Nanhai Chen is a quiet man in his late thirties. He grew up on a collective near Shanghai called the Red Star Farm, where his father was a farmer and where some of his sisters still live. In high school, Chen decided he liked biology, and he went on to have a fast-track career at the Institute of Virology at the Chinese Academy of Preventive Medicine in Beijing, which is probably the top virology center in China. He became an expert in the DNA of vaccinia virus. Mark Buller hired him out of China.
Nanhai Chen has a fuzzy crew cut, hands that work rapidly, wire-rimmed spectacles, and restrained manners. He and his wife, Hongdong Bai, who is also a molecular biologist, have given their children American names, Kevin and Steven. He wears only two outfits, one for winter and one for summer. His winter outfit is a blue cotton sweater, blue slacks, and white running shoes. I spent days with Chen during the time he engineered the mouse supervirus. “It’s not difficult to make this virus,” he said to me one day. “You could learn how to do it.”
A VIRUS that has been engineered in the laboratory is called a recombinant virus. This is because its genetic material—DNA or RNA—has genes in it that come from other forms of life. These foreign genes have been inserted into the virus’s genetic material through the process of recombination. The term construct is also used to describe it, because the virus is constructed of parts and pieces of genetic code—it is a designer virus, with a particular purpose.
The DNA molecule is shaped like a twisted ladder, and the rungs of the ladder—the nucleotides—can hold vast amounts of information, the code of life. A gene is a short stretch of DNA, typically about a thousand letters long, that holds the recipe for a protein or a group of related proteins. The total assemblage of an organism’s genetic code—its full complement of DNA, comprising all its genes—is the organism’s genome. Poxviruses have long genomes, at least for viruses. A pox genome typically holds between 150,000 and 200,000 letters of code, in a spaghettilike knot of DNA that is jammed into the dumbbell structure at the center of the pox particle. The poxvirus’s genome contains about two hundred genes—that is, the pox particle has around two hundred different proteins. Some of them are locked together in the mulberry structure of the particle. Other proteins are released by the pox particle, and they confuse or undermine the immune system of the host, so that the virus can amplify itself more easily. Poxviruses specialize in releasing signaling proteins that derange control systems in the host. For example, insect poxes release signals that cause an infected caterpillar to stop developing and grow into a bag packed with virus.
The human genome, coiled up in the chromosomes of every typical cell in the human body, consists of about three billion letters of DNA, or perhaps forty thousand active genes. (No one is certain how many active genes human DNA has in it.) The letters in the human genome would fill around ten thousand copies of Moby-Dick: a person is more complicated than a pox.
The IL-4 gene holds the recipe for a common immune-system compound called interleukin-4, a cytokine that in the right amounts normally helps a person or a mouse fight off an infection by stimulating the production of antibodies. If the gene for IL-4 is added to a poxvirus, it will cause the virus to make IL-4. It starts signaling the immune system of the host, which becomes confused and starts making more antibodies. But, paradoxically, if too many antibodies are made, another type of immunity goes down—cellular immunity. Cellular immunity is provided by numerous kinds of white blood cells. When a person dies of AIDS, it is because a key part of his or her cellular immunity (the population of CD4 cells) has been destroyed by HIV infection. The engineered mousepox seems to create a kind of instant AIDS-like immune suppression in a mouse right at the moment when the mouse needs this type of immunity the most to fight off an exploding pox infection. An engineered smallpox that triggered an AIDS-like immune suppression in people would be no joke.
TO CREATE a construct virus, you start with a cookbook and some standard ingredients. The basic raw ingredient in Chen’s experiment was a vial of frozen natural wild-type mousepox virus, which sat in a freezer around the corner from his work area. The other basic ingredient was the mouse IL-4 gene. Chen’s cooking, so to speak, involved splicing the gene into the DNA of the poxvirus and then making sure the resulting construct virus worked as it was supposed to.
Chen ordered the IL-4 gene through the Internet. It cost sixty-five dollars, and it came by regular mail at Mark Buller’s lab in November 2001, from the American Type Culture Collection, a nonprofit institute in Manassas, Virginia, where strains of micro-organisms and common genes are kept in archives. The gene arrived in a small, brown glass bottle with a screw top. Inside the bottle was a pinch of tan-colored dry bacteria—E. coli, bacteria that live in the human gut. The bacterial cells contained small rings of extra DNA called plasmids, and the plasmids held the IL-4 gene. The IL-4 gene is a short piece of DNA, only about four hundred letters long, and it is one of the most common genes used in medical research. To date, more than sixteen thousand scientific papers have been written on the IL-4 gene.
The standard cookbook for virus engineering is a four-volume series in ring binders with bright red covers, entitled Current Protocols in Molecular Biology, published by John Wiley and Sons. Nanhai Chen took me to a shelf in the lab, pulled down volume three of Current Protocols, and opened it to section 4, protocol 16.15, which describes exactly how to put a gene into a poxvirus. If anyone puts the IL-4 gene into smallpox, they may well do it by the book. “This cannot be classified,” Chen said, running his finger over the recipe. “No one ever thought this could be used for making a weapon. The only difficult part of it is getting the smallpox. If somebody has smallpox, all the rest of the information for engineering it is public.”
“Are you personally worried about engineered smallpox?”
“Yes, I am,” he answered, holding the cookbook open as he spoke. “I was talking last week with my mentor in China. His name is Dr. Hou, and he’s a very famous virologist in China. He told me the Russians have a genetically modified and weaponized smallpox. My mentor didn’t say where he learned this, but I think he has good access to information, and I think it is probably true. Smallpox was all over the world thirty years ago. It could be anywhere today. It’s not hard to keep back a little bit of smallpox in a freezer.”
I will omit the subtleties of Chen’s work for the sake of general readers, but the outline of a recipe for making the biological equivalent of an atomic bomb is in these pages. I would hesitate to publish it, except that it’s already known to biologists; it just isn’t known to everyone else. It doesn’t take a rocket scientist to make a superpox. You do need training, though, and there is a subtle art to virus engineering. One becomes better at it with experience. Virus engineering takes skill with the hands, and in time you develop speed. Chen felt that with a little luck he could engineer any sort of typical construct poxvirus in about four weeks.
Chen took the little brown glass bottle of dry bacteria that contained the IL-4 gene and cultured the bacteria in vials. Then he added a detergent that broke up the bacteria, and he spun the material in a centrifuge. The cell debris fell to the bottom of the tubes, but the DNA plasmid rings remained suspended and floating in the liquid. He ran this liquid through a tiny filter. The filter trapped the DNA that held the IL-4 gene. He ended up with a few drops of clear liquid.
Next, Chen spliced some short bits of DNA, known as promoters and flanking sequences into the plasmid rings. He did this basically by adding drops of liquid. Promoters signal a gene to begin making protein. The various promoters were going to cause the strains of engineered mousepox to express the IL-4 protein in differing amounts and at different times in the life cycle of the virus as it replicated in cells.
The next step was to put the engineered DNA into the virus, using a genetic-engineering kit called a transfection kit. Transfection is the introduction of foreign DNA into living cells. A transfection kit is essentially a small bottle filled with a reagent, or biochemical mix; a bottle of it cost
s less than two hundred dollars. You can order transfection kits in the mail from a variety of companies. Nanhai Chen used the Lipofectamine 2000 kit from Invitrogen.
Chen grew monkey cells in a well plate, and then he infected them with natural mousepox virus. He waited an hour, giving the virus time to attach to the cells. Then he added the IL-4 DNA, which he’d already mixed with the transfection reagent. He waited six hours. During that time, the IL-4 DNA was taken up into the monkey cells, which were also infected with natural mousepox. Somehow, the IL-4 DNA went into some of the mousepox particles, and the IL-4 gene ended up sitting in the DNA of the mousepox virus.
Chen had long days of work ahead of him, for he had to purify the virus strains. Purification of a virus is a core technique in the art of virus engineering.
A VIRUS is a very small object, and the only way to handle it is to move around cells that are infected with it. A poxvirus growing in the layer of cells at the bottom of a well plate will kill the cells, forming dead spots in the layer. These spots are like the holes in a slice of Swiss cheese, and they are known as plaques. You can remove the dead or dying cells with a pipette. The cells that come out of that spot will contain a pure strain of the virus.
“Would you like to do some plaque picking?” Chen asked me one day. He led me into a small room behind his work area, where there were a couple of laboratory hoods, a couple of incubators (which are warming boxes that keep cell cultures alive), and, tucked away in a corner, a microscope with binocular eyepieces.
Chen put on a pair of latex gloves, opened the door of an incubator, and slid out a well plate. It had six wells, glistening with red cell-culture medium, and a carpet of living cells covered the bottom. He carried the well plate across the room and placed it on the viewing stand of the microscope. You could see with the naked eye the holes in the cell layers. The cells were infected with a strain of engineered IL-4 mousepox.
I sat down at the microscope, and Chen handed me a pipette that had a cone-shaped plastic tip with a hole in it, like a very fine straw. You put your thumb on a button on the pipette, and when you pushed the button you could pick up a small amount of liquid and deposit it somewhere else.
I was beginning to feel a little strange. We were handling a genetically engineered virus with nothing but rubber gloves. “You’re sure it’s not infective?”
“Yes, it is safe.”
I sat down at the microscope and looked into a carpet of monkey cells growing at the bottom of a well. Each cell looked like a fried egg; the yolk in the cell was the nucleus. I started looking for holes in the carpet, where the virus would be growing.
“I can’t find any plaques,” I said. I began moving the well plate around. Suddenly, a huge hole appeared. It was an infected zone, rich with engineered virus. The cells there were dying and had clumped up into sick-looking balls. The cells had caught the engineered pox.
I was holding the pipette in my right hand. I maneuvered the tip into the well plate. “I can’t see the tip,” I said, jabbing it around in the well.
I was wrecking Chen’s careful work, but he made no comment. Then the tip of the pipette heaved into view. It looked like the mouth of a subway tunnel.
“You need to scratch the cells off,” Chen said.
I moved the tip around, scraping it over the sick cells. I let the button go, and a few cells were slurped up into the pipette. Chen handed me a vial, and I deposited a picked plaque of engineered poxvirus into it. “I don’t think I’d make a good virologist.”
“You are doing fine.”
The work of creating four engineered mousepox strains took five months—the work was painstaking, and Chen had to check and double-check every step of the process. He believes that the total cost of laboratory consumables ran to about a thousand dollars for each strain. Virus engineering is cheaper than a used car, yet it may provide a nation with a weapon as intimidating as a nuclear bomb.
IT WAS TIME to infect some mice with the engineered virus, to see what it would do. The mouse colony was kept in a Biosafety Level 3 room on the top floor of the medical school. Mark Buller and I put on surgical gowns, booties, hair coverings, and latex gloves. We pushed through a steel door into a small cinder-block room, where hundreds of mice were living in clear plastic boxes, set on racks behind glass doors. The mice had black fur. They were a purebred laboratory mouse known as the Black 6, which is naturally resistant to mousepox.
Buller opened some boxes, removed some mice, and placed them in a jar that had an anesthetic in it. The mice went to sleep. One at a time, he held a mouse in his hand, stuck the needle of a syringe into its foot, and injected a drop of clear liquid. The liquid contained about ten particles of engineered IL-4 mousepox—an exceedingly low dose of the virus.
Seven days later, my phone rang early in the morning. It was Mark Buller. One of the lab techs had just checked on the mice, he said, and some of them had a hunched posture, with ruffled fur at the neck. “They’re going to go fast,” he said.
The next morning, Buller, Chen, and I put on gloves and gowns and went into the mouse room. There were two boxes of dead mice. Two of the strains of IL-4 mousepox had wiped out the naturally resistant mice. The death rate for those groups was one hundred percent.
Buller carried one box inside a hood and opened it. The dead mice were indeed hunched up, with ruffled fur and pinched eyes. Natural mousepox does not cause a Black 6 mouse to become visibly sick at all.
“Wow. Wow,” Chen said. “They’re all hunched over. This IL-4 has a really funny effect. This is really a strong virus. I’m really surprised.” He hadn’t expected his virus to wipe out all the mice. It disturbed him that he could make such a powerful virus, but he also felt excited.
“It’s really impressive how fast this virus kills the mice at such a low dose,” Buller said.
I sat on a chair before the hood, peering into it beside Buller. He reached in and lifted a dead mouse out of a box, and held the creature in his gloved hand. Without the mouse, there would be no cures for many diseases, and dead mice had been responsible for the saving of many a human life, but what he held in his hand was not a reassuring thing.
Buller showed me the standard way to dissect a mouse: you slit the belly with scissors. He spread open the abdomen with the scissors, looking to see what the pox had done.
The virus had blasted the mouse’s internal organs. The spleen had turned into a bloated blood sausage that was huge (for a mouse’s spleen) and filled much of the mouse’s belly. It was mottled with faint grayish-white spots, which Buller explained is the classic appearance of a mouse’s organs infected with pox. Doctors who opened humans who had died of hemorrhagic smallpox saw the same cloudy effect in their organs. With the tip of the scissors, he pulled out the mouse’s liver. It had turned the color of sawdust, destroyed by the engineered virus. With ten particles of the construct virus in its blood, the pox-resistant mouse had never stood a chance.
THERE ARE TWO WAYS to vaccinate a mouse against mousepox. One way is to infect it with natural mousepox. When it recovers (if you vaccinate a resistant breed of mouse, it will recover), it will be immune. The other way is to vaccinate the mouse with the smallpox vaccine—that is, you infect the mouse with vaccinia, and its immunity to mousepox goes up in the same way that a human’s resistance to smallpox goes up after a vaccinia infection.
Mark Buller and his group began testing IL-4 mousepox on vaccinated mice, and they got strange results. They were not able to completely duplicate the Jackson-Ramshaw experiment. They discovered that mice immunized with natural mousepox become completely immune to IL-4 mousepox—it did not break through their immunity after all. That was very encouraging. It contradicted part of the Jackson-Ramshaw experiment. But in doing preliminary experiments with the smallpox vaccine, they had begun to see something more troubling (the experiments were in progress, and Buller wasn’t able to report any real findings yet). It seemed that the IL-4 mousepox could crash through the smallpox vaccine, killing the mice if the
y had been vaccinated sometime previously. But if their vaccinia vaccinations were very fresh, they were protected against the engineered pox. It suggested that an engineered IL-4 smallpox might be able to break through people’s immunity, but not if the vaccinations were recent, perhaps only weeks old.
Buller didn’t sound as if he thought the world was coming to an end. “We showed that you could find a way to vaccinate mice successfully against the engineered mousepox,” he said to me. “Even if IL-4 variola can blow through the smallpox vaccine, I feel there are drugs we can develop that will nullify the advantage a terrorist might have by using IL-4 variola. We really need an antiviral drug,” he said. He argued that a drug that worked on pox was not only needed as a defense against an engineered superpox, but was also needed in order to cure people who were getting sick from the vaccine during a mass vaccination after a smallpox terror attack.
Any nation or research team that wanted to make a superpox would have to test it on vaccinated humans to see if it worked. “If you’re talking about a country like Iraq,” Buller said, “human experimentation with smallpox is imaginable. If you’ve got a guy like Saddam Hussein, and his scientists tell him they need some humans so they can check out an engineered smallpox, he’ll say, ‘How many do you need?’ There are people like that in every age.”
Nanhai Chen seemed a little less optimistic. “Because the IL-4 mousepox can evade the vaccinia vaccination, it means that IL-4 smallpox could be very dangerous,” he said. “This experiment is very similar to the human situation with the smallpox vaccine. I think IL-4 smallpox is dangerous. I think it is very dangerous.”