Tag Archives: SARS

Genetically Modified Emerging Viruses: Debate over Gain-of-Function Research

Scientists generally loathe restrictions on their freedom to conduct research. Nonetheless, many virologists recognize the need to regulate studies that involve live, potentially pandemic, deadly pathogens, especially when those investigations involve modifying and even creating such pathogens.

We encountered this issue in an earlier posting (1), which told how in 2012 Yoshihiro Kawaoka and Ron Fouchier independently created variants of the H5N1 avian influenza virus that could be transmitted between ferrets (2, 3). They did so by using site-specific mutagenesis to modify the HA protein of the avian virus. Ferrets were used because they are a good model for influenza transmission between humans.

Important to our story, avian influenza viruses are spread in bird populations by the fecal-oral route. And, while H5N1 avian influenza viruses can be extremely pathogenic in humans, they have not yet naturally acquired the ability to be transmissible through the air—an ability that is necessary for influenza viruses to be pandemic in humans. So, to ascertain whether H5N1 avian influenza viruses could adapt to become transmissible via aerosols under natural conditions, both Kawaoka and Fouchier passaged their genetically modified H5N1 avian viruses in ferrets. The genetically modified H5N1 viruses indeed acquired additional mutations during passage in ferrets, which enabled them to become transmissible via the respiratory route. Moreover, each research group reported that the airborne-adapted mutant H5N1 viruses caused lung pathology in the recipient ferrets, none of which died.

The NIH supports studies like the above because of their potential to shed light on the interactions between emerging deadly pathogens and humans, and because they might help to clarify just how threatening these emerging viruses actually are. But regardless of those considerations, the White House Office of Science and Technology Policy (OSTP) and the Department of Health and Human Services were concerned by the potential risks of the avian flu experiments. Consequently, in October 2014 those governmental agencies initiated an assessment of gain-of-function research—that is, studies in which pathogens are manipulated to alter their virulence and transmissibility. Biosafety and biosecurity were the government’s key concerns. Possible risks included the prospects of a potentially pandemic modified virus either escaping from the laboratory, or being stolen from the laboratory and being misused to threaten public health and national security.

With those concerns in mind, the government imposed a temporary suspension of funding of new gain-of-function projects. The OSTP announced that effected studies would include those which “may be reasonably anticipated to confer attributes to influenza, MERS, or SARS viruses such that the virus would have enhanced pathogenicity and/or transmissibility in mammals via the respiratory route.” In addition, the government requested that researchers already carrying out gain-of-function projects should “voluntarily” postpone their studies until the risks might be evaluated by the National Science Advisory Board for Biosecurity (NSABB) and the National Research Council (NRC) of the National Academies.

As expected, these government-imposed restrictions caused quite a bit of controversy in the research community. Some scientists expressed concern that a ban on gain-of-function experiments might be applied too broadly, to include less dangerous types of work, such as development of seasonal influenza vaccines. [In that instance, gain-of-function research might be useful to evaluate the transmissibility of particular influenza strains, and to asses how those strains might mutate to evade candidate vaccines.] The government responded to this concern by modifying its review protocols in order to take public health considerations into account. Yet some researchers still feared that valuable research time could be lost while waiting for an exemption.

But what of experiments such as those of Fouchier and Kawaoka, which do entail a clear and present risk to public safety? The key question in those instances is whether the knowledge gained from the experiments might afford a benefit that is significant enough to justify the danger. Unfortunately, the answer is not always clear, as thoughtful individuals on each side of the debate make valid arguments. And, even if there were a consensus on the merit of a project, the delay in funding imposed by the review process might again cause valuable research time to be lost, or perhaps even destroy the research program, or cause outstanding young scientists to turn to other areas of inquiry, or even end their research careers.

The debate over government imposed restrictions on gain-of-function research waned somewhat after they were first announced. However, it was reignited last month by the announcement by Ralph Baric and co-workers at the University of North Carolina that they had created a chimeric SARS-like virus, which expresses the spike (attachment protein) of a bat coronavirus in a mouse-adapted SARS-CoV backbone (4). As in the cases of the genetically modified H5N1 avian influenza viruses, the newly generated SARS-like virus is potentially an extremely dangerous, possibly pandemic pathogen.

Coronaviruses, showing their characteristic spikes, which give them their characteristic “crown-like” (coronal) appearance
Coronaviruses, showing their characteristic spikes, which give them their characteristic “crown-like” (coronal) appearance

Baric’s team generated the chimeric SARS-like virus using the SARS-CoV reverse genetics system. The justification for the project was to evaluate the risk of SARS coronaviruses emerging from coronaviruses currently circulating in bats. Apropos that, the origin of the SARS coronavirus is not known for certain. However, the genetic diversity of coronaviruses in bats, in which they are avirulent, is consistent with the possibility that bats are a reservoir for SARS-coronaviruses.

The North Carolina group reported that their hybrid SARS-like virus could indeed bind to, and replicate efficiently in human airway cells in vitro. In fact, the chimeric virus replicated as well as epidemic strains of SARS-CoV in the human cells. Moreover, the chimeric virus replicated in, and caused severe pathogenesis in mouse lung in vivo.

Baric and co-workers began their project before the government announced the moratorium. Yet the work was allowed to continue because it was judged not risky enough to be bound by the restrictions; a decision that has since provoked quite a bit of controversy. Moreover, the North Carolina researchers themselves acknowledged the risk of their studies, noting, “Scientific review panels may deem similar studies building chimeric viruses based on circulating strains too risky to pursue…(4)”

Still, the key question is whether Baric’s experimental findings are important enough to justify their risk. At least some in the science community contend that they do not meet that test. In any case, Baric intends to study his new SARS-like virus in non-human primates, for the purpose of better understanding the potential threat of bat coronaviruses to humans.

References:

1. Opening Pandora’s Box: Resurrecting the 1918 Influenza Pandemic Virus and Transmissible H5N1 Bird Flu, posted on the blog April 15, 2014.

2. Imai M, Watanabe T, Hatta M, Das SC, Ozawa M, Shinya K, Zhong G, Hanson A, Katsura H, Watanabe S, Li C, Kawakami E, Yamada S, Kiso M, Suzuki Y, Maher EA, Neumann G, Kawaoka Y. 2012. Experimental adaptation of an influenza H5 HA confers respiratory droplet transmission to a reassortant H5 HA/H1N1 virus in ferrets. Nature 486:420-428.

3. Herfst S, Schrauwen EJ, Linster M, Chutinimitkul S, de Wit E, Munster VJ, Sorrell EM, Bestebroer TM, Burke DF, Smith DJ, Rimmelzwaan GF, Osterhaus AD, Fouchier RA. 2012. Airborne transmission of influenza A/H5N1 virus between ferrets. Science 336:1534-1541.

4. Menachery VD, Yount Jr BL, Debbink K, Agnihothram S, Gralinski LE, Plante JA, Graham RL, Scobey T, Ge XY, Donaldson EF, Randell SH, Lanzavecchia A, Marasco WA, Sh ZL, Baric RS. 2015. A SARS-like cluster of circulating bat coronaviruses shows potential for human emergence. Nature Medicine doi:10.1038/nm.3985

Opening Pandora’s Box: Resurrecting the 1918 Influenza Pandemic Virus and Transmissible H5N1 Bird Flu

The 1918 influenza pandemic killed an estimated 50 million people worldwide, making it the deadliest epidemic in human history. And despite the passage of nearly a century, a number of unexplained mysteries remain concerning the 1918 pandemic virus. A mystery important to our story is that the 1918 virus suddenly and inexplicably disappeared from the world in the early 1920s. And, since influenza virus was not even identified until the 1930s, no samples of the 1918 influenza strain were isolated at the time of the pandemic. Therefore, the 1918 pandemic virus did not exist in the world until it was “resurrected” nearly 80 years later by Jeffery Taubenberger and his colleagues, who used new, state-of-the-art molecular techniques to accomplish that feat.

More recently, in 2011, two independent research groups, one led by Yoshihiro Kawaoka and the other by Ron Fouchier, modified an H5N1 bird flu (see Aside 5) from a form that does not spread between humans, to forms that very well might. The unmodified avian virus has thus far infected only about 600 humans, in almost all instances by close contact with a diseased bird. But, and importantly, the avian virus killed more than half of the infected humans; a fatality rate far greater than that of even the 1918 pandemic virus. Thus, the resurrection of the 1918 pandemic virus, and the creation of transmissible H5N1 avian influenza, may have brought into the world pathogens with the potential to unleash extraordinary devastation.

These stories are compelling scientifically, historically, and for the public policy issues that they raise. As usual, we begin with some background.

The initial outbreak of the 1918 influenza pandemic occurred in March of that year, at an Army training camp outside of Boston. Yet by the fall of 1918 it was being referred to as the “Spanish” flu, probably because Spain, as a non-combatant in World War I, then in its final year, did not censor news of the pandemic. The combatants, on the other hand, fearing that news of the pandemic might cause panic that might undermine their war efforts, repressed news of it.

By the end of the winter of 1918-1919, two billion people around the world contracted the pandemic influenza strain and, as noted above, estimates of the total number of fatalities range as high as 50 million. That amount is about twice as many as would die of AIDS worldwide during the entire first twenty years of the AIDS epidemic. Moreover, the 1918 influenza pandemic killed more people in a single year than the four-year bubonic plague that ravaged Europe from 1347 to 1351. In the United States alone there were an estimated twenty million cases (out of a population of 100 million at the time) and 850,000 dead, including 196,000 people killed during the single month of October1918.

spanish fluRed Cross workers remove victims of the 1918 influenza pandemic from a house in St. Louis.      St. Louis Post-Dispatch

[Aside 1: Influenza virus pandemics also occurred in 1957 (the “Asian” flu) and 1968 (the “Hong Kong” flu). However, those pandemics were much less devastating than the pandemic of 1918. The number of deaths in the United States from those latter flu pandemics is estimated to be 70,000 and 50,000, respectively.]

Bearing in mind the sheer devastation of the 1918 pandemic, consider Taubenberger’s following comments from 1997: “It is curious that the (1918) pandemic doesn’t seem to be part of the cultural memory, at least in the United States, although it was a huge event with a huge impact. Everyone hears about the Black Death in the 1300s, yet here was an infectious disease only 85 years ago that killed 40 million people and for some reason we don’t know about it.”

It also is rather curious that while the 1918 influenza pandemic killed an astonishingly large number of people, it did not cause any public panic. Apropos that, in my last posting, Jonas Salk and Albert Sabin: One of the Great Rivalries of Medical Science, I noted that the annual poliovirus outbreaks, in the pre-vaccine days of the 1940s and 1950s, did cause widespread public panic. Moreover, that was so despite the fact that poliomyelitis actually caused fewer fatalities than were caused by seasonal influenza, to which the public then and now seems rather indifferent.

Another of the mysteries associated with the 1918 pandemic is that the first cases in March 1918 were relatively benign. Then, in August, the mild infection suddenly changed into something astonishingly lethal. Initial outbreaks of the new lethal variant of the virus occurred almost simultaneously in three locations; France, Sierra Leone, and Boston, and then spread worldwide. The changed virus struck with a ferocity that stunned medical professionals.

Influenza’s genetic variability is a well known characteristic of the virus. [Indeed, it is the reason why the flu vaccine needs to be re-formulated each year.] Regardless, it is not clear how the 1918 pandemic virus suddenly became so deadly. Many of the fatalities resulting from our yearly seasonal influenza epidemics are due to pneumonia caused by opportunistic bacterial pathogens. And, while bacterial pneumonia also killed many during the 1918 pandemic, the 1918 virus itself was quickly lethal in many individuals. Some patients had massively hemorrhaged lungs, and were effectively drowning in their own blood; a scenario more reminiscent of the pathology of Ebola virus than of the fevers and aches typically associated with seasonal influenza infections.

Indeed, the 1918 pandemic virus was utterly unique in how quickly it could kill; literally overnight. There are anecdotes of people leaving for work in the morning feeling fine, and then succumbing on their way. One story tells of four women in a bridge group playing together until 11:00 in the evening. By morning, three of them had died.

Another puzzling feature of the 1918 virus was that it tended to kill the hale and hearty; individuals between the ages of 25 and 34, in the primes of their lives. In contrast, seasonal influenza epidemics cause the most fatalities in the elderly, the very young, the chronically ill, and people with weakened immunity.

The lower mortality rates among the elderly during the1918 pandemic is possibly explained by their prior exposure to an influenza strain serologically related to the 1918 pandemic virus, thus providing them with a measure of protective immunity against the pandemic virus. [On this point, and others related to the biology of influenza virus, see chapter 12 of Virology: Molecular Biology and Pathogenesis.]

The higher mortality rate among individuals between the ages of 25 and 34 is sometimes attributed to the fact that the pandemic occurred during the last year of World War I; a time when many individuals in this most susceptible group were living in crowded army camps, which predisposed them to the opportunistic bacterial infections responsible for many of the influenza fatalities in the pre-antibiotic era. Yet the virus itself was extraordinarily lethal, as noted above. Moreover, the “crowded army camp theory” can not explain why the same pattern of disease was seen in the populations of countries that did not participate in the war. So, these mysteries remain.

Recalling that the 1918 pandemic virus was absent from the world after the early 1920s, we now tell the story of Jeffery Taubenberger. In March of 1997, Taubenberger and his colleagues at the Armed Forces Institute of Pathology (AFIP) in Washington, D.C. startled virologists when they reported the sequence of the hemagglutinin (HA) gene of the 1918 pandemic virus. So, how was Taubenberger able to sequence the HA gene of a virus that was nonexistent for nearly 80 years?

[Aside 2: Influenza HA proteins are located in the viral envelope. They bind to the receptor on the target cell, and then promote fusion of the viral envelope with the plasma membrane of the target cell.]

[Aside 3: Jeffery Taubenberger is currently at the National Institute of Allergy and Infectious Diseases. The Armed Forces Institute of Pathology closed its doors in September 2011. It was founded in 1862 as a museum for specimens taken from American Civil War casualties. Over the years, the Institute’s specimen collection became legendary, and it became known for its role in diagnosing difficult civilian, as well as military cases. Moreover, its staff has included some of America’s greatest pathologists.]

Taubenberger was hired by the AFIP to create a state-of-the-art molecular pathology laboratory. Towards that end, his unit, which included molecular biologist Ann Reid, developed new procedures to recover nucleic acids from tissue samples that were fixed in formaldehyde and embedded in paraffin. Although pathologists routinely examine fixed tissues, molecular analysis of those specimens had not been possible, since the fixation can destroy nucleic acids.

Taubenberger’s initial involvement with influenza was not based on an interest in influenza per se. Instead, his intention was merely to showcase his Institute’s new procedures, and also its vast collection of specimens that had been assembled over the past century. With those purposes in mind, Taubenberger and Ann Reid put in a request for fixed tissue samples from soldiers who had succumbed during the 1918 flu pandemic.

Expecting a long wait, Taubenberger and Reid were themselves surprised when the Institute’s automated recovery system successfully retrieved their samples from the 3 million others in the AFIP collection, within a few seconds of receiving their request. The samples contained flecks of tissue from soldiers killed by the flu pandemic 80 years earlier. They were taken by doctors who, of course, had no knowledge at the time of what might be causing the soldiers’ illness.

Their interest now aroused, Taubenberger and Reid began to screen paraffin-embedded, formaldehyde-fixed patient specimens for influenza sequences, using then new, extremely sensitive molecular techniques (reverse- transcription polymerase chain reaction [RT-PCR] amplification of HA gene fragments). They hoped to increase their chance of success by focusing on specimens that showed severe lung disease. The rationale was that these samples would have come from victims who died quickly, before the virus might have cleared. [Influenza generally clears the lungs within days of the infection.] Regardless, they looked in vain for a year, until they came to a sample from Private Roscoe Vaughn, who died in September 1918 at Fort Jackson, SC., during the peak of the pandemic. In Private Vaughn’s fixed cells they found small segments of influenza-like RNA. Then, to be certain that these RNA segments were indeed from the 1918 pandemic virus, they resumed their search for positive samples until they found one from a soldier who died at Camp Upton, NY, also in September 1918. After thus confirming that their samples contained RNA segments from the actual 1918 pandemic virus, they were able to generate the complete sequence of it’s HA gene. Interestingly, the HA gene of the 1918 pandemic virus was unlike that of any other influenza HA gene that had been sequenced to date.

Having thus succeeded at reconstructing the HA gene of the 1918 virus, the next step would be to reconstruct its entire genome. However, from the very small amounts of tissue in the formaldehyde-fixed autopsy samples, Taubenberger doubted ever being able to do so. What follows is my favorite part of the story.

Dr. Johan Hultin, a 73-year-old retired pathologist, unexpectedly provided a solution to the AFIP group’s dilemma. Years earlier, in 1951, when Hultin was a graduate student at the University of Iowa, he attempted to grow live influenza virus from Alaskan Inuit victims of the 1918 pandemic, whose bodies remained buried in the Alaskan permafrost over the subsequent years. It was Hultin’s hope that the virus might have been preserved in those frozen victims. However, all his attempts to grow the virus were unsuccessful.

Hultin’s failure caused him to abandon his graduate studies and, instead, become a pathologist. Then, in 1997, after he was already retired, he happened to read the report from Taubenberger’s group describing how they reconstructed the HA gene of the 1918 pandemic virus. The report rekindled Hultin’s memories of his own earlier attempts in 1951 to grow the virus. Now, excited by his thought that the frozen bodies of the Alaskan victims might contain influenza genome fragments, from which it might be possible to reconstruct the entire genome, he wrote to Taubenberger, offering to return immediately to Alaska to obtain fresh specimens. Taubenberger agreed and, thus, Hultin eagerly returned to Alaska in 1997. There, he deliberately took tissue samples from a particularly obese woman, hoping that the combination of her fat and the permafrost might have preserved the influenza genomes. Hultin’s reasoning may indeed have saved the day, since Taubenberger’s group was able to generate the entire genome of the 1918 virus from these samples and, subsequently, to grow up the virus itself.

After Taubenberger and his co-workers successfully brought the 1918 pandemic virus “back to life,” they then tested its virulence in mice. Not surprisingly, the pandemic virus was extraordinarily lethal in the mouse model. However, the explanation for the exceptional virulence of the virus was not revealed by its genetic sequence per se. But, once the technology was available to recover gene sequences of the 1918 virus, it became technologically feasible to identify which genes of the 1918 virus accounted for its extreme virulence. Some readers may need to read the following brief aside to fully appreciate this part of the story.

[Aside 4: Most viruses contain all of their genes on a single chromosome. In contrast, the influenza genome is comprised of eight distinct single-stranded RNA segments. Five of these segments encode a single protein, while three of these segments encode two different proteins. The segmented nature of influenza genomes has important consequences in nature, as follows.

If a cell were simultaneously infected with two different influenza strains, then the genomic segments of the two strains might randomly re-assort to produce brand new strains. Indeed, this is precisely how pandemic strains are believed to arise in nature. In those instances, a human influenza genome re-assorts with the genome of a zoonotic virus, usually an avian one. In fact, the 1918 pandemic virus is at least partly avian in origin.]

Bearing in mind that influenza viruses contain segmented genomes (Aside 4), and that re-assortment of genomic segments between different strains occurs in nature, several research groups, each working independently, sought to determine which of the genomic segments of the 1918 pandemic virus might be responsible for its extraordinary virulence. In brief, it was possible to experimentally substitute each of the genomic segments of a benign influenza strain with the corresponding genomic segment of the 1918 pandemic virus. [The individual influenza gene segments were reverse transcribed and then inserted into individual plasmids. Recombinant viruses were then generated by microinjecting different combinations of these plasmids into cells in culture.] These viruses were then screened for their virulence in mice.

The results of these experiments showed that several different genes from the 1918 virus contributed to its virulence. These included the viral genes that encode two of the envelope proteins; the HA protein described above and the neuraminidase (NA), which promotes virus release from cells. The viral polymerase also contributed to its virulence.

An early hypothesis to explain the virulence of the 1918 pandemic virus was based on the contention that the virus acquired and expressed the HA gene, and perhaps the NA gene as well, of an avian influenza strain. Consequently, there might have been little if any immunity in the human population against the pandemic virus. However, Taubenberger’s group found that laboratory-generated recombinant viruses, which contained both the HA and the NA proteins of the 1918 pandemic virus, induced higher levels of inflammation in the mouse model than were induced by more benign influenza viruses. That is, the laboratory-generated recombinant viruses were actually more immunogenic than benign influenza strains. While this finding might not have been predicted, it actually is consistent with the extreme lung pathology seen in humans during the pandemic. At any rate, more research still needs to be done to better understand the virulence of the 1918 virus.

Taubenberger’s group also found some important differences between the viruses in samples from individuals infected early in the 1918 pandemic, when the virus was relatively benign, and the viruses in individuals infected after the virus became vastly more virulent. In the earlier cases, the HA protein was more like that found in avian influenza strains, while later cases had an HA protein somewhat more like that found in human influenza strains. Presumably, the avian HA gene underwent changes that adapted the virus to disseminate and spread more easily in its human host.

[Aside 5: There are 16 known serologically distinct types of the influenza HA protein in nature; only three of which, H1, H2, and H3 are found in human influenza strains. There are nine known serologically distinct types of the NA protein, of which N1, N2, and N3 are most commonly found in human strains. The 1918 pandemic virus was an H1N1 strain. Pandemic viruses generally arise when a current seasonal human strain acquires a new HA gene from an avian influenza. Other genes also may be acquired from the avian virus in addition to the HA gene. Thus, the 1957 Asian flu was H2N2, and the 1968 Hong Kong flu was H3N2. See the following aside.]

[Aside 6: In April 2009, a novel H1N1 virus (see the above aside), which originated in swine, was found in humans in the United States, Mexico, Canada, and elsewhere. Although this virus turned out to be relatively benign, its emergence caused widespread panic, due in part to the non-stop updates of new cases in the media, which created the false impression that a killer pandemic was sweeping through the country.

In May, 2009, Vice President Joe Biden told a national TV audience that he would tell members of his own family not to go anywhere where they might be in a confined space, such as an airplane, subway or classroom. But, in fairness to Biden and the media, it was net yet clear that the virus was relatively mild.

Initially, the virus was referred to as the swine flu. But, Biden’s boss, President Barack Obama, in deference to the U.S. pork industry (people were afraid they might catch the virus by eating pork), began to deliberately call this virus “the H1N1 virus.” The new designation stuck. And while it does characterize the 2009 swine flu, it likewise characterizes the vastly more lethal 1918 pandemic virus, as well as a current seasonal influenza strain. Thus, the 2009 virus was hardly the H1N1 virus.

The world was of course fortunate that the 2009 H1N1 swine flu outbreak turned out to be relatively mild. Many millions of people might have been killed. Will the public remember the episode and, consequently, be complacent in the face of a future outbreak, doubting the credibility of government warnings?]

An earlier influenza outbreak, which indeed startled virologists, took place in 1997, when the first cross-species transmission of an avian H5N1 influenza to a human was documented. The patient, a child succumbed, and there were additional lethal human infections that followed. Indeed, the H5N1 virus killed about half of the individuals it infected; a fatality rate far greater than that of even the 1918 pandemic virus. Fortunately, during the past 17 years, the virus has not adapted to spread readily from person to person. Instead, the vast majority of the 600 humans, who were estimated to have been infected, acquired the virus by close contact with diseased birds.

Next, in September 2011, Yoshihiro Kawaoka at the University of Wisconsin and Ron Fouchier of Erasmus Medical Center in Rotterdam, shocked virologists when they announced that they and their colleagues had created variants of the H5N1 virus that could be transmitted between ferrets; often considered a good model for transmission in humans. What’s more, Fouchier’s group deliberately modified the virus so that it might be transmitted through the air; a very significant modification, since transmission of avian influenza viruses between their avian hosts is via the fecal-oral route, whereas mammalian influenza viruses are transmitted via the respiratory route.

Kawaoka’s group randomly mutated the HA gene of the H5N1 virus, until they found mutations that caused it to attach to human receptors, instead of to bird receptors. Then, they replaced the HA gene from the 2009 H1N1 “swine flu” strain (Aside 6) with the mutated H5 HA gene, thereby creating a virus that contained the mutated avian HA gene, and the remaining genes from the 2009 H1N1 virus. In contrast, Fouchier’s group examined the possibility that the H5N1 virus might acquire the ability to transmit via the respiratory route by mutation alone; without re-assortment. They began by giving the H5N1 virus three mutations previously identified in the HA genes of the 1918, 1957, and 1968 pandemic viruses.

Fouchier’s virus indeed was lethal in ferrets. In contrast, Kawaoka’s virus did not kill the animals, and was no more pathogenic in ferrets than the 2009 H1N1 swine virus. But, recombinant viruses that that arise in nature might have unpredictable and very different pathogenicities. And, bear in mind that both research groups in fact demonstrated that H5 avian viruses might acquire the ability to infect mammals.

Now, consider that the resurrected 1918 pandemic virus is essentially identical to the virus that claimed up to 50 million lives during the 1918 pandemic. Moreover, consider that up to now H5N1 viruses have not been able to readily transmit between humans. But, if either of the H5N1 viruses developed in Wisconsin and Rotterdam is indeed transmissible between humans, while retaining a measure of its virulence, it might be even more life-threatening than even the 1918 pandemic H1N1 virus.

In view of the above, one may well ask what reasons could possibly justify creating such potentially dangerous viruses. A common rationalization is that these experiments provide insights into the genetic changes that might happen in nature to generate deadly pandemic viruses. A potential benefit of that knowledge might then be to enable surveillance against the emergence of such viruses, thus providing a window of opportunity to develop strategies to cope with the threat and minimize its consequences.

But regardless of the possibly enormous benefits that might result from the types of experiments described above, one could easily imagine important arguments against doing these experiments. Clearly, resurrecting the 1918 pandemic virus brought an extremely deadly pathogen back to life. And, the experiments in Rotterdam and Wisconsin may likewise have given rise to very lethal viruses. Moreover, the accidental release of these viruses, even from the most secure facility, is not all far-fetched. In this regard, in 2003 and 2004 the SARS virus “escaped” from three different Asian laboratories. Furthermore, while these experiments might be done safely in a very few laboratories in the United States and Europe, there is no global mechanism to insure that they would be done safely elsewhere. What’s more, there is concern that terrorist groups might gain possession of these viruses, or perhaps even replicate the work that gave rise to them.

So what is the bottom line? The issue is not simply whether the research is dangerous. It clearly is. And, the issue is not simply whether the research holds the promise of real and important benefits. While some potential benefits may have been overstated, they yet may one day be considerable. Thus, the real question is whether the potential benefits of the research outweigh its here-and-now risks. Experts have taken opposite positions on this question, and a heated debate goes on.

Yet a new issue arose with regard to the H5N1 experiments; specifically, whether or not the work ought to be reported in scientific journals. This issue arose over concern that the transmissible H5N1 variants might fall into the hands of individuals or groups with evil intentions or, perhaps, even be made by them. Consequently, in December 2011, the U.S. National Science Advisory Board for Biosecurity (NSABB) made the unprecedented recommendation to censor the papers that reported the work of the Rotterdam and Wisconsin groups. The papers were, at the time, under review at Nature and Science. The NSABB worried that publication of “the methodological and other details could enable replication of the experiments by those who would seek to do harm.” Thus, the NSABB recommended that the general conclusions of the papers, but not their methodologies, might be published. Later, in February 2012, a World Health Organization committee recommended that the studies be published in full.

As might be expected, there is no consensus in the scientific community over this censorship issue. On the one hand, constraints on communication are inherently incompatible with free scientific inquiry and would hinder progress in a field that significantly impacts human health. Moreover, would scientists devote years to investigating dangerous viruses, only to have their work censored in the end? On the other hand, should not the scientific community bear at least some responsibility for keeping the fruits of its research from being misused by those who would do harm? Few scientists would prefer to have individuals who are not practicing scientists, and who don’t always understand the science, making these judgments in their place. [I find it interesting that these other individuals are often referred to as bioethics, biosecurity, or bioterror “experts,” and wonder what makes them so.]

References

Herfst S, Schrauwen EJ, Linster M, Chutinimitkul S, de Wit E, Munster VJ, Sorrell EM, Bestebroer TM, Burke DF, Smith DJ, Rimmelzwaan GF, Osterhaus AD, Fouchier RA. 2012. Airborne transmission of influenza A/H5N1 virus between ferrets. Science 336:1534-1541.

Imai M, Watanabe T, Hatta M, Das SC, Ozawa M, Shinya K, Zhong G, Hanson A, Katsura H, Watanabe S, Li C, Kawakami E, Yamada S, Kiso M, Suzuki Y, Maher EA, Neumann G, Kawaoka Y. 2012. Experimental adaptation of an influenza H5 HA confers respiratory droplet transmission to a reassortant H5 HA/H1N1 virus in ferrets. Nature 486:420-428.

Taubenberger JK, Reid AH, Krafft, AE, Bijwaard, KR, and Fanning TG. 1997. Initial genetic characterization of the 1918 “Spanish” influenza virus. Science 275:1793-1796.

 

Carlo Urbani: A 21st Century Hero and Martyr

Carlo Urbani was the first to recognize that the severe acute respiratory syndrome (SARS) epidemic of 2003 signified a new, not seen before, life-threatening, infectious disease. Sadly, Urbani succumbed to SARS while organizing the most effective containment response to a major epidemic in history. Several weeks afterward, the SARS agent was found to be a previously unknown coronavirus, and then aptly named the SARS coronavirus.

Carlo Urbani’s actions during the severe acute respiratory syndrome (SARS) epidemic of 2003 are remarkable on several counts and need to be much better known. But first, we begin with some relevant background

The first known reference to the SARS epidemic dates to February 2003, when the Chinese Ministry of Health announced the mysterious outbreak of an atypical pneumonia in the Guangdong Province of southern China. Chinese authorities reported a total of 305 cases, including five deaths, during the preceding three months. However, these figures were almost certainly an underestimate of the scope of the Chinese epidemic, as discussed below.

A Chinese doctor, who had been treating SARS patients in Guangdong, is believed to have brought the disease to Hong Kong that same February. He developed symptoms during his first day in Hong Kong, where he stayed at the Hotel Metropole. The next day he was transferred to a hospital, and succumbed the day after. However, during his brief stay at the Hotel Metropole, he somehow infected at least 10 other guests. Eight of those infected guests were on the same floor as the doctor, and the two others were two and five flights up from him. Those infected individuals subsequently boarded airplanes that took them to Singapore, Vietnam, Canada, and the United States, thereby spreading an epidemic that lasted more than 100 days.

The World Health Organization (WHO) estimated that there were 8,422 SARS cases worldwide during that 100-day period, resulting in 908 deaths. In all, 29 countries were affected. In the United States there were a total of eight confirmed SARS cases, none of which was fatal. Each of the infected individuals in the United States had shortly before traveled to an area where SARS transmission was occurring. Thus, the SARS outbreak of early 2003 was truly an epidemic of our modern global era, spread by air travel to at least three continents in a period of just a few weeks. Importantly, its consequences might well have been vastly more devastating and, perhaps, less so as well, as described below.

Our story of Carlo Urbani begins in Vietnam, where on February 28, 2003 (a still early time in the epidemic), the Vietnam French Hospital of Hanoi contacted the Hanoi office of the World Health Organization concerning a patient, an American businessman, who seemed to be showing signs of what the Vietnamese doctors feared might be an unusually severe case of the flu. Believing that they might be facing a potentially deadly avian influenza outbreak, hospital officials called on the WHO for help. Urbani, who was an infectious disease specialist, answered the call and quickly determined that the hospital was not facing influenza but, instead, something unusual; a new, previously unknown contagious respiratory disease.

Interestingly, Urbani might not seem to have been the obvious choice to consult on this case, since he was best known as an expert on parasitic infections. Nevertheless, the WHO staff still recommended him to the Vietnamese because of his reputation as a superb clinical diagnostician. Urbani lived up to his reputation, recognizing that SARS was a new and extremely dangerous infectious disease. Moreover, and crucially, he immediately notified the WHO of his findings, thereby without delay setting in motion the most effective global response to a major epidemic in history. His decisive and timely action may have saved millions of lives worldwide!

 urbani

Carlo Urbani, at the Vietnam French Hospital of Hanoi

Disregarding his own safety, Urbani spent the next several days continually at the Vietnam French Hospital of Hanoi, where he organized infection control procedures, while also taking patient samples for analysis. And, as it became clear that the infection was highly contagious and deadly, Urbani worked closely with the hospital staff to maintain morale. Moreover, Urbani, as well as others on the hospital staff, decided not to leave the hospital, so as not to place their families or the community at risk. In doing so, they knowingly placed themselves in jeopardy.

Acutely aware of the danger that the new disease posed to the Vietnamese, Urbani undertook the difficult task of arranging a meeting between WHO officials and the Vietnamese Vice Minister of Health. Urbani was able to bring these parties together largely because of the strong trust he had been building with Vietnamese authorities. At the meeting, Urbani explained the steps that needed to be taken to contain the Vietnamese outbreak. The Vietnamese government responded to Urbani’s recommendations by quarantining the Vietnam French Hospital of Hanoi, and establishing infection control procedures at other hospitals as well. Moreover, the Vietnamese government took the extraordinary step on its part of issuing a public international appeal for expert assistance, despite the possibility of hurting the Vietnamese economy or image by doing so. Specialists who answered the Vietnamese appeal came from the WHO, the CDC of the United States, and Médecins sans Frontières (Doctors without Borders). [Urbani was in fact president of the Italian chapter of Médecins Sans Frontières and was one of the individuals who accepted the 1999 Nobel Peace Prize on its behalf. 1 Speaking at the 1999 Nobel ceremony, Urbani stated that it was the doctors’ duty “to stay close to the victims.”]

Sadly, Urbani began to develop symptoms of SARS during a March 11 flight from Hanoi to Bangkok, where he had planned to attend a conference. He succumbed in a Bangkok hospital on 29 March 2003, not knowing that within several weeks’ time researchers working worldwide would isolate the SARS agent, sequence its genome, and identify it as a newly discovered coronavirus. 2 He was 49 years old. The following is from his obituary in the Guardian, written by Lorenzo Savioli: “His wife Giuliana told me that a few days before falling ill he had argued with her. She was concerned to see him working with patients with such a deadly disease. He said: ‘If I cannot work in such situations, what am I here for – answering emails, going to cocktail parties and pushing paper?’”

Here is another important aspect of the 2003 SARS epidemic to consider; the response of the Vietnamese authorities to the outbreak in their country, versus that of the Chinese authorities to the Chinese outbreak. The Vietnamese responded to their SARS outbreak by taking the unexpected step on their part of promptly issuing an international appeal for expert assistance, which it accepted from U.S. Centers for Disease Control, the WHO, and Médecins sans Frontières. And, by following Urbani’s recommendations, the Vietnamese quickly brought the outbreak in their country under control. In contrast, the Chinese initially tried to cover up their SARS outbreak, and then misrepresented the number of their cases. Indeed, there are reports that they had their SARS patients driven around in taxis to avoid being detected by WHO officials who came to visit their hospitals. News of the Chinese epidemic surfaced in the outside world largely because scientists, who were working in neighboring countries, become aware of what the Chinese authorities knew about, but tried to conceal.

Bearing in mind that the 2003 SARS epidemic may have initially emerged in China, if the Chinese authorities had been more forthcoming, and if they had taken appropriate containment measures at the start of their epidemic, many lives might have been saved worldwide. Moreover, the quarantines that needed to be instituted globally in response to the SARS epidemic, the disruption of international travel, and the worldwide economic consequences, all might have been much less severe. Reacting to international pressure, China finally established strong containment measures in April 2003. [In April 2004, the Chinese Ministry of Health reported several new SARS cases. And, in contrast to its actions of the preceding year, this time China responded aggressively, quickly isolating patients who developed SARS, identifying their nearly 1,000 recent contacts, and sharing information with outside groups such as the WHO.]

 On July 5, 2003, the WHO announced that the cycle of human-to-human transmission of the initial SARS outbreak was broken and that the epidemic had come to an end. The fact that containment of the 2003 outbreak was achieved within 4 months of the first global alert is a tribute to effective public health policy and, what’s more, to the united effort of the international community. As noted above, the global response to the SARS outbreak, first initiated by Urbani in Vietnam, was indeed the most effective response to a major epidemic in history. It also is noteworthy that containment was achieved without the benefit of a vaccine or critical diagnostic reagents; further evidence of the efficacy of good public health policy.

While the public health community well deserves praise for containing the SARS epidemic, the scientific community also merits praise for isolating and identifying the SARS agent within weeks of the initial 2003 outbreak. It happened as follows.

The symptoms of SARS did not suggest any one cause in particular. Thus, researchers tested patient specimens for a broad range of bacterial, chlamydial, rickettsial, and viral agents that were known to target the lower respiratory tract. Viral agents under suspicion included influenza viruses, paramyxoviruses, herpesviruses, and picornaviruses.

In order to amplify potential viral agents, patient samples were inoculated into cell cultures. Then, by means of electron microscopy, a virus, which originated in the respiratory secretions of a SARS patient, was seen which displayed characteristic coronavirus structural features. Then, using electron microscopy in some instances, and serological procedures in others, this virus was detected in additional SARS patients. Subsequent molecular biological and immunologic studies confirmed that the isolated agent was, in fact, a novel coronavirus. Next, the SARS virus isolates that had been grown in cell cultures were found to cause lower respiratory tract disease when inoculated into monkeys.

It is noteworthy, that in the modern genomics era of PCR primers and so forth; the SARS virus was first identified by means of classic tissue-culture amplification and electron microscopy. As noted by the authors, “…electron microscopy proved to be a rapid technique that did not require specific reagents for or prior knowledge of a particular agent but that could nevertheless categorize a pathogen on the basis of its appearance and morphogenesis.” 3

Although the SARS virus was identified using classic virological procedures, the SARS epidemic was the first infectious disease outbreak in which virus researchers took full advantage of the powerful new techniques of the genomics era to analyze the new pathogen. Using these techniques, the SARS virus genome was sequenced less than one month after the virus was first isolated. Within the next three months, genome sequences of 20 independent clinical isolates of the SARS virus were available in the GenBank database for comparison.

Interestingly, the SARS virus’ genealogy initially took researchers in the field by surprise. That was so because coronaviruses were previously known for the mild upper respiratory tract infections they cause in humans; infections which are similar clinically to the innocuous common colds caused by the human rhinoviruses.  In contrast, as many as 40% of individuals infected with the SARS coronavirus required mechanical breathing assistance, and the overall mortality rate for infected individuals was about 10%, rising to as high as 50% in the elderly.

Urbani succumbed to SARS just weeks before the SARS coronavirus was isolated and identified as the cause of the epidemic. So, Urbani never knew of the discovery. But, in tribute to the singular importance of Urbani’s deeds during the epidemic, and his personal sacrifice, the authors of the paper, which reported the identification of the SARS virus, dedicated the paper to Urbani. 3

Urbani also was not forgotten by the Vietnamese government, which conferred upon him two of their highest honorary titles: the medal of Friendship and the medal for People’s Health. What’s more, outside of Hanoi, a hospital has been built in his name. And, in Taiwan, a foundation has been named after him.

Footnotes:

1. The following is excerpted from the “Doctors Without Borders/Médecins Sans Frontières (MSF)” website:

“ Doctors Without Borders/Médecins Sans Frontières (MSF) is an international medical humanitarian organization created by doctors and journalists in France in 1971.Today, MSF provides independent, impartial assistance in more than 60 countries to people whose survival is threatened by violence, neglect, or catastrophe, primarily due to armed conflict, epidemics, malnutrition, exclusion from health care, or natural disasters. MSF provides independent, impartial assistance to those most in need…MSF medical teams often witness violence, atrocities, and neglect in the course of their work, much of which occurs in places that rarely receive international attention…. At times, MSF may speak out publicly in an effort to bring a forgotten crisis into view….For example, in 1985, MSF spoke out against the Ethiopian government’s forced displacement of hundreds of thousands of members of its own population. In 1994, the organization took the unprecedented step of calling for an international military response to the 1994 Rwandan genocide. The following year, MSF condemned the Serbian massacre of civilians at Srebrenica, and four years after that, denounced the Russian military bombardment of the Grozny, the capital of Chechnya. In 2004 and 2005, MSF called on the United Nations Security Council to pay greater attention to the crisis in Darfur. And in 2007, MSF denounced the targeting of civilians in conflict—something that was occurring with greater frequency in the Democratic Republic of Congo, Central African Republic, Chad, and Somalia—and the governments of Thailand and Laos, which were threatening to forcibly return nearly 8,000 Hmong refugees to Laos….”

2. Norkin, L. C., 2010. Virology: Molecular Biology and Pathogenesis, ASM Press, Washington, DC.

3. Ksiazek T. G., D Erdman, C. S. Goldsmith, S.R. Zaki, T. Peret, S. Emery, S. Tong, C. Urbani, J.A. Comer, W. Lim, P.E. Rollin, S. F. Dowell, A.E. Ling, C. D. Humphrey, W. J. Shieh, J. Guarner, C. D. Paddock, P. Rota, B. Fields, J. DeRisi, J. Y. Yang, N. Cox, J. M. Hughes, J. W. LeDuc, W. J. Bellini, L. J. Anderson; SARS Working Group. 2003. A novel coronavirus associated with severe acute respiratory syndrome. N. Engl. J. Med. 348:1953-1966.