Simvastatin and oseltamivir combination therapy does not improve
the effectiveness of oseltamivir alone following highly pathogenic
avian H5N1 influenza virus infection in mice
Jessica A. Belser, Kristy J. Szretter1, Jacqueline M. Katz, Terrence M. Tumpeyn
Influenza Division, National Center for Immunization and Respiratory Diseases, Centers for Disease Control and Prevention, Atlanta, GA 30333, United States
a r t i c l e i n f o
Received 12 December 2012
Returned to author for revisions
21 January 2013
Accepted 23 January 2013
Available online 1 March 2013
a b s t r a c t
Nonspecific anti-inflammatory drugs have been purported to reduce the burden of severe influenza
disease. We demonstrate that, unlike oseltamivir administration, simvastatin administration did not
reduce morbidity, mortality, or viral load of mice infected with H1N1 or H5N1 viruses. No added benefit
to the efficacy of oseltamivir therapy was observed when mice were treated in combination with
simvastatin. Modest reductions in lung cytokine production in H5N1 but not H1N1 virus-infected
simvastatin-treated mice indicate a potential benefit for statin use in mitigating disease following
severe virus infection.
Published by Elsevier Inc.
Statins (3-hydroxy-3-methylglutaryl coenzyme A reductase
inhibitors) are a class of cardioprotective medications which
possess pleiotropic immunomodulatory and anti-inflammatory
properties (Jain and Ridker, 2005; Kwak et al., 2000). The ability of
statins to inhibit cytokine production, improve endothelial cell
function, and modulate host molecular pathways has led to the
hypothesis that statin use could be a preventative therapy against
cellular damage caused by influenza virus infection (Blanc et al.,
2011; Fedson, 2006). As statins represent one of the most widely
prescribed classes of drug in the world, identifying the capacity of
statins to reduce influenza virus morbidity and mortality in the
event of a pandemic or simply to extend the time between onset
of illness and development of severe disease, thus allowing a
greater window for the efficacy of existing antiviral treatments, is
a matter of great public health importance.
Despite conflicting reports, several retrospective observational
studies have identified an association between statin use and
reductions in influenza virus morbidity in humans; however,
these studies are limited by the substantial variability in timing
and duration of drug administration among participants, or the
inclusion of pneumonia morbidity in the absence of laboratory-
confirmed influenza virus infection (Brett et al., 2011; Frost et al.,
2007; Kwong et al., 2009; Mortensen et al., 2005; Vandermeer
et al., 2012). Selected animal studies have further suggested
a protectiverole forstatins
inflammatory drug regimens against acute lung injury, but results
have been mixed (Budd et al., 2007; Ferraro et al., 2011; Gower
and Graham, 2001; Jacobson et al., 2005; Salomon et al., 2007;
Walsh et al., 2011). Recent work has examined the efficacy of
statin treatments following influenza virus infection in mice,
finding only limited benefits of statin treatment in the ameliora-
tion of influenza virus infection, however these studies have not
generally employed contemporary, wild-type viruses and have
not extensively examined statin drugs administered concurrently
with commercially available antiviral treatments (An et al., 2011;
Kumaki et al., 2012; Radigan et al., 2012). Furthermore, incon-
sistencies in statin drug administration, inoculation routes, and a
paucity of studies including influenza-specific antivirals as appro-
priate controls among these previously published studies makes
it difficult to thoroughly assess the efficacy of statin administra-
tion in the context of influenza virus infection. There is a need
to investigate more fully the ability of statin drugs, alone or in
combination with existing antiviral therapeutics, to mitigate
influenza virus disease severity against both mild and virulent
influenza virus strains. To address this question, we utilized
simvastatin, a commonly prescribed statin drug, and oseltamivir,
and othernonspecific anti-
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nCorrespondence to: Influenza Division, National Center for Immunization and
Respiratory Diseases, Centers for Disease Control and Prevention, Mail Stop G-16,
1600 Clifton Road NE, Atlanta, GA 30333, United States. Fax: þ1 404 639 2350.
E-mail addresses: email@example.com, firstname.lastname@example.org,
email@example.com (T.M. Tumpey).
1Current address: Visterra, Inc. One Kendall Square, Suite B3301 Cambridge,
Virology 439 (2013) 42–46
the most widely prescribed antiviral drug for influenza viruses
(Gubareva et al., 2000; Jain and Ridker, 2005).
To determine the susceptibility of influenza viruses to statin
treatment in vivo, we inoculated six-to-eight week old female
BALB/c mice intranasally (i.n.) with two viruses which exhibit
divergent phenotypes in this model. A/Chicken/Korea/Gimje/08
(H5N1) replicates efficiently in the murine respiratory tract,
causing severe morbidity and mortality (D. Wadford and T.M.
Tumpey, unpublished data). A/Mexico/4482/09 (A/H1N1pdm09,
pH1N1) virus, isolated from the 2009 pandemic, does not cause a
lethal infection (Belser et al., 2010). Simvastatin sodium salt
(10 mg/kg body weight; Calbiochem) was administered by oral
gavage once daily for 12 days commencing 72 h before virus
inoculation (Gower and Graham, 2001; Liu et al., 2009). Oselta-
mivir (50 mg/kg body weight; Roche) was administered by oral
gavage once daily for 8 days commencing 24 hours before virus
Fig. 1. Effect of statin treatment on influenza virus disease outcome and replication in mice. Oseltamivir (50 mg/kg), simvastatin (10 mg/kg), or a combination of both was
administered once daily by oral gavage; control mice received vehicle only. (A) Groups of 6–10 mice were inoculated i.n. with 100 MID50of H5N1 virus and monitored
daily for morbidity and mortality. Any mouse which lost 425% initial body weight was euthanized. (B) Groups of 3–5 mice were inoculated i.n. with 100 MID50of pH1N1
or H5N1 virus and lungs were collected days 3 and 6 p.i. for virus titration. Tissues were titrated in cells (pH1N1) or eggs (H5N1) with titers reported as plaque forming
units (PFU) or 50% egg infectious doses (EID50)/ml of tissue, respectively. n, po0.05 compared to control mice by one-way ANOVA with a Bonferroni post-test.
Kinetic analysis of circulating lymphocytes following H5N1 virus challenge in mice.
Percentage of circulating lymphocytes in peripheral blooda
LY NE MO EO BA
2.070.4 220.127.116.11 0.050.3
aAverage percentage of lymphocytes (LY), neutrophils (NE), monocytes (MO), eosinophils (EO), and basophils (BA) in whole blood.
bWBC, total white blood cells in whole blood7standard deviation (K/ml).
npo0.05 compared with control group by one-way ANOVA with a Bonferroni post-test.
J.A. Belser et al. / Virology 439 (2013) 42–46
inoculation (Tumpey et al., 2002). While these doses are above
concentrations typically administered to humans, they are neces-
sary to achieve bioactive doses in rodents and are consistent with
previously published in vivo studies (Ilyushina et al., 2008; Leung
et al., 2003). Control mice received distilled water on the same
schedule. Mice were inoculated with 100 mouse infectious dose
50% (MID50) of each virus (equivalent to a 2 lethal dose 50%
(LD50) of H5N1), and 6–10 mice/group were monitored daily for
2 weeks for morbidity (measured by weight loss) and mortality.
An additional three to five mice/group were euthanized on
days 3 and 6 post-inoculation (p.i.) for collection of whole blood
for analysis of circulating lymphocytes, and lung tissues for
virus titration and analysis of proinflammatory cytokines and
chemokines (Belser et al., 2010). Days 3 and 6 p.i. were chosen
as they represent times of peak viral replication in this model
and are established timepoints for assessment of these para-
meters in murine pathogenesis and antiviral studies (Belser
et al., 2010; Kumaki et al., 2012; Maines et al., 2005; Wong
et al., 2011).
Mice receiving daily statin administration exhibited compar-
able severe morbidity and 100% mortality following a lethal H5N1
virus challenge as untreated control mice (Fig. 1A). In contrast,
oseltamivir treatment protected mice from weight loss and death
following H5N1 virus challenge, and resulted in significantly
decreased viral titers in the lung day 6 p.i. (po0.05) (Fig. 1A
and B). The reductions in H5N1 viral load following oseltamivir
treatment are comparable with prior studies demonstrating a
modest decrease in lung virus titer at these times post-infection
Fig. 2. Effect of statin treatment on proinflammatory cytokine and chemokine production in the lungs of mice following influenza virus infection. Oseltamivir (50 mg/kg),
simvastatin (10 mg/kg), or a combination of both was administered once daily by oral gavage; control mice received vehicle only. Groups of 3–5 mice were inoculated i.n.
with 100 MID50of pH1N1 or H5N1 virus and lungs were collected days 3 and 6 p.i. Clarified lung homogenates were tested via BioPlex (Bio-Rad) or ELISA (R&D Systems)
assays. (A) Cytokine and chemokines detected following pH1N1 virus infection. (B) Cytokines and chemokines that were not significantly altered by statin treatment
following H5N1 virus infection. (C) Cytokines and chemokines following H5N1 virus infection where significant reductions in statin-treated mice compared with control
mice were detected. n, po0.05 compared to control mice by one-way ANOVA with a Bonferroni post-test.
J.A. Belser et al. / Virology 439 (2013) 42–46
(Govorkova et al., 2009; Ilyushina et al., 2007). Weight loss
following pH1N1 virus challenge was not observed in any group
tested (o5% of initial body weight loss in any group p.i.).
However, reductions in viral load in the lung were detected
following daily oseltamivir but not simvastatin administration
in these mice (Fig. 1B). Mice receiving a combination of both
simvastatin and oseltamivir did not exhibit an improvement in
disease outcome compared with oseltamivir treatment alone
following infection with either virus subtype. These results are
in agreement with previous studies which found no improvement
in morbidity or mortality following wild-type H5N1 virus infec-
tion in mice treated with immune modulators (Kumaki et al.,
2012; Salomon et al., 2007).
H5N1 virus infection of mice can cause pronounced leukopenia
and lymphopenia, but pretreatment with oseltamivir was found to
protect mice from these hematopoietic alterations (Table 1) (Maines
et al., 2005). In contrast, simvastatin did not protect against leuko-
penia following H5N1 virus infection and did not significantly reduce
the degree of lymphocyte depletion compared with control mice
(Table 1). Combination therapy did not confer additional benefit
compared to the respective monotherapies. While simvastatin pre-
treatment of mice contributed to maintaining a normal balance of
immune cell subpopulations following non-virus induced acute lung
injury in a prior study, our findings are in accord with a recent study
which detected comparable levels of total leukocyte counts in
bronchoalveolar lavage fluid between statin-treated and untreated
control mice following influenza virus infection (Ferraro et al., 2011;
Radigan et al., 2012).
Previous mouse studies have found that oseltamivir treatment
could reduce lung inflammatory responses induced by influenza
virus infection (Ilyushina et al., 2008; Wong et al., 2011).
Similarly, we found that oseltamivir treatment resulted in the
significant reduction of numerous proinflammatory cytokines and
chemokines associated with influenza virus infection, predomi-
nantly following lethal H5N1 virus infection (Fig. 2A, B, and data
not shown) (Maines et al., 2008). Interestingly, simvastatin treat-
ment significantly reduced (po0.05) the production of IFNg,
IL-10, and TNFa in the lungs of H5N1 virus-infected mice on
day 3 p.i.; all three cytokines are known targets of statin drugs
with roles in the initiation of inflammatory cell infiltration
(Fig. 2C) (Jain and Ridker, 2005). Notably, the observed decreased
production of TNFa in statin-treated mice is in agreement with
previous work which has identified a role for TNFa in influenza
virus morbidity, with reduced production of TNFa in mice
receiving statins following lung injury in mice (Ferraro et al.,
2011; Szretter et al., 2007). While we observed significant reduc-
tions of selected cytokines following statin treatment in H5N1
virus-infected murine lungs, these differences were not observed
following pH1N1 virus-infection (Fig. 2A and data not shown), in
agreement with Radigan et al. who did not observe differences in
cytokine levels in rosuvastatin-treated mice following H3N2 or
H1N1 virus infection (Radigan et al., 2012). No significant differ-
ences in cytokine production were detected between mice receiv-
ing oseltamivir alone and those receiving combination therapy
(Fig. 2). Treatment with simvastatin in the absence of virus
infection did not result in substantial alterations of proinflamma-
tory cytokines and chemokines compared with uninfected control
mice (data not shown).
Host innate immune responses contribute to the severity
of disease induced by H5N1 viruses, and depletion of cytokines
and/or chemokines can influence the resulting course of disease
(Maines et al., 2008; Szretter et al., 2007). The findings in this
report support previous studies which demonstrate limited
improvements in cellular infiltration and reduced cytokine pro-
duction in the lungs of mice following administration of immu-
nomodulatory drugs in the absence of improved survival following
lung injury caused by viral, bacterial, and other stimuli (Boyd et al.,
2012; Ferraro et al., 2011; Walsh et al., 2011). While we did not
observe a striking antiviral effect of simvastatin administration
alone following influenza virus infection, or a measurable improve-
ment of combination therapy over oseltamivir treatment alone in
mice, reduced hypercytokinemia following H5N1 but not pH1N1
virus infection in mice following simvastatin administration sup-
ports a potential benefit of this treatment depending on the virus
used to infect, warranting further study of statin administration in
the mitigation of severe influenza disease. As shown in animal
studies, reductions in influenza virus-induced pulmonary inflam-
mation by statin administration may be of particular benefit in
severe cases of influenza virus infection where mechanical ventila-
tion is employed (Muller et al., 2010).
The findings and conclusions in this report are those of the
authors and do not necessarily reflect the views of the funding
agency. We thank the National Veterinary Research and Quarantine
Service of South Korea for providing the H5N1 virus used in this
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