Abstract
The 1918 influenza pandemic was the most devastating outbreak of infectious disease in human history, accounting for about 50 million deaths worldwide. In addition to a significant number of cases of secondary bacterial pneumonia, this highly pathogenic strain of influenza A virus caused fatal primary viral pneumonia. To identify the viral gene(s) chiefly responsible for the high virulence of the 1918 virus, we generated a series of reassortants between the 1918 virus and a contemporary human H1N1 virus (A/Kawasaki/173/2001; K173) using reverse genetics. We then assessed their virulence properties in ferrets, a model closely resembling humans in terms of sensitivity to influenza virus infection and pattern of spread after intranasal inoculation. Substitution of single genes from the 1918 virus in the genetic background of K173 virus did not markedly alter the pattern of infection. That is, the reassortants grew well in nasal turbinates, but only sporadically (if at all) in the trachea and lungs. One exception was the 1918PB1/K173 reassortant, which replicated efficiently in lung tissues as well as the upper respiratory tract. A reassortant virus expressing the 1918 viral RNA polymerase complex (PA, PB1, and PB2) and nucleoprotein showed virulence properties in the upper and lower respiratory tracts of ferrets that closely resembled those of wild-type 1918 virus. Our findings strongly implicate the viral RNA polymerase complex as a major determinant of the pathogenicity of the 1918 pandemic virus. This new insight may aid in identifying virulence factors in future pandemic viruses that could be targeted with antiviral compounds.
Keywords: pathogenesis, pandemic influenza
In the last century, human populations faced three influenza pandemics: the so-called “Spanish influenza” in 1918/1919, the “Asian influenza” in 1957, and the “Hong Kong influenza” in 1968. Of these, the 1918 pandemic was the most deadly, resulting in clinical infection of approximately 500 million people, or about 30% of the world's population (1). The mortality rate associated with this virus was unprecedented—more than 2.5% among infected persons compared with less than 0.1% in other influenza epidemics—leading to an estimated 20–50 million deaths worldwide (1–3). The 1918 virus preferentially attacked young adults, a group that usually has a very low death rate during influenza epidemics (1, 4). The vast majority of deaths not caused by secondary bacterial pneumonia were attributed to either massive acute pulmonary hemorrhage or pulmonary edema, which often proved fatal within 5 days (1, 5–7).
Unraveling the mechanism(s) of the extraordinary virulence of the 1918 virus has been a daunting task. Taubenberger and colleagues succeeded in amplifying and sequencing all 8 RNA segments of the 1918 virus recovered from preserved lung tissues of 3 victims of the pandemic (8–14). Using plasmid-driven reverse genetics systems, we and others generated viruses bearing all 8 segments of the 1918 strain, enabling study of the molecular properties associated with the unusual virulence of this virus (15, 16). The reconstructed 1918 virus causes a highly pathogenic respiratory infection in mice (15) and macaques (16) that leads to acute respiratory distress culminating in a fatal outcome. It also triggers aberrantly high and sustained expression of genes encoding many proteins involved in the innate immune response, including proinflammatory cytokines and chemokines, suggesting that atypical host innate immune responses may contribute to severe tissue damage, disease, and death (16). Although the HA gene has been linked to the extreme pathogenicity of the 1918 virus in mice (15, 17, 18), it does not contain any motif known to be associated with high virulence, such as multiple basic acid residues at the HA cleavage site (9). Thus, other genes may also play a role in its pathogenicity. Indeed, Tumpey and colleagues (19) recently showed that the PB1 gene has an important role in efficient replication of the 1918 virus in human airway cells and mouse lung.
A unique feature of the 1918 virus found in our study of macaques was that its replicative capacity and tissue tropism differed from those of a contemporary human isolate with low pathogenicity (A/Kawasaki/173/2001; K173). That is, the 1918 virus was present at high titers in both the upper and lower respiratory tract tissues, whereas the K173 virus was isolated primarily from the upper respiratory tract tissues at lower titers (16). In general, human influenza viruses infect and replicate in tissues of the upper respiratory tract (20). Hence, it is assumed that the ability of the 1918 virus to grow in the lower respiratory tract is associated with its high virulence in humans, but the viral genes that support such replication are still poorly understood. To close this gap, we generated a series of reassortant viruses between the 1918 virus and the human K173 virus and assessed their virulence properties in ferrets. This model was selected over mice because the disease manifestation in ferrets to influenza virus infection and the pattern of spread after intranasal inoculation are more representative of human influenza virus infection (21). Here we report that a combination of the 1918 viral polymerase genes (PA, PB1, and PB2), together with the nucleoprotein (NP) gene, was more effective than any single-gene substitution, including the HA or PB1 alone, in transforming the currently circulating K173 virus into a 1918-like strain in terms of replicative efficiency and tissue tropism. These findings may help us identify virulence factors in other emerging pandemic viruses and 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.
Results
Generation of Single-Gene Reassortants Possessing Genes from the 1918 Virus in the Genetic Background of a Contemporary Human H1N1 Virus.
We and others have demonstrated an important contribution from the HA gene to the pathogenicity of the 1918 virus in mice (15, 17, 18). To identify other viral genes that may have supported the virulence of the 1918 virus, we generated a spectrum of reassortants possessing genes of the 1918 virus (A/Brevig Misson/1/18) in the genetic background of a contemporary human H1N1 virus, A/Kawasaki/173/2001 (K173), using plasmid-driven reverse genetics (22). The wild-type 1918 and K173 viruses were also generated by reverse genetics.
To confirm that the replacement of a gene from K173 virus with that from the 1918 virus does not cause significant growth defect, we compared the growth kinetics of reassortants with those of the wild-type viruses in cell culture. Madin-Darby canine kidney (MDCK) cells were infected with viruses at a multiplicity of infection (MOI) of 0.001 and their growth kinetics monitored for 72 h. As shown in Fig. 1, the wild-type 1918 virus replicated well, reaching a maximum titer at 24 h postinfection [p.i.; 3.9 × 108 plaque-forming units (PFU)/ml], which was one log higher than that of the K173 virus (1.3 × 107 PFU/ml at 48 h p.i.). Reassortant viruses possessing the PB1 or HA gene of the 1918 virus grew more efficiently in MDCK cells than did the K173 virus, reaching maximum titers of more than 108 PFU/ml. In contrast, the titers of reassortant viruses possessing the 1918 PA, PB2, NP, M, or NS gene were comparable to that of the wild-type K173 virus, although a virus expressing the 1918 neuraminidase (NA) gene replicated slightly slower than did the K173 virus (Fig. 1). These results suggest that the reassortant viruses replicated reasonably well in MDCK cells without any significant growth defect.
Fig. 1.
Growth kinetics of reassortant viruses in MDCK cells. MDCK cells were infected with reassortant, wild-type 1918, or K173 virus at an MOI of 0.001. At the indicated times after infection, virus titers in the supernatant were determined with MDCK cells. The reported values are means ± SD from five experiments.
Evaluation of the 1918 and K173 Wild-Type Viruses in Ferrets.
In a macaque model, we found that the 1918 virus replicated well in tissues of both the upper and lower respiratory tracts, whereas the K173 virus was isolated mainly from the upper respiratory tissues at considerably lower titers (16). To identify the viral gene(s) of the 1918 virus that promotes efficient spread to the lower respiratory tract tissues, we evaluated a panel of reassortant viruses in our ferret model. To validate this model, we compared the growth properties of the wild-type 1918 and K173 viruses in ferrets intranasally infected with 105 PFU of each virus. On day 3 p.i., both strains replicated well in nasal turbinates (Table 1) and produced marked pathologic changes in the nasal mucosa (supporting information (SI) Table S1 and Fig. S1). By contrast, only the 1918 virus was recovered from the lungs and trachea of infected animals (Table 1). In the lungs of the 1918 virus-infected ferrets, we observed macroscopic pathologic changes such as severe lesions and hemorrhage (Fig. S2C), as well as severe peribronchitis and bronchopneumonia (Fig. 2C). Viral antigens were mainly detected on peribronchial glands and rarely on terminal bronchial epithelia (Fig. 2D and Table S1). Virus was not detected in either the spleen or kidneys. K173 virus, on the other hand, produced only mild peribronchitis and bronchopneumonia in some lung lobes (Fig. 2A); viral antigen could not be detected (Fig. 2B and Table S1). These results are consistent with our previous findings in a macaque model (16), verifying that ferrets are a reliable model for testing the contributions of individual viral genes to lung pathology.
Table 1.
Replication efficiency of 1918/K173 recombinant viruses in ferrets*
| Virus | Virus titer (mean log10PFU ± SD/g) in: |
||
|---|---|---|---|
| Nasal turbinates | Trachea | Lungs | |
| 1918 WT | 7.43 ± 0.13 | 6.65 ± 0.73 | 6.40 ± 0.40 |
| K173 WT | 5.92 ± 0.67 | —† | — |
| 1918(3P + NP)/K173 | 6.69 ± 0.37 | 5.21 ± 0.62 | 5.98 ± 0.28 |
| K173(3P + NP)/1918 | 6.90 ± 0.38 | 2.72 | — |
| 1918PA/K173 | 4.26 ± 0.11 | — | — |
| 1918PB1/K173 | 5.62 ± 0.32 | 2.84, 1.85 | 4.31, 5.36 |
| 1918PB2/K173 | 5.24 ± 0.28 | — | — |
| 1918NP/K173 | 5.55 ± 0.65 | — | 3.58 |
| 1918HA/K173 | 5.74 ± 0.11 | — | — |
| 1918NA/K173 | 4.44 ± 0.02 | — | — |
| 1918M/K173 | 5.35 ± 1.39 | — | — |
| 1918NS/K173 | 5.55 ± 0.37 | — | 2.36 |
* Ferrets, anesthetized with ketamine and xylazine, were infected intranasally with 105 PFU/500 μl of virus. Three ferrets from each group were euthanized on day 3 p.i. for virus titration. Individual titers were recorded when virus was not recovered from all three ferrets.
† —, Virus not isolated.
Fig. 2.
Pathologic examination of lungs of ferrets infected with viruses. On day 3 p.i., lung samples were collected from ferrets infected with 105 PFU of the 1918, K173, or 1918(3P+NP)/K173 virus for pathologic examination. (A) In lungs of a K173 virus-infected ferret, peribronchitis and bronchopneumonia were observed in some lung lobes, but (B) viral antigen was not detected. In lungs of animals infected with (C) the 1918 virus or (E) the 1918(3P+NP)/K173 reassortant, we observed moderate to severe peribronchitis and bronchopneumonia. Viral antigens were mainly detected in peribronchial gland (arrows) and rarely in terminal bronchial epithelium (D and F). (Scale bars, 100 μm.) The distribution of viral antigen and bronchitis/bronchopneumonia is shown schematically on the left. The left lower lobes of the lungs (gray) were used for virologic examination.
The 1918 Viral RNA Polymerase Complex Promotes Optimal Spread of Influenza Virus to the Lower Respiratory Tract of Ferrets.
Table 1 shows the replication efficiencies of our panel of reassortant viruses in a ferret model. Most of the single-gene reassortants replicated similarly to the K173 virus: that is, they showed reasonably efficient growth in nasal turbinates but poor or no growth in trachea and lungs. Even the 1918HA/K173 virus lacked increased growth potential in lower respiratory tract tissues (Table 1). It also did not cause appreciable macroscopic pathologic changes in lungs and produced only limited histopathologic changes (Table S1 and Figs. S2D and S3 C and D). By contrast, the 1918PB1/K173 virus was isolated from the trachea and lungs of two of three ferrets as well as nasal turbinates (Table 1). Pathologic changes were detected in the tracheas of infected ferrets, notably migration of neutrophils into tracheal epithelial cells, focal disarrangement of epithelial cells, and focal inflammation of tracheal glands (Fig. S4). In the lungs of ferrets infected with the 1918PB1/K173 virus, we observed peribronchitis and bronchopneumonia and detected viral antigens in the epithelial cells of bronchial glands (Fig. S3 E and F).
The viral RNA polymerase complex, consisting of three polymerase proteins (PA, PB1, and PB2) and NP, is responsible for the transcription and replication of the influenza viral RNA genome (23). In view of our findings for PB1 (Table 1) and of a recent report indicating an important role for this gene in replication of the 1918 virus (19), we asked whether a recombinant virus expressing all four genes encoding the polymerase complex (1918[3P+NP]/K173) would possess a high-growth phenotype reminiscent of the 1918 pandemic strain. In MDCK cells, this reassortant replicated very efficiently, reaching a titer that was comparable to that of the wild-type 1918 virus (1.4 × 108 PFU/ml; Fig. 1). Additionally, we also examined the growth property of a recombinant virus, K173(3P+NP)/1918, which contained three polymerase and NP genes from K173 and the remaining four genes (HA, NA, M and NS) from the 1918 virus, and found that the K173(3P+NP)/1918 virus grew slower than the K173 virus (Fig. 1). In ferrets, the 1918(3P+NP)/K173 virus replicated more efficiently in both trachea and lungs as well as nasal turbinates on day 3 p.i., as compared with the wild-type K173 virus and each of the single-gene reassortants (Table 1). Pathologic changes were also detected in these tissues (Table S1). Small macroscopic lesions (Fig. S2F) and moderate-to-severe peribronchitis and bronchopneumonia with viral antigens were observed in the lungs of ferrets infected with this four-gene reassortant (Fig. 2 E and F and Table S1). By contrast, replication of the K173(3P+NP)/1918 virus was limited to nasal turbinates despite the presence of both the 1918 HA and NA (Table 1). Thus, the viral RNA polymerase complex appears to be essential to efficient replication of the 1918 virus in both the upper and lower respiratory tracts of ferrets and therefore to its pathogenicity. As shown in Tables S2–S5, sequence comparison analysis showed that there are 21, 25, 32 and 34 aa differences between the PA, PB1, PB2, and NP of the 1918 virus and those of K173 virus, respectively. Therefore, it is likely that those amino acid differences are responsible for the growth difference between the 1918 and K173 viruses observed in the respiratory tracts of ferrets.
Discussion
Here we used a ferret model of influenza virus infection to gain insight into genes responsible for the high-growth phenotype and extraordinary virulence of the 1918 pandemic virus. Our results, obtained by replacing genes from a currently circulating human influenza virus with those from the 1918 virus, show that both the 1918 polymerase subunit (PA, PB1, and PB2) and NP genes are required for optimal replication of the contemporary K173 virus in the nasal turbinates, trachea, and lungs of ferrets. This result suggests that the viral RNA polymerase complex played an important role in the spread of the 1918 virus from the upper to the lower respiratory tract.
In general, human influenza viruses infect and replicate in the upper respiratory tract (20). Because of their poor replication in the lower respiratory tract, they rarely cause fatal pneumonia, which tends to be associated with secondary bacterial infection (20, 24). As in humans, the replication of human influenza viruses in ferrets is mainly limited to the upper respiratory tract (20, 25, 26). We recently showed, in macaques, that the 1918 virus differs from a contemporary human virus (K173) in both its pathogenicity and tissue tropism (16). Unlike the K173 strain, the 1918 virus was present at high titers in both the upper and lower respiratory tract tissues, whereas the K173 virus was isolated mainly from the upper respiratory tract at lower titers (16). This pattern of infection was reiterated in ferrets, in which the highly pathogenic 1918 virus grew well in trachea, lungs, and nasal turbinates, while the less pathogenic contemporary K173 virus replicated only in nasal turbinates (Table 1), indicating a close correlation between the ability of the 1918 virus to infect the lower respiratory tract and its pathogenicity. Indeed, a distinct feature of the 1918 virus was its ability to cause fatal primary viral pneumonia in persons infected during the 1918 pandemic (1, 5, 27). We suggest that the viral RNA polymerase complex of the 1918 virus was a major contributor to the efficient spread of virus from the upper to the lower respiratory tract and therefore to the exceptional severity of the “Spanish” influenza.
How does the 1918 viral RNA polymerase complex contribute to the efficient spread of virus to the lower respiratory tract? One possible explanation is simply that it contributes to the increased replication ability of the virus, a characteristic which likely allows the virus to overcome the host immune responses, facilitating efficient virus replication in the lower respiratory tract. This increased ability to replicate could result from the enhanced polymerase activity of viral polymerase complex. The PB1 segment within the polymerase complex is particularly interesting because both the 1957 and 1968 pandemic viruses contained this segment, in addition to the HA and/or NA segments, from avian viruses (28). Since the 1918 pandemic virus is thought to have originated from spread and adaptation of an avian virus to humans (13, 29), it likely contained the avian PB1 gene. Moreover, in a minireplicon system, an avian virus PB1 supported virus-like RNA replication and transcription better than a human virus PB1 (30), suggesting that the avian gene confers an important replicative advantage in mammals. This property may reflect the polymerase activity of the PB1 protein itself or the recently discovered proapoptotic viral protein, PB1-F2, which is encoded by the PB1 gene (31). Recent studies have implicated PB1-F2 in the enhanced pathogenicity of the 1918 virus (32, 33), although the molecular basis for this involvement remains unclear. Mazur, et al. (34) have proposed a novel function for PB1-F2, whereby this protein indirectly regulates polymerase activity through its interaction with PB1. A different tissue tropism controlled by the polymerase complex could be another possible explanation. For example, the 1918 polymerase complex may be able to better use a lung-specific cellular factor to support virus replication or to circumvent a cellular factor that inhibits virus replication, leading to efficient viral replication in the lung tissues. As shown in Table S3, we found that there are 25 aa differences between the PB1 of the 1918 virus and that of K173 virus. Thus, some of the amino acid differences found between the two PB1 proteins may be responsible for the difference in viral replication in the lower respiratory tract of ferrets.
Earlier evaluations in mice suggested that the HA gene exerts a critical influence on the pathogenicity of the 1918 virus (15, 17, 18). In one study, we showed that a recombinant influenza virus possessing the 1918 HA gene strongly induced proinflammatory cytokines in infected mice (18). These data also suggested that the 1918 HA is a critical determinant of macrophage activation, especially early in infection, and of the production of chemoattractants for neutrophils, leading to the trafficking of neutrophils and acute lung injury (18). We subsequently showed that infection of nonhuman primates with the 1918 virus triggered high, sustained expression of genes involved in innate immune responses, such as proinflammatory cytokines and chemokines (16), but induced fewer IFN-α genes, which likely enhanced viral replication (16). “Overactive” innate immune responses have also been observed in mice inoculated with the 1918 virus (35), as well as in hosts infected with avian H5N1 influenza viruses (36–39). These findings implicate the HA gene in the strong immune responses associated with infection by the 1918 virus; however, in ferrets, a single-gene reassortant virus expressing the 1918 HA gene failed to establish infection in lung tissues (Table 1 and Fig. S3 C and D). Thus, in hosts with relative resistance to influenza virus infection, the 1918 and H5N1 HAs may not be sufficient alone to confer a high-virulence phenotype.
Reassortant virus possessing the 1918 NA gene, designated 1918NA/K173 virus, replicated less efficiently both in vitro and in vivo than did the K173 virus (Fig. 1 and Table 1), despite evidence that this gene is required for optimal replication and virulence of the 1918 virus (19). The NA protein facilitates the mobility of virions by removing sialic acid residues from viral glycoproteins and infected cells during both entry and release from cells (40–43). The balance between the receptor-binding activity of the HA and the sialidase activity of the NA is crucial for efficient virus replication in host cells (44). Hence, replacement of a currently circulating NA gene with the 1918 NA gene would be expected to disrupt the usual balance between the HA and NA, leading to attenuation of the reassortant, as was seen with the 1918NA/K173 virus.
Understanding the molecular basis of the high-virulence phenotype of the 1918 pandemic virus is important, as it could identify useful targets for drug intervention when new pandemic viruses begin to emerge. Our data, obtained in a ferret model closely simulating influenza virus infection in humans, suggest that the viral RNA polymerase complex contributes importantly to the efficient spread of virus to the lower respiratory tract and may be required, together with a specific HA, to induce fatal pneumonias, such as those encountered during the 1918pandemic.
Materials and Methods
Cells.
293T human embryonic kidney cells and MDCK cells were maintained in DMEM supplemented with 10% FCS and in MEM (MEM) containing 5% newborn calf serum, respectively. All cells were maintained at 37 °C in 5% CO2.
Plasmid-Driven Reverse Genetics.
All reassortant viruses and the parental 1918 and K173 viruses were generated from plasmids. Each reassortant expressed one of the eight viral RNA segments or the PA, PB1, and PB2 plus NP (3P+NP) genes, as described by Neumann, et al. (22). Forty-eight hours posttransfection, viruses were harvested and used to inoculate MDCK cells for the production of stock viruses. Eight genes of each transfectant virus were partially sequenced to confirm the origin of the gene. All experiments with live viruses and with transfectants generated by reverse genetics were performed in an enhanced biosafety level 3 (BSL3) containment laboratory approved for such use by the Centers for Disease Control and Prevention and the U.S. Department of Agriculture.
Replicative Properties of the Transfectant Viruses in MDCK Cells.
MDCK cells were infected with the 1918, K173, or reassortant viruses at an MOI of 0.001, overlaid with MEM medium containing 1.0 μg of trypsin per ml, and incubated at 37 °C. At select times, supernatants were assayed for infectious virus in plaque assays on MDCK cells.
Ferret Experiments.
Four- to six-month-old female ferrets (Triple F Farms and Marshall Farms) that were serologically negative by hemagglutination inhibition assay for currently circulating influenza viruses were used in this study. They were anesthetized with ketamine and xylazine (5 mg and 0.5 mg per kg of body weight, respectively) intramuscularly and infected intranasally with 500 μl (105 PFU) of virus. On day 3 p.i., the animals were euthanized for virologic and pathologic examinations. Virus titers in the organs were determined by plaque assay on MDCK cells.
Pathologic Examination.
Ferrets infected with the K173, 1918HA/K173, 1918PB1/K173, 1918(3P+NP)/173, or 1918 virus were euthanized on day 3 p.i. for pathologic examination. Excised tissues of the nasal turbinates, trachea, and lungs were preserved in 10% phosphate-buffered formalin. Tissues were then processed for paraffin embedding and cut into 5-μm-thick sections. A section from each tissue sample was stained with standard hematoxylin and eosin and another was processed for immunohistologic staining with rabbit anti-H1N1 influenza virus polyclonal antibody (anti-A/WSN/33). Specific antigen-antibody reactions were visualized by 3, 3′ diaminobenzidine tetrahydrochloride staining with a Dako EnVision system (Dako Co. Ltd.).
Supplementary Material
Acknowledgments.
We thank M. McGregor and K. Wells for technical assistance and J. Gilbert for editing the manuscript. This work was supported by a grant-in-aid for Specially Promoted Research and by a contract research fund for the Program of Funding Research Centers for Emerging and Reemerging Infectious Diseases from the Ministry of Education, Culture, Sports, Science and Technology, and by grants-in-aid from the Ministry of Health, Labor, Welfare of Japan.
Footnotes
The authors declare no conflict of interest.
This article is a PNAS Direct Submission. H.-D.K. is a guest editor invited by the Editorial Board.
This article contains supporting information online at www.pnas.org/cgi/content/full/0806959106/DCSupplemental.
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