection and symptoms, so “healthy” animals might be exposed without having fallen sick yet. “Some of the ‘healthy’ samples came from an aquarium where all the animals later died,” Hewson adds.
“The authors present persuasive evidence that they have identified an agent associated with SSWS,” says Ian Lipkin, a virus hunter from the Mailman School of Public Health. “Nonetheless, as they note themselves, there is much more work to be done before we will know whether the densovirus they describe is necessary and sufficient to cause disease.”
Vincent Racaniello, a virologist from Columbia University, agrees that the evidence is strong. “The crucial experiment that remains to be done is to isolate infectious virus in cell culture, inoculate it into sea stars, and show that it causes wasting disease,” he says.
Hewson agrees that this step is crucial, but he also thinks that it will be very difficult. Ideally, he’d like to infect starfish in the absence of all other microbes to show that the virus is truly responsible for the disease. But these animals pump seawater through their bodies and they are naturally riddled with microbes. The alternative is to grow starfish tissues in the lab, but no such cultures exist for marine invertebrates. “That’s a real stumbling block,” says Hewson. “We’re trying to isolate a cell culture of a sea star but that’ll be a long and tedious process.
He also suspects that the virus may not actually cause the symptoms of SSWS directly. It could, for example, disrupt the sea stars’ ability to control the bacteria that they normally co-exist with. “The lesions are probably just the native bacteria taking advantage of an immunocompromised host,” he speculates. “So, associating the virus with those lesions may be a challenge.”
There’s also another mystery: why is the current outbreak of SSWS so dramatic when the newly discovered virus is actually an old presence? The team found its DNA within starfish that had been collected from the Pacific coast as far back as 1942, and that have been sitting in museum jars ever since. So why weren’t North America’s starfish melting away while World War II was raging?
“Viruses do smoulder in populations,” says Hewson. Familiar names like HIV and Ebola were affecting humans at small scales long before they triggered huge scary epidemics. The same may apply to SSaDV. In recent years, booming sea star populations in the Pacific Northwest may have given the virus newfound impetus. “There were mountains of sea stars underwater, tens of metres high,” says Hewson. “If you talk to crab-fishers in the region, their crab pots were full of these stars and they were getting annoyed.” The virus could have more easily jumped from host to host, or developed mutations that made it more transmissible or virulent.
Environmental changes may be important. Carol Blanchette from the University of Santa Barbara says, “We have been sampling sea stars across southern California throughout this epidemic and it is likely that in our region, as well as in others, environmental causes like increased temperatures have played an important role.” She finds the virus evidence convincing, but thinks it “may only be one part of the story”.
SSaDV may just be part of a natural cycle that controls sea star populations if they grow too big. Then again, some starfish act as keystone species—they wield disproportionate influence over their habitats by controlling populations of mussels and barnacles that would otherwise takeover. Their loss could dramatically reshape the coastlines of the Pacific Northwest, from thriving communities into black mussel monocultures.
SSaDV is also found in other related animals like sea urchins and brittle stars. No one knows if it causes disease in these groups, but the worry is that the virus could use its sea star hosts as a platform for building in numbers and launching outbreaks in other species.
Even if that was true, Hewson doubts that anything can be done. Even if scientists could develop a cure or vaccine, it would be impractical to inoculate the animals on a wide scale. “We’re just trying to understand this as a natural phenomenon,” he says.
The most common viruses in your body don’t make you ill. Instead, they infect the legions of microbes that live in your gut. These bacteriophages, or phages for short, number in their trillions. And the most common of them might be a newly discovered virus called crAssphage.
No one has seen crAssphage under the microscope, but we know what its genome looks like—Bas Dutilh from Radboud University Medical Centre pieced it together using fragments of DNA from the stools of 12 individuals. He found crAssphage in all of them. Then, he found it in hundreds more.
To study the microbes that live in a person’s guts, scientists will typically collect a stool sample, break all the DNA within into small fragments, and sequence these pieces. The result is a metagenome: a mish-mashed collection of DNA from all the local bacteria, viruses and other microbes.
Dutilh’s team, led by Rob Edwards at San Diego State University, analysed 466 metagenomes that have been added to public databases and found crAssphage in three-quarters of them. It’s there in stool samples from people in the USA, Europe and South Korea. It actually accounted for 1.7 percent of all the sequences that the team analysed—six times more than all the other known phages put together. You probably have it inside you right now.
The work highlights just how much we don’t know about the viruses in our guts and “what exciting times these are for viral discovery”, says Lesley Ogilvie from the Max Planck Institute for Molecular Genetics.
But how could such a common virus go undiscovered for so long, especially considering how popular the study of gut microbes has become? It’s as if zookeepers suddenly realised that most of their zoos contain a giant grey animal with tusks and a trunk, which no one had noticed before.
For one thing, the viruses in our guts are hard to study. “To study a virus, normally you have to make heaps of it, which isn’t possible if you can’t grow the host,” says Martha Clokiefrom the University of Leicester.And since mostgut bacteria won’t grow easily in a lab, the viruses that infect them are similarly hard to rear.
The alternative is to use metagenomics to analyse a microbe’s genes without having to grow it. But first, you have to assemble your mish-mash of sequences, which come from different organisms, into a complete genome. It’s a bit like putting all the pieces of a thousand jigsaw puzzles into one bag, and trying to solve just one.
The usual strategy is to work off what you know by aligning these new sequences to those in databases. But this approach doesn’t work very well for our inner viruses because most of them are unknown. The sequences in the databases represent the tip of the iceberg. According to Dutilh, around 75 percent of the DNA from any new stool sample—and as much as 99 percent—won’t match any of these known sequences.
So what’s in that other 75 percent?
Well, crAssphage for starters.
Dutilh’s team found it by using a different approach based on a simple idea: that fragments which repeatedly turn up in the same samples are more likely to be parts of the same genome. They used a technique called cross-assembly to identify one such group of co-occurring sequences, in stool samples from 12 people. They then assembled these sequences into a single genome.
The genome had several distinctive features which told the researchers that it belonged to a phage, albeit one that’s very different to any we currently know of. They called it crAssphage after the cross-assembly method that revealed its existence.
They used the same technique to work out what the virus infects: if there’s lots of crAssphage DNA in a sample, there should also be lots of DNA from its host. Based on this logic, the most likely hosts are a group of bacteria called Bacteroides.
The team checked this result with a second technique. They looked at CRISPR sequences—a kind of bacterial immune system that recognises DNA from infecting phages. The team scanned all known bacterial genomes for CRISPR sequences that matched crAssphage and found that the closest matches came from two groups of gut bacteria, one of which was Bacteroides.
Bacteroides are major players in our guts. They help us break down our food, control the development of our immune system, and protect us from disease-causing bacteria. Their numbers change depending on the food we eat, and they correlate with our risk of different diseases. If crAssphage infects these microbes, it could also be an important player in our daily dramas.
It’s too early to speculate what its role might be, says Dutihl. Still, we know that phages are generally important. By killing off the most abundant bacteria in the gut, they ensure that no single species can monopolise the space. And last year, Jeremy Barr, who was involved of this new study, showed that phages could even act as part of our own immune system.
Many scientists had assumed that viruses in the gut are caught up in fast-paced evolutionary battles with local bacteria. This leaves people with very different collections, and explains why most of the viral sequences that we find don’t match anything in the databases. But the existence of crAssphage challenges this concept: it was part of the pool of unknowns but it’s also incredibly common. “It definitely changes the idea we had about viruses being very individual-specific,” says Dutihl. The study of human gut bacteria followed a similar path: early studies highlighted the differences between us but important similarities started emerging as our techniques became more sophisticated.
There are probably many more common viruses waiting to be discovered. “The biggest contribution of this work is the method they used,” says David Pride from the University of California, San Diego. “It provides a blueprint for further viral discovery.”
“What are we missing when we are unable to classify a sequence? What do we do with all of the sequence reads that we can’t classify? These are tough questions that we’ve been thinking about for years,” says Kristine Wylie from Washington University in St Louis. “This paper demonstrates that the community is developing clever approaches that can be used to mine those data.”
Giant viruses have turned up in the strangest places. The first one–Mimivirus–was discovered infecting amoebas in an English water tower. Others have been found in other water towers, in the oceans, and in an Australian pond. Now, the French team behind many of these discoveries has thawed out the latest and biggest virus yet from 30,000-year-old Russian ice. It looks like a corked urn, it’s unlike any other virus thus far discovered, and it can still infect modern amoebas despite its stint in the freezer.
HIV is a virus that kills by crippling our defences against other infections. It sends our immune system into a creeping decline. Germs that were once easy to fight off now become debilitating and lethal threats. A simple cold can kill. Tumours start to grow.
This is AIDS. It was formally described in 1981 and now, over 30 years later, we’re finally starting to understand why it happens.
HIV can infect many different types of white blood cell, but chief among them are the CD4 T-cells. These are the bugle-players of the immune system—they mobilise other immune cells, which actively kill viruses and other invaders. HIV prevents these troops from entering the fray, because it slowly destroys the CD4 T-cells.
Only a minority fall to the virus directly. More than 95 percent don’t seem to be infected, but die anyway. This collateral damage is what leads to the symptoms of AIDS; it’s what makes HIV so lethal. If we want to know why this virus has killed 34 million people since its discovery, we need to know why these bystander CD4 cells die… and we don’t. “In many ways, the question of why these cells die after HIV infection has been neglected, and it’s at the heart of what the virus does—it kills CD4 cells,” says Gary Nabel, Chief Scientific Officer at Sanofi.
Warner Greene from the Gladstone Institute of Virology and Immunology has been trying to solve this mystery for years, and he thinks he has finally cracked it. In two papers, published simultaneously in Science and Nature, his team lays out why HIV kills so many bystander cells and, better still, a possible way of stopping it.
In 2010, Greene’s team, led by Gilad Doitsh, showed that HIV actually tries to infect the bystander CD4 cells, but fails. Ironically, it’s their botched attempt that kills the cell.
During an infection, HIV fuses with a CD4 cell, and releases its genetic material, in the form of RNA molecules. These are converted into DNA, and inserted into the cell’s genome. When the cell divides, it copies its own genes and duplicates the hitchhiking viral DNA too. But in the bystander CD4 cells, which are in a resting state, the process that coverts RNA into DNA repeatedly stalls. Rather than producing the complete HIV genome, it churns out small fragments of viral DNA, and the infection can’t continue.
That’s great, except the cell now has bits of viral DNA floating about. Three years back, the team suggested that some sensor inside the CD4 cells detects this DNA and triggers a self-destruction programme.
Now, Kathryn Monroe at the Gladstone Institutes has discovered the sensor. She used a piece of HIV DNA to fish for molecules in CD4 cells that might stick to it. She caught several bites, but the most enticing one was a protein called IFI16. When Monroe removed this protein from resting CD4 cells, they didn’t overreact to the DNA pieces left behind by the virus’s bungled attempts at infection. They didn’t die.
IFI16 evolved as an antiviral DNA sensor. It’s meant to launch a defensive programme that kills infected cells before they can contaminate their neighbours. But when it comes to HIV, this protective response just kills the host faster. IFI16 turns into a general who gets false intelligence, panics, and pushes the big, red button anyway. “CD4 cell death is more a suicide than a murder,” says Greene.
The cells don’t go out quietly either.
In many cases, cells commit suicide through a gentle process called apoptosis. They shrink and break up into neat parcels, which are tidied away by cleaner cells. They die with a whimper; they don’t leave a mess. Everyone assumed that bystander CD4 cells die in this way.
Instead, Doitsh, together with student Nicole Galloway, showed that they die through a more violent process called pyroptosis. They swell instead of shrinking. Their membranes rupture, and their innards leak out through the holes.
These escaping molecules include interleukin-1 beta (IL1β), which summons more CD4 cells to the site of infection. The result is a massive amount of inflammation, and a vicious cycle—emphasis on vicious. HIV tries to infect a few CD4 cells, which go through pyroptosis in response. Their leaked remains summon more CD4 cells, which also get abortively infected, and also go through explosive suicide. Their deaths summon yet more cells, and so on.
“We think this is the major driver that depletes the CD4 T-cells,” says Greene. “It’s at the heart of AIDS.”
“The two papers provide substantial insights into how HIV depletes CD4 T-cells,” says Dan Barouch from Harvard University. “We didn’t have a clear mechanism for how that happened before, and it’s a central aspect of HIV pathogenesis.”
Greene thinks that pyroptosis (or the lack of it) could explain why HIV usually causes AIDS in humans but its relatives, the SIVs, barely sickens the apes and monkeys that they infect. SIVs can kill CD4 cells directly, but they can’t trigger the same pyroptosis response in other primates. They kill a few cells but the majority survive, and the immune system stays strong. “That’s the evolutionary solution—not to control the virus but to control the host response,” says Greene. “I think if we had another million years, we’d evolve in the same way.”
Thankfully, his team is working to a tighter schedule. They’ve already found a molecule that can stop pyroptosis, at least in lab-grown cells.
The whole messy process depends on a protein called caspase-1. Without it, you don’t get any mature IL1β, and without that, you don’t trigger the vicious cycle of CD4 cell death. Caspase-1 plays many other roles in the body, and several pharmaceutical companies have tried to make drugs that block it, for the purposes of treating other diseases. One of these, VX-765, was developed to treat chronic epilepsy and autoimmune diseases.
Greene’s team showed that it completely prevents HIV from killing the bystander CD4 cells. No caspase-1 activity. No IL1β signals. No inflammation. No mass cell death.
No AIDS? That remains to be seen. These are only lab experiments, after all, and the drug still needs to be tested in actual HIV patients.
Encouragingly, it has already gone through early phase II clinical trials, which means that we know it’s safe and well-tolerated. “Maybe it could be repurposed for HIV infection,” says Greene. He imagines a joint attack: current antiretroviral treatments would target HIV itself, while caspase-1 blockers would stop the patient’s immune system from overreacting to the virus.
Greene is now in talks with the drug’s manufactuer—Vertex Pharmaceuticals—about launching a proper HIV trial. There are other options too—several other caspase-1 inhibitors have been developed, although they haven’t done enough in their respective diseases to justify taking them to market and seeking FDA approval. If Greene can’t get the go ahead for VX-765, he’ll just look somewhere else.
He also wants to see if caspase-1 blockers could have other benefits. Since they target the host rather than the virus, he thinks it’s less likely that you’d get resistance to them. They could also give people more time while they wait for antiretrovirals. “For every 10 people we put on antiretrovirals today, 16 more become infected,” says Greene. “There are 16 million people who should be on these drugs but aren’t, and are progressing to AIDS and dying. Maybe these caspase-1 inhibitors could be used as a bridge therapy while they wait.”
And, in the lab experiments, the caspase-1 blockers also prevented the inflammation that goes hand-in-hand with CD4 cell death. Greene suspects that this inflammation accelerates the ageing process in HIV patients. “It’s why they’re dying of heart attacks, liver diseases, dementia and cancer at an earlier age than anticipated,” he says. “Maybe we could restore their normal lifespan or improve their quality of life?”
Meanwhile, other scientists have discovered more cellular sensors that detect HIV in other types of cells. Nabel’s team showed that a protein called DNPK-1 senses HIV DNA once it has been inserted into a CD4 cell’s genome, which triggers a different self-destruct sequence. But this only happens in the small proportion of CD4 cells where the infection process is truly underway.
Another protein called cGAS can also detect HIV DNA, but in a different group of white blood cells. It’s not found in the CD4 cells that Greene examined.
This baffling variety comes as no surprise to Andrew Bowie from Trinity College Dublin, who studies how the immune system detects viruses. He was the one who discovered that IFI16 is a DNA sensor back in 2010. “Since then, we suspected that these sensors would have very cell-type specific roles in sensing viruses,” he says.
And scientists have made tremendous strides in understanding these roles just this year. The cGAS discovery was announced in February, DNAPK-1 in June, and now IFI16 in December! “We’re seeing a Renaissance of our understanding of the fundamentals of HIV infection,” says Nabel. “The more we know, the better off we’ll be with controlling it.”
References: Monroe, Yang, Johnson, Geng, Ditosh, Krogan & Greene. 2013. IFI16 DNA Sensor Is Required for Death of Lymphoid CD4 T Cells Abortively Infected with HIV. Science. Tbc.
Doitsh, Galloway, Geng, Monroe, Zepeda, Yang, Hunt, Hatano, Sowinski & Greene. 2013. Pyroptosis drives depletion of CD4 T cells in HIV-infected lymphoid tissues. Nature. Tbc.
It’s been more than ten years since the first cases of SARS (severe acute respiratory syndrome) were identified in southern China. It spread to four continents and infected at least 8,200 people before heroic efforts finally brought its progress to a halt. Since then, scientists have sequenced the genome of the virus behind the disease—a coronoavirus called SARS-CoV—and teased apart its infectious tricks. But one lingering question remained: Where did it come from?
At first, the answer seemed straightforward. When investigators swept Chinese animal markets, they found SARS-CoV in palm civets. The animals were quickly culled in their thousands, but later studies showed that their wild or farmed relatives don’t actually carry the virus. They may have acted as a stepping stone for SARS-CoV, but they weren’t its natural reservoir.
Then in 2005, two teams of scientists found that Chinese horseshoe bats harbour a wide range of SARS-like coronaviruses, and SARS-CoV itself belongs to f this diverse family. It seemed that bats, not civets, were the source of SARS—a conclusion that was supported by several later studies.
“But there were doubters,” says Peter Daszak from EcoHealth Alliance, who was involved in one of these studies. “We were among them.”
There were a few niggling problems. The genes of the new coronaviruses had been sequenced but no one had managed to grow one in a laboratory and take a look at it. None of them looked like they could attack the same target that SARS-CoV does—a human protein called ACE2. And although they were all similar to SARS-CoV, none was a good enough match to actually be a direct ancestor. “It would have taken around 100 mutations to turn those into something that could directly infect people,” says Daszak. “That’s not something you’d expect from a bat in a wildlife market.”
The team have now quelled these doubts. They’ve been spending the last few years travelling to 20 countries and searching for new viruses among groups of mammals that are notorious as virus-carriers. Bats are very much on the list, and Chinese bats in particular.
Led by Xing-Yi Ge, Jia-Lu Li and Xing-Lou Yang, the team spent a year in the southern city of Kunming, repeatedly taking faecal samples and anal swabs from a colony of horseshoe bats. They identified seven strains of coronaviruses, including two new ones that were 95 percent identical to SARS-CoV. That’s a far closer match than any virus thus far discovered.
The team even manage to isolate one of the new viruses and grow it in the lab. It could infect human lung cells, as well as cells from pigs and horseshoe bats. And it used the ACE2 protein as a gateway for breaking into its hosts. It’s the most SARS-like virus ever found.
This is the strongest evidence yet that SARS-CoV originated in bats. “Maybe a bat with this virus or something similar got into a market, along with dozens of mammals it had never got into contact with before. Civets, ferret-badgers, rabbits, you name it,” says Daszak. Those other species could have acted as stepping stones on the virus’s path from bats to humans, but since the new strains can stick to ACE2 and infect humans cells, they may have been able to jump directly. “This tells me that we didn’t need civets, or even markets, for SARS to emerge,” says Daszak.
Ksiazek thinks that’s unlikely. “If you look at bat-borne diseases, instances of direct transmission to humans aren’t common, with exceptions like Ebola and Marburg,” he says. Likewise, Christian Drosten from the University Hospital at Bonn still believes that civets were an intermediate host. “That doesn’t make the paper less valuable,” he says. “It means that not every virus sitting in bats is dangerous, but there are some which probably are.
Coronaviruses were mostly neglected until SARS came along, but they seem to be an increasing problem. There’s SARS-CoV itself. There’s the new virus that’s responsible the emerging threat of Middle East Respiratory Syndrome (MERS). And there are goodness knows how many undiscovered strains, which could infect humans. “They’re very evolvable,” says Daszak. “They don’t correct errors in their genome when they make copies of themselves, so you get a lot of mutations, which allows them to lock onto new receptors in new hosts. They’re very good at jumping hosts.”
The team are now checking people who work in Chinese wildlife markets and farms to see if they’ve already been infected by the new SARS-like viruses. And they’re starting pragmatic programmes to minimise the risk of future spillovers. No matter your feelings on China’s wildlife trade, it’s a cultural phenomenon that’s not going away any time soon. So rather than calling for bans, the team are trying to educate farmers about the species most likely to harbour deadly viruses, and steer them towards safe choices. “We know a guy who farms bamboo rats, while other farmers bring bats in from the wild,” says Daszak. “We’d suggest that bamboo rats are a better alternative to bats right now.”
They’re also pushing for bigger projects to identify undiscovered viruses. “It’s pretty sad that it’s taken so long to find these [new corona]viruses,” says Daszak. “We as a species are pretty pathetic about getting ready for pandemics. We sit here and wait for them to emerge, and then we say, Wow, where did that come from?”
Sick of being caught on the backfoot, Daszak and his colleagues are spearheading a new approach to identify every single mammalian virus before they have a chance to spread to humans. They reckon that there are around 320,000 of them, and that it would take USD $1.4 billion to find them all. That’s a trivial cost compared to the price of a pandemic—SARS alone is estimated to have cost USD 16 billion.
This is one the most extraordinary and convoluted evolutionary tales that I have ever heard. It’s the origin story of a group of viruses called REVs. It’s the tale of how naturalists and scientists inadvertently created a bird virus out of a mammalian one through zoo-collecting and medical research.
To understand it, we need to go back to 1957, when the very first REV was isolated from a turkey in America.
Or we should travel back to the years after World War II, when REVs spread around the world in vaccines that were meant to stop poultry diseases rather than cause them.
Better still, we should go back to the 1930s, when scientists analysed the blood of a beautiful Asian pheasant (captured from New Guinea and living in what’s now the Bronx Zoo) and found a new species of malaria parasite (which was a huge boon for malaria research but is now lost to science).
Actually, forget all that. Let’s start with the mongoose.
The mongoose and the turkey
The ring-tailed mongoose, a native of Madagascar, looks like a cross between a ferret and a red panda. It has a sinuous, rusty body and a fuzzy tail with black and red stripes. A few years ago, virus hunters Anna Maria Niewiadomska and Robert Gifford were scanning the mongoose’s DNA when they noticed the complete genome of an ancient virus.
Many viruses—the retroviruses, in particular—have a habit of inserting their genes into the genomes of their hosts. These sequences sometimes get passed down the generations and turn into genetic “fossils”—remnants of ancient infections that are now permanent parts of their hosts. An incredible 8 percent of your genome consists of these “endogenous retroviruses” or ERVs.
Niewiadomska and Gifford specialise in studying these fossilised viruses, and they’ve found more than anyone ever expected. Finding another one in the ring-tailed mongoose wasn’t odd. But its closest relative turned out to be a bird virus known as reticuloendotheliosis virus (REV), which was first isolated from a turkey in 1957. REV and its relatives infect a wide range of poultry, including chickens, ducks and geese. It stunts their growth, weakens their immune system, and occasionally causes cancer.
Niewiadomska and Gifford screened the genomes of other animals for similar fossilised viruses, but they couldn’t find any in birds. The only hits came from the narrow-striped mongoose (another Madagascan mammal), and the short-beaked echidna—a spiny, egg-laying mammal from Australia.
Now, that was odd. Why is a widespread bird virus absent from the DNA of any birds, but present in the DNA of three mammals that are separated by the Indian Ocean? And how does it move from mammals to birds (or vice versa) anyway? Retroviruses shouldn’t be able to do that. They might occasionally jump between distantly related animals, but sustained transmission in the new host is incredibly rare, if it ever happens. What was going on?
To find out, Niewiadomska and Gifford reviewed every published report of a REV outbreak, and got their hands on as many archived samples as they could. They sequenced everything and used those sequences (combined with the ERVs from the mongooses and echidnas) to create a family tree of the REV dynasty.
The tree unequivocally showed that REVs were originally mammal viruses. They arose no earlier than 25 million years ago. Between 18 and 8 million years ago, one of them entered the genome of a Madagascan carnivore and stayed there. The others continued to infect mammals and at some point, one of them hopped over into birds. This must have happened very recently—decades ago, rather than centuries. We know this because retroviruses change very quickly, but the genetic diversity of bird REVs is incredibly narrow.
Fortunately, Niewiadomska and Gifford’s viral family tree also gives us clues about when, where and how this jump took place.
The pheasant and the parasite
In 1935, a French scientist called Emile Brumpt identified a new parasite called Plasmodium gallinaceum, which causes malaria in poultry. Lowell T. Coggeshall, an American tropical disease specialist, saw the potential in this discovery. If the parasite could be easily raised in domestic birds, it would provide a convenient way of studying malaria. The problem was that he couldn’t get any—Brumpt’s parasite came from a Sri Lankan chicken and US laws frowned upon importing poultry diseases from foreign lands.
But the solution was already in America. Around 10 years earlier, an intrepid naturalist called Lee Saunders Crandall had travelled to New Guinea and, barring one unfortunate shipwreck, returned to the US with hundreds of captive animals. His collection lived in the New York Zoological Park, now Bronx Zoo. Coggeshall reasoned that one of these imported birds might be carrying a parasite similar to P.gallinaceum, and he was right. In June 1937, he found Plasmodium lophurae in the blood of a stunning Borneo firebacked pheasant.
That was the first and only time that P.lophurae has ever been isolated, but it was enough. Coggeshall and others kept it going by repeatedly injecting it into chicken, duck and turkey chicks. It became a mainstay of malaria research, especially in the post-war years when the search for anti-malarial drugs reached fever pitch. But eventually, scientists started using other malaria parasites instead, and P.lophurae left the limelight. The stocks were finally exhausted in the 1980s and no one has ever managed to find it again. It’s probably still out there, infecting wild birds in south-east Asia, but for now, this once-fashionable parasite is lost to science.
But during its heyday, P.lophurae acted as a stepping stone for REV, on its crossing from mammals into birds.
As early as 1941, scientists suspected that P.lophurae stocks had been contaminated by… something. Infected animals were becoming anaemic, independently of their malarial symptoms. In 1959, William Trager identified the cause as a virus that was hiding in the stocks, which he called spleen necrosis virus (SNV). In 1972, a second contaminating virus was discovered—duck infectious anaemia virus (DIAV), found in P.lophurae from five different laboratories. Both of these were REVs.
At the time, everyone thought that SNV and DIAV were natural duck viruses that had somehow got into the parasite stocks. Niewiadomska and Gifford think otherwise. Their analysis says that SNV and DIAV came from the stocks themselves, which provided repeated opportunities for these mammalian viruses to sneak into birds.
Which mammal did they come from? No one knows. Laboratory mammals like mice or rabbits don’t harbour live REVs. Instead, Gifford suspects that “maybe one of the small south-east-Asian mammals that was housed in the Bronx Zoo was the source of the avian isolates.” Since REVs have never been found in a wild mammal, it could have been any of the zoo’s residents, although the smart money’s on bats. “The paper highlights the risks we create by bringing animals from disparate geographical origins into close proximity,” says veterinary virologist Glenn Browning.
Maybe this mystery mammal infected the firebacked pheasant that Coggeshall examined, so that the P.lophurae stocks were contaminated right from the start. Alternatively, it had plenty of opportunities to sneak in later, when the parasite was being bred and passaged from chick to chick. Either way, every time scientists injected P.lophurae into a bird, they gave the mammalian REVs a new opportunity to switch hosts.
The pox and the vaccine
REV genes turn up in the strangest places. They’ve also been found in the DNA of two completely unrelated bird viruses—fowlpox and gallid herpesvirus-2 (GHV-2). The latter causes Marek’s disease, which was a huge problem for the intensive poultry farms of post-war America and prompted an intense search for a vaccine. Progress was slow until the 1960s, when scientists developed ways of growing bird cells in laboratory cultures. Soon, we had created vaccines against both fowlpox and Marek’s, both using weakened versions of the respective viruses. They have saved countless numbers of domestic birds.
At the same time, malaria research was ramping up, and P.lophurae samples were whizzing around the country. Niewiadomska and Gifford think that these parallel streams of research allowed REVs to hop from the contaminated parasite stocks into the two bird viruses. “It’s likely that there were people working with both organisms,” says Gifford. “It’s circumstantial, but there was at least one company laboratory in the US that produced a commercial vaccine against fowlpox and worked with lophurae.”
When they got the chance, the REVs did what retroviruses like to do—they inserted their genes into a foreign genome. Only, in this case, those genomes belonged to the two other viruses—the weakened ones that were used to create the fowlpox and Marek’s vaccines. Aboard these vaccines, REVs hitchhiked around the world—a virus stowing away inside another virus, stowing away inside poultry, like the world’s worst turducken.
Occasionally, as earlier work has shown, they pop out to create REV outbreaks among birds that are vaccinated against fowlpox or Marek’s. This explains why all modern REVs are so genetically similar. They’re not circulating freely among the world’s birds, evolving as they go. They represent repeated incursions from the same stable staging ground.
There’s one possible exception. A REV that was recently isolated from China looks a bit different to the others, and might actually be an independently circulating virus. If that’s the case, it would be the icing on this extraordinary tale—of a mammalian virus that became confined with a malaria parasite, insinuated itself into other viruses, hitchhiked round the world in vaccines, and finally resumed its free-living existence, but this time as a bird virus.
“What an amazing paper!” says Vincent Racaniello from Columbia University. “Not only is the work an incredible detective story but it’s another example of how we can be so blind to exactly what viruses can do, even when we are as careful as possible. What we don’t know will always come back to haunt us.”
There are other similar stories of viruses arising or spreading through unexpected and ironic means. Last year, I wrote about a chicken virus called ILTV, which arose when two vaccines made from weakened viruses merged to create a new live one.
That’s unlikely to ever happen in humans (read the post for why) but our history isn’t short of accidents either. While trying to treat Egyptian people for schistosomiasis, a disease caused by parasitic worm, well-meaning healthcare workers accidentally injected them with needles contaminated by hepatitis C, kickstarting an epidemic that continues to this day. HIV has spread through Africa through similar means.
“I wonder how many other viral pathogens have we inadvertently spread?” asks Racaniello. “Fortunately, as this story shows, we now have the tools to determine what is in every virus stock that we produce, so such inadvertent infections should be a thing of the past.”
Gifford agrees that the story he discovered is unlikely to play out again but he points out that none of the scientists at the time could have anticipated what happened. They didn’t really know how retroviruses worked, or their propensity for smuggling themselves into their hosts, or even other viruses. “There could be things that we aren’t anticipating now that could be a threat,” Gifford says.
But let’s not throw the baby out with the bathwater. There are unquestionable benefits to doing basic research with pathogens like P.lophurae, or developing vaccines using live weakened viruses. The risks may be hard to assess, but there are ways of mitigating them.
Better surveillance—virus-hunting, in other words—tops Gifford’s list. “You’d have a few sentinel sites in key locations around the world that would routinely sequence viruses in their local environment and share their data.” This exists for some prominent infections like HIV and influenza, but Gifford thinks it’s time to expand such efforts to other viruses, including those that affect other animals and plants. “You wouldn’t need a lot of data to realise that something really strange like this is going on.”
It takes just two shots of the MMR vaccine to protect a child against measles, mumps and rubella for life. The same is true for polio and hepatitis B, a few injections grant life-long immunity against these viral diseases. By showing samples of the viruses to our immune system, we teach it to store a permanent memory of these enemies and guard against them in perpetuity.
Influenza is a different matter. There is a vaccine, but we have to take it every year. That’s because flu viruses evolve at tremendous speed. They copy themselves with surprising sloppiness, producing thousands of slightly different daughter viruses. If different strains infect the same cell, they can carry out the viral version of sex by mingling their genetic material to make hybrid daughters. And occasionally, entirely new strains that we’ve never encountered before can spill over into humans from animals.
In order to prepare the immune system in advance for this constantly changing enemy, scientists have to predict the strains that are going to pose the most problems in the coming season. Dead or weakened versions of these viruses are then incorporated into a vaccine, which prepares the immune system for the year ahead. As the viruses evolve, the vaccine must be re-made and the immune system re-educated, at the cost of 2 to 4 billion US dollars every year. This strategy does save lives, but it’s not foolproof. Its effectiveness is much lower in people over the age of 65, and predictions can be wrong, leading to seasons where the vaccine underperforms. Annually, flu kills between 250,000 and 500,000 people around the world, and pandemics have the potential to kill many more. “We can do better,” says Sarah Gilbert from the University of Oxford, UK. “The vaccines we use for flu are really using decades-old technology. There’s nothing else we vaccinate against every year.”
You could argue that we’d be in better straits if it wasn’t for the emergence of another virus that is adept at outmanoeuvring the immune system – HIV. Once HIV was discovered in the early 1980s, “the immunologists who’d done really interesting work on flu in the 1970s started working on that instead,” says Gilbert. “Nothing really very innovative was done with flu for a while.” The presence of the annual vaccine eased some of the pressure and, after all, there hadn’t been a flu pandemic since 1978.
But the last two decades have shaken us out from under this blanket of false security. In 1997, an outbreak in China heralded the spread of H5N1 bird flu into humans. To our knowledge, the virus has so far infected 622 people and killed 60% of them. In 2009, a strain of H1N1 leapt from pigs into humans, triggering the first pandemic for 30 years. And just this year, H7N9 has emerged out of nowhere to infect more than 130 people in China. “We can’t really predict what’s going to come,” says Ian Wilson from the Scripps Research Institute in La Jolla, USA. “But if we had a universal flu vaccine, we could counter all of [those strains] and not worry about a pandemic.”
When flu viruses enter our bodies, they recognise and enter cells using a protein that studs the surface like pins in a cushion called haemagglutinin (HA). When our immune cells see these intruders, they start producing antibodies that recognise HA and stick to it, disabling it and preventing infections. This is why flu vaccines use real viruses – so the immune system can make the right antibodies ahead of time. The problem is that there are 17 different types of HA (an H5N1 virus, for example, carries the 5th subtype) and many different strains within each type. An antibody that neutralises one may not work against another.
There are exceptions though. Some parts of HA are almost always the same, no matter the strain or subtype. They are hard to mutate without compromising the entire protein. But since these conserved regions are found on inaccessible parts of the protein that the immune system cannot reach, it’s hard to make antibodies against them. Hard, but not impossible.
In 1993, Japanese scientists isolated an antibody from a mouse that had been immunised against H2N2 flu. It protected against viruses from the H2 subtype, but also those from H1, H5, H6 and H9. Fifteen years later, other teams found similar “broadly neutralising antibodies” in human patients. The most exciting of these was discovered in 2011 by a group led by immunologist Antonio Lanzavecchia at the Institute for Research in Biomedicine in Bellinzona, Switzerland. Known simply as F16, it’s a super-antibody that binds to all 16 types of HA that were known at the time (the 17th was only found last year).
But F16 isn’t a vaccine in itself. You cannot mass-produce it and inject it into healthy people – their immune systems must learn to make the antibody for themselves. “The good news is that we know where to target and we know human antibodies have done it,” says Wilson. The next step is to design molecules that mimic the part of HA that F16 recognises, to stimulate the immune system into making similar antibodies. “You want it to look like what it does on the virus, but much more exposed,” says Wilson. “You want to focus the immune response on that particular area.”
Another approach might be to display natural HA molecules in a more accessible way. Gary Nabel, formerly at the US National Institute of Allergy and Infectious Diseases, did it by fusing HA molecules to ferritin, a protein that naturally assembles into spheres. Twenty-four of these fused proteins will spontaneously merge into a ferritin ball with eight HA spikes protruding from it. These spikes are far more accessible than they would be on a real flu virus, where some 450 HA molecules are crowded together alongside other proteins. For this reason, the ferritin particles raise 10 to 40 times more antibodies against flu than licensed vaccines, including ones that recognise the HA stem and protect against many strains.
Meanwhile, other scientists are trying to create universal flu vaccines by tapping into a different branch of the immune system. During a bout of flu, our bodies produce swarms of defenders called killer T-cells, which destroy infected cells. These swarms can counter a broad range of flu viruses but they’re short-lived. Once their job is done, they die off. “To a certain extent, we produce universal protection, but it doesn’t last long,” says Gilbert.
But our immune system doesn’t suffer from total amnesia. A type of immune cell called central memory T-cells lurks within our lymph nodes, carrying a record of infections past. They can generate fresh squads of killer T-cells if we get infected by an old virus. Unfortunately, this response is slow and flu is fast. Infections can come and go before T-cell reinforcements are summoned. “The central memory T-cells are very good for mopping up and helping with recovery but they don’t prevent illness,” says Gilbert.
Gilbert’s approach is to rouse the memory T-cells ahead of time, creating fresh T-cell patrols that can guard against incoming viruses. Her vaccine uses two of a flu virus’ most common proteins—NP and MP1. These are smuggled into a cell by a different virus that cannot make copies of itself. The cell never dies, but it looks like it has been infected by a flu virus. In small, preliminary trials, Gilbert’s team showed that the vaccine is safe and boosts T-cell numbers by 10 times. Unlike licensed vaccines, it’s just as effective in older people as it is in the young. “That’s not surprising,” she says. The current vaccines force the immune system to mount a fresh response every year – an ability that falters with age. “We’re trying to boost memory that already exists.” Now, they have to check whether this protects against fresh flu infections, and how long this protection lasts for.
Even if this vaccine works, it won’t last for life. The T-cell army will have to be continually reinforced by stimulating the memory T-cells again and again. You cannot just give someone one or two shots as a child and expect them to be protected indefinitely. “You’d have to get it regularly, but not every year,” says Gilbert. “I think if we got to the point where you needed to vaccinate every 5 years, we’d be doing very well.”
Sculpture is all about deliberation. You painstakingly chip marble from a block, or slowly assemble Lego bricks into a shape, or carefully pile clay upon clay. But we’re now entering a world where people can sculpt with proteins, creating amazingly intricate nano-scale shapes just a few billionths of a metre across. And when it comes to these nano-sculptures, the deliberation lies in selecting your materials in the first place. Once that’s done, you throw them all together and watch them assemble themselves.
Masaru Kanekiyo and Gary Nabel used this approach to create a new breed of flu vaccine that (in animal tests) provides better and broader protection than the ones we currently have. I’ve written more about this over at Nature, so head over there for the details.
Over here, I’m going to lay out exactly how the vaccine is built because it’s just so damn cool.
Flu vaccines are caught in a seemingly endless struggle against an ever-changing enemy. Flu viruses mutate all the time, and every year brings a slightly different set of seasonal strains. Immunising against one strain won’t necessarily protect against the others, so flu vaccines must be re-made every year to protect against the (usually three) strains that are predicted to cause the most problems in the upcoming season.
The traditional vaccine uses actual flu viruses that have been killed or inactivated. The idea is to give the immune system a sneak preview of next year’s likely blockbuster strains, so it can prepare by raising an army of defensive antibodies. Kanekiyo’s vaccine trains the immune system in the same way, but does it much more effectively. And it uses faux-viruses.
It’s made of two proteins. The first—haemagglutinin (HA)—is used by flu viruses to recognise and break into our cells. It studs the outer coat of a flu virus like pins in a pincushion. Each pin is made of three HA molecules, which line up in parallel to form a three-sided cylinder.
The second protein—ferritin—is involved in shuttling and storing iron molecules. It’s completely irrelevant to flu infections; it’s there for its handy ability to assemble into a sphere. Each sphere looks a bit like a volleyball and is made from 24 ferritin molecules.
Here’s the beautiful bit. Kanekiyo fused the two proteins together at just the right point that when he mixed 24 of them together, they automatically assembled into a ferritin sphere surrounded by eight HA spikes. Once he got the components right, they could sculpt themselves.
It gets better. The ferritin sphere has eight small triangular gaps on its surface, which are exactly the same width as the HA spikes—28 nanometres, no more and no less. This means that the HA spikes don’t just attach themselves to the sphere in random places. Instead, they become evenly spaced out in very specific positions. Under the microscope, the finished nanoparticle looks like a jack.
Compared to a traditional vaccine (which, if you’ll remember, are actual viruses)., these nanoparticles raise anywhere from 10 to 40 times more antibodies against flu. And the antibodies work against a far more diverse range of strains.
It’s all in the presentation. A real flu virus has around 450 HA spikes crowding its surface, along with other proteins. The nanoparticles have just 8 HA spikes, which are regularly spaced and have nothing else getting in the way. It screams, “LEARN THIS!” to the immune system and then gives it a really good look.
Nanoparticles built using the HA from one particular strain seem to protect against many others. So, if they prove their worth in human test, the hope is that they won’t need to be updated so regularly, or could better prepare us against future pandemics. (Again, read more over at Nature.)
But for the moment, I’m just in awe of how neatly the numbers work. Twenty-four ferritin molecules make a sphere with eight 28-nanometre-wide gaps. Twenty-four HA molecules make eight spikes that are 28 nanometres wide, and fit onto that sphere in exactly the right places.
It’s a testament to the importance of structural biology—knowing the precise shapes of proteins. That knowledge was the key to designing this new vaccine.
Every May, a herd of viruses thunders through East Africa in one of the greatest migrations in the natural world.
For a few months, 1.3 million wildebeest head north through the Serengeti in search of food and water. There are so many of them that their lines can stretch from one horizon to the next. And almost every one of these animals is infected by a virus called acelaphine herpesvirus 1 (AlHV-1).
Wildebeest calves pick up the virus from their mothers within their first few months of life, and carry the infection until they die. The virus causes no symptoms, and doesn’t harm the wildebeest. But domestic cows aren’t so fortunate. If they contract the infection, they develop a disease called malignant catarrhal fever (MCF)—a cancer-like illness where white blood cells start dividing out of control. Once a cow shows symptoms, it’s almost always dead within a couple of weeks.
MCF is a huge threat to the cattle-farming Maasai people of east Africa—the annual wildebeest migration spells death for their livestock. It’s also a problem for any zoos that keep wildebeest, which might infect other hoofed residents. It doesn’t help that there is no treatment. The only way of controlling the disease is to corral animals to prevent them from straying near infected wildebeest.
But Benjamin Dewals from the University of Liege is working on a different solution. By studying how AlHV-1 infects its hosts, he has developed a neutered version that can’t cause MCF and might be able to immunise animals against the wild viruses. It’s a preliminary step towards a vaccine.
The AlHV-1 virus doesn’t cause MCF straight away; it persists in its host for a long time before they start to show symptoms. Dewals found that when calves start coming down with MCF, the virus is unusually passive. It only switches on around 10 percent of its genome, compared to the 45 percent that gets used when it’s actually copying itself and infecting new cells. While many viruses cause disease by infecting and killing new cells, it seems that AlHV-1 does not.
Instead, it persists within the white blood cells of its hosts as free-floating rings of genetic material called episomes. Dewals found that it’s the long-term presence of these rings that causes the cells of the immune system to divide uncontrollably, and triggers MCF. Several other herpes viruses, such as Epstein-Barr virus (EBV) and Kaposi sarcoma-associated herpes virus (KSHV) can cause cancer in humans in a similar way. They don’t kill the cells they infect, but encourage them to divide.
This persistent state, known as “latency”, depends on a viral protein called ORF73. When the host cell divides, ORF73 tethers the virus’ genetic material (which normally floats freely) to the DNA of the host. This ensures that the virus ends up in both daughters.
When Dewals deleted the ORF73 protein from synthetic versions of the virus, it could infect cells easily enough but it couldn’t become latent. It could invade, but it couldn’t stay. It couldn’t nudge white blood cells into wanton growth, and it didn’t trigger MCF. Get rid of one protein, and suddenly the virus can’t cause disease.
Best of all, when Dewals exposed rabbits to this neutered virus, they mounted a strong immune response and were later protected against the fully competent version of AlHV-1. Of five rabbits infected with the normal virus, four were dead within a month. But all of those infected by the synthetic virus survived.
It’s not quite a vaccine yet – the team will have to test their neutered virus on a larger number of animals, and on actual cows rather than rabbits. But it’s certainly a first step towards safeguarding domestic animals that are caught in the path of AlHV-1’s annual migration through east Africa.
Reference: Palmeira, Soerl, Van Campe, Boudry, Roels, Myster, Reschner, Coulie, Kerkhofs, Vanderplasschen & Dewals. 2013. An essential role for γ-herpesvirus latency-associated nuclear antigen homolog in an acute lymphoproliferative disease of cattle. PNAS http://dx.doi.org/10.1073/pnas.1216531110