Citation: Watanabe T, Kawaoka Y (2011) Pathogenesis of the 1918 Pandemic Influenza Virus. PLoS Pathog7(1): e1001218. https://doi.org/10.1371/journal.ppat.1001218
Editor: Hiten D. Madhani, University of California San Francisco, United States of America
Published: January 27, 2011
Copyright: © 2011 Watanabe, Kawaoka. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported, in part, by U.S. National Institute of Allergy and Infectious Diseases Public Health Service research grants, by a grant-in-aid for Specially Promoted Research (Japan), by funding from the Program of Founding Research Centers for Emerging and Reemerging Infectious Diseases from the Ministry of Education, Culture, Sports, Science and Technology (Japan), and by ERATO (Japan Science and Technology Agency). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
At the height of World War I, the human population was assaulted by a powerful, but very small, foreign agent that rapidly appeared seemingly from nowhere. Ultimately identified as the “Spanish flu”, this agent wreaked havoc on anyone in its path. Prostrating vast numbers of victims worldwide with severe pneumonia, which often progressed to a fatal outcome, the “Spanish flu” caused an estimated 20–50 million deaths worldwide . The resultant 1918 pandemic was one of the most formidable foes faced by humankind. In this brief review, we discuss some recent insights into the pathogenicity of its causative agent, the 1918 pandemic influenza virus.
The “Spanish” Influenza Pandemic of 1918–1919
In early March of 1918, several severe cases of influenza were reported in the United States. This would mark the beginning of the first wave of the “Spanish” influenza pandemic (reviewed in ). As massive numbers of US military troops were deployed in Europe, the virus spread too, leading to outbreaks throughout the United States, Europe, and possibly Asia. Spain was neutral during the war and its press was therefore uncensored. Accordingly, the Spanish newspapers were filled with reports of the disease, especially when the king became ill. It is believed that these published accounts of the spread of the disease explain why the virus became known as the “Spanish” influenza. During the first wave of this, like other, pandemics, mortality rates were in the normal range, though morbidity rates were high. However, while the first wave killed relatively few, by the time the next wave came, in the fall of 1918, the virus had undergone numerous passages through humans, had changed dramatically, and was now lethal. Together with a third wave, which occurred in the winter of 1918–1919, approximately 30% of the world's population (500 million people) is thought to have been clinically affected by the 1918 pandemic . Unexpectedly, the 1918 pandemic attacked particularly young adults, who usually have a low death rate during influenza epidemics. As a result, influenza and pneumonia death rates for 15- to 34-year-olds were more than 20 times higher in 1918 than in the previous year (the mortality rate associated with the 1918 virus was more than 2.5% among infected persons compared with less than 0.1% in other influenza epidemics; ). Most deaths were caused from secondary bacterial pneumonia due to a lack of antibiotics . The 1918 pandemic virus, however, also killed quickly and directly with a violent viral pneumonia, often with either massive acute pulmonary hemorrhage or pulmonary edema. The disease course was frequently less than 5 days .
Efforts to Unmask the Deadly 1918 Pandemic Virus
The identity of the 1918 pandemic virus remained a mystery in the absence of technology to isolate viruses. By the early 1930s, American and British researchers succeeded in establishing a system to isolate swine and human influenza viruses , . With this “new” technique in hand, researchers in the 1950s tried to isolate the 1918 pandemic virus from victims of the pandemic who were buried in permafrost graves in Alaska. However, they were unable to recover virus from the available specimens.
In the late 1990s, the development of molecular biotechnology brought about a breakthrough. Taubenberger's group at the US Armed Forces Institute of Pathology succeeded in isolating viral genome RNA fragments from formalin-fixed, paraffin-embedded autopsy tissues of American army soldiers who had died in the 1918 pandemic . They also isolated viral RNAs from an Alaskan Inuit woman who had been buried in permafrost in Alaska . With these samples, they were able to determine the coding sequences of all eight RNA segments of the 1918 pandemic virus –. This 10-year endeavor finally led them to identify the pathogenic agent that had caused the great 1918 pandemic: that is, influenza A virus subtype H1N1. Further sequencing analyses suggested that the 1918 virus may be of avian origin and transmitted from birds to humans directly or indirectly, although this is remains controversial. In the process of adaptation to humans through mutations, the 1918 virus is thought to have acquired its high virulence.
The Resurrection of the “Spanish” Influenza Virus
Although the complete sequences of the viral RNAs of the 1918 pandemic virus have been determined, the viral genome does not contain any motifs known to be associated with high virulence . Therefore, to understand the extraordinary virulence of the 1918 pandemic virus, it was important to re-create the virus and examine its pathogenicity in animals. The recent technological advancement of reverse genetics, which allows the generation of infectious influenza viruses entirely from cDNAs , made possible the re-creation of the 1918 pandemic virus. Tumpery's group at the US Centers for Disease Control and Prevention  and we  succeeded in rescuing viruses bearing all eight RNA segments of the 1918 virus by using reverse genetics. Now that we had all of the materials required, we could study the molecular properties associated with the unusual virulence of the 1918 pandemic virus.
Aberrant Immune Responses in Animals Infected with the Reconstructed 1918 Virus
To derive a molecular mechanism of pathogenicity for the 1918 virus, the reconstructed 1918 virus was characterized in mouse and non-human primate models –. In the infected animals, the 1918 virus replicated to high levels and spread rapidly throughout the respiratory tract. Severe damage, including extensive edema and hemorrhagic exudates, was observed in the lungs of the virus-infected animals , as reported for patients who succumbed to the “Spanish” influenza, leading to acute respiratory distress and death (Figure 1). Host responses to the 1918 virus were also investigated by using microarray technology , . Strikingly, infection of non-human primates with the 1918 virus triggered high, sustained expression of genes involved in innate immune responses, such as proinflammatory cytokines and chemokines . On the other hand, the virus induced fewer type I interferon genes , likely leading to enhanced viral replication. Such uncontrolled innate immune responses have also been observed in mice inoculated with the 1918 virus  and in hosts infected with avian H5N1 influenza viruses. These findings suggest that strong immune responses are a hallmark of highly pathogenic influenza virus infections.
Taubenberger's group isolated viral genome RNA fragments from formalin-fixed, paraffin-embedded autopsy tissue from victims of the 1918 pandemic and determined the coding sequences of all eight RNA segments of the 1918 pandemic virus. Based on these sequences, a virus bearing all eight RNA segments of the 1918 virus was generated by reverse genetics and its pathogenicity was determined in a non-human primate model. The 1918 virus caused a highly pathogenic respiratory infection in non-human primates that culminated in acute respiratory distress and a fatal outcome. The infected animals mounted an immune response, characterized by dysregulation of the antiviral response, indicating that atypical host innate immune responses may contribute to lethality. The infection of nonhuman primates with the 1918 pandemic virus caused severe damage in the lungs (a), whereas no appreciable lesions were observed in the lungs of animals infected with a contemporary human H1N1 virus (b).
Determinants of the High Virulence of the 1918 Virus in Animals
Which viral factor(s) contribute to the extraordinary virulence of the 1918 virus? One of the key players is hemagglutinin (HA), which is the viral surface glycoprotein that has two functions in the early stage of virus replication: receptor binding and membrane fusion. The HAs of highly pathogenic avian influenza viruses play a critical role in virulence as they typically contain a specific motif (i.e., a series of basic amino acids at the cleavage site , ) that contributes to pathogenicity. This motif, however, is not found in the 1918 HA sequence. Nonetheless, we and others demonstrated that a reassortant virus possessing the 1918 HA gene in the genetic background of a contemporary human virus replicated to a high titer in the lungs and caused severe lung damage accompanied by a significant influx of neutrophils and alveolar macrophages into the lung , (Figure 2). Similar findings were observed with the authentic 1918 virus  with severe morbidity and eventual death. These results suggest a critical role for the HA gene in the pathogenicity of the 1918 virus . The region(s) of HA responsible for high virulence has not yet been identified; however, Taubenberger's group has demonstrated that HA receptor binding specificity plays a role in pathogenesis in mice, although their results also suggest the presence of additional virulence determinant(s) . The importance of the 1918 HA, especially two amino acids that are responsible for receptor binding specificity  and efficient virus transmission, has also been demonstrated in ferrets , .
Mice infected with a contemporary human H1N1 influenza virus showed no symptoms. In contrast, a virus possessing the HA gene derived from the 1918 virus in the genetic background of the contemporary human H1N1 virus was lethal to mice.
The other important player in the virulence of the 1918 virus is the viral RNA polymerase complex. A unique feature of the 1918 pandemic was that many people died from viral pneumonia; human influenza viruses typically replicate poorly in the lungs of infected individuals and rarely cause fatal viral pneumonia. We reported efficient replication of the 1918 virus in the lungs of infected ferrets and non-human primates, which led to viral pneumonia , . In contrast, a contemporary human H1N1 virus was not detected in the lungs of infected animals, although it replicated in the nasal turbinates. Hence, we assume that the ability of the 1918 virus to grow in the lungs is associated with its high virulence in humans. We also found that high virus titers in the lungs of infected ferrets were dependent on the 1918 polymerase and NP genes (Figure 3) . The importance of the polymerase genes in pathogenicity in mice  and transmission in ferrets  was also shown. These findings strongly implicate the viral RNA polymerase complex in the efficient spread of virus to the lower respiratory tract and suggest it may be required, together with a specific HA, to induce the type of fatal pneumonia encountered during the 1918–1919 pandemic.
The 1918 wild-type virus and a virus possessing the 1918 viral RNA polymerase complex genes (i.e., PA, PB1, PB2, and NP) replicated efficiently in both the upper and lower respiratory tracts of ferrets, whereas the replication of the contemporary human H1N1 virus was restricted to the upper respiratory tract, suggesting a role for the viral RNA polymerase complex in the optimal growth of the 1918 virus in the lower respiratory tract of ferrets. +++, high replicative ability; ++, moderate replicative ability; −, no virus detected.
Other viral factors may also be involved in the pathogenicity of the 1918 pandemic virus, such as PB1-F2, NA, and NS1 –. PB1-F2, a proapoptotic viral protein, requires only a single amino acid change at position 66 to increase the pathogenicity of the 1918 virus . Expression of the 1918 PB1-F2 promotes lung pathology in primary viral infection and secondary bacterial infection . A potential role for the NA gene in optimal replication and virulence of the 1918 virus has also been suggested . For NS1, an antagonist of cellular innate immune defenses, the 1918 protein is better able to block the expression of IFN-regulated genes than is the NS1 protein of other human influenza viruses . Moreover, the C-terminal of NS1 is known to contribute to enhanced virulence and pathogenesis by a mechanism distinct from its IFN-antagonistic properties . Despite these important functions of NS1, substitution of the NS gene of the 1918 virus for that of a parental virus caused attenuation of the parental virus in mice , .
The first influenza pandemic of the 21st century was caused by a novel swine-origin H1N1 influenza virus that emerged in early 2009. This virus is substantially less virulent than the 1918 influenza virus, but it has the potential to acquire amino acid changes in its viral proteins that would increase its pathogenicity. To prepare for such events and future pandemics, we need to understand the molecular basis of the high-virulence phenotype of the 1918 pandemic virus to help identify virulence factors in other emerging pandemic viruses. Additionally, the fact that more than 97% of the people infected with the 1918 virus survived raises the intriguing possibility of some contribution of host genetics to the consequences of influenza (i.e., survival or death). Thus, it is also important to explore host factors that are involved in resistance or susceptibility to influenza virus infection. Such information could accelerate the development of new antiviral drugs for prophylaxis and treatment, which are urgently needed given the obstacles to rapid development of an effective vaccine against pandemic influenza.
- 1. Taubenberger JK, Reid AH, Janczewski TA, Fanning TG (2001) Integrating historical, clinical and molecular genetic data in order to explain the origin and virulence of the 1918 Spanish influenza virus. Philos Trans R Soc Lond B Biol Sci 356: 1829–1839.JK TaubenbergerAH ReidTA JanczewskiTG Fanning2001Integrating historical, clinical and molecular genetic data in order to explain the origin and virulence of the 1918 Spanish influenza virus.Philos Trans R Soc Lond B Biol Sci35618291839
- 2. Shope RE (1931) Swine Influenza : Iii. Filtration Experiments and Etiology. J Exp Med 54: 373–385.RE Shope1931Swine Influenza : Iii. Filtration Experiments and Etiology.J Exp Med54373385
- 3. Smith W, Andrewes C, Laidlaw P (1933) A virus obtained from influenza patients. Lancet 66–68.W. SmithC. AndrewesP. Laidlaw1933A virus obtained from influenza patients.Lancet6668
- 4. Taubenberger JK, Reid AH, Krafft AE, Bijwaard KE, Fanning TG (1997) Initial genetic characterization of the 1918 “Spanish” influenza virus. Science 275: 1793–1796.JK TaubenbergerAH ReidAE KrafftKE BijwaardTG Fanning1997Initial genetic characterization of the 1918 “Spanish” influenza virus.Science27517931796
- 5. Reid AH, Fanning TG, Hultin JV, Taubenberger JK (1999) Origin and evolution of the 1918 “Spanish” influenza virus hemagglutinin gene. Proc Natl Acad Sci U S A 96: 1651–1656.AH ReidTG FanningJV HultinJK Taubenberger1999Origin and evolution of the 1918 “Spanish” influenza virus hemagglutinin gene.Proc Natl Acad Sci U S A9616511656
- 6. Taubenberger JK, Reid AH, Lourens RM, Wang R, Jin G, et al. (2005) Characterization of the 1918 influenza virus polymerase genes. Nature 437: 889–893.JK TaubenbergerAH ReidRM LourensR. WangG. Jin2005Characterization of the 1918 influenza virus polymerase genes.Nature437889893
- 7. Reid AH, Fanning TG, Janczewski TA, Lourens RM, Taubenberger JK (2004) Novel origin of the 1918 pandemic influenza virus nucleoprotein gene. J Virol 78: 12462–12470.AH ReidTG FanningTA JanczewskiRM LourensJK Taubenberger2004Novel origin of the 1918 pandemic influenza virus nucleoprotein gene.J Virol781246212470
- 8. Reid AH, Fanning TG, Janczewski TA, McCall S, Taubenberger JK (2002) Characterization of the 1918 “Spanish” influenza virus matrix gene segment. J Virol 76: 10717–10723.AH ReidTG FanningTA JanczewskiS. McCallJK Taubenberger2002Characterization of the 1918 “Spanish” influenza virus matrix gene segment.J Virol761071710723
- 9. Reid AH, Fanning TG, Janczewski TA, Taubenberger JK (2000) Characterization of the 1918 “Spanish” influenza virus neuraminidase gene. Proc Natl Acad Sci U S A 97: 6785–6790.AH ReidTG FanningTA JanczewskiJK Taubenberger2000Characterization of the 1918 “Spanish” influenza virus neuraminidase gene.Proc Natl Acad Sci U S A9767856790
- 10. Basler CF, Reid AH, Dybing JK, Janczewski TA, Fanning TG, et al. (2001) Sequence of the 1918 pandemic influenza virus nonstructural gene (NS) segment and characterization of recombinant viruses bearing the 1918 NS genes. Proc Natl Acad Sci U S A 98: 2746–2751.CF BaslerAH ReidJK DybingTA JanczewskiTG Fanning2001Sequence of the 1918 pandemic influenza virus nonstructural gene (NS) segment and characterization of recombinant viruses bearing the 1918 NS genes.Proc Natl Acad Sci U S A9827462751
- 11. Neumann G, Watanabe T, Ito H, Watanabe S, Goto H, et al. (1999) Generation of influenza A viruses entirely from cloned cDNAs. Proc Natl Acad Sci U S A 96: 9345–9350.G. NeumannT. WatanabeH. ItoS. WatanabeH. Goto1999Generation of influenza A viruses entirely from cloned cDNAs.Proc Natl Acad Sci U S A9693459350
- 12. Tumpey TM, Basler CF, Aguilar PV, Zeng H, Solorzano A, et al. (2005) Characterization of the reconstructed 1918 Spanish influenza pandemic virus. Science 310: 77–80.TM TumpeyCF BaslerPV AguilarH. ZengA. Solorzano2005Characterization of the reconstructed 1918 Spanish influenza pandemic virus.Science3107780
- 13. Kobasa D, Jones SM, Shinya K, Kash JC, Copps J, et al. (2007) Aberrant innate immune response in lethal infection of macaques with the 1918 influenza virus. Nature 445: 319–323.D. KobasaSM JonesK. ShinyaJC KashJ. Copps2007Aberrant innate immune response in lethal infection of macaques with the 1918 influenza virus.Nature445319323
- 14. Kash JC, Tumpey TM, Proll SC, Carter V, Perwitasari O, et al. (2006) Genomic analysis of increased host immune and cell death responses induced by 1918 influenza virus. Nature 443: 578–581.JC KashTM TumpeySC ProllV. CarterO. Perwitasari2006Genomic analysis of increased host immune and cell death responses induced by 1918 influenza virus.Nature443578581
- 15. Klenk HD, Garten W (1994) Host cell proteases controlling virus pathogenicity. Trends Microbiol 2: 39–43.HD KlenkW. Garten1994Host cell proteases controlling virus pathogenicity.Trends Microbiol23943
- 16. Steinhauer DA (1999) Role of hemagglutinin cleavage for the pathogenicity of influenza virus. Virology 258: 1–20.DA Steinhauer1999Role of hemagglutinin cleavage for the pathogenicity of influenza virus.Virology258120
- 17. Kobasa D, Takada A, Shinya K, Hatta M, Halfmann P, et al. (2004) Enhanced virulence of influenza A viruses with the haemagglutinin of the 1918 pandemic virus. Nature 431: 703–707.D. KobasaA. TakadaK. ShinyaM. HattaP. Halfmann2004Enhanced virulence of influenza A viruses with the haemagglutinin of the 1918 pandemic virus.Nature431703707
- 18. Tumpey TM, Garcia-Sastre A, Taubenberger JK, Palese P, Swayne DE, et al. (2005) Pathogenicity of influenza viruses with genes from the 1918 pandemic virus: functional roles of alveolar macrophages and neutrophils in limiting virus replication and mortality in mice. J Virol 79: 14933–14944.TM TumpeyA. Garcia-SastreJK TaubenbergerP. PaleseDE Swayne2005Pathogenicity of influenza viruses with genes from the 1918 pandemic virus: functional roles of alveolar macrophages and neutrophils in limiting virus replication and mortality in mice.J Virol791493314944
- 19. Perrone LA, Plowden JK, Garcia-Sastre A, Katz JM, Tumpey TM (2008) H5N1 and 1918 pandemic influenza virus infection results in early and excessive infiltration of macrophages and neutrophils in the lungs of mice. PLoS Pathog 4: e1000115.LA PerroneJK PlowdenA. Garcia-SastreJM KatzTM Tumpey2008H5N1 and 1918 pandemic influenza virus infection results in early and excessive infiltration of macrophages and neutrophils in the lungs of mice.PLoS Pathog4e1000115
- 20. Qi L, Kash JC, Dugan VG, Wang R, Jin G, et al. (2009) Role of sialic acid binding specificity of the 1918 influenza virus hemagglutinin protein in virulence and pathogenesis for mice. J Virol 83: 3754–3761.L. QiJC KashVG DuganR. WangG. Jin2009Role of sialic acid binding specificity of the 1918 influenza virus hemagglutinin protein in virulence and pathogenesis for mice.J Virol8337543761
- 21. Glaser L, Stevens J, Zamarin D, Wilson IA, Garcia-Sastre A, et al. (2005) A single amino acid substitution in 1918 influenza virus hemagglutinin changes receptor binding specificity. J Virol 79: 11533–11536.L. GlaserJ. StevensD. ZamarinIA WilsonA. Garcia-Sastre2005A single amino acid substitution in 1918 influenza virus hemagglutinin changes receptor binding specificity.J Virol791153311536
- 22. Van Hoeven N, Pappas C, Belser JA, Maines TR, Zeng H, et al. (2009) Human HA and polymerase subunit PB2 proteins confer transmission of an avian influenza virus through the air. Proc Natl Acad Sci U S A 106: 3366–3371.N. Van HoevenC. PappasJA BelserTR MainesH. Zeng2009Human HA and polymerase subunit PB2 proteins confer transmission of an avian influenza virus through the air.Proc Natl Acad Sci U S A10633663371
- 23. Tumpey TM, Maines TR, Van Hoeven N, Glaser L, Solorzano A, et al. (2007) A two-amino acid change in the hemagglutinin of the 1918 influenza virus abolishes transmission. Science 315: 655–659.TM TumpeyTR MainesN. Van HoevenL. GlaserA. Solorzano2007A two-amino acid change in the hemagglutinin of the 1918 influenza virus abolishes transmission.Science315655659
- 24. Watanabe T, Watanabe S, Shinya K, Kim JH, Hatta M, et al. (2009) Viral RNA polymerase complex promotes optimal growth of 1918 virus in the lower respiratory tract of ferrets. Proc Natl Acad Sci U S A 106: 588–592.T. WatanabeS. WatanabeK. ShinyaJH KimM. Hatta2009Viral RNA polymerase complex promotes optimal growth of 1918 virus in the lower respiratory tract of ferrets.Proc Natl Acad Sci U S A106588592
- 25. Pappas C, Aguilar PV, Basler CF, Solorzano A, Zeng H, et al. (2008) 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 U S A 105: 3064–3069.C. PappasPV AguilarCF BaslerA. SolorzanoH. Zeng2008Single 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 U S A10530643069
- 26. Conenello GM, Zamarin D, Perrone LA, Tumpey T, Palese P (2007) A single mutation in the PB1-F2 of H5N1 (HK/97) and 1918 influenza A viruses contributes to increased virulence. PLoS Pathog 3: e141.GM ConenelloD. ZamarinLA PerroneT. TumpeyP. Palese2007A single mutation in the PB1-F2 of H5N1 (HK/97) and 1918 influenza A viruses contributes to increased virulence.PLoS Pathog3e141
- 27. McAuley JL, Hornung F, Boyd KL, Smith AM, McKeon R, et al. (2007) Expression of the 1918 influenza A virus PB1-F2 enhances the pathogenesis of viral and secondary bacterial pneumonia. Cell Host Microbe 2: 240–249.JL McAuleyF. HornungKL BoydAM SmithR. McKeon2007Expression of the 1918 influenza A virus PB1-F2 enhances the pathogenesis of viral and secondary bacterial pneumonia.Cell Host Microbe2240249
- 28. Geiss GK, Salvatore M, Tumpey TM, Carter VS, Wang X, et al. (2002) Cellular transcriptional profiling in influenza A virus-infected lung epithelial cells: the role of the nonstructural NS1 protein in the evasion of the host innate defense and its potential contribution to pandemic influenza. Proc Natl Acad Sci U S A 99: 10736–10741.GK GeissM. SalvatoreTM TumpeyVS CarterX. Wang2002Cellular transcriptional profiling in influenza A virus-infected lung epithelial cells: the role of the nonstructural NS1 protein in the evasion of the host innate defense and its potential contribution to pandemic influenza.Proc Natl Acad Sci U S A991073610741
- 29. Jackson D, Hossain MJ, Hickman D, Perez DR, Lamb RA (2008) A new influenza virus virulence determinant: the NS1 protein four C-terminal residues modulate pathogenicity. Proc Natl Acad Sci U S A 105: 4381–4386.D. JacksonMJ HossainD. HickmanDR PerezRA Lamb2008A new influenza virus virulence determinant: the NS1 protein four C-terminal residues modulate pathogenicity.Proc Natl Acad Sci U S A10543814386