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The Fear of the Known

Publishing the genetic sequence of a transmissible influenza virus might be scary, but harder decisions are yet to come

Robert L. Dorit

Ferreting Out the Truth

As I write, a new controversy surrounding the publication of research on the influenza virus has erupted. This time the work involves the identification of a small number of mutations in the influenza genome that affect transmissibility. Two investigators—Yoshihiro Kawaoka, from the University of Tokyo and the University of Wisconsin, and Ron Fouchier of the Erasmus Medical Center in Rotterdam—independently obtained similar results. Their papers (one of which I have not yet seen) report the creation and characterization of a strain of avian influenza (H5N1) that, in carefully controlled laboratory settings, evolved to exhibit airborne transmissibility between mammalian hosts. The experiments used ferrets as the host species, at least partially because ferrets show symptoms similar to those exhibited by humans after contracting the flu. When they get the flu, ferrets get runny noses, spike fevers, cough and, we have to assume, feel lousy. The starting point of the Imai et al. experiment was a deliberately created H5N1 strain. Influenza viruses are classified based on their two principal surface proteins: hemagglutinin (HA) and neuraminidase (NA). These two proteins define the outer appearance of the virus, and mediate the virus’s interaction with the host’s cells and its encounters with the host immune system. The H5 class HA of the starting strain meant that it was particularly effective at targeting surface receptors on the cells of birds, but comparatively inefficient at sneaking into human cells with their characteristic mammalian cell surface receptors. Taking advantage of the fragmented nature of the viral genome (more about this below), the investigators created a deliberately weakened laboratory strain of human influenza capable of expressing the H5 surface protein. Equipped only with the H5 coat protein, this strain was initially not transmissible between mammalian hosts. In order to examine whether this feeble chimera could be turned into a strain capable of ferret-to-ferret transmission, the investigators used an ingenious strategy that mimicked and accelerated the evolutionary process. To do so, they first deliberately introduced mutations in the region of the HA protein that directly interacts with host cell receptors, thus increasing the raw material on which evolution depends. They then triaged millions of viruses—each with a slightly different version of the HA protein—based on their ability to bind to engineered cells that were coated with the human form of the influenza receptor. Eight—and only eight—variants of the original strain emerged from this rigorous screen, each of them capable of binding tightly to the human form of the receptor in vitro. Eventually, only one of those variants, now placed against the genetic background of a more aggressive pandemic strain, would prove capable both of binding to the mammalian-type receptor in the lab and of successfully replicating in a deliberately infected ferret. This variant (HA N158D/N224K/Q226L/CA04) was then deliberately inoculated into the nasal cavity of ferrets, and healthy ferrets were placed in nearby cages. The naive ferrets (a description that suggests they had never seen the selected influenza strains before) could not contact or interact directly with the sickened ferrets, but the cages were designed to allow air to flow between them. Six days later, two of six naive ferrets had developed influenza: The strain had evolved the capacity to survive, reproduce and be transmitted via airborne droplets from one ferret to another. In subsequent experiments, this newly transmissible strain would acquire additional mutations that increased its infectivity and transmissibility.

These experiments showed that an H5N1 virus that was previously transmitted only between birds could, after a few generations of selection, travel between mammals in airborne droplets (such as those generated by ferret sneezes). What disconcerts is the ease with which this lock was picked: Only a handful (four to five) of recurrent mutations were necessary to account for this evolution.

Impressive as these results appear, however, context is important. Ferrets, after all, are not an exact counterpart of human beings. Like all model systems, they capture important aspects of the system we are interested in (ourselves), but they are not precise replicas. Although there can be no question about the inherent value of identifying mutations associated with increased transmissibility, we cannot assert that these ferret-adapted strains are capable of infecting humans. Also lost amid the public and regulatory anxiety surrounding these results was the observation that the lethality of these viruses was comparable to, if not lower than, that of seasonal influenza (which kills less than 0.1 percent of those it infects), a fact for which the ferrets are surely grateful. Finally, it is worth remembering that the evolved version of the HA protein resulted in successful airborne transmissibility in the context of the rest of the influenza genome in which it occurs: Other strain-specific mutations in other genes likely play a critical role in the success of this engineered virus.

In 2011, when the ferret influenza papers were submitted for publication, they triggered a review by the NSABB. This time, the review board was far from comfortable with publication. Their concern, at least initially, centered on the possibility that knowledge of the sequence changes—changes presumably tied to increased transmissibility—could be misused by enemy states or other entities to engineer a particularly threatening strain of influenza. Reconstituting a virus with the 1918 genome from scratch had always been seen as a fairly daunting task that would require considerable technical expertise. In contrast, engineering a handful of mutations into an existing avian influenza strain appeared comparatively straightforward.

In December 2011, the NSABB recommended significant changes in the manuscripts and urged that critical details of the experiments be omitted. Following a significant debate in the scientific community, the committee revisited the issue. By March of 2012, it had reversed its recommendation, arguing that the benefits of the work outweighed its potential risks. Publication has been further delayed by concerns raised (and hurdles imposed) by both the U.S. and Dutch governments. On May 2nd, the first of the papers was finally published.

To be sure, the transmissibility work, the sequencing of the 1918 influenza strain, and an increasing amount of other research in the life sciences can, in principle, be misused. Fields that deal with infectious disease, in particular, often undertake research and generate results that could be used—by agents so inclined—to design more effective biological weapons, to reduce the effectiveness of current antiviral and antibiotic therapies, or to create conditions favoring the spread of infection. But the general anxiety surrounding the publication of these latest influenza papers, much as the anxiety that surrounded the publication of the 1918 influenza genome, rests on three fundamental misunderstandings: one about the nature of the scientific enterprise, one about the character of viral evolution and one about the causes of epidemic outbreaks.

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