A narrow escape—at least this time

Leigh MacMillan, Ph.D.
Published: April, 2004

Mark Denison, M.D., seems to be engulfed in a projected image of cells infected with a mouse coronavirus.
Photo illustration by Dean Dixon
For Julia, the 13-year-old daughter of Mark Denison, M.D., SARS was a passing fancy. In February 2003, before the world confronted a strange new disease—Severe Acute Respiratory Syndrome—she asked her Dad why he didn’t work on an important virus. After all, he’d been studying a coronavirus for nearly 20 years, and who had ever heard of coronaviruses?

Within a month, the world had heard: a new coronavirus was causing SARS. Denison, one of a handful of scientists who had studied the biology of this virus family, became a sought-after source of information for both public health officials and the media. Julia enjoyed that her Dad finally was working on something important, but advised him that he was getting “kind of full of himself,” he recalls.

By the fall, SARS was gone. An unprecedented global public health response had halted the virus’ spread, continuing surveillance efforts were in place to quickly spot and address new cases of SARS, and scientists were searching for effective treatments and pursuing vaccine strategies. For Denison, professor of Pediatrics and Microbiology & Immunology at Vanderbilt University Medical Center, the experience was an unsettling near miss.

SARS, he says, represented “potentially the worst pandemic virus in the last 100 years. When you look at the overall severity and mortality rate of SARS and the rapidity of its spread, I’d say the bomb had started going off.”

A well-organized international public health response defused the crisis. In July—just four months after the World Health Organization first issued global alerts about SARS—WHO announced that the last chain of human transmission of SARS had been broken, ending the epidemic. The final tally: 8,422 cases of SARS and 916 deaths.

Even though its spread was stopped, SARS is still around. Swift isolation of this year’s few victims and their contacts has quelled the virus—so far, there has been no transmission to contacts and the SARS patients have all recovered. But increased vigilance must continue, Denison says. SARS was a primer for the future’s lurking viral threats: from it, we learned that global public health and rapid intervention mechanisms must be in place.

We were lucky with SARS, Denison says. Despite its high pandemic potential, the SARS coronavirus also had an Achilles heel that made it succumb to the infection control barriers erected against it. The next time around, we might not be so lucky.

Crown of spikes

In images from the electron microscope, coronaviruses look like the suns of preschool drawings—large circles surrounded by crowns of smaller dots. The crown, formed by the “spike” protein on the viral surface, gives the family its name.

Inside these spike-covered spheres is the coronavirus genome, one long chain of nucleic acids. While most organisms have DNA as a genetic material, coronaviruses—and other viruses including HIV and influenza—use RNA, a fact that makes them prone to mutation.

The coronavirus genome, the largest-known RNA molecule, is translated in infected cells into a replicase polyprotein, which is snipped into smaller proteins that together mediate all of the different steps of making new viruses.

The replicase polyprotein captured Denison’s interest nearly 20 years ago. He and Stanley Perlman, M.D., Ph.D., professor of Pediatrics and Microbiology at the University of Iowa, were the first to identify coronavirus proteins that were required for the virus to reproduce. Using tools such as monoclonal antibodies, antibodies made in the laboratory to recognize specific targets, Denison has continued to work toward a complete understanding of what the proteins are, how they’re lined up within the larger polyprotein, and how they’re cut apart.

“Sometimes it’s been like working on a jigsaw puzzle where all the pieces are square, and they’re all black, and I’m in a closet with the lights turned off,” Denison muses. Adding to the difficulty, he says, has been the need over the course of his career to explain why he would choose to work on mouse hepatitis virus, one member of the coronavirus family. He recalls hallway conversations with colleagues that went something like: “You’re a smart guy, Mark, why don’t you study a different virus?”

It turned out that those studies were critical.

“When the SARS epidemic hit, Mark was among the first to realize that of the coronaviruses, SARS was most like mouse hepatitis virus,” says Barton Haynes, M.D., director of the Duke University Human Vaccine Institute, and leader of the Southeast Regional Center of Excellence for Emerging Infections and Biodefense. The consortium of six universities, including Vanderbilt, is charged with developing the next generation of vaccines, drugs and diagnostic tests against emerging infections such as SARS, and for defense against organisms such as smallpox that might be used in bioterrorist attacks.

Denison “had already made many monoclonal antibodies against (mouse hepatitis virus) replicase,” Haynes says. “Remarkably (they) reacted with SARS, and Mark had the first SARS monoclonal antibodies in the world. He continues to make major contributions to our understanding of SARS pathogenesis, and is already regarded as a world leader in the field of both coronaviruses and SARS in particular.”

Confocal immunofluorescence image of cultured mouse brain tumor cells that have been infected with the mouse hepatitis virus, a coronavirus. The culture was mixed with a fluorescence-labeled antibody that specifically attaches to a viral enzyme. Confocal microscopy visualizes infected cells and virus-induced syncytia, or cell fusion, which enables the virus to slip from one cell to another.
Sarah Brockway
Besides the inherently interesting biological features of the coronaviruses, Denison says, they are important and widespread pathogens. Coronaviruses have long been known to cause severe disease in animals, particularly pigs, calves, and chickens. Two human coronaviruses are responsible for between 20 and 30 percent of cases of the common cold.

“They were important human pathogens; they just weren’t severe or critical human pathogens,” Denison says of coronaviruses before SARS.

But coronavirologists like Denison recognized the capacity of these viruses for trans-species adaptation. Over the last decade, he says, accumulating evidence has shown that coronaviruses can move between species “without too much fuss.”

So when an ordinary coronavirus took a leap to human beings—most likely from a still unidentified animal source—and caused SARS, “I think coronavirologists were amazed, but not surprised,” Denison says.

The leap appears to have happened in the southern Chinese province of Guangdong, where SARS-like illnesses occurred before the epidemic was acknowledged. Retrospective studies of patient records by Chinese and WHO epidemiologists have identified independent clusters of cases in seven Guangdong municipalities between November 2002 and January 2003.

The absence of a link between these clusters adds weight to theories that the virus jumped to human beings from an animal species or other environmental reservoir in southern China, according to WHO.

The civet connection

In Guangdong China, wild animal markets and restaurants cater to the population’s penchant for exotic fare—a made-to-order situation for putting people in contact with unusual animal viruses. Suspect animals in the SARS jump include the masked palm civet, a relative of the mongoose, and the raccoon dog, which are both consumed as delicacies in southern China and have been confirmed to be infected with the SARS coronavirus. In fact, the genetic sequence of the virus isolated from captive civets was nearly identical to that from the first confirmed SARS patient this year, prompting Chinese officials to order the killing of all civets—estimated at 10,000 animals—in the region to protect against further SARS cases.

The masked palm civet is a small mammal related to the mongoose that is sold in the animal markets of southern China and consumed as a delicacy. It has been implicated as a source of the coronavirus that has been transmitted to humans.
Norman Owen Tomalin/Bruce Coleman Inc.
Civets or raccoon dogs may have served as the conduit for transmission of the SARS coronavirus to human beings, Denison says, but the evidence is only circumstantial. “They may have just been bystanders—they happened to be in cage number three from the top, and whatever was on top was dropping virus all over everything below it.”

Even if the civet is confirmed to be the culprit in transmitting the SARS coronavirus to human beings, eliminating contact with the animal will only go so far to prevent future outbreaks. The potential will still exist for animal coronaviruses—and other classes of viruses as well—to jump from animals to human beings.

RNA viruses, with their propensity for mutation, are especially likely to cause emerging infectious diseases, Denison says. The polymerase enzyme that copies the coronavirus genome, for example, has a high error rate—it makes lots of mistakes, resulting in virus particles with mutations. Some of these random changes may result in dead viruses, others may have no effect, and still others may make the virus better at infecting another species.

“Viruses don’t ‘respond’ to things like antiviral drugs, antibodies, temperature … they’re making changes all the time,” Denison explains. “Viruses are incredibly adaptive.

“I like to picture a kind of ‘king of the hill’ model,” he says. “You’ve got the dominant viral population at the top, and all the time these other viruses are being produced. It’s like a coup d’etat-in-waiting -- if circumstances change, there’s another virus group there ready to depose the king.”

A study published this winter in the journal Science demonstrates just how adaptive the SARS coronavirus proved to be. A consortium of Chinese scientists tracked the virus’ evolution—the changes in its genetic code—during last year’s epidemic by analyzing the viral genome in tissue samples from patients infected during the early, middle and late phases of the epidemic.

They found multiple SARS coronavirus strains present during the early phase, with wide variation in the outer spike protein used for viral attachment to host cells. As the epidemic progressed, the spike protein sequence stabilized, presumably to the form with the greatest capacity for infecting human cells, according to the team led by Dr. Guoping Zhao of the Chinese National Human Genome Center in Shanghai.

The remarkable speed with which the SARS coronavirus adapted to human hosts underscores the importance of having robust public health systems in place that can recognize and defeat emerging viral threats before they sharpen their human attack skills.

Since the end of last year’s epidemic, there have been five confirmed SARS cases—two researchers handling laboratory samples and three members of the public in China’s Guangdong province. Quarantine measures have apparently been successful -- none of the cases became the focus of a new epidemic, and all of the victims recovered.

Much to learn

“We really don’t understand why SARS came up in the first place, or where it’s gone,” Denison says. “There is certainly still a risk that it will reemerge as a severe pandemic disease, and based on that, there’s a need to understand the virus and its emergence, biology, pathogenesis, treatment and prevention.”

SARS has been called a respiratory illness -- patients have usually presented with flu-like symptoms of fever, chills, aches, and coughing or breathing difficulty. Some developed hypoxia, with 10 to 20 percent of cases requiring mechanical ventilation. Most developed pneumonia. It appeared to spread by close person-to-person contact, probably involving respiratory droplets. But other features—a high incidence of diarrhea, the prolonged (seven- to 10-day) incubation period, and the mild disease in children—suggest to Denison that SARS might be a systemic disease, like measles, with a severe respiratory manifestation.

“We don’t fully understand the pathology of this disease,” he says.

So the world watches and waits. Surveillance programs, especially in regions that were hardest hit by SARS, aim to swiftly detect and isolate suspected SARS cases. In Hong Kong, for example, where SARS sickened 1,755 people and killed 299, every passenger entering or leaving the city—by any route—has been required since last summer to fill out health forms and pass in front of infrared cameras that measure the temperature of skin and clothing. Anyone with a fever must see a doctor.

But protracted surveillance of this level is an arduous prospect, and it may not catch the single case that starts a new epidemic.

“Surveillance is a difficult thing; formal surveillance programs are often not located in the right place at the right time,” says Larry Anderson, M.D., director of the Division of Viral Diseases at the U.S. Centers for Disease Control and Prevention. Instead, for SARS and other emerging infectious diseases, the CDC and WHO rely on what’s called the “astute clinician concept”—the idea that practicing physicians notice something odd, talk about it, pursue it and bring the information to the public health community.

“We learned from SARS that global interaction and the rapid exchange of information are very important for containing and controlling emerging diseases.”
Larry Anderson, M.D., chief of the Respiratory and Enteric Virus Branch at the U.S. Centers for Disease Control and Prevention.
James Gathany/CDC
“In light of that, what we’ve tried to do with SARS is provide opportunities to inform clinicians about when they should be concerned,” says Anderson, who has been overseeing the CDC’s SARS task force. The task force is responsible for coordinating the CDC’s SARS activities, which include developing guidance, training and education documents, working on SARS diagnostic tools, interacting with the international public health community, and providing experts on site to assist in investigations.

“We learned from SARS that global interaction and the rapid exchange of information are very important for containing and controlling emerging diseases,” Anderson says.

Only time will tell whether or not SARS will continue to be a pandemic threat. Denison suggests that awareness should be maintained for at least five years, maybe even 10 or more. In the meantime, funding has flooded into coronavirus research and the search is on for earlier diagnostic tools, treatments and vaccines.

Why invest in treatment and vaccine development if the SARS threat is uncertain? “It’s about what we don’t know, not what we do know,” Denison says. “We don’t know if SARS will reemerge as a more severe disease. It’s too early to say.”

Vaccine candidates

Current attempts to develop a SARS vaccine are pursuing multiple vaccine types (see “A Vaccine Primer). These include a live, attenuated virus, an inactivated virus, purified viral proteins such as the spike protein, and recombinant virus vectors harboring one of the SARS proteins.

Denison and colleagues favor the live, attenuated virus approach based on the history of coronavirus vaccines in animals. Among multiple approaches that have been tried in different animal species, live, attenuated vaccines have been the most effective at generating a protective immune response in animal models. But because the virus is still capable of infecting cells, it can have undesired effects, among them reversion to a virulent strain or recombination with other viruses to make a new virus of unknown disease-causing capacity.

Denison argues that all of the various vaccine strategies must be pursued because we don’t know how studies of animal viruses will translate to human beings. “Other investigators and I agree that inactivated virus strategies are likely the safest and may work,” he says. “But they haven’t worked anywhere else (in animal studies), so it’s not wise to only pursue that approach—you’d put yourself way behind the curve.”

Using a genetic system they developed for modifying the mouse hepatitis virus, Denison and colleagues plan to introduce mutations into the SARS coronavirus genome and assess the effects of these mutations on the ability of the virus to infect cells, reproduce and cause disease. Their goal is to create viruses that grow well in culture but do not cause disease, and which could be candidates for a vaccine. They’ve had success with this approach using the mouse hepatitis virus as a model.

If inactivated viruses turn out to be a viable strategy for vaccinating humans against the SARS coronavirus, Denison says their technique for modifying the viral genome would likely still be useful for safely growing the large quantities of virus needed to produce the vaccine. By January 2004, China was already moving to human studies of an inactivated virus vaccine, a government official announced.

Denison smiles about the wealth of resources now being devoted to coronavirus research; it makes his scientific life easier, after all. But he hasn’t forgotten his days in the trenches studying a virus no one thinks about.

“What this outbreak taught us was not just about coronaviruses,” he says. “We need to understand the capacity of all kinds of viruses to move between species and the mechanisms by which they cause disease. We need to make sure that there are fundamental things that we know about all identified viruses—their genomic sequences, for example, and some basics about their biology.”

Now that influenza has claimed the headlines, Denison says his daughter Julia wonders why he’s not working on the flu virus instead. “I think she reflects the general attention span of the public for newly emerging viruses,” he says. “But I live in this world because I understand that if we’re successful—if we prevent disease through vaccination and other public health measures—people will say, ‘What was the big deal?’”

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