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Proc Natl Acad Sci U S A. Feb 26, 2008; 105(8): 3064–3069.
Published online Feb 19, 2008. doi:  10.1073/pnas.0711815105
PMCID: PMC2268585
Microbiology

Single gene reassortants identify a critical role for PB1, HA, and NA in the high virulence of the 1918 pandemic influenza virus

Abstract

The 1918 influenza pandemic was exceptionally severe, resulting in the death of up to 50 million people worldwide. Here, we show which virus genes contributed to the replication and virulence of the 1918 influenza virus. Recombinant viruses, in which genes of the 1918 virus were replaced with genes from a contemporary human H1N1 influenza virus, A/Texas/36/91 (Tx/91), were generated. The exchange of most 1918 influenza virus genes with seasonal influenza H1N1 virus genes did not alter the virulence of the 1918 virus; however, substitution of the hemagglutinin (HA), neuraminidase (NA), or polymerase subunit PB1 genes significantly affected the ability of this virus to cause severe disease in mice. The 1918 virus virulence observed in mice correlated with the ability of 1918 recombinant viruses to replicate efficiently in human airway cells. In a second series of experiments, eight 1918 1:7 recombinants were generated, in which each Tx/91 virus gene was individually replaced by a corresponding gene from 1918 virus. Replication capacity of the individual 1:7 reassortant viruses was assessed in mouse lungs and human airway cells. Increased virus titers were observed among 1:7 viruses containing individual 1918 HA, NA, and PB1 genes. In addition, the 1918 PB1:Tx/91 (1:7) virus showed a distinctly larger plaque size phenotype than the small plaque phenotype of the 1918 PA:Tx/91 and 1918 PB2:Tx/91 1:7 reassortants. These results highlight the importance of the 1918 HA, NA, and PB1 genes for optimal virus replication and virulence of this pandemic strain.

Keywords: human airway cells, mice, pathogenesis

Influenza A viruses regularly circulate in humans, causing annual epidemics and sporadic pandemics. Interpandemic (seasonal) influenza results in over 200,000 hospitalizations and causes an average of 36,000 deaths each year in the United States alone (1, 2). During pandemic years, the death rate is significantly higher. It is estimated that the “Spanish” influenza pandemic of 1918 was responsible for more than 20 million deaths worldwide, a vastly higher number than the approximate combined total of 100,000 for the Asian influenza pandemic of 1957 and the Hong Kong influenza pandemic of 1968 (35). The genetic composition of the influenza A viruses responsible for the 1957 (H2N2) and 1968 (H3N2) human pandemics is largely known based on retrospective sequencing and phylogenic analyses (6). Of importance, both pandemic viruses acquired a novel hemagglutinin (HA) gene and a polymerase subunit PB1 gene of wild waterfowl origin (7). The major functions of the HA protein are the receptor host cell binding and subsequent fusion of virus and host membranes in the endosome after the virus has been taken up by endocytosis (8). A novel HA protein has a replication advantage in a population immunologically naive to the antigen (8). The PB1 subunit is a key component of the viral RNA polymerase complex and catalyzes the sequential addition of nucleotides during RNA chain elongation (812). In addition to the HA and PB1 proteins, the 1957 pandemic influenza virus acquired a neuraminidase (NA) gene of avian origin (7). The NA protein of influenza A virus functions largely in the release of progeny particles (8), thus promoting viral spread, and a pandemic virus bearing a novel avian NA may have a selective advantage by evading immune detection and maintaining compatibility with the newly acquired avian HA (13, 14). Much less is known about the origin of the H1N1 virus responsible for the catastrophic influenza pandemic of 1918. Although virus sequencing and phylogenetic analysis suggest it to be an avian-like influenza virus (15), there are still unanswered questions about the origin of the virus and the molecular properties that confer its exceptional virulence.

To better understand the molecular basis for the unprecedented virulence of the 1918 pandemic virus, a reconstructed influenza virus containing eight 1918 virus genes was generated in cultured cells (16). The reconstructed 1918 pandemic virus displays a high-growth phenotype in human bronchial epithelial cells and replicates efficiently in mice, ferrets, and macaques, causing death in all three species (1619). Mice intranasally infected with the 1918 virus had a sudden onset of severe illness and succumbed to infection as early as 3 days after infection (16). Accompanying the high virus titers were significant increases in the numbers of macrophages and neutrophils detected in the mouse lung after 1918 virus infection and their sustained presence in lung tissue distinguished this virus from a contemporary A/Texas/36/91 (Tx/91) virus infection (L.A.P., A.G.-S., J.M.K. and T.M.T., unpublished data). In mice, the virulence of the 1918 virus was largely determined by the HA and, to a lesser extent, by the polymerase gene complex (16, 17, 21). However, it is not known which of the three polymerase genes contribute to the exceptional virulence of the 1918 virus or whether other virus genes also contribute to its virulence.

During the peak of the 1918 pandemic, attending pathologists noted severe disease of the respiratory tree, and, for the majority of cases, confirmed death was due to pneumonia and respiratory failure (22, 23). Although most deaths were attributed to secondary bacterial pneumonia (24), the disease process most likely began with severe acute viral infection resulting from efficient replication of the pandemic strain. A thorough understanding of the molecular mechanisms involved in virus replication of the 1918 virus may help reveal virulence factors used by other influenza viruses with pandemic potential. Our approach has been to reconstruct recombinant viruses, in which genes of the 1918 virus are replaced with genes from a contemporary human influenza (Tx/91) virus in attempts to understand which of the eight virus gene segments contribute to its high virulence. In a reciprocal experimental approach, eight Tx/91-like recombinant viruses were generated, in which each Tx/91 virus gene was individually replaced by a corresponding gene from the 1918 virus. The rescued viruses, referred to as 7:1 or 1:7 recombinant viruses, have revealed the importance of the 1918 HA, NA, and PB1 genes for optimal virus replication of this pandemic strain. The identification of the precise pandemic virus genes associated with replication may help elucidate virulence factors for other influenza viruses with pandemic potential and, thereby, help identify targets for drug intervention.

Results

Construction and Characterization of Recombinant Viruses with 1918 Influenza Virus Genes.

To determine which of the eight 1918 virus gene segments contribute to its high virulence, we generated a series of eight single-gene recombinant influenza viruses, each possessing seven gene segments of the 1918 virus and one segment from a seasonal human H1N1 influenza virus, Tx/91. The 1918 (7:1) recombinant viruses were compared with the parental eight-gene 1918 virus for replication efficiency in human respiratory cells and for virulence in mice. In a similar fashion, we generated eight 1918 (1:7) recombinant viruses, in which each Tx/91 virus gene was individually replaced by a corresponding gene from 1918 virus. All 1918 recombinant viruses had high infectivity titers (>107 pfu/ml) in MDCK cells, similar to the parental rescued 1918 and Tx/91 viruses (Tables 1 and and22).

Table 1.
Properties of 7:1 recombinant and parental influenza viruses used in this study
Table 2.
Properties of 1:7 recombinant and parental influenza viruses used in this study

We initially evaluated the pathogenicity of each 1918 7:1 recombinant virus by intranasally inoculating BALB/c mice with 105 pfu of virus for determination of morbidity (measured by weight loss), mortality, and virus replication. We also determined the LD50 titers in mice infected with each 1918 7:1 recombinant virus and compared them with titers in groups of mice infected with the parental eight-gene 1918 virus or Tx/91 H1N1 virus, which previously were shown to be of high and low virulence, respectively (16). Infection of mice with the eight-gene 1918 virus resulted in lung virus titers on days 3 and 5 postinoculation (p.i.) that were at least 15,000-fold higher than those of mice infected with the Tx/91 virus (Fig. 1A). However, virus replication in the lungs of mice inoculated with single-gene recombinant viruses containing NP, PB2, PA, M, or NS from Tx/91 did not differ significantly from that in mice inoculated with the parental 1918 virus (Fig. 1A). Furthermore, each of the these five 7:1 viruses caused severe signs of illness and weight loss that were comparable with those caused by the parental 1918 virus (Fig. 1 B and C and Table 1). Mice infected with the Tx PB2:1918, Tx PA:1918, or Tx M:1918 virus displayed a 2- to 3-day delay in death compared with mice infected with the eight-gene 1918 virus; however, these 7:1 viruses were still considered highly virulent, and all mice succumbed to infection by day 9 p.i. (Fig. 1C). In contrast to the lethal outcome in mice infected with the 1918 virus, mice infected with the 7:1 recombinant viruses containing the Tx/91 HA or NA were significantly attenuated, and none of the mice infected with 105 pfu died (Fig. 1 B and C and Table 1). The reduced disease and weight loss of the Tx/91 HA:1918- and Tx/91 NA:1918-infected mice correlated with the lower levels of virus replication in mouse lungs on days 3 and 5 p.i. (Fig. 1A). Of interest, replacing the 1918 PB1 gene resulted in an attenuated virus (Tx/91 PB1:1918) with a lethality (LD50, 105.5) that was 170 times less than that of the 1918 virus (Table 1). Tx/91 PB1:1918-infected mice lost significant weight (10.5%) by day 8 p.i., but most of the mice infected with 105 pfu of virus survived the infection (Fig. 1C).

Fig. 1.
Pathogenicity of 7:1 recombinant H1N1 influenza viruses. Comparison of lung virus titers (A), lethality (B), and weight loss (C) in mice (12 per virus group) intranasally inoculated with 105 pfu of 1918 (♦), Tx/91 PA:1918 (□), Tx/91 PB2:1918 ...

The 1918 HA, NA, and PB1 Genes Contribute to Replication Efficiency in Human Airway Cells.

To determine whether the 1918 virus genes responsible for the high virulence in vivo also contribute to optimal virus replication in human airway cells, we assessed the growth and release of virus in primary human bronchial epithelial (NHBE) cells (25) and in Detroit 562 epithelial cells derived from a human pharyngeal carcinoma (26). Culture medium from virus-inoculated NHBE cells was collected from the apical and basolateral chambers of the cell monolayers at different times after inoculation and examined for virus production in the presence and absence of trypsin. With all viruses tested, titers of progeny virus progressively increased during the first 24 h p.i., and virus was detected almost exclusively in the apical supernatant (Fig. 2A). Only trace amounts of each virus tested (<101.2 pfu/ml) were released into the basolateral reservoir. All 1918 recombinant viruses replicated with similar kinetics and to similar titers in polarized NHBE and Detroit 562 cells with (Fig. 2) or without (data not shown) the addition of exogenous trypsin, suggesting that these epithelial cells possess protease activity that can support H1N1 virus HA cleavage. Regardless of the presence or absence of trypsin, infectivity titers of Tx/91 HA:1918, Tx/91 NA:1918, and Tx/91 PB1:1918 viruses, like the parental Tx/91 virus, were significantly lower than virus titers detected in the 1918 virus-infected cultures at 12, 24, and 48 h p.i. (Fig. 2A). Next, the replication kinetics of 7:1 viruses was determined in Detroit 562 cell cultures. For comparison, cultures were infected with an H5N1 virus isolated in 2004, A/Viet Nam/1203/04 (VN/1203), which was cultivated from a patient who died (27). Remarkably, the H5N1 virus replicated in human airway cells derived from the upper respiratory tract to titers similar to those of the 1918 virus (Fig. 2B). At 12, 16, and 24 h p.i., 1918 and VN/1203 virus production was at least 7-fold higher than that observed in cultures infected with Tx/91 or the 1918 7:1 virus containing the PB1 from Tx/91 virus. As was observed in NHBE cells, infectivity titers of Tx/91 HA:1918 and Tx/91 NA:1918 viruses were significantly lower than virus titers released in the 1918 virus-infected cultures at 12, 24, and 48 h p.i. (data not shown), further demonstrating that the 1918 HA, NA, and PB1 are essential for maximal replication of the pandemic virus in human cells derived from upper and lower respiratory tract epithelium.

Fig. 2.
Release of 7:1 recombinant H1N1 influenza viruses from apically infected NHBE cells (derived from autopsy specimens from adults) and from Detroit 562 epithelial cells. NHBE (A) and Detroit 562 (B) cells were grown on Transwell inserts. Cells were infected ...

Generation of 1918 Recombinant 1:7 Viruses.

To further prove that the HA, NA, and PB1 genes contribute to the high virulence of the 1918 pandemic virus, we generated reciprocal recombinant (1:7) viruses in which each of the contemporary Tx/91 virus genes were replaced with the corresponding gene from the 1918 virus. The reciprocal constellation of 1918 PB1 on the parental Tx/91 background genes (1918 PB1:Tx/91 virus) displayed larger plaque size morphology on MDCK cells (Fig. 3A) and greater virus replication in NHBE cells compared with that displayed by the 1918 PA:Tx/91, 1918 PB2:Tx/91, and Tx/91 viruses (Fig. 3B). As early as 12 h p.i., the 1918 PB1:Tx/91 virus produced 8-fold greater virus release in apical supernatants compared with virus production by the other polymerase reassortants and the wild-type Tx/91 virus. Virus replication in culture supernatants from NHBE cells inoculated with 1:7 recombinant viruses (in which the Tx/91 NP, PB2, M, and NS genes were substituted with the corresponding 1918 virus genes) was not significantly different from virus replication in cells inoculated with the parental Tx/91 virus (data not shown). However, the Tx/91 virus with the recombinant virus expressing the 1918 NA or HA, in the presence and absence of 1918 NA, increased the replication efficiency of the Tx/91 virus in NHBE cells (Fig. 3C) and in mouse lungs (Fig. 4). Furthermore, both 1918 HA:Tx/91 and 1918 HA/NA:Tx/91 viruses caused severe illness and death, whereas the remaining 1:7 viruses did not cause death in mice, even at the highest obtainable titer used for virus inoculation (106 pfu). Taken together, these results demonstrate that the HA, NA, and PB1 genes of the 1918 influenza virus contribute to the virulence of this virus in mice. Moreover, the virulence observed in mice correlates with the ability of the 1918 recombinant viruses to replicate efficiently in mouse lungs and human airway cells.

Fig. 3.
Plaque morphology and release of 1:7 recombinant H1N1 viruses from apically infected NHBE cells. Confluent monolayers of MDCK cells were inoculated with 1:7 polymerase recombinants and Tx/91 virus, and plaque morphology was visualized at 48 h p.i. (A ...
Fig. 4.
Replication of 1:7 recombinant H1N1 influenza viruses in mouse lungs. Comparison of lung virus titers in mice infected with 106 pfu of the indicated 1:7 virus. For comparison, a group of mice was infected with the 2:6 virus containing the 1918 HA and ...

Discussion

The emergence of another pandemic virus is considered likely, if not inevitable (28). The molecular characterization of the reconstructed 1918 pandemic influenza virus may shed light on the threat posed by new influenza virus strains with pandemic potential. The factors responsible for the high lethality associated with the 1918 virus are complex and poorly understood; however, the ability of the virus to replicate efficiently in the host most likely contributed to its unusual virulence. Because the coding sequences of the 1918 viral RNA segments did not reveal obvious genetic features that have been associated with virulence (15, 29), it is crucial to study 1918 recombinant viruses in suitable animal models to better understand the genetic markers responsible for virus replication and virulence of this pandemic strain. Our results suggest that multiple 1918 virus genes contribute to optimal virus replication efficiency in human airway cells and in lungs of mice. By comparing the highly replication-competent eight-gene 1918 virus with the single-gene 7:1 recombinant viruses, we determined the role of each 1918 virus gene in replication and in virulence. Similarly, the ability of each 1918 virus gene was tested for its ability to enhance the replication efficiency of a contemporary human H1N1 virus. These data demonstrated that the HA, NA, and PB1 virus genes are essential for maximal replication and virulence of the 1918 virus.

Because human airway epithelium is the primary site for infection and replication of influenza viruses, we have used two airway epithelial (Detroit 562 and NHBE) cells to determine the replication efficiency of the 1918 recombinants. NHBE cells have been more recently used as an in vitro human airway epithelium model to evaluate interactions between influenza A virus and the human host (25, 30). The concept that efficient viral growth in the upper respiratory tract of humans can facilitate virus excretion by coughing and sneezing, prompted us to evaluate the growth and release of the virus in polarized oropharyngeal (Detroit 562) cells. We found that the replication efficiency of the 1918 recombinants in mouse lungs correlated to the replication efficiency in both airway epithelial cells. In addition, we also observed that an avian influenza H5N1 virus isolated from a human in 2004 displayed a high-growth phenotype in Detroit 562 cells derived from the upper respiratory tract. It has been postulated that the lack of sustained human to human transmission of avian H5N1 viruses is due to their α2,3 sialic acid receptor binding preference and thus to the presumed inability of the virus to replicate efficiently at this site (31, 32). However, we found that the H5N1 virus replicated to similarly high infectivity titers as the 1918 virus in these cells. These data are in agreement with a recent report that demonstrated efficient H5N1 virus replication in ex vivo cultures of human nasopharyngeal tissues (33), suggesting that avian H5N1 viruses may have a broader tropism for the human respiratory tract than initially reported.

Infection with 1:7 reassortants in which the 1918 NP, M, NS, or polymerase subunits PB2, PA genes were individually substituted into the background of the Tx/91 virus did not result in increased virus replication compared with the parental Tx/91 virus. The NS1 protein was of particular interest because it has been shown to antagonize type I IFN production (34) leading to the concept that a strong IFN-antagonist NS1 protein may contribute to enhanced influenza virus virulence, in general and in particular to the exceptional virulence of the 1918 virus. However, the introduction of the 1918 virus NS1 gene into a WSN virus background (1918 NS1:WSN) resulted in a virus that was not more virulent in mice (35). Similarly, in the current study, 1918 NS1:Tx/91 virus did not confer a more virulent virus in mice or increase the replication efficiency of the parental Tx/91 virus in human airway cells. These data suggested that the NS1 protein is not a crucial virulence factor of the 1918 virus or that the model systems used in this study were not ideal to study human NS1 virulence. The 1918 virus genes that were able to enhance replication efficiency of the Tx/91 virus were the HA, NA, or PB1 genes. Interestingly, among all eight gene segments tested in the current study, the HA was the only 1918 virus gene able to confer a virulent phenotype when rescued in the genetic background of Tx/91 virus. The 1918 HA gene was shown to be essential for maximum virus replication and for eliciting a heightened host inflammatory response (16, 17, 21). The contribution of the 1918 NA gene may, in part, represent the need for optimal balance between sialidase and 1918 HA receptor binding activities and/or previously observed HA cleavage properties (13, 14). The increased replication efficiency of the 1918 PB1:Tx/91 (1:7) virus, observed in NHBE cells, was also observed in MDCK cells as distinctly larger plaque size phenotype in comparison with the small plaque phenotype of the 1918 PA:Tx/91 and 1918 PB2:Tx/91 (1:7) reassortants. The PB1 subunit is a key component of the viral RNA polymerase complex and contains multiple active sites critical for the polymerization of RNA chains and also for association with PA and PB2 to form a heterotrimer (911). The contribution of the PB1 gene is particularly significant in the context of the 1957 and 1968 pandemic viruses, which each acquired PB1 together with HA, NA, or both genes from the avian gene pool in wild ducks by genetic reassortment, retaining other virus genes from circulating human strains (6, 7). The 1918 PB1 protein differs from the conserved avian influenza consensus sequence by only seven amino acid residues (15) and an avian-like PB1 gene may provide increased transcriptional activity of the RNA-dependent RNA polymerase (12) and greater virus replication.

The increased virulence associated with the 1918 PB1 might be due to the PB1-F2 protein generated by an alternate reading frame (36). PB1-F2 is truncated in Tx/91 and all contemporary human H1N1 viruses but is functional in the 1918 virus and may contribute to virulence by functioning as a proapoptotic protein (37, 38). Recent work where a point mutation was introduced at amino acid 66 of PB1-F2 revealed the importance of this protein in the virulence of a H5N1 virus and the 1918 pandemic virus (37). A better understanding of the contribution of polymerase proteins in virulence will aid in designing drugs that target the key intersubunit binding sites of the polymerase complex and diminish the high replication efficiency of pandemic virus strains (39). For currently circulating H5N1 influenza viruses, the PB2 polymerase subunit appears to play a greater role in the high growth phenotype and increased virulence associated with these highly pathogenic viruses in mammals (40, 41).

Methods

Generation of 1918 Recombinant Viruses by Reverse Genetics.

Genes encoding the 1918 pandemic influenza virus were reconstructed from deoxyoligonucleotides corresponding to the reported 1918 virus coding sequences. The noncoding regions of each segment are identical to that of the corresponding segment of influenza A/WSN/33 (H1N1) virus. All 1918 recombinant viruses were generated by using 1918 virus gene cDNAs described in refs. 1618 and the described reverse genetics system (35, 42, 43). Transfection supernatants were passaged onto MDCK cells and virus stock prepared and titrated. The 1918 viruses were handled under biosafety level 3 enhanced (BSL-3+) containment in accordance with guidelines of the National Institutes of Health and the Centers for Disease Control and Prevention (CDC) (available at http://www.cdc.gov/OD/ohs/biosfty/bmbl5/bmbl5toc.htm) and in accordance with requirements of the U.S. Department of Agriculture/CDC Select Agent Program. This research was done by staff taking antiviral prophylaxis and using stringent biosafety precautions to protect the researchers, the environment, and the public. The identity of the 1918 influenza virus genes in the recombinant viruses was confirmed by RT-PCR and sequencing.

Infection of Mice.

Female BALB/c mice, 6–8 weeks old (Charles River Laboratories), were anesthetized with an i.p. injection of 0.2 ml of Avertin (Aldrich) and 50 μl of infectious virus diluted in PBS was inoculated intranasally (i.n.) (43). LD50 titers were determined by inoculating groups of three mice i.n. with serial 10-fold dilutions of virus. LD50 titers were calculated by the method of Reed and Muench (20) and are expressed as the log10 pfu required to give 1 LD50. For comparison of morbidity (measured by weight loss), mortality, and lung virus titers, additional mice were infected with inoculating doses of 105 or 106 pfu of virus. On days 3 and 5 p.i., three mice from each group were killed and whole lungs were removed and homogenized in 1 ml of sterile PBS. Fifty percent egg infectious dose (EID50/ml) titers were calculated by the method of Reed and Muench (20).

Human Airway Cells and Viral Infection.

Primary human bronchial epithelial (NHBE) cells (Cambrex Bio Science) (25) and Detroit 562 epithelial cells (American Type Culture Collection) (26), were grown in MEM as described in ref. 25. Briefly, cells (5 × 105) were seeded onto Corning 24-mm diameter semipermeable membrane inserts with 0.4-μm pore size and cultured for 1 week to achieve a stable transepithelial resistance of >1,000 Ω·cm2. Monolayers were washed with MEM supplemented with 0.3% BSA (MEM/BSA). Virus was diluted in MEM/BSA and added to the apical surface of cells at a multiplicity of infection (moi) of 0.01 for 1 h at 37°C. Monolayers were then washed and 2 ml of MEM/BSA was added to both apical and basolateral reservoirs. Cultures were set up with or without TPCK-treated trypsin (1 μg/ml; Sigma). Apical and basolateral supernatants were collected at the indicated times, and virus content was determined in a standard plaque assay (25). The values shown represent the mean virus titer of supernatants from three replicate infected cultures. One avian H5N1 virus isolated from fatal human case in early 2004, A/Vietnam/1203/2004 (VN/1203), was used in this study; virus stocks were grown as described in ref. 27.

ACKNOWLEDGMENTS.

This work was partially supported by National Institutes of Health Grant P01 AI058113 (to A.G.-S.), the Northeast Biodefense Center Grant U54 AI057158, and the Center for Investigating Viral Immunity and Antagonism Grant U19 AI62623. C.F.B. was supported by Northeast Biodefense Center-Lipkin Grant U54 AI057158, Center for Investigating Viral Immunity and Antagonism-Moran Grant U19 AI62623, and National Institutes of Health Grant P01 AI058113. P.V.A. was supported by a fellowship awarded by Northeast Biodefense Center-Lipkin Grant U54 AI057158.

Footnotes

The authors declare no conflict of interest.

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