Re: Researchers unlock secrets of 1918 flu pandemic
Viral RNA polymerase complex promotes optimal
growth of 1918 virus in the lower respiratory
tract of ferrets
Tokiko Watanabe
a, Shinji Watanabea, Kyoko Shinyab, Jin Hyun Kima, Masato Hattaa, and Yoshihiro Kawaokaa,b,c,d,1
a
Department of Pathobiological Sciences, School of Veterinary Medicine, University of Wisconsin-Madison, 2015 Linden Drive, Madison, WI 53706; bDivision
of Zoonosis, Department of Microbiology and Infectious Diseases, Graduate School of Medicine, Kobe University, Kobe 650-0017, Japan; and cDivision of
Virology, Department of Microbiology and Immunology, and dInternational Research Center for Infectious Diseases, Institute of Medical Science, University
of Tokyo, Tokyo 108-8639, Japan
Edited by Hans-Dieter Klenk, Philipps-Universitat Marburg, Marburg, Germany, and accepted by the Editorial Board November 13, 2008 (received for review
July 18, 2008)
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 wildtype
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.
pathogenesis
pandemic influenza
I
n 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 theHAcleavage
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 singlegene
substitution, including the HA or PB1 alone, in transforming
the currently circulating K173 virus into a 1918-like strain in
Author contributions: T.W. and Y.K. designed research; T.W., S.W., K.S., J.H.K., and M.H.
performed research; T.W., S.W., K.S., and Y.K. analyzed data; and T.W. and Y.K. wrote the
paper.
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.
1
Towhomcorrespondence should be addressed. E-Mail: kawaokay@svm.vetmed.wisc.edu.
This article contains supporting information online at www.pnas.org/cgi/content/full/
0806959106/DCSupplemental.
? 2008 by The National Academy of Sciences of the USA
January 13, 2009 vol. 106 no. 2 587?591
MICROBIOLOGY
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.
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.
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
Hours postinfection
1918WT
K173WT
1918(3P+NP)/K173
K173(3P+NP)/1918
1918PA/K173
1918PB1/K173
1918PB2/K173
1918NP/K173
1918HA/K173
1918NA/K173
1918M/K173
1918NS/K173
Virus titers (log
10 PFU/ml)
1
2
3
4
5
6
7
8
9
10
0 20 40 60 80
Fig. 1.
Growth kinetics of reassortant viruses inMDCKcells.MDCKcells 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.
Table 1. Replication efficiency of 1918/K173 recombinant viruses
in ferrets*
Virus
Virus titer (mean log
10PFU 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 10
5 PFU/500 l 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.
588
www.pnas.orgcgidoi10.1073pnas.0806959106 Watanabe et al.
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
10
8 PFU/ml; Fig. 1). Additionally, we also examined the growth
property of a recombinant virus, K173(3PNP)/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(3PNP)/1918 virus grew slower than
the K173 virus (Fig. 1). In ferrets, the 1918(3PNP)/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-tosevere
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(3PNP)/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
Terminal bronchus
A
C
E F
Peribronchial gland
K173
1918
1918(3P+NP)/K173
Peribronchial gland
Terminal bronchus
D
: bronchitis/bronchopneumonia without viral antigen
: viral antigen
Left Right
Left Right
Left Right
B
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(3PNP)/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(3PNP)/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 m.) 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.
Watanabe
et al. PNAS January 13, 2009 vol. 106 no. 2 589
MICROBIOLOGY
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- 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 in5%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 (3PNP)
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 withMEMmedium containing 1.0g 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 l (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(3PNP)/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-m-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 anti-
590
www.pnas.orgcgidoi10.1073pnas.0806959106 Watanabe et al.
gen-antibody reactions were visualized by 3, 3
diaminobenzidine tetrahydrochloride
staining with a Dako EnVision system (Dako Co. Ltd.).
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.
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