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Departments of Microbiology and Medicine I, Kumamoto University School of Medicine, Kumamoto 860-0811, Japan;
* Department of Viral Infection, Institute of Medical Science, University of Tokyo, Tokyo 108-0071, Japan; and
DNAVEC Research Institute, Tsukuba, Japan
1Correspondence: Department of Microbiology, Kumamoto University School of Medicine, Kumamoto 860-0811, Japan. E-mail: takakaik{at}gpo.kumamoto-u.ac.jp
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: NO • peroxynitrite • oxidative stress
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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2
3
4
5)
. The resultant
reactive molecular species such as peroxynitrite
(ONOO-) nonselectively affect the host’s cells
and tissues (6
, 7)
. Such host defense effectors are
originally produced to injure the intruding pathogens, which will then
suffer oxidative stress because of the host. Oxidative stress has been
suggested to be involved in mutagenesis and hence carcinogenesis
(8
9
10
11)
. Also, RNA virus mutation was reported to be
increased by chemical mutagens, including nitrous acid
(HNO2) (12
13
14
15)
, although the degree
of mutation appears to be slight (16)
. Therefore, it may
be that mutagenesis of various viruses is occurring naturally in
biological systems during infections as a result of host defense.
Although involvement of NO-induced oxidative stress in viral
pathogenesis is now well recognized (4
, 5)
, the role of NO
and NO-derived reactive nitrogen intermediates in viral mutation is
only poorly understood.
RNA viruses share high mutation rates ranging from
10-5 to 10-3
misincorporation/nucleotide site/round of copying because of the
error-prone nature of their replicases. This high mutation rate
accounts for their quasispecies nature, as they exist as a mixture of
heterogeneous populations (17)
. Numerous methods exist for
estimating viral mutation, including measurement of mutation
frequencies of phenotypic variations such as temperature-sensitive
growth, plaque morphology, host range, and pathogenicity. The mutations
cannot be assessed precisely and quantitatively by these criteria,
however, because the variant viruses often contain multiple base
substitutions in different genes (18)
. Escape of a virus
from a particular neutralizing monoclonal antibody occurs by a single
base substitution, leading to a single codon change on the epitope.
Thus, investigation of viral genome mutation has been based on
selection of the escape mutants. The frequency of generation of escape
mutants in vitro in cultured cells is reported to be
~10-4.5 for four different negative-strand RNA
viruses, i.e., Sendai virus (SeV), vesicular stomatitis virus,
Newcastle disease virus, and influenza A virus (19
, 20)
.
Nevertheless, the selection procedures are not fully developed because
the frequencies fluctuate greatly, even within a given virus species,
depending on the antibodies used for selection (18)
. Also,
any such mutation analysis is not truly applicable to the estimation of
mutation rates occurring in vivo in infected host organisms.
Performing a green fluorescent protein (GFP) -indicated viral mutation
assay with the use of a recombinant SeV constructed with GFP genome as
a tool for estimating mutation frequency occurring in vivo,
in the present study we verified for the first time that oxidative
stress induced by high-output NO accelerates RNA virus mutation
in vivo.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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) (21
, 22)
. The final construct,
pSeV18+bGFP(+), produced recombinant GFP-SeV antigenome (16.25 kb). GFP-SeV was harvested from
pSeV18+bGFP(+)-transfected LLCMK2 cells to obtain
fully infectious particles and then was propagated in embryonated
chicken eggs.
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Assessment of GFP-indicated mutation
Mutation frequency of GFP-SeV was determined by measuring
incidence of fluorescence-negative viral plaques among
fluorescence-positive plaques formed by GFP-SeV replication as
identifying the plaques by hemadsorption test. Specifically, viral
plaques were formed on monolayers of CV-1 cells (a monkey kidney cell
line) cultured in 6-well plates (35 mm diameter; Falcon, Oxnard,
Calif.) overlaid with 0.4% agarose containing 0.5 µg/ml trypsin
(Sigma, St. Louis, Mo.) in Dulbecco’s minimum essential medium (DMEM)
(Life Technologies, Inc., Grand Island, N.Y.) + 0.2% bovine serum
albumin (BSA) (Sigma), and the mutation was identified and quantified
by hemadsorption and fluorescence. A hemadsorption test was performed
by incubating the monolayer cells with 2% guinea pig erythrocyte
suspension in DMEM (37°C; 30 min) after removal of the agarose
overlay of the cell culture (4 days). Hemadsorption foci with clear
contour were observed only in the viral plaques, when nonadsorbed
erythrocytes were removed by washing with 10 mM phosphate-buffered
saline (PBS; pH 7.4) (Fig. 1B, C
). Simultaneously, GFP
expression was detected via its strong fluorescence under microscopic
observation with an inverted phase-contrast fluorescence microscope
(Fig. 1B, C
). Hemadsorption-positive foci were also detected
by use of a color reaction: erythrocyte’s hemoglobin-catalyzed
peroxidation of diaminobenzidine with 3.5%
tert-butylhydroperoxide as substrate (23)
;
hemadsorption foci show brown colors.
In vivo study of GFP-SeV mutation
GFP-SeV was administered to C57BL/6 (B6) and B6
iNOS-/- mice (4 wk old; male) by inhalation of
viral suspension (2)
. B6 inducible nitric oxide synthase
(iNOS) knockout mice were produced in Jackson Laboratories (West Grove,
Pa.; 24
). At various times after viral infection, the lung
tissues were obtained and homogenized in ice-cold PBS (pH 7.4) as
described earlier (2)
. The supernatant of homogenates
prepared by centrifugation (10, 000 g for 10 min) was
serially diluted and inoculated onto CV-1 cells to form viral plaques
for GFP-indicated mutation assay. The mutation frequency of GFP-SeV
isolated from the lung was measured after formation of viral plaques on
CV-1 cell monolayers, as just mentioned. For accurate determination of
mutation frequencies, the number of fluorescence-negative plaques was
counted, with the cell culture plate having less than 200 plaques/well
(35 mm diameter) after 4 days of culture. NO production in the
GFP-SeV-infected lung tissue was identified directly by using electron
spin resonance (ESR) spectroscopy.
N-Dithiocarboxy(sarcosine)
(DTCS)–Fe2+ complex, a spin trap for NO, was
administered subcutaneously and the lung was subjected to X-band ESR
spectroscopy (Bruker ESP 380E) at 110K (3)
.
Immunohistochemistry for iNOS induction and nitrotyrosine formation in
the lung was performed as described previously (3)
.
Briefly, after the lung was fixed in 2% periodate/lysine/paraformaldehyde, 6 µm sections were prepared,
followed by immunostaining with either an anti-iNOS antibody (1:500;
Santa Cruz Biotechnology, Santa Cruz, Calif.) or a polyclonal
anti-nitrotyrosine antibody (1:500; Upstate Biotechnology, Lake Placid,
N.Y.) by the indirect immunoperoxidase method, using diaminobenzidine
as substrate. The lung sections were also stained with hematoxylin and
eosin (H&E) for histopathological examination.
Treatment of GFP-SeV with peroxynitrite
GFP-SeV was treated in vitro with a constant-flux
system of peroxynitrite (25)
prepared by infusing 250 µM
peroxynitrite solution (in 10 mM NaOH) at 120 µl per 30 s into
1.2 ml of 0.5 M PBS (pH 7.4) containing 1 x
107 PFU/ml GFP-SeV at room temperature, so that
the concentration of peroxynitrite, which decays within a few seconds
at neutral pH, was maintained at 0.8 µM. An aliquot (120 µl) of the
reaction mixture was withdrawn every 30 s after initiation of the
peroxynitrite infusion and inoculated onto CV-1 cells to form viral
plaques so that the mutation frequency of GFP-SeV was determined as
described above. Peroxynitrite used in this study was synthesized from
acidified nitrite and hydrogen peroxide by use of a quenched flow
reactor, as reported previously (25
, 26)
, and its purity
was > 90% with < 10% nitrite and < 0.1% hydrogen
peroxide contaminations. During constant infusion of peroxynitrite, pH
of the reaction mixture gradually increased up to pH 7.8 at 5 min after
initiating infusion.
Analyses for GFP and SeV virion expression
The expression of GFP and GFP-SeV virion was examined by Western
blotting. Each virus strain was inoculated onto CV-1 cell monolayers at
a multiplicity of infection (moi) of 5 PFU/cell. After 2 days of
culture with 0.5 µg/ml trypsin, the total protein (15 µg) extracted
from the cells with 0.5% sodium dodecyl sulfate (SDS) in 50 mM
Tris-HCl buffer (pH 7.4) containing a protease inhibitor mixture
(2)
was subjected to SDS-polyacrylamide gel
electrophoresis (PAGE) with or without 8 M urea under reducing
conditions. After transfer to Immobilon polyvinylidene difluoride
membrane (Millipore, Bedford, Mass.), the protein was immunostained
with anti-GFP polyclonal antibody (Clontech) or anti-SeV antiserum as
described previously (21)
. Also, mRNA expression of GFP
and each virion was tested by reverse transcriptase-polymerase chain
reaction (RT-PCR). Specifically, after total RNA was extracted from the
virus-infected CV-1 cells by a guanidine thiocyanate lysis method with
Trizol reagent (Gibco, BRL, Grand Island, N.Y.), 0.3 µg of the RNA
was subjected to RT-PCR, which was performed according to our protocol
reported previously (27)
. The oligonucleotide primers used
were: sense 22 mer, 5'-TGAGCAAGGGCGAGGAGCTGTT-3'; antisense 23 mer,
5'-TACAGCTCGTCCATGCCGAGAGT-3' to obtain a 712 bp GFP cDNA fragment, and
sense 24 mer, 5'-AAGATAGCTGGATCCCACGAATCG-3'; antisense 30 mer,
5'-AGGCTTTGATGAGCGCTATGTCTCTTTTGG-3' to obtain a 334 bp F protein cDNA
fragment, including a coding region for the amino acid sequence of
trypsin cleavage site. Similarly, mRNA for glyceraldehyde-3-phosphate
dehydrogenase was examined as an internal control for mRNA expression
in the cells with use of the primers as described elsewhere
(27)
.
Detection of chymotrypsin-sensitive SeV mutants
Wild-type SeV was generated by reverse genetics without GFP
insertion in a same manner as GFP-SeV and treated with bolus injections
of peroxynitrite. GFP-SeV (1x107 PFU/ml) was
reacted with peroxynitrite at room temperature by bolus additions of 10
µl of peroxynitrite solution (0.1 M NaOH) to the 1.0 ml of viral
suspension (final concentration of peroxynitrite: 8.0 µM) in 0.5 M
PBS (pH 7.4). The addition was repeated three times at 1 min intervals;
8 min after the last peroxynitrite addition, an aliquot of the reaction
mixture was inoculated onto CV-1 cells, followed by plaque formation.
Plaque was formed in agarose-overlaid CV-1 cell monolayers containing
2.0 µg/ml chymotrypsin (N
-tosyl-L-lysyl
chloromethyl ketone treated; free of trypsin; Sigma) in DMEM +
0.2% BSA.
Cloning of GFP-SeV mutants and sequence analysis for GFP and F
protein genomes
GFP-SeV mutants were cloned by picking up a single plaque formed
on the CV-1 cell monolayer. For cloning of spontaneously occurred
mutants, 5000–10,000 plaques of GFP-SeV without peroxynitrite
treatment were produced at the density of 50–100 plaques/well (35 mm
diameter; Falcon), and fluorescence-negative plaques identified
microscopically by hemadsorption test were isolated carefully and
suspended in 100 µl DMEM. The plaque of GFP-SeV mutants generated
after peroxynitrite treatment was isolated from the plaque formation
plates (50–100 plaques/well) showing apparently high mutation
frequencies (>2%), usually obtained later than 2.0 min after
initiating the constant flux of peroxynitrite (cf. Fig. 4
). The mutant
plaques of GFP-SeV generated by peroxynitrite was picked up in five
different experiments of peroxynitrite treatment. The viral suspension
prepared from a single plaque was then inoculated onto CV-1 cells in
12-well plates (22 mm diameter; Falcon). After 48 h culture in
DMEM + 0.2% BSA in the presence of 0.5 µg/ml trypsin, total RNA
extracted from the virus-infected cells was subjected to RT-PCR to
obtain a cDNA fragment of GFP as mentioned above. Similarly, F protein
cDNA was generated by RT-PCR from the viral clone of
chymotrypsin-sensitive SeV mutant. Subsequently, cDNA fragments
sequences for GFP and SeV F protein were determined by a dideoxy method
using ALF Express (Pharmacia, Piscataway, N.J.) with a Cy5 AutoRead
Sequencing Kit (Pharmacia) to verify the genetic basis of GFP and F
protein mutations.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). It is also
important that the present GFP-indicated mutation assay can be done
with GFP-SeVs grown in mice, the natural host, with different genetic
backgrounds.
The effect of NO-induced oxidative stress on GFP-SeV mutation was
investigated in an in vivo system. A lethal dose (2
LD50) of GFP-SeV was administered to B6 mice or
B6 iNOS-/- mice by inhalation of viral aerosol.
The GFP-SeV yield in the lung on days 2, 4, 6, and 7 after infection
was isolated and the GFP mutation was measured. The GFP-SeV mutation
frequency in wild-type B6 mice increased significantly as infection
proceeded, whereas that in iNOS knockout mice was elevated only
slightly (Fig. 2A
). A mutation frequency almost six- to sevenfold higher was
observed on days 4, 6, and 7 in wild-type B6 mice than in
iNOS-deficient mice, although the viral growth in the lung became
plateau at these time points (Fig. 2B
). No significant
difference between the two groups was found for virus production
throughout the course of infection (Fig. 2B
), indicating
that enhanced mutation frequencies of GFP-SeV is not necessarily
dependent on the cycles of viral replication. An ESR study showed that
overproduction of NO was evident in the GFP-SeV-infected lung of the
wild-type mice, and its time profile was correlated with that of the
mutation frequency (Fig. 2C
). In contrast, no appreciable NO
production was observed in GFP-SeV-infected iNOS knockout mice.
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When GFP-SeV was replicated in CV-1 cells in culture, the mutation frequency increased per a single-step replication was estimated to be ~0.1%. If we assume that viral mutation was occurring in the lung cells at a same rate as in the CV-1 cells, excess generation of NO in wild-type B6 mice seems to enhance viral mutation to a great extent, similar to that achieved after more than 10 cycles of viral multiplication in cells without NO exposure in that the difference in mutation frequency (1.5%; on days 6 and 7) between wild-type mice and iNOS knockout mice was almost 15-fold higher than the frequency increased per single viral replication.
NO-induced oxidative stress in GFP-SeV-induced pneumonia
We reported earlier that overproduction of both NO and superoxide,
possibly through peroxynitrite formation, contributes to the
pathogenesis of influenza virus-induced pneumonia in mice
(1
2
3)
. The contribution of high-output NO to influenza
pathogenesis is also verified by Karupiah et al. (28)
using mice lacking iNOS. Therefore, a similar pathogenetic mechanism
involving NO-induced oxidative stress may operate in SeV-induced
pneumonia. The immunohistochemical analysis showed that iNOS was
expressed by inflammatory phagocytic cells such as exudate macrophages
infiltrating alveoli and interstitial tissues of the GFP-SeV-infected
lung in wild-type B6 mice (Fig. 3A
). Similar distribution of strong immunostaining for
nitrotyrosine was observed in the virus-infected lung of the wild-type
mice (Fig. 3B
), but nitrotyrosine formation was not
identified significantly in iNOS-deficient mice (Fig. 3C
).
Thus, it is quite likely that high production of NO in vivo
accelerates RNA virus mutation through formation of reactive nitrogen
oxides such as peroxynitrite. It is important that the inflammatory
tissue injury of the lung infected with GFP-SeV was significantly
reduced in the iNOS-deficient mice compared with wild-type mice (Fig. 3D, E
), suggesting involvement of NO-induced oxidative
stress in the pathogenesis of SeV pneumonia.
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Elevation of mutation frequency of SeV by peroxynitrite
To further clarify the NO-dependent enhanced mutation, GFP-SeV was
treated with peroxynitrite in vitro and its GFP mutation was
tested after propagation on CV-1 cells monolayers (plaque-forming
assay). Specifically, peroxynitrite-induced mutation was evaluated with
a constant-flux system of peroxynitrite (0.8 µM). The mutation
frequency was enhanced at 1.5 min after peroxynitrite infusion: a
2.6-fold increase (0.68±0.07%) compared with the background value
(0.26±0.02%), when no appreciable virucidal effect of peroxynitrite
was observed (Fig. 4
). A time-dependent increase in mutation frequency was obtained,
although the virus infectivity was reduced inversely. A surprisingly
high mutation frequency was observed after a 4 min infusion period
(Fig. 4
, inset): 100% of the GFP-SeV became mutated. In contrast, the
mutation frequency was not affected by the constant flux of decomposed
peroxynitrite, which was prepared by incubation of peroxynitrite in
neutral solution, indicating that remarkable elevation of GFP mutation
frequency was induced by peroxynitrite, but not by hydrogen peroxide
and nitrite contaminated in the peroxynitrite preparation if any.
Specific proteolytic cleavage of SeV F protein by a trypsin-like enzyme
is a prerequisite for the virus to be infectious (29)
.
Altered protease-activated mutants can be selected by chymotrypsin
(30)
. This conventional selection method was also used
here to assess the effect of peroxynitrite on SeV mutation. SeV treated
with peroxynitrite or vehicle was subjected to a plaque formation
assay; in the agarose overlay containing chymotrypsin, as much as a
10-fold increase in chymotrypsin-requiring plaque formation was found
after peroxynitrite treatment (Fig. 5
), although viral infectivity was reduced moderately (by 42%). The
sequence analysis of F protein RNA showed that each chymotrypsin mutant
isolated had an amino acid change at the P1 cleavage site of the F
protein (Arg 
Ile). This result suggests that peroxynitrite enhances
mutation not only of the artificially inserted GFP genome, but also of
the natural SeV genome.
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In a separate experiment, we examined the GFP-SeV mutation treated with
an NO donor propylamine NONOate
(CH3N[N(O)NO]-(CH2)3NH2+CH3),
which releases spontaneously pure NO in solution at neutral pH
(T1/2, 7.6 min) (31)
, for 30 min at
37°C under ambient condition. As a result, NO per se did
not show any significant mutagenetic potential, even when the
concentration of propylamine NONOate was increased up to 1 mM.
According to the data shown in Fig. 2C
, the level of NO
production in the virus-infected lung was estimated to be less than 20
µM at maximum. Thus, we concluded that NO per se, even if
it is converted to the oxidized intermediates such as
NO2 and
N2O3 under ambient
condition, cannot induce biological relevant GFP-SeV mutation.
Characterization of various GFP-SeV mutants
The GFP-SeV mutants were cloned (19 clones for control; 25 clones
for peroxynitrite treatment), and GFP and mRNA expression of each clone
was examined. Western blot analysis indicated that all nonfluorescence
GFP variants possessed the same immunoreactivity and mobility as did
the native GFP (Fig. 6
). Mutant GFPs, however, showed slightly slower mobility than did native
GFP on gels without urea, suggesting alteration of their primary
structures. In contrast, expression of each virion protein was not
impaired for all GFP-SeV mutants tested. GFP mRNA synthesis of all
mutants was as efficient as that of the parent GFP-SeV, as revealed by
RT-PCR (Fig. 6)
.
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Sequence analysis of GFP-SeV mutants
GFP genes in various GFP-SeV clones were sequenced. Neither
deletion nor insertion was found for any clone, and all mutations were
point mutations. A G transition (in viral negative sense)
predominated over other transitions and transversions among 25 GFP-SeV
mutants obtained after peroxynitrite treatment (Table 1
). The overall substitution pattern found in the 19
peroxynitrite-untreated spontaneous mutant clones was essentially the
same as that of peroxynitrite-induced mutations, except that the
incidence of A G substitution was higher in the latter than in the
former and U C transition was higher in the former than in the
latter. Almost all these point mutations led to amino acid
substitutions. Also, there are apparently hot spots of point mutations
in both mutants. For example, A G transition frequently occurred at
nucleotide positions of 496, 530, 547, 601, and 623 (nucleotide number
in viral cDNA), and these substitutions were observed with 10/19 clones
of spontaneous mutants and 14/25 clones of peroxynitrite-induced
mutants. This bias in the site of point mutations may be due to
selection of the GFP-SeV mutants based on the phenotypic change, i.e.,
loss of fluorescence emission of GFP.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, 27)
.
A previous study showed that human leukocytes producing superoxide
exert a mutagenic potential on Salmonella typhimurium TA100
in vitro (32)
. Recent work by Gal and Wogan
(33)
demonstrated mutagenicity associated with NO in
vivo by using lacZ gene-containing plasmid transgenic
mice. It is therefore of paramount importance to investigate a link
between NO-induced oxidative stress and viral mutation. Beck et al.
(34)
show that the pathogenicity of coxsackievirus B3 is
strongly potentiated in vivo in mice fed a
selenium-deficient diet. Intriguingly, an avirulent strain of the virus
is converted to a potent cardiotoxic variant during infection in
selenium-depleted animals. The deficiency of selenium may result in an
ineffective antioxidant system, e.g., low levels of glutathione
peroxidase. These studies were extended to vitamin E- and glutathione
peroxidase-deficient animals, and the results suggest that oxidative
stress facilitates selection and generation of virulent mutants due to
impairment of immunological clearance of the virus (35)
.
Because peroxynitrite can permeate a biological phospholipid bilayer
membrane efficiently (36
, 37)
, enhanced mutation of
GFP-SeV may be explained by the action of peroxynitrite through
nitration, oxidation, or other modifications of the viral genome.
Peroxynitrite causes mutagenesis of prokaryotic DNA (38
, 39)
. Also, increased mutagenesis of the hprt gene of
activated murine macrophages expressing iNOS was recently documented;
this NO-associated mutation spectrum, however, did not differ from that
arising spontaneously (40)
. Similarly, in our current
study, the mutation profile of GFP-SeV mutants induced by peroxynitrite
resembled that of spontaneously occurring mutants. However,
peroxynitrite treatment in vitro preferentially elicits A
G transition in the GFP genome. Our preliminary study suggests that
the GFP-SeV mutants recovered from the wild-type mice had a mutation
pattern similar to that of peroxynitrite-induced mutants: the base
substitutions found in six different clones of GFP-SeV mutants were A
G, 78%; U C, 11%; C U, 11%. Therefore, the
mutational spectrum of GFP-SeV mutants seems not to be consistent with
that of prokaryotic and eukaryotic DNA treated with peroxynitrite, in
which G T transversion is often obtained via depurination of
nitrated deoxyguanosine (10
, 38
, 39)
. In a separate
experiment, we found that nitro G formed in viral RNA by peroxynitrite
is chemically more stable than that in DNA (data not shown). Thus, the
G-specific point mutation might not occur in peroxynitrite mutagenesis
of viral RNA. Because the SeV genome is a highly integrated complex of
a single-stranded RNA and RNA polymerase as well as nucleoproteins, it
is possible that peroxynitrite affects the integrity of the viral
RNA–protein complex, leading to augmentation of the error-prone nature
of the RNA replicase. In any case, more detailed experiments (e.g.,
analysis using a cell-free viral RNA replication system) may be needed
to clarify the molecular events of peroxynitrite-induced RNA
mutagenesis.
It is believed that because of the absence of proofreading and repair functions of viral polymerases, RNA viruses exist as a mixture of heterogeneous genomic populations, termed quasispecies, and evolve rapidly under selective pressures. The present study provides direct evidence that host-derived oxidative stress promotes point mutations. This result expands heterogeneity and increases the number and repertories of variants from which a particular genotype will then evolve more readily under selective pressure. Induction of NO and peroxynitrite generation is a common phenomenon in virus-infected hosts. Therefore, our results and the concepts described here probably have great relevance to viral evolution in general, including the rapid generation of drug-resistant and neutralization escape variants as well as cell tropism-altered variants of human immunodeficiency viruses in vivo.
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ACKNOWLEDGMENTS |
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Received for publication August 30, 1999.
Revision received November 18, 1999.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-dependent clearance of influenza A virus and protection from consolidating pneumonitis in nitric oxide synthase 2-deficient mice. J. Exp. Med. 188,1541-1546This article has been cited by other articles:
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