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Departments of Infectious Diseases and Molecular Biotechnology, St. Jude Children's Research Hospital, Memphis, Tennessee
Secondary bacterial pneumonia is a common cause of death during influenza epidemics. We hypothesized that virus-specific factors could contribute to differences in annual excess mortality. Recombinant influenza viruses with neuraminidases from representative strains from the past 50 years were created and characterized. The specific level of their neuraminidase activity correlated with their ability to support secondary bacterial pneumonia. Recombinant viruses with neuraminidases from 1957 and 1997 influenza strains had the highest level of activity, whereas a virus with the neuraminidase from a 1968 strain had the lowest level of activity. The high level of activity of the neuraminidase from the 1957 strain, compared with that of other neuraminidases, more strongly supported the adherence of Streptococcus pneumoniae and the development of secondary bacterial pneumonia in a mouse model. These data lend support to our hypothesis that the influenza virus neuraminidase contributes to secondary bacterial pneumonia and subsequent excess mortality.
Influenza A virus causes epidemics annually and pandemics several times a century. Two subtypes of influenza A virus, H3N2 and H1N1, are presently circulating in the human population. Epidemics caused by H3N2 are associated with higher mortality in human populations than are epidemics caused by H1N1 or influenza B virus [1, 2]. Viruses that cause pandemics are defined as those that acquire a new hemagglutinin (HA), have a high attack rate, and spread globally. Infection with them is generally considered to result in high mortality. Understanding what factors are associated with increased mortality will help us prepare for the next influenza pandemic.
It has been suggested that antigenic novelty accounts for increased mortality during pandemics, but a comparison of mortality estimates between different pandemic years and between interpandemic years indicates that other factors are also involved. The capability of influenza viral strains to predispose to bacterial superinfection may be a factor that determines mortality during an epidemic. Secondary bacterial pneumonia is an important cause of influenza-associated death during both pandemic and interpandemic periods. Results of studies performed during the influenza pandemics of 1957 and 1968 revealed that there was a bacterial etiology in 70% of patients with fatal or life-threatening pneumonia [3, 4]. During interpandemic periods, 44%57% of patients hospitalized with influenza have bacterial pneumonia [58], andalthough the estimate varies from year to year, depending on the viral strain that is circulatingon average, 25% of all influenza-associated deaths are due to secondary bacterial pneumonia [9].
Streptococcus pneumoniae is the leading cause of community-acquired pneumonia [10] and is a major cause of pneumonia and other bacterial complications of influenza. We developed a mouse model of synergism between influenza virus and S. pneumoniae [11, 12], and, using this model, we established that viral neuraminidase (NA) is an important factor in viral-bacterial synergism [13, 14]. NA activity is needed by an influenza virus to release newly synthesized virus by cleaving sialic acid both from host cell glycoconjugates and from oligosaccharides of viral HA and NA. This action of NA also promotes adherence and invasion of S. pneumoniae, because cleavage of sialic acid from the surface of host cells exposes cryptic receptors for S. pneumoniae [1518]. Bacteria that can successfully invade the lower respiratory tract typically express NA for this purpose. That generally higher levels of NA activity are found in modern H3N2 than in H1N1 viruses [19] is consistent with the hypothesis that high levels of NA activity lead to higher mortality from secondary bacterial pneumonia. We sought to define the viral-specific NA activity in several influenza viruses within a subtype and correlate it with the biological characteristics of secondary bacterial pneumonia.
MATERIALS AND METHODS
Generation of recombinant influenza viruses.
Recombinant influenza viruses were produced using an established 8-plasmid reverse-genetics system [20] in a coculture of 293T and MDCK cells and parental viruses from the influenza virus repository at St. Jude Children's Research Hospital. Internal gene segments of A/Puerto Rico/8/34 (subtype H1N1; hereafter referred to as "PR8") [21], the HA gene segment of the human influenza virus A/Hong Kong/1/68 (subtype H3N2; hereafter referred to as "HK68"), and the HA and NA gene segments of the human influenza virus A/Fujian/411/02 (subtype H3N2; hereafter referred to as "Fuj02") had been cloned into the pHW2000 plasmid previously. NA gene segments of the human H2N2 influenza viruses A/Singapore/1/57 (Sing57) and A/England/12/62 (Eng62); the human H3N2 influenza viruses HK68, A/Memphis/102/72 (Mem72), A/Leningrad/516/86 (Len86), and A/Sydney/5/97 (Syd97); and the chicken H9N2 influenza virus A/Chicken/Hong Kong/WF2/99 (WF2) were amplified by polymerase chain reaction (PCR) and cloned into the pHW2000 plasmid. HA and NA genes were sequenced, and their identities were confirmed by comparison of their sequences with those of the parental strain. Viral stocks were grown in embryonated chicken eggs, centrifuged on a 25%/75% sucrose cushion, pelleted, and resuspended in PBS.
Pneumococci.
A type 3 strain of S. pneumoniae that had been transformed with the lux operon (provided by Kevin Francis and Jun Yu, Xenogen Corp.) was used in mouse studies. R6T, an unencapsulated laboratory strain of S. pneumoniae, was used in adherence assays.
Characterization of recombinant influenza viruses in cell culture, embryonated eggs, and mice.
Allantoic fluid and concentrated viral stocks were titrated in MDCK cells and embryonated chicken eggs by standard methods to obtain the TCID50 and EID50. Groups of 4 mice were infected with serial dilutions of the allantoic fluid stocks to obtain an MLD50 by the method of Reed and Muench [22]. For lung viral titers, mouse lung homogenates were titrated in MDCK cells as described elsewhere [12].
NA activity.
The total level of NA activity in concentrated influenza virus diluted in calcium saline buffer to a final substrate concentration of 10 mol/L was determined by measuring the fluorescence of 4-methylumbelliferone cleaved from 2-(4-methylumbelliferyl)-N-acetylneuraminic acid (Mu-NANA; Sigma) as described elsewhere [23]. The relative linkage specificity of NA was determined using N-acetylneuraminic acid (NANA) bound to lactose through either (23) or (26) linkage as a substrate. Fetuin was used as a substrate to measure the level of NA activity against a large molecule with both (23) and (26) sialic acid linkages. The amount of sialic acid released from NANA-lactose (substrate concentration, 0.1 mmol/L) or fetuin (substrate concentration, 6.1 mmol/L) was measured by the thiobarbituric acid assay, as described elsewhere [24]. All reactions were performed for 30 min at 37°C. To relate NA activity to the predominant viral proteins, virus concentrates were run on 10% Tris-HCl gels. After staining with Sypro orange (Amersham Pharmacia), HA and nucleoprotein (NP) bands were quantified using a laser-excited gel scanner.
Immunoelectron microscopy (IEM).
IEM was performed, as described elsewhere [25], using a mixture of monoclonal antibodies against 7 different N2 NAs representing the range of NAs studied. Anti-NA monoclonal antibodies 152/6 (A/Japan/1/57), 25/4 (A/Tokyo/3/67), 5/2 (A/Aichi/2/68), 1/1 (A/Udorn/307/72), and E12-8 (A/Memphis/12/85) were provided by Dr. Robert Webster (St. Jude Children's Research Hospital) [26, 27], and Mem4 and Mem5 (A/Memphis/31/98) were provided by Dr. Gillian Air (University of Oklahoma Health Sciences Center) [28]. Negative staining was performed with 2% phosphotungstic acid, and positive staining was performed with ethanolic uranyl acetate. Gold particles representing the amount of NA per virion were counted in positively stained samples.
Adherence assay.
Adherence assays were performed using standard methods, as described elsewhere [13], using S. pneumoniae strain R6T after a 30-min incubation with influenza virus at 37°C. Controls were treated identically, except that influenza virus was not added. For inhibition of viral NA, 30 min before incubation with monolayers, the oseltamivir prodrug Ro 64-0796 (Roche Products) was added to the viral suspension at a concentration of 10 mol/L (the concentration of the active metabolite oseltamivir carboxylate was not determined).
Mice and infection model.
Experimental procedures were performed after the mice (810-week-old female BALB/c; Jackson Laboratory) were anesthetized with inhaled 2.5% isoflurane (Baxter Healthcare). Infectious agents were administered intranasally in a volume of 100 L of PBS. For synergism between influenza virus and S. pneumoniae, mice were infected first with influenza virus and then 7 days later with S. pneumoniae and were monitored at least daily for illness and mortality. Groups of 910 mice were infected with 0.01 MLD50 of influenza virus and 100 cfu of S. pneumoniae and were imaged daily. Instead of using a fixed number of infectious particles, we calculated doses of influenza virus in relation to the MLD50, so that weight loss, viral lung titers, and damage to the respiratory tract before bacterial challenge were the same between groups. Mice found to be moribund were euthanized and were considered to have died that day. All animal experiments were approved by the St. Jude Children's Research Hospital Animal Care and Use Committee and were performed under biosafety level 2 conditions.
Imaging of live mice.
Anesthetized mice were imaged for 20 s by an IVIS CCD camera (Xenogen Corp.). The total emission of photons from selected and defined areas within the images of each mouse was quantified by LivingImage software (version 2.20; Xenogen Corp.), as described elsewhere [29], and was expressed as relative light units (rlu). Pneumonia was defined as detection of >20,000 rlu/min from the thorax.
Statistical analysis.
Comparison of survival rates in the groups of mice was performed using the Mantel-Cox 2 test on Kaplan-Meier survival data, and comparisons of bacterial adherence were made using 1-way analysis of variance followed by Dunn's test. P < .05 was considered to be statistically significant.
RESULTS
Recombinant influenza viruses differing only in NA.
Influenza viruses with an N2 NA have been circulating in the human population since 1957. Representative NA genes were selected from 6 human pandemic and interpandemic H2N2 and H3N2 influenza viruses isolated between 1957 and 2004. Recombinant influenza viruses that differed only in their NAs were rescued on the common background of 7 gene segments (6 internal gene segments from PR8 and the HK68 HA gene). For comparison with these human influenza viruses, an influenza virus that had an NA gene from a 1999 chicken H9N2 influenza virus was also rescued. All the recombinant viruses grew to high titers in embryonated eggs (table 1). They caused morbidity and mortality in mice and thus were suitable for further study of viral-bacterial synergism in a mouse model. The MLD50 of the influenza viruses that had a human NA gene were similar, whereas the MLD50s of the influenza virus that had the chicken virus NA gene and of the mouse-adapted parental strain PR8 were 34 logs lower. An influenza virus that had the Fuj02 NA gene could not be rescued when the Fuj02 NA gene was paired with the HK68 HA gene, and it was therefore excluded from comparison with other isogenic influenza viruses. However, it was rescued when it had its own HA gene (Fuj02 HA), and a comparison influenza virus containing the Fuj02 HA gene and the Mem72 NA gene was also rescued. Because mutations may be generated during rescue of influenza viruses when the 8-plasmid system is used and during passage of influenza viruses in eggs, NA gene sequences of the recombinant influenza viruses were compared with published NA gene sequences of the parental viruses. Site-directed mutagenesis was used to ensure that the NA gene of all influenza viruses matched the GenBank sequence of the NA gene of the target influenza viruses.
NA activity in recombinant influenza viruses.
To characterize this set of viral tools, the level of NA activity in concentrated influenza viruses was measured using Mu-NANA as the substrate [23] and was expressed as a function of the amount of NA protein (figure 1). The level of NA activity was highest in the pandemic strain containing the Sing57 NA. The level of NA activity decreased and was lowest in the influenza virus containing the HK68 NA. From 1968 to 1997, a general increase in the level of NA activity was seen, such that the influenza virus containing the Syd97 NA had the second-highest level of NA activity. Although a direct comparison with the isogenic set of influenza viruses was not possible, both the wild-type Fuj02 influenza virus and the recombinant influenza virus that had the Fuj02 NA had NA activity levels that were similar to that of the influenza virus containing the Syd97 NA (data not shown). WF2 NA from a recent chicken strain of the H9N2 subtype had a very low level of NA activityonly 2% of that of the influenza virus containing the Sing57 NA. A similar pattern of NA activity was seen when values were expressed relative to the amount of the most abundant viral proteins, HA and NP, or when fetuin was used as a substrate: a decline in NA activity in the influenza viruses isolated from 1957 to 1968, then a gradual increase in those isolated from 1968 to 2002 (data not shown).
Changes in the amino acid sequences of the NAs of influenza viruses isolated between 1957 and 1962 have been associated with a decrease in the level of NA activity, which suggests that these structural changes are responsible for the observed differences [30]. To determine whether these differences might reflect different preferences for (23) or (26)linked substrates, we performed additional experiments. IEM was used to quantify the amount of NA protein per virion (figure 2A and 2B). The differences in the amount of NA protein were minor (figure 2B) and did not correlate with the differences in the level of NA activity. Thus, it seems unlikely that changes in expression over time contributed significantly to changes in the level of NA activity. In addition to demonstrating the relatively stable amount of NA protein per virion, IEM studies confirmed the earlier finding of a clustered distribution of NA protein on virions [25].
Next, the substrate preference of the recombinant influenza viruses was examined. The ability of viral NA to liberate sialic acid from (23)sialyllactose and (26)sialyllactose as substrate was determined. All influenza viruses had the majority of NA activity against (23) linkage of sialic acid. As expected, the influenza virus containing the Sing57 NA, as well as the chicken influenza virus WF2 NA, had activity almost solely (98%) against substrate with an (23) linkage, which is the primary linkage of sialic acid in the gastrointestinal tract of aquatic birds [31]. As is consistent with previous findings [30, 32], the ratio of (26) activity : (23) activity increased in the influenza viruses isolated from 1957 to 1986. Even for NAs from influenza viruses circulating in 1986 or 1997, however, only 11% of their activity was against the (26) linkage. Thus, it seems unlikely that substrate preference is responsible for differences in the level of N2 NA activity. It is noteworthy that passaging influenza viruses in eggs causes selective pressure in favor of (23) NA activity, because only (23)linked sialic acid is found in chicken egg allantoic cells [33].
Adherence of S. pneumoniae to human respiratory cells and NA activity.
We next tested the effect that the level of NA activity has on adherence of S. pneumoniae strain R6T. Before incubation on A549 cell cultures, the recombinant influenza viruses were diluted until they had the same amount of viral protein. At a viral protein concentration of 1 g/mL, all influenza viruses clearly increased the adherence of S. pneumoniae strain R6T to cells, but incubation with the influenza virus containing the HK68 NA, which has a relatively low level of NA activity, increased the adherence of S. pneumoniae strain R6T by only 2.1-fold, compared with a 3.8-fold increase after incubation with the influenza virus containing the Sing57 NA and a 2.9-fold increase after incubation with the influenza virus containing the Syd97 NA. At a lower viral protein concentration of 0.2 g/mL, the influenza virus with the most active NA, the one containing the Sing57 NA, still increased the adherence of S. pneumoniae strain R6T by 3.6-fold, whereas, at this concentration, the other influenza viruses caused only 1.31.7-fold increases in adherence (figure 3). The effect of all influenza viruses on the adherence of S. pneumoniae strain R6T was reversed when oseltamivir was added to the incubation mixture (only data related to the higher concentration of influenza virus are shown). Oseltamivir carboxylate is a specific inhibitor of influenza virus NA and has no effect on bacterial NAs [13]. This reversal by oseltamivir in the effect of all influenza viruses on the adherence of S. pneumoniae strain R6T, together with a 30-min incubation time that is too short for the completion of mechanisms involved in viral replication, suggest that differences between influenza viruses in their effects on the adherence of S. pneumoniae are due to their intrinsic level of NA activity.
Higher levels of NA activity and higher mortality from secondary bacterial pneumonia in mice.
Infection with influenza virus primes mice for subsequent lethal pneumonia caused by S. pneumoniae [11, 12]. We infected mice with influenza virus and, 7 days later, infected them with a strain of S. pneumoniae that had been transformed with the luciferase-expressing lux operon that permits bioluminescent imaging of pneumonia in live anesthetized mice [13]. We compared pairs of recombinant influenza viruses that differed 2-fold in their level of NA activity (pair 1, the influenza virus containing the Sing57 NA and the influenza virus containing the Syd97 NA matched with the HK68 HA; pair 2, the influenza virus containing the Fuj02 NA and the influenza virus containing the Mem72 NA matched with the Fuj02 HA). The mean weight loss at the time of infection with S. pneumoniae was similar in paired groups of mice7.9% in mice infected with the influenza virus containing the Sing57 NA versus 8.9% in mice infected with the influenza virus containing the Syd97 NA and 0.1% in mice infected with the influenza virus containing the Fuj02 NA versus 1.3% in mice infected with the influenza virus containing the Mem72 NAwhich indicated that morbidity relating to infection with the 2 different influenza viruses was similar. However, a significant difference in survival after infection with S. pneumoniae was observed: 8 (42%) of 19 mice infected with the influenza virus containing the Sing57 NA versus 15 (79%) of 19 mice infected with the influenza virus containing the Syd97 NA (P < .05) lived (figure 4A), and 3 (33%) of 9 mice infected with the influenza virus containing the Fuj02 NA versus 8 (89%) of 9 mice infected with the influenza virus containing the Mem72 NA (P < .05) lived (figure 4D). Daily imaging showed the development of pneumococcal pneumonia 23 days before death (figure 4A, 4C, and 4E; 2 representative mice are shown). Results of studies using several other recombinant influenza viruses indicated that there were similar differences when influenza viruses with high and low levels of NA activity were compared (data not shown). These data establish that even small differences in the level of NA activity are reflected in differences in the ability of influenza viruses to permit the development of secondary bacterial pneumonia in a mouse model. Data for the influenza virus containing the Syd97 NA (figure 4A and 4B) and the influenza virus containing the Fuj02 NA (figure 4C and 4D) are not directly comparable, despite the similarities in measured levels of NA activity, because the HA differs between the 2 pairs of viruses and could independently contribute to secondary bacterial pneumonia.
The experiment using the influenza viruses containing the Sing57 and Syd97 NAs was repeated, and lung viral titers were measured 3 and 7 days after infection to make certain that the kinetics of viral infection in the 2 groups of mice were comparable. On day 3 after infection, groups of 4 mice infected with the influenza virus containing the Sing57 NA had mean ± SD viral titers of 6.5 ± 0.4 log10 TCID50/mL, compared with 6.3 ± 0.3 log10 TCID50/mL in mice infected with the influenza virus containing the Syd97 NA. On day 7, the mean ± SD viral titers were 2.4 ± 0.7 log10 TCID50/mL and 2.5 ± 0.4 log10 TCID50/mL, respectively, and there was no detectable influenza virus in the lungs of 1 mouse from each group of 4 mice.
DISCUSSION
Results of a previous study using a panel of reassortant duck influenza viruses containing human N2 NAs suggested that the level of NA activity in influenza viruses decreased markedly between 1957 and 1968 [30]. We have confirmed and extended this observation by use of human influenza viruses that were generated by reverse genetics to contain N2 NAs representative of influenza viruses isolated between 1957 and 2004. The level of NA activity decreased in influenza viruses isolated from 1957 to 1968 and increased again in those isolated from 1968 to 1997. This trend in NA activity correlates with the observed historic mortality caused by H3N2 influenza viruses, which was highest in 1957, decreased during the next decade, but increased again during the 1990s (table 2). Low levels of NA activity in the influenza viruses circulating in 1968 is consistent with lower mortality from this pandemic, compared with that in the 1957 pandemic or during the epidemics caused by H3N2 influenza viruses during the 1990s. It has been suggested that conservation of the N2 NA in the influenza viruses circulating in 1968 resulted in relatively low mortality during this pandemic, but this does not explain why influenza viruses circulating later that had no antigenic shift in HA or NA caused higher mortality. The N2 NA in the influenza virus circulating in 1997 had the second highest level of NA activity, and this influenza virus caused the highest mortality during an epidemic since the 1957 pandemic [2, 34, 36].
Although the correlation between historical excess mortality in humans and the NA activity and secondary bacterial pneumonia found in our model is intriguing, other factors must be involved. A large proportion of excess deaths related to influenza are coded as cardiovascular disease, cerebrovascular disease, or diabetes [37], and most of these conditions are presumably not related to bacterial superinfection. Virus-specific factors contribute to excess morbidity and mortality, and differences in these virulence factors account for differences in morbidity and mortality from season to season. Our data suggest that NA activity is such a factor and that its impact can be seen in deaths from secondary bacterial pneumonia. Other virulence factorssuch as the antigenic novelty of the HA, the modulation of interferon [38] or cytokine expression by nonstructural protein1 [39], or interactions between several genesmay be involved, and NA may have effects on the host unrelated to cleavage of sialic acid, as is suggested by its ability to activate tumor growth factor [40].
Results of our previous studies indicated that pharmacological inhibition of viral NA improves survival in secondary pneumococcal pneumonia after influenza [13, 14]. However, these studies were performed using a single influenza virus and could not establish whether a dose effect based on the activities of different influenza viruses might exist. In the present study, we created a set of recombinant influenza viruses that differed from each other only in their level of NA activity. A hierarchy of support for bacterial adherence and secondary bacterial pneumonia could be seen when recombinant influenza viruses were compared in cell culture and animal models of viral-bacterial interactions. The NA that had the highest level of activity (from Sing57, the strain that caused the 1957 pandemic) was capable of inducing more adherence by S. pneumoniae to cultured respiratory epithelial cells and higher mortality from secondary bacterial pneumonia in mice than an NA with 2-fold less activity (from Syd97). Similar weight loss and lung viral titers during the influenza phase of the infections indicated that differences in viral kinetics were not interfering in the comparisons. An influenza virus containing the NA of the Fuj02 strain, which has a level of NA activity comparable to that of an influenza virus containing the Syd97 NA, more effectively supported secondary bacterial pneumonia than did an influenza virus that had another 2-fold decrease in relative activity (Mem72) when tested on the same background (paired with the Fuj02 HA). An H3N2 virus (Fuj02) caused high mortality during the 20032004 influenza season and contributed to a number of well-publicized deaths from methicillin-resistant Staphylococcus aureus [41]. The activity of the NA from the influenza virus that caused the 1918 pandemic has not been reported, and activities of N1 NAs from influenza viruses circulating before 1957 have not been studied in a comprehensive fashion. Our data provide direct evidence that NA activity in influenza viruses is a predictor of mortality from secondary bacterial pneumonia.
These results, together with our previous data showing that oseltamivir treatment prevents secondary bacterial pneumonia in mice even when it is administered late during the course of the viral infection [13], warrant clinical studies of the prevention and treatment of secondary bacterial infections after influenza with NA inhibitors. Effectiveness studies of these drugs in the treatment of influenza have already shown that they decrease the number of complications, although the populations studied have most often not included those individuals who are most vulnerable, because of underlying illnesses or extremities of age, to secondary bacterial infections [4244]. Preparations for the next influenza pandemic must take into account the possibility that many deaths will be caused by secondary bacterial pneumonia, and an increased focus on viral NAincluding stockpiling of NA inhibitorsis therefore essential.
Acknowledgments
We thank G. Air, for providing us with Mem4 and Mem5 monoclonal antibodies; E. Hoffmann, D. R. Perez, L. Widjaja, and R. G. Webster, for providing us with A/Puerto Rico/8/34, A/Hong Kong/1/68 hemagglutinin, and A/Fujian/411/02 hemagglutinin and neuraminidase plasmids; R. McKeon, for technical support; and R. G. Webster and E. I. Tuomanen, for critical reading of the manuscript.
References
1. Simonsen L, Clarke MJ, Williamson GD, Stroup DF, Arden NH, Schonberger LB. The impact of influenza epidemics on mortality: introducing a severity index. Am J Public Health 1997; 87:194450. First citation in article
2. Thompson WW, Shay DK, Weintraub E, et al. Mortality associated with influenza and respiratory syncytial virus in the United States. JAMA 2003; 289:17986. First citation in article
3. Hers JFP, Masurel N, Mulder J. Bacteriology and histopathology of the respiratory tract and lungs of fatal Asian influenza. Lancet 1958; 2:11413. First citation in article
4. Lindsay MI Jr, Herrmann EC Jr, Morrow GW Jr, Brown AL Jr. Hong Kong influenza: clinical, microbiologic, and pathologic features in 127 cases. JAMA 1970; 214:182532. First citation in article
5. Scadding JG. Lung changes in influenza. Quart J Med 1937; 6:42565. First citation in article
6. Stuart-Harris CH, Laird J, Tyrrell DA, Kelsall MH, Franks ZC. The relationship between influenza and pneumonia. J Hyg (Lond) 1949; 47:43448. First citation in article
7. Tyrrell DAJ. The pulmonary complications of influenza as seen in Sheffield in 1949. Quart J Med 1952; 21:291306. First citation in article
8. Nicholson KG. Human influenza. In: Nicholson KG, Webster RG, Hay AJ, eds. Textbook of influenza. London: Blackwell Science, 1998:2223. First citation in article
9. Simonsen L. The global impact of influenza on morbidity and mortality. Vaccine 1999; 17(Suppl 1):S310. First citation in article
10. Bartlett JG, Mundy LM. Community-acquired pneumonia. N Engl J Med 1995; 333:161824. First citation in article
11. McCullers JA, Webster RG. A mouse model of dual infection with influenza virus and Streptococcus pneumoniae. In: Osterhaus ADME, Cox N, Hampson AW, eds. Options for the control of influenza IV. Amsterdam: Elsevier Science, 2001:6017. First citation in article
12. McCullers JA, Rehg JE. Lethal synergism between influenza virus and Streptococcus pneumoniae: characterization of a mouse model and the role of platelet-activating factor receptor. J Infect Dis 2002; 186:34150. First citation in article
13. McCullers JA, Bartmess KC. Role of neuraminidase in lethal synergism between influenza virus and Streptococcus pneumoniae. J Infect Dis 2003; 187:10009. First citation in article
14. McCullers JA. Effect of antiviral treatment on the outcome of secondary bacterial pneumonia after influenza. J Infect Dis 2004; 190:51926. First citation in article
15. Tong HH, McIver MA, Fisher LM, DeMaria TF. Effect of lacto-N-neotetraose, asialoganglioside-GM1 and neuraminidase on adherence of otitis media-associated serotypes of Streptococcus pneumoniae to chinchilla tracheal epithelium. Microb Pathog 1999; 26:1119. First citation in article
16. LaMarco KL, Diven WF, Glew RH. Experimental alteration of chinchilla middle ear mucosae by bacterial neuraminidase. Ann Otol Rhinol Laryngol 1986; 95:3048. First citation in article
17. Linder TE, Lim DJ, DeMaria TF. Changes in the structure of the cell surface carbohydrates of the chinchilla tubotympanum following Streptococcus pneumoniae-induced otitis media. Microb Pathog 1992; 13:293303. First citation in article
18. Tong HH, Grants I, Liu X, DeMaria TF. Comparison of alteration of cell surface carbohydrates of the chinchilla tubotympanum and colonial opacity phenotype of Streptococcus pneumoniae during experimental pneumococcal otitis media with or without an antecedent influenza A virus infection. Infect Immun 2002; 70:4292301. First citation in article
19. Wagner R, Wolff T, Herwig A, Pleschka S, Klenk HD. Interdependence of hemagglutinin glycosylation and neuraminidase as regulators of influenza virus growth: a study by reverse genetics. J Virol 2000; 74:631623. First citation in article
20. Hoffmann E, Neumann G, Kawaoka Y, Hobom G, Webster RG. A DNA transfection system for generation of influenza A virus from eight plasmids. Proc Natl Acad Sci USA 2000; 97:610813. First citation in article
21. Hoffmann E, Krauss S, Perez D, Webby R, Webster RG. Eight-plasmid system for rapid generation of influenza virus vaccines. Vaccine 2002; 20:316570. First citation in article
22. Reed LJ, Muench H. A simple method for estimating fifty percent endpoints. Am J Hyg 1938; 27:4937. First citation in article
23. Warner TG, O'Brien JS. Synthesis of 2-(4-methylumbelliferyl)-alpha-D-N-acetylneuraminic acid and detection of skin fibroblast neuraminidase in normal humans and in sialidosis. Biochemistry 1979; 18:27837. First citation in article
24. Warren L. The thiobarbituric acid assay of sialic acids. J Biol Chem 1959; 234:19715. First citation in article
25. Murti KG, Webster RG. Distribution of hemagglutinin and neuraminidase on influenza virions as revealed by immunoelectron microscopy. Virology 1986; 149:3643. First citation in article
26. Webster RG, Brown LE, Laver WG. Antigenic and biological characterization of influenza virus neuraminidase (N2) with monoclonal antibodies. Virology 1984; 135:3042. First citation in article
27. Air GM, Els MC, Brown LE, Laver WG, Webster RG. Location of antigenic sites on the three-dimensional structure of the influenza N2 virus neuraminidase. Virology 1985; 145:23748. First citation in article
28. Gulati U, Hwang CC, Venkatramani L, et al. Antibody epitopes on the neuraminidase of a recent H3N2 influenza virus (A/Memphis/31/98). J Virol 2002; 76:1227480. First citation in article
29. Francis KP, Yu J, Bellinger-Kawahara C, et al. Visualizing pneumococcal infections in the lungs of live mice using bioluminescent Streptococcus pneumoniae transformed with a novel gram- positive lux transposon. Infect Immun 2001; 69:33508. First citation in article
30. Kobasa D, Wells K, Kawaoka Y. Amino acids responsible for the absolute sialidase activity of the influenza A virus neuraminidase: relationship to growth in the duck intestine. J Virol 2001; 75:1177380. First citation in article
31. Ito T, Couceiro JN, Kelm S, et al. Molecular basis for the generation in pigs of influenza A viruses with pandemic potential. J Virol 1998; 72:736773. First citation in article
32. Baum LG, Paulson JC. The N2 neuraminidase of human influenza virus has acquired a substrate specificity complementary to the hemagglutinin receptor specificity. Virology 1991; 180:105. First citation in article
33. Ito T, Suzuki Y, Takada A, et al. Differences in sialic acid-galactose linkages in the chicken egg amnion and allantois influence human influenza virus receptor specificity and variant selection. J Virol 1997; 71:335762. First citation in article
34. Housworth WJ, Spoon MM. The age distribution of excess mortality during A2 Hong Kong influenza epidemics compared with earlier A2 outbreaks. Am J Epidemiol 1971; 94:34850. First citation in article
35. Lui KJ, Kendal AP. Impact of influenza epidemics on mortality in the United States from October 1972 to May 1985. Am J Public Health 1987; 77:7126. First citation in article
36. Simonsen L, Clarke MJ, Schonberger LB, Arden NH, Cox NJ, Fukuda K. Pandemic versus epidemic influenza mortality: a pattern of changing age distribution. J Infect Dis 1998; 178:5360. First citation in article
37. Reichert TA, Simonsen L, Sharma A, Pardo SA, Fedson DS, Miller MA. Influenza and the winter increase in mortality in the United States, 19591999. Am J Epidemiol 2004; 160:492502. First citation in article
38. Garcia-Sastre A, Egorov A, Matassov D, et al. Influenza A virus lacking the NS1 gene replicates in interferon-deficient systems. Virology 1998; 252:32430. First citation in article
39. Guan Y, Poon LL, Cheung CY, et al. H5N1 influenza: a protean pandemic threat. Proc Natl Acad Sci USA 2004; 101:815661. First citation in article
40. Schultz-Cherry S, Hinshaw VS. Influenza virus neuraminidase activates latent transforming growth factor beta. J Virol 1996; 70:86249. First citation in article
41. Centers for Disease Control and Prevention. Update: influenza-associated deaths reported among children aged <18 yearsUnited States, 200304 influenza season. MMWR Morb Mortal Wkly Rep 2004; 52:12545. First citation in article
42. Treanor JJ, Hayden FG, Vrooman PS, et al. Efficacy and safety of the oral neuraminidase inhibitor oseltamivir in treating acute influenza: a randomized controlled trial. US Oral Neuraminidase Study Group. JAMA 2000; 283:101624. First citation in article
43. Whitley RJ, Hayden FG, Reisinger KS, et al. Oral oseltamivir treatment of influenza in children. Pediatr Infect Dis J 2001; 20:12733. First citation in article
44. Randomised trial of efficacy and safety of inhaled zanamivir in treatment of influenza A and B virus infections. The MIST (Management of Influenza in the Southern Hemisphere Trialists) Study Group. Lancet 1998; 352:187781. First citation in article