Jeffery K. Taubenberger is Chief of the Division of Molecular
Pathology, Department of Cellular Pathology, Armed Forces Institute of Pathology,
Washington, D.C
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Seeking the 1918 Spanish Influenza Virus
Gene sequences of the virus from the 1918 influenza pandemic are yielding insights into its origin but little about virulence
Jeffery K. Taubenberger
In the fall of 1918, as World War I was ending, an influenza pandemic of unprecedented virulence swept the globe, leaving some 40 million dead in its wake. A search for the responsible agent began in earnest that year, leading to the first isolation of an influenza virus by 1930. However, the lethal influenza virus that circulated in 1918 was not isolated and thus seemed lost for further study.
Nonetheless, and thanks to the incredible foresight of the U.S. Army Medical Museum, the persistence of pathologist Johan V. Hultin, and advances in the molecular genetic analyses of fixed tissue specimens as applied by the Molecular Pathology Division at the Armed Forces Institute of Pathology, we are now studying the genetic features of that deadly 1918 influenza virus.
These studies are not just products of historical curiosity. Since influenza viruses continually evolve by mechanisms of antigenic shift and drift, new influenza strains, as emerging pathogens, continue to threaten human populations. For example, pandemic influenza viruses have emerged twice since 1918--in 1957 and 1968. Moreover, the risk for future influenza pandemics is thought to be high. An understanding of the genetic makeup of the most virulent influenza strain in history may facilitate prediction and prevention of such future pandemics.
The 1918 Influenza Pandemic
The influenza pandemic of 1918 was exceptional in both breadth and depth. Unlike most subsequent influenza strains, which first appeared in Asia, the initial wave of the 1918 pandemic seemingly arose in the United States. The first wave of influenza in the spring and summer of 1918 was highly contagious but caused few deaths.
In late August, however, a virulent form of the disease emerged and swept the globe in six months. The main wave of the global pandemic occurred in September through November of 1918, killing over 10,000 people per week in some U.S. cities. Outbreaks of the disease not only swept North America and Europe but also spread as far as the Alaskan wilderness and the most remote islands of the Pacific. (The disease was dubbed the ``Spanish flu'' because it was first widely reported in Spanish newspapers; news reports of the disease were suppressed by wartime censorship in many countries engaged in World War I.) Large proportions of the population became ill; 28% of the U.S. population is estimated to have been infected. The disease was also exceptionally severe, with mortality rates of over 2.5% among the infected, compared to less than 0.1% in other influenza epidemics. Some isolated populations had mortality rates grater than 70%.
Furthermore, in the 1918 pandemic most deaths occurred among young adults, a group that usually has a very low death rate from influenza. Influenza and pneumonia death rates for 15- to 34-year-olds were more than 20 times higher in 1918 than in previous years, with 99% of excess deaths among people under 65 years of age (Fig. 1A). Thus, the influenza epidemic of 1918 killed an estimated 675,000 Americans, including 43,000 servicemen mobilized for World War I. The impact was so profound as to depress average life expectancy in the U.S. by over 10 years (Fig. 1B), and it may have played a significant role in ending the war.
Indirect Information about the Virus
Analyses of antibody titers of 1918 flu survivors from the late 1930s and inferences based on phylogenetic analyses of past influenza outbreaks suggest that the 1918 strain was an H1N1-subtype influenza A virus, probably closely related to what is now known as ``classic swine'' influenza virus. This swine connection also relates directly to the 1918 pandemic, during which simultaneous outbreaks of influenza in humans and pigs were reported around the world. While historical accounts suggest that the 1918 flu spread from humans to pigs, the relationship of these two species in the development of the 1918 flu has not been resolved.
Whether there was an avian association with the 1918 flu has not been determined. The natural reservoir for influenza virus is thought to be wild waterfowl. Periodically, genetic material from avian strains emerges in strains infectious to humans. Since pigs can be infected with both avian and human strains, they are thought to be an intermediary in this process. For example, in 1979, an avian influenza A virus (without reassortment) entered the swine population in northern Europe, forming a stable viral lineage.
Influenza strains with recently acquired genetic material have been responsible for pandemic influenza outbreaks in 1957 and 1968. Until recently, however, there was no evidence that a wholly avian influenza virus could directly infect humans. This barrier was dramatically broken in 1997 in Hong Kong, when 18 people were infected with an avian H5N1 influenza virus and 6 died of complications after infection.
Determining the relationship of the 1918 influenza virus to swine and avian viral strains is one of the primary goals of our project. How influenza viruses move between species is extremely important for our understanding of the emergence of pandemic influenza strains.
Finding Virus Samples for Study
In 1951, scientists from the University of Iowa exhumed bodies of victims of the 1918 flu that had been buried in permafrost in Teller Mission, an Inuit fishing village on the Seward Peninsula of Alaska. This village, now called Brevig Mission, suffered extremely high mortality rates during the influenza pandemic in November 1918. According to available records, influenza spread through the village in about 5 days and killed 72 people, representing about 85% of the adult population.
During the 1951 expedition, samples of lung, brain, and other organs were taken for histologic analyses and viral culture. All attempts to culture live influenza virus from these specimens were unsuccessful. Molecular genetic analyses of the samples were impossible at that time--indeed, the double-stranded structure of DNA, with its implications for molecular genetics, was not determined until 1953.
In 1995, my laboratory initiated a project to characterize the 1918 influenza virus genetically using archival formalin-fixed, paraffin-embedded autopsy tissues of 1918 flu victims stored in the National Tissue Repository of the Armed Forces Institute of Pathology (AFIP). A search of the archives revealed over 70 autopsy cases of U.S. soldiers who died of influenza pneumonia in the fall of 1918. We used techniques to isolate RNA that had been optimized for fixed tissue specimens based on previous studies.
In total, 78 autopsy cases of victims of the lethal fall wave of the 1918 pandemic were examined for this study, and 74 of these consisted of fixed tissue samples. The majority of these individuals died of secondary bacterial pneumonia. Because they often had clinical courses longer than one week, it was extremely unlikely that any of the tissue samples from these cases would still retain influenza RNA.
However, a subset of individuals died within one week, with very unusual and characteristic lung pathology including massive pulmonary edema or hemorrhage. These patients literally drowned in their own serum or blood, often in as little as 48 hours. While these pathologic changes have been seen in other influenza outbreaks (including during the 1957 ``Asian'' flu outbreak), its prominence in 1918 is one of the cardinal features of the Spanish flu. We then concentrated our efforts on specimens from this subset of patients.
Examination of 1918 Autopsy Samples
In 1996 we found the first positive case, an autopsy lung sample from a soldier who died on 26 September 1918 at Fort Jackson, S.C. He presented with pneumonia and influenza symptoms, was admitted to the camp hospital, and expired six days later. At autopsy (confirmed by reexamination of the microscopic slides in 1996), he suffered a lethal bacterial pneumonia of his left lung, with only very early inflammatory changes in his right lung. The changes in the right lung, acute focal bronchiolitis and alveolitis, are consistent with primary influenza viral pneumonia (Fig. 2A). Indeed, no influenza RNA was recovered from his left lung.
The influenza RNA recoverable from the right lung was fragmented into pieces no longer than 150 bases in length. Our initial genetic characterization of the 1918 influenza involved determining the sequence of fragments of five influenza genes from this sample.
In order to confirm that the sequences derived from this case represented those of the lethal fall wave of the 1918 flu, additional cases were sought. A second archival autopsy case was identified in 1997. This case also involves a soldier--one who died on 26 September 1918 at Camp Upton, N.Y. He also presented with pneumonia and had a very rapid clinical course, with death in just three days after onset of symptoms. Microscopic examination of sections of his lung reveals massive acute pulmonary edema (Fig. 2B). Influenza RNA fragments in this case were also no greater than 150 bases in length.
Pathologist Johan V. Hultin submitted a third case to the AFIP in 1997. Hultin was a team member of the 1951 University of Iowa project. After reading about our results with fixed tissue specimens, he contacted us about obtaining new frozen lung biopsies of 1918 flu victims from the same mass grave sampled in 1951. He obtained permission of the Brevig Mission City Council and obtained in situ frozen lung biopsies of four victims of the 1918 flu. These samples were placed in fixatives for histologic analysis and RNA isolation. No attempt to culture virus was made.
Lung tissue samples of one of these cases, an Inuit female nicknamed Lucy by Hultin, was positive for influenza RNA. Histologically, sections of her lung show evidence of massive pulmonary hemorrhage, consistent with a rapid clinical course. Unfortunately, influenza RNA fragments in this case are no longer than those obtained from the fixed cases.
HA Sequence Analysis: 1918 Influenza Resembles Swine, Human Strains
There are two broad goals of this project: (i) to determine where the 1918 influenza virus came from and how it moved into humans, and (ii) to learn whether specific features of its nucleotide sequence provide insights into the virulence of this or other strains of influenza.
The best available technique to analyze the relationships among influenza viruses is phylogeny, whereby hypothetical family trees are constructed by computer programs that use available sequence data to assign ancestral relationships among various influenza strains. Since influenza genes are encoded by eight discreet RNA segments that can move independently between strains by the process of reassortment, such evolutionary studies must be performed independently for each gene segment.
We compared the sequence of the gene for hemagglutinin (HA) from the 1918 strain to those of numerous human, swine, and avian sequences. The 1918 HA gene always groups with the strains that infect humans and swine, never with the avian sequences. While it has sequence similarities with avian HA genes, we believe that the HA gene was adapted for life in mammals prior to 1918. Whether that process occurred in a human or a pig host is not known. The 1918 HA gene sequence is probably very similar to that of the viral ancestor of both human and swine H1 strains.
HA Sequence Analysis Says Little So Far About Virulence
Little is known about how genetic features of influenza viruses affect virulence. Virulence is a complex phenomenon that involves several features, including host adaptation, viral transmissibility, tissue tropism, and replication efficiency. Moreover, each of these features is likely polygenic in nature. There are, however, several identified mutations among influenza strains that radically change their behaviors. We have screened for several of these mutations in our 1918 influenza isolates.
The influenza gene sequences that have been generated from the three case specimens available to us have been, with rare exceptions, identical to one another. The HA protein is found on the external surface of the virus, where it plays a key role in binding and entry into target cells. It is also the principal target of the host humoral immune system response to an infection.
The complete coding sequence of the gene was generated from the South Carolina case and was confirmed using RNA from the New York and Alaska cases. Of the 981 bases of the HA1 domain of the gene, only two nucleotide differences were noted among the three case specimens. One base difference would not have changed the amino acid coded for at that site.
However, another base change, which was detected in the New York case, would change an amino acid as compared to the sequence of the South Carolina and Alaska cases. Moreover, that change occurs at one of the critical amino acids involved in receptor binding. The overall receptor binding pattern for the 1918 hemagglutinin is most similar to those of classic swine influenza strains, suggesting that, if intact, the New York isolate could bind both avian- and mammalian-type receptors, a property of classic swine influenza viruses.
A prototypical example of a genetic change in influenza that leads to increased virulence is the cleavage site mutation seen in some avian influenza strains. For viral activation, the HA protein must be cleaved into two pieces by a host protease. Some avian influenza strains can cause systemic disease in birds, instead of the normally benign infection that typically is limited to the gastrointestinal tracts of host birds. These mutant strains, however, are associated with exceptionally high rates of mortality among infected birds.
This mutation has never been seen in H1 subtype HAs. However, if present, it would have offered a ready explanation of the unusual virulence of the 1918 influenza. Hence, as part of our initial analysis, we carefully examined this site in all three 1918 case specimens. Nonetheless, our analysis indicates that the 1918 strains do not carry a mutation at this site.
Complete Neuraminidase Analysis Expected Soon
In mid-1999, our efforts to determine the complete sequence of the gene encoding the second surface protein of the virus, neuraminidase (NA), are complete. Like the HA gene, a mutation has been observed in the NA gene which could allow the virus to cause a systemic infection. Hideo Goto and Yoshihiro Kawaoka of the University of Wisconsin-Madison recently proposed that such a change in NA might help account for the virulence of the 1918 flu.
In this model, a change of a single amino acid in NA may allow flu viruses carrying this gene a ``back door'' to ubiquitous HA cleavage and systemic infection by sequestering plasminogen on the surface of infected cells. Plasminogen, a normal precursor protein of the blood's clotting system, can be bound by this mutant NA. When converted to its active form, plasmin, it can function as a protease to cleave HA.
However, like the HA cleavage site mutation, this change was also not observed in any of the three 1918 influenza specimens that we have analyzed. In both circumstances, this is not that surprising. The pathology of 1918 flu victims does not support the hypothesis that the virus could cause a systemic infection. Additionally, the HA cleavage site mutation has only been observed in H5 or H7 subtype viruses associated with infections in domestic poultry. The NA mutation has never been observed in a wild-type influenza isolate, only in an unusual, laboratory-derived flu strain adapted for growth in mouse brain.
Meanwhile, we have analyzed partial sequences of all the remaining gene segments of the 1918 viral isolates and will soon determine their full-length sequences. We anticipate completing the sequences of both the matrix and nonstructural gene segments this year. Such information will enable us to conduct phylogenetic analyses of each segment and will help elucidate the origin of the 1918 virus. Each of these genes has features that may play crucial roles in explaining the uniqueness of the 1918 influenza. Whether any particular genetic features of the virus can be related directly to its exceptional virulence is yet unclear, but genetic analysis is still likely to yield significant insight into the behavior of this virus.
Even as the genetic structure of the Spanish flu virus is becoming fully known, however, other questions may never be answered due to the passage of 80 years. For instance, how may differences in immunity have affected mortality rates among different age groups during the 1918 pandemic? Nonetheless, knowledge gained by studying this all-too-successful human pathogen can still be applied to understanding and perhaps preventing, or at least predicting, the emergence of future influenza viruses with pandemic potential.
ACKNOWLEDGMENTS
I thank Ann H. Reid and Thomas G. Fanning for helpful discussions and fruitful collaboration.
This work was partially supported by grants from the American Registry of Pathology and the Department of Veterans Affairs and by the intramural funds of the Armed Forces Institute of Pathology. The opinions or assertions contained herein are the private views of the author and are not to be construed as official or as reflecting the views of the Department of the Army or Department of Defense. This is a U.S. government work; there are no restrictions on its use.
SUGGESTED READING
Crosby, A. W. 1989. America's forgotten pandemic. Cambridge University Press, Cambridge.
Gorman, O. T., W. J. Bean, and R. G. Webster. 1992. Evolutionary processes in influenza viruses: divergence, rapid evolution, and stasis. Curr. Top. Microbiol. Immunol. 176:75-97.
Goto, H., and Y. Kawaoka. 1998. A novel mechanism for the acquisition of virulence by a human influenza A virus. Proc. Natl. Acad. Sci. USA 95:10224-10228.
Kilbourne, E. D. 1997. Perspectives on pandemics: a research agenda. J. Infect. Dis. 176(Suppl.):S29-S31.
Reid, A. H., and J. K. Taubenberger. 1999. The 1918 flu and other influenza pandemics: `over there' and back again. Lab. Invest. 79:95-101.
Reid, A. H., T. G. Fanning, J. V. Hultin, and J. K. Taubenberger. 1999. Origin and evolution of the 1918 ``Spanish'' influenza virus hemagglutinin. Proc. Natl. Acad. Sci. USA 96:1651-1656.
Simonsen, L., M. J. Clarke, L. B. Schonberger, N. H. Arden, N. J. Cox, and K. Fukuda. 1998. Pandemic versus epidemic influenza mortality: a pattern of changing age distribution. J. Infect. Dis. 178:53-60.
Taubenberger, J. K. 1998. Cleavage of influenza virus hemagglutinin into HA1, HA2: no laughing matter. Proc. Natl. Acad. Sci. USA 95:9713-9715.
Taubenberger, J. K., A. E. Reid, A. E. Krafft, K. E. Bijwaard, and T. G. Fanning. 1997. Initial genetic characterization of the 1918 `Spanish' influenza virus. Science 275:1793-1796.
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