GLOBAL COLLABORATION ON SARS BEARS FRUIT
26 May 2003
Source: New York Times, May 26, 2003
RISE OF A VIRUS
Global Collaboration on SARS Bears Fruit
By DENISE GRADY and LAWRENCE K. ALTMAN
There are biohazard signs on the doors and airflow systems designed to keep viruses from wafting out of laboratories. In one laboratory, a researcher wears gloves, gown and mask. In another, the dress code is a full spacesuit with its own air supply. A visitor, allowed to peer in through glass panels, is warned, only half jokingly, never to shake hands with anyone here who is wearing latex gloves.
This is Building 15, home of the special pathogens branch of the federal Centers for Disease Control and Prevention in Atlanta.
The most feared diseases, caused by deadly, highly contagious viruses, are studied in this building. Hemorrhagic fevers like Ebola and Lassa qualify; malaria and hepatitis do not.
Early in the outbreak of severe acute respiratory syndrome, or SARS, when it was still being called a mystery disease, it became apparent that the microbe causing it was acting like what the C.D.C. terms a special pathogen: flashing through hospitals, swiftly cutting down scores of health workers, killing an alarming number of victims. Whatever this germ was, it belonged in Building 15.
If infectious disease experts have a worst nightmare, it is that a new, very contagious disease with a high death rate reaches an international airline hub — a place like Hong Kong, for instance — and hops to other countries before anyone even notices it. That is precisely what SARS has done.
The potential of SARS to seed itself rapidly around the world and touch off a chain reaction of deadly epidemics galvanized the World Health Organization to start an international collaboration to identify the cause of the disease and to try to develop ways to diagnose, contain, treat and prevent it.
The effort got results, and with extraordinary speed.
The investigation has ranged from universities and besieged hospitals to live-animal markets in southern China to high-tech genetics laboratories and the sprawling C.D.C. complex.
When SARS emerged, many laboratories and infectious disease experts, in the aftermath of the anthrax attacks of 2001, were already in a heightened state of alert and on the lookout for unusual diseases.
SARS first came to the world's attention in mid-March, and only a week later, scientists isolated the virus that appeared to be causing it. A few weeks after that, two teams decoded the viral genome, providing information that could help to develop diagnostic tests, vaccines and antiviral drugs and to find out where the virus came from. Last week, scientists pinpointed a possible source of SARS — civets, badgers and raccoon dogs being sold for meat in China's Guangdong Province — and were able to compare the gene sequence of the animal virus with the one found in people.
But the campaign had to begin with a simple, essential question: what exactly was causing SARS?
A Surprising Find
Before the world learned about SARS, specimens from infected patients were hard to come by. Some international shippers balked at handling them.
In a hospital in Vietnam, where autopsies are frowned upon, it took delicate negotiations for a pathologist to get permission to remove lung tissue from a patient who had died.
Finally, in the first half of March, specimens packed in dry ice began to arrive at the special pathogens branch from Hong Kong, Thailand and Vietnam — vials of blood, sputum, lung and throat washings, bits of tissue.
Researchers hoped that the specimens, dropped into cell cultures and injected into baby mice, would grow enough virus or bacteria to let them identify the cause of the new disease.
On March 18, researchers noticed that something was happening in a flask of cells that had been seeded with a sample from the throat of a patient. The flask contained a culture of monkey kidney cells, known as Vero cells, which are fertile ground for certain viruses. Clear zones had begun to form, meaning that something was killing the Vero cells.
"That's a sign something is growing," said Dr. Thomas G. Ksiazek, chief of the special pathogens branch. "Then you need to find out, well, what is this?"
The next step was to process the contents of that flask so that they could be looked at with an electron microscope. On Friday morning, March 21, an electron microscopist named Cynthia Goldsmith examined a type of sample, called a thin section, of infected cells that were magnified about 14,000 times.
Almost immediately, telltale features stood out: black, beadlike dots of genetic material inside spherical viruses, the distinctive way the viruses lined up along the cell surface and the region where they clustered inside the cells, a structure called the endoplasmic reticulum.
"My first reaction was coronavirus," Ms. Goldsmith said.
It took her by surprise, she said. Although coronaviruses made animals very sick, in people they were known to cause only colds and gut trouble, not serious diseases like pneumonia. They had not even been mentioned as a possible culprit in SARS. And most did not even grow in Vero cells.
Ms. Goldsmith spent the next 20 or 30 minutes examining more cells, thinking about whether she might be mistaken and looking at images of other coronaviruses for comparison.
At 12:11 p.m., she sent her supervisor, Dr. Sherif R. Zaki, an e-mail message saying that she could not believe it herself but she was looking at a coronavirus.
Ms. Goldsmith and Dr. Zaki met with other C.D.C. scientists that afternoon. The microscope image, all agreed, was just the beginning.
"We have a coronavirus," Dr. Ksiazek said. "So what?"
Finding the virus in a sample did not prove that it caused the disease. Other tests were needed to verify that it was indeed a coronavirus, determine which coronavirus it was, see whether it turned up in other patients with SARS and whether it could cause the same disease if given to test animals.
One way to get clues about the identity of an unknown virus is to expose it to antibodies, proteins made by animals to fight off specific viral infections. If the unknown virus matches a particular antibody, they will stick together, and dyes will detect them.
When pathologists at the disease centers learned that Ms. Goldsmith had found a coronavirus, they tested SARS virus samples against 35 to 40 different coronavirus antibodies.
Antibodies from pigs and people gave positive tests. But by far the strongest reaction was to a coronavirus antibody from cats.
"That doesn't mean SARS comes from a cat," said Dr. Wun-Ju Shieh, a member of the pathology team.
Indeed, last week, researchers in Hong Kong said they had found a virus nearly identical to the SARS virus in other animals, civets, badgers and raccoon dogs in the market stalls of China's Guangdong Province.
Confirming a Theory
At the University of California at San Francisco, Dr. Joseph DeRisi, an assistant professor of biochemistry and biophysics, could not wait to get his hands on virus samples from the disease centers.
"We literally begged the C.D.C.," Dr. DeRisi said. "We were salivating."
On Saturday, March 22, he got his samples, the day after Ms. Goldsmith had made the preliminary identification of a coronavirus.
Dr. DeRisi and his colleagues were eager to see if they, too, could identify the SARS virus with a tool they had created, a DNA chip or microarray. The chip is essentially a microscope slide spotted with gene fragments from 1,000 viruses. If a sample being tested has a stretch of genetic material that matches one on the slide, it will stick to that spot and light up when the slide is put into a scanning device.
The array of spots is then displayed on a computer screen, and sliding a cursor over any spot brings up the name of the virus whose DNA made the spot.
By Sunday morning, Dr. DeRisi and Dr. David Wang had found several lit-up spots on their microarray. Within a few hours, they felt sure that the samples they had been sent were a coronavirus. It had genetic similarities to three coronaviruses that infect birds, cows and people, but it was not identical to any of them. In fact, it was not any known coronavirus.
"We thought it was a completely new coronavirus, between bird and animal viruses," Dr. DeRisi said.
By mid-April, two laboratories had mapped the genome of the SARS virus. First to finish was the British Columbia Cancer Agency in Vancouver followed by the C.D.C. The findings confirmed what initial studies had suggested: the virus was different from any known coronavirus, different enough, in fact, to become the first member of a new grouping of coronaviruses.
A 'Sleepy' Specialty at the Fore
Before SARS, few researchers studied coronaviruses. Known for making chickens cough and giving pigs diarrhea, coronaviruses were not seen as a path to scientific glory. In people they were thought to cause only mild diseases, and many researchers found them unexciting, difficult to grow and generally not worth the bother. Before SARS, one scientist said, coronaviruses were a "sleepy little corner of virology."
Now, coronavirus experts are eagerly sought by the disease centers and the W.H.O. "Suddenly they're rock stars," said Dr. Donald Ganem, a virologist at the University of California at San Francisco.
The expert most often named by other researchers is Dr. Kathryn V. Holmes, a professor of microbiology at the University of Colorado Health Sciences Center in Denver. She has studied coronaviruses for more than 20 years, and is now turning her attention to the one believed to cause SARS.
"I'm doing it because people are sick and I want to help," Dr. Holmes said.
One focus of her research has been investigating how coronaviruses get into cells. All viruses must enter cells to survive because they cannot replicate on their own and need to take over the cell's equipment to make copies of themselves.
"That's a hijacking step," Dr. Holmes said, adding that one invaded cell can churn out a thousand or more viruses. "It's an incredible assembly line."
Eventually, the process kills the cell. The question of how viruses penetrate cells is central to understanding viral diseases. It can explain why a virus attacks only certain species and invades certain types of cells.
"Why is there a coronavirus of chickens, and one of dogs, and one of rats, and why don't they infect each other?" Dr. Holmes asked. "We don't understand how a virus chooses a host, and we understand precious little about how they cause disease."
It is known that an essential first step is for the virus to lock on to a molecule on the cell surface called a receptor. Cells have receptors of various types that link up with body chemicals like insulin and other hormones. Sometimes a virus happens to have a molecule that can grab a cell's receptors.
"The virus is the opportunist," Dr. Holmes said. "The host is not putting out a receptor to get infected. The receptor has a job of its own, and the cell needs it. But the virus can exploit it."
For coronaviruses, the burglar tools are the spikes that stud the surface of the virus, giving it a crownlike shape on certain types of electron micrographs. The spikes, made of proteins, are what grab onto cell receptors. Each type of coronavirus has a distinct spike protein. If the spike changes, because of a genetic mutation, the virus might, in theory, be able to invade a new host.
"Is it a changed spike, or just a terrible coincidence that somebody got infected with an animal virus, by coming in touch with something that normally nobody comes in touch with?" Dr. Holmes asked. "We know the people getting SARS now are catching it from people, not from being exposed to unusual animals. But where did it come from?"
Weeks before scientists working in China found the animals carrying a virus much like the one linked to SARS, Dr. Holmes had suggested that the disease was probably caused by an animal virus that acquired the ability to infect humans, perhaps because of a changed spike. If that is the case, and if the virus can still infect its original animal host, it may be impossible to eliminate, particularly if the host is a wild animal rather than a domesticated one that can be rounded up and slaughtered.
But until that is known, Dr. Holmes said, it makes sense to study the SARS spike protein and to try to find its receptor. If a receptor is found, Dr. Holmes said, it may be possible to develop a treatment to keep the virus from getting into cells.
Finding the origin of SARS, whether it is the civets and other animals or some other host, may also help researchers figure out how the virus evolved and how it found its way into people, Dr. Holmes said. That question extends far beyond SARS, to the larger problem of emerging infectious diseases, a category that includes scores of infections like West Nile encephalitis, hantavirus, Lyme disease and AIDS.
"What makes these things appear and disappear?" Dr. Holmes asked. "What makes the host-pathogen pair? What makes virulence? People are trying to understand emerging infectious diseases."