Two studies of H5N1 avian influenza viruses that had been genetically engineered to render them transmissible between ferrets have proved highly controversial. Divergent opinions exist about the importance of these studies of influenza transmission and about potential ‘dual use’ research implications. No consensus has developed yet about how to balance these concerns. After not recommending immediate full publication of earlier, less complete versions of the studies, the United States National Science Advisory Board for Biosecurity subsequently recommended full publication of more complete manuscripts; however, controversy about this and similar research remains.
Knowledgeable observers operating within a legitimate framework for the public good have expressed divergent opinions about the importance and public safety implications of two papers, one recently published1 and one soon-to-be published2, describing the production of ferret-transmissible H5N1 influenza viruses, and about related influenza transmission and pathogenesis research3,4,5,6,7. Some have emphasized that understanding the underlying principles of influenza virus host adaptation and transmission can lead to better prevention and control of viruses that arise naturally, whereas others have drawn attention to ‘dual use’ implications—that is, bioterrorism—or to accidental release of potentially deadly viruses. The most commonly mentioned public safety concerns relate to three assumptions: (1) H5N1 viruses are currently highly lethal to humans but are poorly transmissible; (2) genetic manipulation of H5N1 viruses to increase transmissibility in mammals such as ferrets will create variant viruses that remain highly pathogenic and that become transmissible in humans; and (3) if accidentally or intentionally released, such a virus could precipitate a historically severe influenza pandemic. How do these assumptions hold up against scientific data? In this perspective, we address research evidence related to the epidemic/pandemic potential of genetically engineered H5N1 viruses, and discuss limitations in understanding how influenza viruses become pathogenic, transmissible and potentially pandemic in humans.
Influenza is among the leading global infectious causes of death, periodically causing pandemics that can kill millions of people. Countless influenza A viruses circulate globally in a reservoir that consists of hundreds of avian species. Rarely, one of these viruses undergoes changes that enable it to switch hosts to infect mammals, including humans, although it is not clear whether human transmission can result directly from adaptation of an avian influenza virus (this has not been documented to occur), or only indirectly via further adaptation of pre-existing human or mammalian-adapted viruses, the mechanism that has been associated with all known pandemic and seasonal viruses after 1918. The factors underlying all such emergences are poorly understood8. In the past 80 years of influenza virology, three pandemics have resulted from reassortments of pre-existing human-adapted or mammalian-adapted viruses with one or more avian-influenza-derived genes, but no purely avian influenza virus has emerged to cause a pandemic or human outbreak, or has even become stably adapted to humans. However, because avian influenza viruses have adapted to other mammals, it is considered plausible that such an emergence could occur in humans.
Among many other important research areas related to influenza, it is therefore critical to study the mechanisms by which influenza viruses emerge from birds to become adapted to mammals and ultimately humans, and to learn how the phenotypic properties of such evolving viruses may be associated with human transmission and disease. Among the many subtypes and strains of avian influenza A viruses that exist in nature, those that have at least occasionally infected mammals (for example, H5N1, H7N7 and H9N2) are of interest because they might theoretically be more likely than other influenza A viruses to adapt directly or indirectly to humans. Highly pathogenic avian influenza (HPAI) H5N1 viruses have been of particular interest with respect to theoretical pandemic potential because they have been unusually pathogenic in domestic poultry and have infected and killed several hundred people over a 15-year period.
In seeking to understand such influenza viruses, a research approach used widely in virology is to engineer specific genetic mutations into naturally occurring viruses, and then study the resulting viral phenotypic properties in animals, including infectivity, cell tropism, viral replication, pathogenicity and transmissibility. These types of experiments can potentially provide clues about whether and how a virus might adapt to humans, and what prevention and control options might be useful if that virus did emerge. Much H5N1 research of this type has already been published, including viral genetic engineering to evaluate properties such as pathogenicity and transmissibility in ferrets and other animal models. In the context of this published research literature, we comment on questions relevant to the two papers under discussion1,2.
H5N1 infectivity for humans
The ongoing HPAI H5N1 enzootic continues to cause ‘spill-over’ human infections. World Health Organization (WHO) data indicate that since 2003, HPAI H5N1 viruses have infected 603 people and killed 356 (ref. 9). Technically, the term ‘highly pathogenic’ refers only to the effects of certain H5 and H7 influenza viruses in poultry, not in humans or other mammals; most such viruses either cannot infect, or are relatively harmless in, humans. HPAI H5 and H7 phenotypes are both associated with mutations in the haemagglutinin (HA) gene that usually result from insertion of a sequence of codons encoding multiple basic amino acids at the location where the two linked protein domains comprising the mature HA are cleaved during infection10. This cleavage site change leads to disseminated viral replication in multiple organs of avian species, resulting in high mortality. However, in humans, who cannot be easily infected with most low pathogenicity or HPAI viruses, if they can be infected at all, efficient replication outside the respiratory tract generally does not occur. Therefore, despite the current unusual situation with H5N1 viruses in humans since 2003 (see below), neither H5N1 nor other HPAI viruses would necessarily replicate systemically or cause extreme pathogenicity should human adaptation occur. Although the basis of HPAI H5N1 viral pathogenicity in severe and fatal human cases remains unknown, there is no evidence suggesting that it results from changes known to be associated with viral adaptation to gallinaceous poultry; in fact, no human-adapted or pandemic influenza virus contains genetic changes indicative of prior poultry adaptation.
Solely on the basis of publicly available information about pathogenicity of intranasally inoculated H5N1 virus (the model for natural human and animal infection), the laboratory-derived H5N1 viruses produced in the two papers under discussion1,11,12,13,14,15 do not have enhanced pathogenicity in ferrets compared to the 2009 pandemic H1N1 virus, which is considered to be mildly pathogenic for humans14,15. An apparent misconception has nevertheless arisen in recent public discussions of these studies, namely that the engineered, ferret-transmissible H5N1 viruses were extremely pathogenic in ferrets after intranasal inoculation or aerosol transmission. This notion seems to have resulted in part from misunderstandings about a technique—intratracheal inoculation—used in a separate sub-study reported in the manuscript by the Fouchier group14,15, a method that is not directly relevant to viral transmissibility or natural pathogenesis. As documented since the 1940s16, intratracheal inoculation of influenza viruses is not a ‘model’ for natural viral pathogenicity; influenza viruses that are otherwise considered to be of low pathogenicity often induce severe and even fatal disease in animals when administered by this route, including the 2009 pandemic H1N1 virus15. The presentation of transmissibility studies alongside high-dose intratracheal inoculation pathogenesis studies in the Fouchier manuscript seems to have suggested (incorrectly) to some that the engineered transmissible H5N1 virus is deadly after intranasal inoculation or aerosol transmission between ferrets and, by extension, might be both transmissible and deadly for humans, that is, a virus of deadly pandemic potential. No evidence has yet been presented to support this, although the possibility that additional unspecified genetic changes might do so cannot be excluded.
Potential for human adaptation of H5N1 viruses
It is questionable to what extent HPAI H5N1 is adapted to, or capable of adapting to, humans. It is not clear why one evolving lineage of avian HPAI H5N1 viruses, out of a large and genetically divergent pool of H5 and other avian influenza viruses that rarely infect humans (much less cause severe human disease), has recently infected hundreds or perhaps thousands of people. It may be that the human cases are a result of unusual high-dose exposures or rare individual genetic susceptibilities. Alternatively, H5N1 viruses may be beginning to do something no other HPAI virus has ever been documented to do—adapt directly to humans. And if H5N1 did adapt, could it cause a pandemic?
No HPAI virus in the historic record has ever been efficiently transmitted between humans, let alone caused a pandemic. Even when avian influenza virus genes have been imported by reassortment into existing human influenza viruses, as happened for example in 1957 with H2N2 influenza and in 1968 with H3N2 influenza, the sources seem to have been circulating low-pathogenicity avian viruses, not poultry-adapted viruses such as HPAI viruses10. Conceivably, the considerable host-switching mutations associated with adaptation of wild bird viruses to gallinaceous poultry, or at least of wild viruses to HPAI poultry viruses, represent an evolutionary pathway divergent from those pathways associated with mammalian adaptation, seemingly presenting an additional challenge for poultry-adapted influenza viruses to achieve efficient mammalian adaptation17. After 15 years of high-density enzootic circulation in domestic poultry around the world, no human-adapted H5N1 virus has emerged from a natural reservoir, suggesting the existence of unknown biological barriers.
Despite circulation of influenza A viruses of 16 HA subtypes in billions of birds over a very long time span, the four pandemics in the last century have been restricted to influenza viruses bearing HA subtypes H1, H2 or H3. Decades ago, many experts predicted that influenza pandemics could be explained by ‘recycling’ of a small number of HAs in new human generations; more recently, this belief has been expanded to posit that the other HA subtypes (including H5) are fundamentally incapable of adapting to humans, being selected against by biological constraints or unappreciated selection pressures18,19,20. Despite widespread influenza virus circulation and dynamic evolution at the human–animal interface, with many billions of quasispecies, mutations and gene constellations circulating, only four influenza pandemics have occurred in the last century, and in the three of those with a known viral origin the viruses resulted from reassortment of pre-existing human or swine viruses21, not by mutation or adaptation of existing avian viruses.
This suggests that de novo emergence of a human pandemic influenza virus is an extremely rare event that is not easily achieved in nature10, and presumably would not be easily achieved by engineering a small number of laboratory mutations. As some of the key engineered H5N1 mutations in the two studies occur spontaneously during normal laboratory passage22, or have been found singly or in combination in natural H5N1 and in other influenza viruses23,24,25,26, including strains from wild birds, it remains unclear whether or how the engineered viruses in question create or increase the risk of a pandemic.
Engineering H5N1 phenotypic changes
Serial passage of a virus in intact animals or in tissues derived from a particular species often results in enhanced species-specific virulence, which can be applied to establish an animal model with measurable morbidity and/or mortality outcomes useful for evaluating antiviral therapeutics, passive immunization and vaccines. Influenza viruses, SARS coronavirus and Ebola virus have all been passaged in mice to enhance virulence; the resulting host-adapted viruses have been studied biologically and used to evaluate strategies for control and prevention. However, adaptational mutations resulting from serial passage tend to be host-specific and may not produce the same outcomes in other species. For example, the classical swine influenza virus, A/swine/Iowa/1930 (H1N1), is very pathogenic in ferrets and mice but poses no threat to humans27. Another example is mouse-adapted Ebola virus, which is lethal for mice and guinea pigs but attenuated for nonhuman primates28,29. Ferrets are susceptible to a wide range of viruses including influenza viruses, SARS coronavirus, canine distemper virus and some parvoviruses, many of which do not infect humans or other mammals. A number of influenza viruses that replicate efficiently in ferrets30,31,32,33 seem poorly able, or unable, to infect humans, even after experimental challenge34. Thus, pathogenicity and transmissibility of any influenza virus in ferrets cannot be used directly to predict what type and severity of disease the same virus might produce in humans and human populations.
Predicting human transmissibility
It is unclear whether genetic manipulation of an H5N1 virus to achieve transmissibility in a particular mammal such as a ferret can predict human transmissibility. Because natural history and viral challenge studies cannot always be performed in humans, they have been conducted in experimental animals including mice, guinea pigs, ferrets, non-human primates and various other mammals. Unfortunately, there is no perfect animal model capable of reproducing all of the important variables involved in human influenza infection, although each animal model may be useful in understanding some aspect of influenza biology. Unlike most other mammals, ferrets generally can be infected with many or most avian, mammalian and human influenza viruses without prior viral adaptation, and often transmit efficiently between them35, providing useful general information about the viral genetic basis of phenotypic properties such as infectivity, pathogenicity, transmissibility and immune responses35, even though the findings cannot necessarily be directly applied to human infections36,37,38. Furthermore, in decades of research, using a large number of different avian and mammalian influenza viruses, severe or fatal disease has not often been observed in ferrets following intranasal inoculation or aerosol exposure.
These useful traits of easy infectability and mildly symptomatic infection have rendered the ferret a ‘permissive’ influenza model. Specifically, many naturally occurring influenza viruses that infect, and often transmit between, ferrets are not known to infect people or cause human disease27,30,31,32,33,39,40. Ferrets are thus an imperfect model for predicting human infectivity or transmissibility, let alone the high level of transmissibility characteristic of pandemic spread. On the basis of public presentations by the senior authors of the two studies in question, neither of the engineered H5N1 viruses was as efficiently transmissible in ferrets as the human-adapted 2009 pandemic H1N1 virus14,15. Phenotypic properties such as replication, pathogenicity and transmissibility are likely to be polygenic traits driven by mutations that are independent and possibly competing10,41. Transmissibility is a complex phenotype that probably requires cooperative changes in more than one gene segment, and these may differ greatly between different viruses that become transmissible. Mutations that confer transmissibility in a ferret may be species-specific and irrelevant to other hosts42. There are probably multiple unique virus-specific pathways to transmissibility for particular viruses infecting particular hosts43. For example, transmissibility of the 1918 pandemic H1N1 virus has been linked to changes in the genes encoding HA and PB2 proteins36,37, whereas transmissibility of the 2009 pandemic H1N1 virus, which has a closely related HA, has been linked to gene segments encoding the neuraminidase and matrix proteins44.
Moreover, because determinants of viral pathogenicity may be lost upon adaptation to a new host, H5N1 viruses, whether or not transmissible, do not always cause severe disease in ferrets or non-human primates45,46,47. For these reasons viral phenotypes observed in animal models like the ferret may not predict what would happen in humans. Indeed, given that many influenza viruses that are non-pathogenic for humans easily transmit and may cause illness in ferrets, the ‘ferret model’ does not reliably predict human transmissibility or pathogenicity, although the model remains valuable for understanding the principles and dynamics of infection.
In addition to data from experiments in mammals, it is noteworthy that of the many mammalian-adapted influenza viruses that infect and transmit explosively among pigs, among horses and among dogs, few infect humans and none are transmitted between them48. Although swine influenza viruses caused sporadic human infections before 200949,50, and caused the 2009 H1N1 pandemic21, outbreaks associated with human influenza viruses are rare in pigs. It is even conceivable that H5N1 viruses have already become adapted to mammals without causing severe disease or onward transmission to humans. Evidence from China’s Qinghai Lake, for example, shows 13.4% H5N1 seropositivity and 3.4% active infection in apparently healthy, live-caught rodents called pikas51. Clearly, adaptation of an influenza virus to a specific mammalian host does not necessarily predict its infectiousness, pathogenicity or transmissibility in other mammals, even though valuable insights into mechanisms of disease, host responses, and prevention and treatment may be obtained by studying these particular animals. Such insights can provide valuable clues in designing countermeasures against deadly epidemics and pandemics.
H5N1 case-fatality rate
Belief that an H5N1 virus could produce a 59% pandemic case-fatality rate is the most frightening aspect of the current controversy surrounding aerosol transmission of H5N1 virus in ferrets. In 500 years of observation, no influenza pandemic is believed to have caused a case-fatality rate over about 2% (ref. 52); pandemic and seasonal circulation of H1, H2 and H3 influenza viruses over the past century have all produced lower overall mortality rates53. The widely cited 59% figure is not a mortality rate but a case-fatality rate computed from cumulative cases reported to WHO since 20039. (Case fatality refers to fatal cases divided by all fatal plus non-fatal cases combined.) By general consensus, the WHO figure probably overestimates actual mortality. Among other concerns common to epidemiological data, the WHO case definition54 features diagnostic severity criteria (evidence of an acute pneumonia on chest radiograph plus evidence of respiratory failure) that constitute a self-fulfilling prophecy for fatality; as with many illnesses studied epidemiologically, severe diseases are more likely to receive optimal diagnostic work-up (‘detection bias’); and most H5N1 cases have been reported from countries with limited resources for identifying milder cases, if they occurred. These factors together could combine to erroneously inflate case-fatality calculations by over-counting severe cases and under-counting mild cases55.
However, potentially more important clues to actual H5N1 pathogenicity and human case-fatality rates come from epidemiological studies, which taken together suggest to us that H5N1 may not be highly lethal except in people with rare susceptibilities. Forty-six published H5N1 seroprevalence studies of various exposure categories (household contacts, healthcare workers, poultry workers, and so on) show generally low H5N1 seroprevalence (mean, 1.7% of 21,435 persons examined in all 46 studies combined (a bibliography of these studies is available from the authors on request)). Given intense poultry and other exposures in many study areas, these low rates at first seem perplexing, especially when compared to the much higher seroprevalence rates in humans for other avian influenza viruses such as H9N2 (ref. 56). When such information is considered in light of statistically significant clustering of non-human-transmitted (that is, presumably avian-acquired) household cases in genetically related versus unrelated persons57,58, a reasonable explanation seems to us to be that H5N1 is so poorly adapted to humans that exposure does not normally lead to infection or even the development of a detectable immune response57,59, except in persons with specific but undefined genetic susceptibilities, many of whom become cases60,61,62. There are few data on what the basis of such genetic susceptibilities may be, although recent evidence has linked severe human influenza to a minor IFITM3 allele63, supporting the suspicion that genetic determinants of influenza infection and replication in humans do exist.
A published meta-analysis of selected seroprevalence studies implies that the actual H5N1 case-fatality rate may be far below 1% (ref. 56), and thus probably far below the case-fatality rates for seasonal influenza. This has been disputed because it has been difficult to find mild cases, and because of the possibility that some low-level antibody titres (<1:80) might be false positives5. On the other hand, rapid disappearance of human H5N1 vaccine-induced antibody64 suggests that the opposite problem of false negatives could be occurring and, if so, might be especially problematic in cross-sectional studies in which the time since infection is not known, and which could in some cases be long enough for antibody titres to wane to sub-threshold or undetectable levels.
Given such confusing information, there has been little agreement so far on the important question of asymptomatic and undetected H5N1 infections. But whatever the case, unless healthy seropositive people detected in seroprevalence studies temporally and geographically associated with H5N1 cases are all falsely seropositive, their addition to exposure denominators greatly decreases case-fatality determinations. For example, the 1997 Hong Kong H5N1 outbreak case-fatality rate of 33.3% (ref. 65) drops to around 3% with the addition of exposed seropositive persons detected in the related seroprevalence studies. Similar recalculations of other data would yield far lower rates, and wider seroprevalence studies would undoubtedly lower case-fatality rates even further.
Thus, an explanation for the apparent case-fatality rate/seroprevalence paradox may not be purely one of missing cases. Like other poorly adapted viruses that rarely infect humans34, the H5N1 virus may simply be productively infecting too few of the people exposed to it, even in situations of widespread human contact, leaving minimal immunological evidence of exposure at the population level, while at the same time ‘finding’ and infecting those occasional persons with unusual susceptibilities to it; that is, cases59. Even so, it should be remembered that limited spread of a deadly H5N1 virus, or pandemic H5N1 spread associated with a far lower case-fatality rate, would still be of public health concern.
The dangers of information release
Owing to global concern over a possible H5N1 influenza pandemic, the pathogenicity, immunogenicity and transmissibility of naturally occurring and laboratory-derived H5N1 viruses have been examined extensively and safely using high-containment facilities and appropriate regulatory and safety oversight (see later). The two H5N1 studies under discussion1,2 build upon and are the logical extensions of dozens of similar published studies performed in the wake of the 1997 Hong Kong H5N1 outbreak. This research includes another published study in which genetic engineering of the H5N1 virus was able to newly create transmissibility in ferrets66, a similar study in which increased ferret transmissibility was not documented67, and a study in which transmissibility was restored and arguably increased in guinea pigs68. None of these publications, including the prior publication of engineered H5N1 transmissibility in ferrets, led to concern among scientists, federal agencies or the public.
Such studies feature numerous pathogenicity-associated, and sometimes transmissibility-associated, mutations affecting the HA-receptor-binding site, including changes that enhance receptor affinity for α2-6-linked sialic acid receptors, thought to be important for human adaptation25,69,70,71. Other studies have examined mutations associated with changing antigenicity72,73, changes associated with fusion25,74, changes associated with the polybasic HA cleavage site75,76,77, and virulence factors in the polymerase proteins, crucial for viral replication24,68, and in the non-structural protein (NS1), involved in antagonizing host type I interferon responses78,79,80. This widely available body of published research complicates determination of what to do with these two and with similar research manuscripts that seem likely to continue to appear. Withholding or redacting them does not prevent anyone from piecing together the basic information that they contain. Most of this information is generally known and relatively obvious, has already been published, and is now being widely publicized and discussed as a result of increased attention11,13,14,81,82.
Some would argue that even this background research should not have been done, or should henceforth be classified and made available only to ‘approved’ scientists who would be vetted by yet-to-be-determined mechanisms83,84. But had these former studies not been made available in the open literature, the field of influenza research would have been considerably impeded and our current state of knowledge and readiness for responding to future outbreaks and/or pandemics would be lessened. Some proposed that ‘censoring’ this information actually increases the risk of bioterrorism85. The two studies under discussion1,2 can help augment surveillance to detect naturally emerging viruses with pandemic potential and expand our knowledge of the principles underlying host adaptation. Although the dangers of ‘information release’ in the case of these two studies is probably small or nil—because all or most of the critical information is widely available anyway—it nevertheless remains important to rethink larger questions about balancing safety (accidental or deliberate release of an influenza virus or any dangerous pathogen) with the need to study such viruses to learn enough of their biology to prevent and control them. These are important issues that should be discussed broadly among scientists, policy makers and the public.
Biosafety and biosecurity concerns
As novel pathogens emerge, scientists must be able to continue to work with them safely and appropriately in teams using the talents of many highly trained researchers. Numerous layers of robust biosafety and biosecurity protection and oversight are in place to safeguard the scientists and the public alike, including rigorous safety training, biocontainment practices, regulations and oversight, select agent rules, background investigations and biosurety oversight86. The H5N1 studies under discussion1,2 were both performed in high containment laboratories with rigorous and appropriate oversight and biosecurity measures15, as is the case for all such research in the US.
Few disagree that it is crucial to continue research with H5N1 and other emerging infections, including investigation of how emerging pathogens adapt to new hosts and cause disease. However, it is important to ask whether some types of infectious diseases research should not be done, or not published openly. If so, we need criteria to identify such research in advance, and processes to balance the importance of the research knowledge with the importance of preventing adverse consequences of the research87. Even with the eventual publication of the two H5N1 studies, questions about how such research should be approved, evaluated and made public remain unanswered83,84. The biomedical field is built on more than a century of openness and full publication/broad discussion of all findings; it is unclear how redacted publications of future scientific data can be accomplished, and what effect such a system would have on science and scientific progress.
These complex questions have been asked and answered in the past87, and are being asked again in the context of these two papers. Continued discussion and decisions about how to deal with this research will be of importance to scientific progress and public health. We believe that it is important to consider the broader context of research aimed at understanding how influenza viruses adapt to humans. H5N1 is only one of many avian influenza viruses. If, as we believe existing data suggest, pathways to human adaptation are many, virus-specific, and with few common denominators, it will be important to study not just H5N1 but a wide range of avian and mammalian- and human-adapted viruses, including studies that feature backward genetic engineering to remove phenotypic determinants of adaptation, studies in nonhuman primates and, when safe and appropriate to do so, in human challenge studies88.
The H5N1 controversy underscores how little is known about determinants of human influenza pathogenicity and transmissibility, which are among the most fundamentally important questions in infectious disease research because of the huge burden of influenza.
In the past two decades the question of pursuing and publishing potential ‘dual use’ infectious disease research has always been decided in favour of conducting and publishing the research; for example, delineating the genomes of smallpox89 and SARS viruses90, defining the pathogenicity of neuraminidase-inhibitor-resistant influenza viruses91,92, genetically altering and making ferret transmissible both a more pathogenic pandemic H2N2 influenza virus93 and an H9N2 avian influenza virus40, and resurrecting from RNA fragments, recreating and studying in vivo the 1918 pandemic influenza virus94,95,96. In the latter case, important findings already have markedly enhanced our understanding of the emergence, transmissibility and pathogenicity of that important virus, helping us to prepare for and respond to the emergence of other influenza viruses. Examples include using the 1918 HA crystal structure in vaccine design97, investigating the role of the host immune response in disease98,99, identification of mutations associated with pathogenicity and host adaptation36,100,101, understanding influenza evolution21, and helping guide and target the response to the 2009 H1N1 pandemic53,102. All of this work has been done safely with appropriate oversight, and without negative consequences.
In considering the threat of bioterrorism or accidental release of genetically engineered viruses, it is worth remembering that nature is the ultimate bioterrorist. Indeed, H5N1 mutations, including some of those made in the two studies under discussion1,2, occur spontaneously in nature, probably at a high rate, although they have not yet led to a pandemic. Given the relative rarity of pandemics caused by newly emerging influenza viruses, their explosive transmissibility may result from unique and virus-specific mutational changes that arise at very low frequency. For past pandemics, we have had limited ability to detect such changes by surveillance or by animal model experimentation. Thus, our best hope in preventing and controlling the microbial agents that continually challenge us is to increase fundamental knowledge about the mechanisms by which they emerge, spread and cause disease, so that we can develop countermeasures such as enhanced surveillance, better diagnostics, vaccines and drug therapies. In moving forward we need to be safety conscious and to have consensus safety measures and policies in place, while at the same time using all available tools to seek broad understanding about the complex relationships between viruses and hosts. It is only this knowledge that stands between us and the devastation of future influenza pandemics. In reconsidering the proper balance between progress and safety, the critical importance of advancing scientific knowledge needs to be kept front and centre.
Imai, M. et al. Experimental adaptation of an influenza H5 HA confers respiratory droplet transmission to a reassortant H5 HA/H1N1 virus in ferrets. Naturehttp://dx.doi.org/10.1038/nature10831 (this issue)
Herfst, S. et al. Aerosol transmission of avian influenza A/H5N1 virus. Science (22 June 2012)
Doherty, P. C. & Thomas, P. G. Dangerous for ferrets: lethal for humans? BMC Biol. 10, 10 (2012)
Fouchier, R. et al. Preventing pandemics: the fight over flu. Nature 481, 257–259 (2012)
Osterholm, M. T. & Kelley, N. S. Mammalian-transmissible H5N1 influenza: facts and perspective. MBio 3, 2 (2012)
Palese, P. Don’t censor life-saving science. Nature 481, 115 (2012)
Webster, R. G. Mammalian-transmissible H5N1 influenza: the dilemma of dual-use research. MBio 3, 1 (2012)
Parrish, C. R. et al. Cross-species virus transmission and the emergence of new epidemic diseases. Microbiol. Mol. Biol. Rev. 72, 457–470 (2008)Authors review data on viral host-switch events.
WHO. Cumulative number of confirmed human cases of avian influenza A(H5N1) reported to WHO. http://www.who.int/influenza/human_animal_interface/H5N1_cumulative_table_archives/en/index.html (2012)
Taubenberger, J. K. & Kash, J. C. Influenza virus evolution, host adaptation, and pandemic formation. Cell Host Microbe 7, 440–451 (2010)A review of influenza host adaptation and pandemic formation.
Brown, T. H5N1 research discussed in special session at ASM. http://www.medscape.com/viewarticle/759465 (2012)
Fouchier, R. A. et al. Pause on avian flu transmission research. Science 335, 400–401 (2012)
Kawaoka, Y. H5N1: Flu transmission work is urgent. Nature 482, 155 (2012)
Osterholm, M. T., Fauci, A. S., Alberts, B. & Fouchier, R. A. M. Discussion of NSABB’s publication recommendations for the NIH-funded research on the transmissibility of H5N1. http://www.asmbiodefense.org/index.php/program-information/nsabbs-recommendations-for-h5n1-research (2012)
Kawaoka, Y. & Fouchier, R. A. Royal Society meeting. H5N1 research: biosafety, biosecurity and bioethics. http://royalsociety.org/events/2012/viruses/ (2012)
Wilson, H. E., Saslaw, S., Doan, C. A., Woolpert, O. C. & Schwab, J. L. Reactions of monkeys to experimental mixed influenza and Streptococcus infections: an analysis of the relative roles of humoral and cellular immunity, with the description of an intercurrent nephritic syndrome. J. Exp. Med. 85, 199–215 (1947)
Swayne, D. E. Understanding the complex pathobiology of high pathogenicity avian influenza viruses in birds. Avian Dis. 51, 242–249 (2007)A review of highly pathogenic avian influenza viruses.
Francis, T., Jr Influenza: the new acquayantance. Ann. Intern. Med. 39, 203–221 (1953)
Masurel, N. & Marine, W. M. Recycling of Asian and Hong Kong influenza A virus hemagglutinins in man. Am. J. Epidemiol. 97, 44–49 (1973)
Hilleman, M. R. Realities and enigmas of human viral influenza: pathogenesis, epidemiology and control. Vaccine 20, 3068–3087 (2002)
Morens, D. M., Taubenberger, J. K. & Fauci, A. S. The persistent legacy of the 1918 influenza virus. N. Engl. J. Med. 361, 225–229 (2009)
Bogs, J. et al. Reversion of PB2-627E to -627K during replication of an H5N1 Clade 2.2 virus in mammalian hosts depends on the origin of the nucleoprotein. J. Virol. 85, 10691–10698 (2011)
Chen, H. et al. Properties and dissemination of H5N1 viruses isolated during an influenza outbreak in migratory waterfowl in western China. J. Virol. 80, 5976–5983 (2006)
Hatta, M., Gao, P., Halfmann, P. & Kawaoka, Y. Molecular basis for high virulence of Hong Kong H5N1 influenza A viruses. Science 293, 1840–1842 (2001)A study showing the key role that changes in a viral polymerase gene has in H5N1 virulence in a mammalian model.
Stevens, J. et al. Structure and receptor specificity of the hemagglutinin from an H5N1 influenza virus. Science 312, 404–410 (2006)A study describing the haemagglutinin crystal structure of an H5N1 influenza virus and studies of mutations associated with receptor-binding specificity.
Yen, H. L. et al. Inefficient transmission of H5N1 influenza viruses in a ferret contact model. J. Virol. 81, 6890–6898 (2007)
Memoli, M. J. et al. An early ‘classical’ swine H1N1 influenza virus shows similar pathogenicity to the 1918 pandemic virus in ferrets and mice. Virology 393, 338–345 (2009)
Bray, M., Davis, K., Geisbert, T., Schmaljohn, C. & Huggins, J. A mouse model for evaluation of prophylaxis and therapy of Ebola hemorrhagic fever. J. Infect. Dis. 178, 651–661 (1998)
Bray, M., Hatfill, S., Hensley, L. & Huggins, J. W. Haematological, biochemical and coagulation changes in mice, guinea-pigs and monkeys infected with a mouse-adapted variant of Ebola Zaire virus. J. Comp. Pathol. 125, 243–253 (2001)
Chen, G. L. et al. Evaluation of replication and cross-reactive antibody responses of H2 subtype influenza viruses in mice and ferrets. J. Virol. 84, 7695–7702 (2010)
Hinshaw, V. S., Webster, R. G., Easterday, B. C. & Bean, W. J., Jr Replication of avian influenza A viruses in mammals. Infect. Immun. 34, 354–361 (1981)
Joseph, T. et al. Evaluation of replication and pathogenicity of avian influenza a H7 subtype viruses in a mouse model. J. Virol. 81, 10558–10566 (2007)
Song, H., Wan, H., Araya, Y. & Perez, D. R. Partial direct contact transmission in ferrets of a mallard H7N3 influenza virus with typical avian-like receptor specificity. Virol. J. 6, 126 (2009)
Beare, A. S. & Webster, R. G. Replication of avian influenza viruses in humans. Arch. Virol. 119, 37–42 (1991)
Belser, J. A., Katz, J. M. & Tumpey, T. M. The ferret as a model organism to study influenza A virus infection. Dis. Model Mech. 4, 575–579 (2011)A review of the ferret as a model organism in influenza virus pathogenicity studies.
Tumpey, T. M. et al. A two-amino acid change in the hemagglutinin of the 1918 influenza virus abolishes transmission. Science 315, 655–659 (2007)A study elucidating the importance of the haemagglutinin-receptor-binding region in conferring ferret transmissibility in the 1918 pandemic influenza virus.
Van Hoeven, N. et al. Human HA and polymerase subunit PB2 proteins confer transmission of an avian influenza virus through the air. Proc. Natl Acad. Sci. USA 106, 3366–3371 (2009)A study describing the importance of both the 1918 haemagglutinin and polymerase PB2 genes in conferring ferret transmissibility in viruses expressing 1918 pandemic influenza virus genes.
Watanabe, T. et al. Viral RNA polymerase complex promotes optimal growth of 1918 virus in the lower respiratory tract of ferrets. Proc. Natl Acad. Sci. USA 106, 588–592 (2009)
Kimble, J. B., Sorrell, E., Shao, H., Martin, P. L. & Perez, D. R. Compatibility of H9N2 avian influenza surface genes and 2009 pandemic H1N1 internal genes for transmission in the ferret model. Proc. Natl Acad. Sci. USA 108, 12084–12088 (2011)
Sorrell, E. M., Wan, H., Araya, Y., Song, H. & Perez, D. R. Minimal molecular constraints for respiratory droplet transmission of an avian-human H9N2 influenza A virus. Proc. Natl Acad. Sci. USA 106, 7565–7570 (2009)
Koster, F. et al. Exhaled aerosol transmission of pandemic and seasonal H1N1 influenza viruses in the ferret. PLoS ONE 7, e33118 (2012)
Yamanaka, T. et al. No evidence of horizontal infection in horses kept in close contact with dogs experimentally infected with canine influenza A virus (H3N8). Acta Vet. Scand. 54, 25 (2012)
Mänz, B., Brunotte, L., Reuther, P. & Schwemmle, M. Adaptive mutations in NEP compensate for defective H5N1 RNA replication in cultured human cells. Nature Commun. 3, 802 (2012)
Lakdawala, S. S. et al. Eurasian-origin gene segments contribute to the transmissibility, aerosol release, and morphology of the 2009 pandemic H1N1 influenza virus. PLoS Pathog. 7, e1002443 (2011)
Chen, Y. et al. Pathological lesions and viral localization of influenza A (H5N1) virus in experimentally infected Chinese rhesus macaques: implications for pathogenesis and viral transmission. Arch. Virol. 154, 227–233 (2009)
Govorkova, E. A. et al. Lethality to ferrets of H5N1 influenza viruses isolated from humans and poultry in 2004. J. Virol. 79, 2191–2198 (2005)
Maines, T. R. et al. Avian influenza (H5N1) viruses isolated from humans in Asia in 2004 exhibit increased virulence in mammals. J. Virol. 79, 11788–11800 (2005)
Morens, D. M. & Taubenberger, J. K. Historical thoughts on influenza viral ecosystems, or behold a pale horse, dead dogs, failing fowl, and sick swine. Influenza Other Respir. Viruses 4, 327–337 (2010)
Myers, K. P., Olsen, C. W. & Gray, G. C. Cases of swine influenza in humans: a review of the literature. Clin. Infect. Dis. 44, 1084–1088 (2007)
Shinde, V. et al. Triple-reassortant swine influenza A (H1) in humans in the United States, 2005–2009. N. Engl. J. Med. 360, 2616–2625 (2009)
Zhou, J. et al. Characterization of the H5N1 highly pathogenic avian influenza virus derived from wild pikas in China. J. Virol. 83, 8957–8964 (2009)
Morens, D. M. & Taubenberger, J. K. Pandemic influenza: certain uncertainties. Rev. Med. Virol. 21, 262–284 (2011)A historical review of influenza pandemics.
Morens, D. M., Taubenberger, J. K. & Fauci, A. S. The 2009 H1N1 pandemic influenza virus: what next? MBio 1, 4 (2010)
WHO. WHO case definitions for human infections with influenza A(H5N1) virus. http://www.who.int/influenza/resources/documents/case_definition2006_08_29/en/index.html (2006)
Palese, P. & Wang, T. T. H5N1 influenza viruses: facts, not fear. Proc. Natl Acad. Sci. USA 109, 2211–2213 (2012)
Wang, T. T., Parides, M. K. & Palese, P. Seroevidence for H5N1 influenza infections in humans: meta-analysis. Science 335, 1463 (2012)
Aditama, T. Y. et al. Risk factors for cluster outbreaks of avian influenza A H5N1 infection, Indonesia. Clin. Infect. Dis. 53, 1237–1244 (2011)A study describing human H5N1 case clusters in Indonesia.
Aditama, T. Y. et al. Avian influenza H5N1 transmission in households, Indonesia. PLoS ONE 7, e29971 (2012)
Roos, R. No sign of missed H5N1 cases in Bangladesh study. http://www.cidrap.umn.edu/cidrap/content/influenza/avianflu/news/mar1312seroprev.html (2012)
Horby, P. et al. What is the evidence of a role for host genetics in susceptibility to influenza A/H5N1? Epidemiol. Infect. 138, 1550–1558 (2010)
Trammell, R. A. & Toth, L. A. Genetic susceptibility and resistance to influenza infection and disease in humans and mice. Expert Rev. Mol. Diagn. 8, 515–529 (2008)
Zhang, L., Katz, J. M., Gwinn, M., Dowling, N. F. & Khoury, M. J. Systems-based candidate genes for human response to influenza infection. Infect. Genet. Evol. 9, 1148–1157 (2009)
Everitt, A. R. et al. IFITM3 restricts the morbidity and mortality associated with influenza. Nature 484, 519–523 (2012)
Brady, R. C. et al. Safety and immunogenicity of a subvirion inactivated influenza A/H5N1 vaccine with or without aluminum hydroxide among healthy elderly adults. Vaccine 27, 5091–5095 (2009)
Katz, J. M. et al. Antibody response in individuals infected with avian influenza A (H5N1) viruses and detection of anti-H5 antibody among household and social contacts. J. Infect. Dis. 180, 1763–1770 (1999)
Chen, L. M. et al. In vitro evolution of H5N1 avian influenza virus toward human-type receptor specificity. Virology 422, 105–113 (2012)
Maines, T. R. et al. Effect of receptor binding domain mutations on receptor binding and transmissibility of avian influenza H5N1 viruses. Virology 413, 139–147 (2011)
Steel, J., Lowen, A. C., Mubareka, S. & Palese, P. Transmission of influenza virus in a mammalian host is increased by PB2 amino acids 627K or 627E/701N. PLoS Pathog. 5, e1000252 (2009)
Ilyushina, N. A., Govorkova, E. A., Gray, T. E., Bovin, N. V. & Webster, R. G. Human-like receptor specificity does not affect the neuraminidase-inhibitor susceptibility of H5N1 influenza viruses. PLoS Pathog. 4, e1000043 (2008)
Yamada, S. et al. Haemagglutinin mutations responsible for the binding of H5N1 influenza A viruses to human-type receptors. Nature 444, 378–382 (2006)
Yen, H. L. et al. Changes in H5N1 influenza virus hemagglutinin receptor binding domain affect systemic spread. Proc. Natl Acad. Sci. USA 106, 286–291 (2009)
Rudneva, I. A. et al. Antigenic epitopes in the hemagglutinin of Qinghai-type influenza H5N1 virus. Viral Immunol. 23, 181–187 (2010)
Wang, W. et al. Glycosylation at 158N of the hemagglutinin protein and receptor binding specificity synergistically affect the antigenicity and immunogenicity of a live attenuated H5N1 A/Vietnam/1203/2004 vaccine virus in ferrets. J. Virol. 84, 6570–6577 (2010)
Sui, J. et al. Structural and functional bases for broad-spectrum neutralization of avian and human influenza A viruses. Nature Struct. Mol. Biol. 16, 265–273 (2009)
Bogs, J. et al. Highly pathogenic H5N1 influenza viruses carry virulence determinants beyond the polybasic hemagglutinin cleavage site. PLoS ONE 5, e11826 (2010)
Gohrbandt, S. et al. Amino acids adjacent to the haemagglutinin cleavage site are relevant for virulence of avian influenza viruses of subtype H5. J. Gen. Virol. 92, 51–59 (2011)
Suguitan, A. L., Jr et al. The multibasic cleavage site of the hemagglutinin of highly pathogenic A/Vietnam/1203/2004 (H5N1) avian influenza virus acts as a virulence factor in a host-specific manner in mammals. J. Virol. 86, 2706–2714 (2012)
Seo, S. H., Hoffmann, E. & Webster, R. G. Lethal H5N1 influenza viruses escape host anti-viral cytokine responses. Nature Med. 8, 950–954 (2002)
Spesock, A. et al. The virulence of 1997 H5N1 influenza viruses in the mouse model is increased by correcting a defect in their NS1 proteins. J. Virol. 85, 7048–7058 (2011)
Zielecki, F. et al. Virulence determinants of avian H5N1 influenza A virus in mammalian and avian hosts: role of the C-terminal ESEV motif in the viral NS1 protein. J. Virol. 84, 10708–10718 (2010)
MacKenzie, D. Five easy mutations to make bird flu a lethal pandemic. New Sci. http://www.newscientist.com/article/mg21128314.600-five-easy-mutations-to-make-bird-flu-a-lethal-pandemic.html (2011)
WHO. Public health, influenza experts agree H5N1 research critical, but extend delay. http://www.who.int/mediacentre/news/releases/2012/h5n1_research_20120217/en/index.html (2012)
NSABB. National Science Advisory Board for Biosecurity Findings and Recommendations March 29–30, 2012. http://oba.od.nih.gov/biosecurity/biosecurity_documents.html (NSABB, 2012)
WHO. Report on technical consultation on H5N1 research issues. http://www.who.int/influenza/human_animal_interface/consensus_points/en/index.html (2012)
MacKenzie, D. Censoring flu data could raise bioterror threat.. New Sci http://www.newscientist.com/article/dn21675-censoring-flu-data-could-raise-bioterror-threat.html (2012)
CDC. in Biosafety in Microbiological and Biomedical Laboratories (BMBL) (eds Chosewood, L. C. & Wilson, D. E. ) Ch. 8 (Government Printing Office, 2009)
National Research Council. in Science and Security in a Post 9/11 World: A Report Based on Regional Discussions Between the Science and Security Communities Ch. 4, 57–68 (NAS, 2007)
Killingley, B. et al. Potential role of human challenge studies for investigation of influenza transmission. Lancet Infect. Dis. 11, 879–886 (2011)
Massung, R. F. et al. Potential virulence determinants in terminal regions of variola smallpox virus genome. Nature 366, 748–751 (1993)
Marra, M. A. et al. The genome sequence of the SARS-associated coronavirus. Science 300, 1399–1404 (2003)
Herlocher, M. L. et al. Influenza viruses resistant to the antiviral drug oseltamivir: transmission studies in ferrets. J. Infect. Dis. 190, 1627–1630 (2004)
Memoli, M. J. et al. Multidrug-resistant 2009 pandemic influenza A(H1N1) viruses maintain fitness and transmissibility in ferrets. J. Infect. Dis. 203, 348–357 (2011)
Pappas, C. et al. Receptor specificity and transmission of H2N2 subtype viruses isolated from the pandemic of 1957. PLoS ONE 5, e11158 (2010)
Taubenberger, J. K. & Kash, J. C. Insights on influenza pathogenesis from the grave. Virus Res. 162, 2–7 (2011)
Taubenberger, J. K. et al. Characterization of the 1918 influenza virus polymerase genes. Nature 437, 889–893 (2005)
Tumpey, T. M. et al. Characterization of the reconstructed 1918 Spanish influenza pandemic virus. Science 310, 77–80 (2005)
Fleishman, S. J. et al. Computational design of proteins targeting the conserved stem region of influenza hemagglutinin. Science 332, 816–821 (2011)
Kash, J. C. et al. Genomic analysis of increased host immune and cell death responses induced by 1918 influenza virus. Nature 443, 578–581 (2006)
Kobasa, D. et al. Aberrant innate immune response in lethal infection of macaques with the 1918 influenza virus. Nature 445, 319–323 (2007)
Pappas, C. et al. Single gene reassortants identify a critical role for PB1, HA, and NA in the high virulence of the 1918 pandemic influenza virus. Proc. Natl Acad. Sci. USA 105, 3064–3069 (2008)
Qi, L. et al. The ability of pandemic influenza virus hemagglutinins to induce lower respiratory pathology is associated with decreased surfactant protein D binding. Virology 412, 426–434 (2011)
Morens, D. M., Taubenberger, J. K., Harvey, H. A. & Memoli, M. J. The 1918 influenza pandemic: lessons for 2009 and the future. Crit. Care Med. 38, e10–e20 (2010)
This work was supported by the National Institutes of Health and the National Institute of Allergy and Infectious Diseases. The authors declare no competing financial interests. We thank L. Qi for translation and discussion of a Chinese language publication.
The authors declare no competing financial interests.
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Morens, D., Subbarao, K. & Taubenberger, J. Engineering H5N1 avian influenza viruses to study human adaptation. Nature 486, 335–340 (2012). https://doi.org/10.1038/nature11170
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