Biology is the epitome of a "dual use" technology. All of the tools and techniques that promise progress in basic science and that enable new vaccines can be put to nefarious uses with equal ease.
Our response to infectious organisms such as Influenza and the SARS virus are excellent scientific and policy test cases of our readiness for future threats. Only by openly studying pathogens that cause epidemics, publicly discussing the results, and publicly preparing our defense, can we hope to be ready for both human creations and natural surprises.
How the Flu Virus is Put Together
Influenza viruses have for many years been difficult and dangerous to work with at the laboratory bench. The ability of these viruses to cause disease in other organisms - their "pathogenicity" - remains poorly understood. Appreciating the danger and value of the methods used to study infectious viruses requires another cartoon-level exploration of the relevant technology (Figure 9.1).
Influenza is an RNA virus; its genes are encoded not on double-stranded DNA, but rather in a genome composed of single-stranded RNA. Complicating matters further, the genome is "negative strand RNA". The genome must be copied into "positive strand" RNA - mRNA - before it is translated into protein. Molecular biology as a science is just now producing tools that enable scientists to manipulate, and thus investigate, negative strand viruses; it is still very difficult work.
Influenza viruses have just eleven genes distributed on eight chromosomes (sometimes called "gene segments", with more than one gene per segment). Those chromosomes are packaged, along with several kinds of viral proteins, within an envelope composed largely of a membrane stolen from a host cell in which the virion - a single infective viral particle - was produced. Among the proteins carried along by the virus is an RNA polymerase that copies the negative strand genome into mRNA that host cells then translate into proteins. The complexity of both the viral packaging and the coding strategy have long made doing any molecular biology with influenza extremely arduous.
Contributing further to the challenge is the speed with which the virus changes in the wild. The most frequent ways for the virus to evolve appear to be through 1) mutation of individual bases, and 2) "reassortment", the exchange of whole chromosomes made possible when more than one virus infects the same cell at the same time. In addition, the viral RNA polymerase makes frequent copying mistakes, which means that many sequence variants are produced during infection, further enhancing the virus' ability to evolve in the face of pressure from drugs and vaccines.
The first paper demonstrating a "reverse genetics" system for constructing influenza viruses was published in 1990. The authors introduced a method to build an RNA virus from constituent parts, which could be of synthetic origin. Because of the difficulties inherent in working with RNA in the laboratory, the technological strategy developed to handle RNA viruses revolves around simplifying the problem by working with DNA instead. Typically, researchers first construct plasmids - that ubiquitous bit of biological technology introduced in Chapter 2 - that contain DNA versions of viral genes, which are then transcribed into viral RNA (vRNA) in vivo in cultured cells (Figure 9.2).
The authors of the 1990 paper had the goal of eliminating the "Difficulty in modifying the genomes of negative-strand RNA viruses [that] has slowed our progress in understanding the replication and the pathogenicity of the negative-strand virus groups." Looking ahead, they also noted that, "The ability to create viruses with site-specific mutations will allow the engineering of influenza viruses with defined biological properties."
Improvements in reverse genetics were published throughout the 1990's. A 1999 paper introduced "packaging plasmids" that simplified viral assembly. The specific sequences on the packaging plasmids constituted a program that 1) choreographed the behavior of proteins within the host cell to first make viral proteins necessary for transcribing the DNA into vRNA and 2) then directed the construction of additional proteins that, in effect, act to mimic viral infection by packaging the vRNA into active viral particles. A further advance in 2005 reduced the number of required packaging plasmids from two to twelve, thereby dramatically improving the efficiency of building artificial viruses.
The Consequences of Reincarnating a Pandemic Virus
In the fall of 2005, several high profile academic papers described the genomic sequence of the 1918 "Spanish" Flu, responsible for more than 40 million deaths worldwide between 1917 and 1919. This feat was possible because Jefferey Taubenberger at the Armed Forces Institute of Pathology in Rockville, Maryland, rebuilt the virus using RNA fragments he recovered from tissue samples stored in government repositories and from the lung of a victim buried in the Alaskan permafrost. The flu genome publications were reviewed by the US National Science Advisory Board for Biosecurity (NSABB), composed of knowledgeable members of academia and government agencies, and determined to be in the best interest of the public.
Critics denounced publication of the reconstructed sequence as the height of folly, asserted that the project was of questionable scientific and public health benefit, and that electronic, rather than laboratory, study of the sequence information would be sufficient to discover the virus' secrets. Nonetheless, within a year of the flu reconstruction, articles appeared that validated the decision to rebuild the virus. In particular, the experimental results, "Indicated a cooperative interaction between the 1918 influenza genes and show that study of the virulence of the 1918 influenza requires the use of the fully reconstructed virus." In early 2007, this result was extended to primates in a study that investigated in monkeys the molecular mechanisms that the host mounts in defense against the virus. That paper described the important role of a vigorous early innate immune response in both controlling the virus and causing tissue damage to the host, which may begin to explain why the Spanish Flu killed otherwise healthy young adults at such high rates. Research continues into why the 1918 strain of the flu was so deadly.
At the time the 1918 flu sequence was announced, several high visibility editorials and Op-Ed pieces questioned the wisdom of releasing that information into the public domain, suggesting the sequence could be used to create bioweapons.
From the perspective of "LEGO-style" biology, it would appear that all you have to do is plug the appropriate DNA sequence into the packaging plasmids, dump those into a pot of mammalian cells, and wait for infectious viruses to spread throughout the culture. In reality, the process is full of high art and skill, and it is no simple matter to take synthetic DNA and from it create live, infectious negative-strand RNA viruses such as influenza. This is a crucial point. I have discussed this issue with a number of RNA virus experts, including some involved in sequencing and building flu strains, and they universally say reproducing the flu genome is presently quite difficult even for experts. Unfortunately, there has been far too little public discussion by scientists involved of the threats posed by reconstituted pathogens.
More importantly, however, the threat from a modern release of the 1918 Flu virus is not as dire as many fear. During the 2007-2008 season, the CDC found that 26% of samples positive for influenza viruses contained the "H1N1" subtype, named for proteins on the outside of the virus and identical to the proteins on the 1918 strain. Therefore, the subtype continues to circulate widely in the population, and most people now have some immunity to it. Without minimizing any illness that might result from release of the original 1918 flu virus, suggestions that any such event would inevitably be as deadly as the first go round appear to be overstated.
Nonetheless, as with every other biological technology described in this book, it is inevitable that the technology to build RNA viruses will become widespread. Although it is challenging right now for even experts to recreate live, pathogenic influenza viruses from synthetic DNA, I have no doubt that over time the relevant skill set will eventually reach individuals with considerably less good sense about how to safely handle the resulting organism. There will even probably be a kit available someday that reduces the expertise to following a recipe, and there may well be an automated platform that implements the recipe with minimal human participation.
The consequent threat to public health and safety from proliferating skills and technology will be substantial. We will eventually require constant vigilance and the ability to detect threats, and to either preempt or remediate them, on short notice. As I argue in the next sections of this chapter, guaranteeing public health and safety requires significant and rapid maturation of technologies that enable biological engineering and, consequently also enable increased threats.
We're stuck; there are no two ways about it. We require new technology to deal with threats, technology that can only be developed within the context of a diverse and capable bio-economy. Yet, the existence of that technology will enable widely distributed use for both beneficial and nefarious purposes.
Dealing with emerging biological threats will require better communication and technical ability than we now possess, as directly revealed by progress resulting from rebuilding and publishing the 1918 influenza genome. Open discussion and research are crucial tools to create a safer world.
To be clear, I do not dismiss the potential of DNA synthesis technologies to be used for nefarious purposes, but rather have come to the conclusion that it is a looming rather than imminent threat. A recent survey of the difficulty of constructing pathogens de novo concluded that, "Any synthesis of viruses, even very small or relatively simple viruses, remains relatively difficult." The report then acknowledges that the risk is likely to increase; it is a virtual certainty that some day a synthetic pathogen will emerge as a threat.
We don't have to wait for that day to find out how might respond to such threats; we are presently at the mercy of many emerging diseases. While the threat of artificial pathogens appears minimal at present, there exists a clear and present danger from naturally occurring pathogens against which we are demonstrably incapable of defending ourselves. The combination of increased intrusion of humans into previously undisturbed environments and rapid transportation of people and goods around the globe is raising red flags for observers who are concerned about public health and national security. With respect to reconstructing the 1918 flu, Jefferey Taubenberger concludes that:
There can be no absolute guarantee of safety. We are aware that all technological advances could be misused. But what we are trying to understand is what happened in nature and how to prevent another pandemic. In this case, nature is the bioterrorist.
It is this latter problem, immediate and pressing, that must define the path of our scientific and technological investment in the near future.
For more, see Biology is Technology (Amazon). At the time of this posting, the hardback edition is sold out, the Kindle edition is available, and the paperback is on the way.
1. Enami, M., et al., Introduction of Site-Specific Mutations Into the Genome of Influenza Virus. Proceedings of the National Academy of Sciences 1990. 87(10): p. 3802-3805.
2. Neumann, G., et al., Generation of influenza A viruses entirely from cloned cDNAs. PNAS, 1999. 96(16): p. 9345-9350.
3. Neumann, G., et al., An improved reverse genetics system for influenza A virus generation and its implications for vaccine production. Proceedings of the National Academy of Sciences, 2005. 102(46): p. 16825-16829.
4. The 1918 flu virus is resurrected. Nature, 2005. 437(7060): p. 794.
5. Kash, J.C., et al., Genomic analysis of increased host immune and cell death responses induced by 1918 influenza virus. Nature, 2006. 443(7111): p. 578.
6. Kobasa, D., et al., Aberrant innate immune response in lethal infection of macaques with the 1918 influenza virus. Nature, 2007. 445(7125): p. 319.
7. Loo, Y.-M. and Gale, M., Influenza: Fatal immunity and the 1918 virus. Nature, 2007. 445(7125): p. 267.
8. Repeated requests to Jefferey Taubenberger for clarification on the issue of ease of influenza viral reconstruction went unmet, even when mediated by one of his collaborators. While somewhat frustrating from my perspective, his reticence to discuss the issue is entirely understandable on a personal and professional level and may well be part of official U.S. government or military strategy to contain proliferiation. But more open discussion about how hard it is would benefit both policy debates and basic science
9. "Influenza Activity --- United States and Worldwide, 2007-08 Season", Morbidity and Mortality Weekly Report. June 26, 2008, Centers for Disease Control: Atlanta, GA.
10. Garfinkel, M., et al., "Synthetic Genomics: Options for Governance", October, 2007, J. Craig Venter Institute, CSIS, MIT:
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