Risen from the dead: Can 1918 virus help defeat bird flu threat?

The road to hell is usually paved with good intentions’

In a high-containment laboratory along Clifton Road in Atlanta last summer, a mouse became the first living thing to inhale the 1918 pandemic influenza virus since it killed millions of people and vanished from the face of the earth. Predictably, it died.

After a decade of research that included digging up tissue from a flu victim buried in the Alaskan permafrost, the most infamous infectious disease agent in human history has been brought back to life.1,2 Its legendary virulence apparently came back with it. The research mice — which are not particularly fazed by human flu strains — died in three to five days. Unlike typical human flu, the infecting 1918 H1N1 strain replicated deep within the mice lungs, prompting an immune system hyperreaction — the so-called "cytokine storm" — which researchers suspect is a key to the virus’ virulence.

"There is something unique about the 1918 virus," says Terrence Tumpey, PhD, the senior microbiologist at the Centers for Disease Control and Prevention who recreated the virus and "intranasally inoculated" the mice. "It is one of the [research] areas we really want to get into. Why does the virus target the deeper areas of the lung and kill mice so quickly?"

Ferrets actually are the preferred research animal in terms of mimicking human response to influenza, but the smaller mammals were chosen due to the extreme conditions required in the laboratory. Tumpey worked under enhanced biosafety level three conditions in a high-security lab at the CDC’s Clifton Road facility. The lab precautions were elaborate and redundant, including working with safety cabinets, isolation cabinets, gloves, gowns and wearing a powered air-purifying respirator body suit. He was required to change clothes and "shower out" of the lab, provide routine weekly written reports and notify agency officials immediately of any concerns related to biosafety or biosecurity.

Using "reverse genetics" — essentially reassembling genetic fragments into a whole, functioning microorganism — Tumpey and his fellow researchers built a virus that is to influenza what the Titanic is to ocean liners. When done, it contained the complete coding sequences of the eight viral gene segments from the 1918 pandemic strain. There below the electron microscope was the Spanish flu, so-called because of the popular misconception that it originated in Spain. For anyone who works in infectious diseases, this is akin to finding the Ark of the Covenant. The 1918 influenza pandemic still is retold like a ghost story, wherein a pathogen of unknown origin arises to sweep the earth and mysteriously disappears after killing as many as 50 million people worldwide, including an estimated 675,000 in the United States.

With warring nations under press censorship, Spain’s free press publicized the emerging 1918 pandemic and became forever branded as its country of origin. Ironically — considering that the virus now resides here again — the United States may have actually birthed the 1918 flu. Soldiers training at Fort Riley, KS, in March 1918 were among the first to fall ill and die. It was off to war for their comrades, many of whom may have been incubating the virus when they hit the trenches. Ultimately, the 1918 flu would kill nearly as many soldiers as their human enemies. Indeed, the 1918 influenza pandemic’s most striking feature was the unusually high death rate among healthy adults aged 15 to 34 years. Epidemiologists still look in puzzlement at the age/mortality charts, rising to a grim peak in the middle years when one should have maximum immunity to infectious diseases.

A soft-spoken, bespectacled 43-year-old, Tumpey paused for a second when asked what his first thoughts were when he realized he had successfully recreated the notorious 1918 influenza virus. In his answer he used the word "rescue," a concept apparently not unusual to virologists, but a striking verb choice when one is bringing a natural-born killer back to molecular life.

"This represents an exciting time to be able to work with these viruses, to understand their molecular and biological properties and identify what additional targets could be looked at for the development of antivirals," he replies. "Not only did I want to rescue the virus, but I also wanted to show that certain genes — certain viral proteins — associated with the 1918 virus were important for causing this virus to be so lethal. I thought of it from that standpoint. That we could see some clear benefits of rescuing this virus. We are [now] actually working on trying to prepare better vaccines against the 1918 virus that would give us information about vaccines against pandemic viruses."

Ghoulish work

Tumpey was able to rebuild the virus because he had viral building blocks provided by Jeffery K. Taubenberger, MD, PhD — the Armed Forces Institute of Pathology researcher who spent 10 years reconstructing the 1918 pandemic flu strain bit by genetic bit. Two of the viral strains Taubenberger used came from autopsy remains of two U.S. soldiers who died in the great pandemic. The other genetic fragments required more ghoulish work in 1997, when investigators exhumed the frozen body of an Inuit woman who fell victim to the pandemic in a remote village on the Seward Peninsula of Alaska. As they hoped, viral fragments remained in the semipreserved body. Coincidentally, 1997 also marked the first appearance in Hong Kong of the bird flu strain causing much current anxiety: avian H5N1.

Looking at the reassembled 1918 virus, Taubenberger sees strong avian similarities, but it appears the deadly strain jumped into humans without going through the typical re-assortment with a circulating human flu strain. That is not particularly encouraging since it runs counter to the conventional wisdom that the H5N1 bird flu will not become transmissible in humans until it reassorts with a human flu strain. While the 1918 flu strain appears avian, it is unlike anything researchers have ever seen.

"Wherever it came from it came as a whole virus," Taubenberger says. "This is true for all the segments of the virus. It looks kind of like a matched set. It suggests to us that it came from some animal that is a reservoir for influenza that we just don’t know about. It could be some other kind of wild bird, but it doesn’t look like your typical duck influenza viruses that people have been studying for the last 30 years. We don’t know where it came from, which is yet another tremendous mystery."

The pig is the usual suspect, the classic "mixing vessel" for avian and human flu strains. Yet the genetic signature of the recreated virus suggests that pigs infected in 1918 actually may have acquired the virus from humans, he explains. Could another animal — one not typically associated with influenza mutation — have served as the intermediate host? The soldiers at Fort Riley, KS, were encamped with herds of horses and mules and all the manure that came with them. The manure was routinely burned to dispose of it, so plenty of opportunities for animal, human, and viral contact existed.

"Whether there was an intermediate host involved, like another mammal, unfortunately we just don’t know," Taubenberger admits. "We think it is unlikely that the pig was the so-called mixing vessel or the intermediate host, but I can’t rule out some other animal — a horse? I can’t. I don’t know."

The controversy surrounding his research is no mystery to Taubenberger. He is well aware of the accusations that it is irresponsible, but argues eloquently that finding the key to the virulence and transmissibility of the 1918 pandemic strain may help thwart the rise of H5N1 or future pandemic strains. "The key is to figure out how animal viruses adapt to humans and gain the ability to cause pandemics," he says.

It is nature that is coming after us’

While extremely controversial, the achievement is staggering. One of the few precedents is the creation of a polio virus entirely from synthetic (e.g., nonliving) materials in 2002.1 That paper chillingly concludes, "Our results show that it is possible to synthesize an infectious agent by in vitro chemical-biochemical means solely by following instructions from a written sequence." The lead researcher of the polio paper says the same thing could now be done for the 1918 H1N1 flu virus by following the recipe in the recently published papers.

"Yes, it can be done," says Eckard Wimmer, PhD, professor in the department of molecular genetics and microbiology at the State University of New York in Stony Brook. "You could take this information in the public domain and recreate this virus. It would be difficult. Flu virus is much more complicated than polio virus. It would not be a piece of cake."

Nevertheless, Wimmer argues that publication was the right strategy, noting that it will set off an explosion of research similar to the response when severe acute respiratory syndrome (SARS) suddenly appeared in 2002.

"This [SARS] corona virus came out of China," he says. "When that virus first hit, nobody had an inkling what it was. It was fairly lethal. Depending on the age of the infected person it had a [mortality rate] of between 15% to 30%. There were no drugs, no vaccines. We really didn’t know how to grow this virus. People didn’t panic, but people could have panicked because it was really very scary."

The World Health Organization quickly asked for global research on the emerging SARS virus, encouraging all clinical, molecular and virological research to be rapidly shared on the Internet. "Within a very short time — six or seven months — the virus was isolated, identified and sequenced," Wimmer emphasizes. "Everything was put on the Internet and experiments for antiviral research and vaccines were initiated."

Of course, like the 1918 virus, SARS disappeared, but the point is that the transparency with which it was met with was the absolute right scientific response, he emphasizes. "This [1918 research] will enormously stimulate research on influenza virus and open new avenues to protect us," he says. "We are actually facing a threat, but not from bioterrorists who could synthesize this virus. It is nature that is coming after us. The bird flu that is coming out of Asia will eventually learn how to infect people. The enormous importance of this work on the 1918 flu is that it also was a bird flu virus. Research to develop new drugs against the influenza virus is of the utmost importance."

Almost lost in the debate are the bioethical questions Wimmer faced when he used similar reverse genetic techniques to build a polio virus from scratch. Essentially a parasite, a virus replicates by invading a cell and using its existing biochemical materials to continue replication. Moreover, Wimmer "synthesized" the poliovirus genome, making an organism capable of replication and infection out of nonliving materials. Are such researchers creating life? A bioethicist who questions the wisdom of such research emphasizes that such research — even though viral in nature — is essentially cloning.

"There is a lot of discussion about human cloning and the ethics of it, but the science is very primitive," says Arthur Caplan, PhD, director of the Center for Bioethics at the University of Pennsylvania in Philadelphia. "Engineering viruses, engineering microbes, reconstructing nasty pathogens — people are doing that. It is not hypothetical. People are getting very good at microbial genetics. I think it is only our vanity — thinking that we are important — that makes us forget that microbes can be more important. This is a very dangerous attitude to take."

Unfortunately, it is an ideal weapon’

Others see danger of a more calculated variety, particularly since the 1918 viral genome was published and can now be recreated by other laboratorians using entirely synthetic materials. Among those questioning the wisdom of the research is Kenneth Alibek, MD, PhD, DSc, former chief scientist and deputy director of bioweapons research in the former Soviet Union.

Born Kanatjan Alibekov in the Soviet Republic of Kazakhstan, Alibek oversaw bioweapons research and development involving such pathogens as smallpox, anthrax, and viral hemorrhagic fevers. Now a U.S. citizen, he changed his name when he defected to the United States in 1992. Weaponizing influenza — particularly the 1918 strain — was discussed by Soviet researchers, says Alibek, now a distinguished professor in the department of molecular and microbiology at the National Center for Biodefense at George Mason University in Washington, DC.

"It was always under consideration," he says. "The Soviet Union was trying to start some things, but it never came to developing actual weapons because the viruses that were available at that time had very low virulence and a low mortality rate. But when we talk about this type of virus [1918], of course there is a much a higher mortality rate. The probability that some other country will be interested in possible development of this as a biological weapon is very high. I don’t think that terrorists would be able to do something in terms of success with this virus, but for rogue states, it could be the case."

It has been argued that influenza would not make a good bioweapon since it could not be controlled once released, and even if you developed a vaccine the weaponized virus still would be subject to ongoing mutation. Nevertheless, Alibek says the sheer virulence and transmissibility of the 1918 strain make it attractive as a bioweapon.

"This virus has a very high transmission rate — the highest rate known to man [for influenza]," he says. "Second, it can be grown easily using different techniques. The mortality rate for regular influenza virus is not so high, but for this virus the mortality rate was between 2% to 5%. Unfortunately, it is an ideal weapon."

Given his unique background, Alibek is difficult to dismiss as an alarmist. Yet his warnings about the research were not heeded, he says. "My position was always absolutely obvious and clear," he tells Hospital Infection Control. "It was a really bad idea. I had a discussion about this a couple of years ago with some of the scientists involved in the study of this virus. I said it is not a good idea, but if a decision was made to finish this work it shouldn’t be published."

Prior to publication, the 1918 virus research was reviewed by the National Science Advisory Board for Biosecurity, an advisory committee to the U.S. government. Julie Gerberding, MD, MPH, director of the Centers for Disease Control and Prevention, and Anthony Fauci, MD, director of the National Institute for Allergy and Infectious Diseases, issued a joint statement that read in part: "The rationale for publishing the results and making them widely available to the scientific community is to encourage additional research at a time when we desperately need to engage the scientific community and accelerate our ability to prevent pandemic influenza. . . . Moving forward with research conducted by the world’s top scientists and openly disseminating their research results remain our best defense against H5N1 avian influenza virus and other dangerous pathogens that may emerge or re-emerge, naturally or deliberately."

Alibek is skeptical that insights gained into the 1918 virus can be used to thwart the emerging bird flu threat. "It’s a different virus," he says. "It is not H5N1. I am kind of doubtful how much information we will get from this Spanish flu virus. There would be a much higher probability [of success] just to start with all possible isolates of H5N1 virus, which are already available. That might produce real knowledge about how to fight this [avian] influenza, but not a virus that was infecting people a century ago."

Advised that the CDC confirmed for this report that it already is working on a 1918 vaccine, Alibek could hardly contain the incredulity that rang through his thick Russian accent.

"First, we create the problem and then we use a huge amount of money to solve the problem," he says. "If this work had not been done nobody would be developing a vaccine. It’s like, Let’s create the disease and then we will try and fight it.’ It is absolutely insane. People can say they had good intentions and they will develop a new vaccine and so forth. Of course, their explanation is that they had good intentions, but I say the road to hell is usually paved with good intentions."

It’s not Ebola’

Taubenberger, the man who has dedicated his career to restoring the virus and solving its mysteries, says the risks were weighed against the rewards.

"Certainly, we can not say there is no risk," he says. "We thought about that at the very beginning when we decided to do this project. But the risk of that [bioterrorism] is pretty low compared with the benefits that could come out of studying the 1918 virus. Here was a naturally occurring virus that killed millions of people. Pandemics occur on a similar regular basis and we don’t understand how they work. It would very beneficial if we could understand the basic rules of how this happened, so that we could ultimately prevent it from happening [again]. We feel that the public health good from this project far, far outweighs the risk."

Besides, the world has changed considerably since 1918, when there were no antibiotics, no influenza vaccines, no antivirals and viruses themselves were as yet undiscovered. The 1918 H1N1 flu strain would not find its human host as defenseless today. The virus is susceptible to the four currently available antiviral medications, which can minimize flu effects if taken shortly after infection. In addition, antibiotics could presumably stave off some portion of the bacterial coinfections that were responsible for so many of the 1918 deaths. Current vaccines appear to muster at least partial immunity to the strain, and humans may now even have some residual immunity through successive exposures to natural recurring flu and receipt of the annual seasonal vaccine.

"The ideal thing would be to develop a seed stock [vaccine] against 1918, but even the modern human virus vaccine would provide some protection," Taubenberger says. "It’s not perfect, but there would be some protection. Certainly, 1918 was an incredibly lethal flu virus. The numbers of dead are astounding, but you also have to understand that basically everybody on earth breathed in this virus. The case fatality rate in the U.S. in 1918 — without vaccines, without antivirals, without antibiotics — was around 2%. That is terrible — the worst flu ever — but it’s not Ebola."

The CDC is sufficiently concerned about the virus that it recently added it to its list of select agents and toxins. Acknowledging that it now is possible for those with knowledge of reverse genetics to reconstruct this virus, the CDC stated in making the announcement that it had been advised that the "molecular properties that enabled the 1918 pandemic influenza virus to cause such widespread illness and death are not completely understood and that it is not known how virulent the reconstructed virus would be in the population today."

There currently are 41 other agents and toxins listed as select agents under the Public Health Security and Bioterrorism Preparedness and Response Act of 2002. As with the other agents, all scientists and researchers that possess, use or transfer the 1918 strain of influenza or the eight key gene regions of the 1918 virus are required to register with the CDC. The CDC also announced it will not distribute any of its 1918 viral isolates, but may allow other researchers to work with them at the CDC.

Created from scratch’ in months

The U.S. restrictions are well and good, but do not address Alibek’s concern that researchers in another country could simply follow the published "recipe" and create the 1918 strain.

"If — as should be the case — transfer of the plasmids is restricted, then it would be necessary for someone attempting reconstruction to generate the plasmids again," explains Richard H. Ebright, PhD, a professor of chemistry at Rutgers University in New Jersey. "The plasmids could be generated by de novo synthesis. There are a host of corporations that provide commercial gene and genome synthesis. To construct eight plasmids — each about 4,000 base pairs in size — would be a matter of a month or two months and moderate costs. They also could be constructed easily in major research labs. To go from scratch’ to live fully infectious particles would be a matter of months."

Beyond that there is always the possibility that well intended researchers may accidentally allow the virus to escape from the lab, he notes. Ebright, who is doubtful that sufficient residual immunity remains in the human population to provide much protection, points out there is no current direct vaccine match and antivirals are in extremely short supply. "I believe this research should not have been done," he says. "The researchers have deliberately reconstructed a virus that has been extinct for more than eight decades, and when last present killed 1% of the world’s population."

A scholar with a penchant for succinct analysis, Ebright lines his points up like dominos. "Overall, this poses three sets of risks," he begins. "It poses a risk of accidental release, it poses a risk of deliberate release by a disturbed, disgruntled or extremist employee of a laboratory, and it poses the risk of clandestine reconstruction and deliberate release by a hostile party. In any one of those three scenarios, there would be the potential for significant loss of life and significant economic disruption."

Echoing Alibek’s sense of irony, he adds, "It will be possible to build an effective vaccine, but the remarkable point here is to create a pathogen for the expressed purpose of developing a countermeasure. There is a logical lapse. The ethical barrier that would prohibit somebody from doing this has been breached by the fact that the work already has been performed and approved. It is a threat that must be considered. It would be prudent to produce and stockpile vaccine against this threat."

Indeed, bioterrorism fears have prompted massive smallpox immunization campaigns among the military and health care workers in recent years, even though that virus has been completely eradicated in nature. Now, with restoration of the 1918 influenza virus we have another "demon in the freezer," as the frozen stores of smallpox virus at the CDC and in Russia have been described. That raises the possibility of future mass immunizations against the 1918 flu, a pathogen that is so unusual that even its fully recreated genetic sequence doesn’t explain its origins. Is it possible that the 1918 strain was some kind of viral anomaly that was very unlikely to arise again in nature? "I guess that is certainly possible," Taubenberger concedes.

Yet even if it had never recurred in nature, some other group of researchers may have eventually pieced the 1918 virus together. The Everest of the infectious disease world was not going to stay unclimbed for lack of trying.

"We knew all along that the [genetic] sequence would eventually be published, so we wanted to provide a clear public health benefit to rescuing this virus and understanding it." Tumpey argues. "If it wasn’t done by Jeffery Taubenberger, there were others that were interested in it. Others had tissues and could do this work. We knew this day would come."

References

  1. Taubenberger JK, Reid AH, Lourens RM, et al. Characterization of the 1918 polymerase genes. Nature 2005 Oct 6; 437(7,060):889-893.
  2. Tumpey TM, Basler CF, Aguilar PV, et al. Characterization of the reconstructed 1918 Spanish influenza pandemic virus. Science 2005; 310(5,745):77-80.
  3. Jeronimo C, Paul AV, Wimmer E. Chemical Synthesis of Poliovirus cDNA: Generation of Infectious Virus in the Absence of Natural Template. Science 2002; 1,018:1,016-1,018.