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RESEARCH ARTICLE
Cryptoporus volvatus Extract Inhibits
Influenza Virus Replication In Vitro and
In Vivo
Li Gao
1.
, Yipeng Sun
2.
, Jianyong Si
1
, Jinhua Liu
2
, Guibo Sun
1
, Xiaobo Sun
1
,
Li Cao
1
*
1. Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences and Peking Union
Medical College, Beijing, China, 2. Key Laboratory of Animal Epidemiology and Zoonosis, Ministry of
Agriculture, College of Veterinary Medicine, China Agricultural University, Beijing, China
*lcao@implad.ac.cn
.These authors contributed equally to this work.
Abstract
Influenza virus is the cause of significant morbidity and mortality, posing a serious
health threat worldwide. Here, we evaluated the antiviral activities of Cryptoporus
volvatus extract on influenza virus infection. Our results demonstrated that the
Cryptoporus volvatus extract inhibited different influenza virus strain replication in
MDCK cells. Time course analysis indicated that the extract exerted its inhibition at
earlier and late stages in the replication cycle of influenza virus. Subsequently, we
confirmed that the extract suppressed virus internalization into and released from
cells. Moreover, the extract significantly reduced H1N1/09 influenza virus load in
lungs and dramatically decreased lung lesions in mice. And most importantly, the
extract protected mice from lethal challenge with H1N1/09 influenza virus. Our
results suggest that the Cryptoporus volvatus extract could be a potential candidate
for the development of a new anti-influenza virus therapy.
Introduction
Influenza is a serious public health problem that causes severe illnesses and deaths
for higher risk populations. Influenza A viruses are responsible for seasonal
epidemics and have caused three pandemics in the 20
th
century (1918, 1957, and
1968) as well as the 2009 H1N1 pandemic. Annually, up to 10% of the U.S.
population is affected by symptomatic influenza infection. It had been reported
that more than 220,000 persons are hospitalized, of which 24,000 die due to
influenza-associated illness each year [1]. The highest hospitalization rate occurs
OPEN ACCESS
Citation: Gao L, Sun Y, Si J, Liu J, Sun G,
et al. (2014) Cryptoporus volvatus Extract Inhibits
Influenza Virus Replication In Vitro and In
Vivo. PLoS ONE 9(12): e113604. doi:10.1371/
journal.pone.0113604
Editor: Ravi Jhaveri, University of North Carolina
School of Medicine, United States of America
Received: June 24, 2014
Accepted: October 26, 2014
Published: December 1, 2014
Copyright: ß2014 Gao et al. This is an open-
access article distributed under the terms of the
Creative Commons Attribution License, which
permits unrestricted use, distribution, and
reproduction in any medium, provided the original
author and source are credited.
Data Availability: The authors confirm that all data
underlying the findings are fully available without
restriction. All relevant data are within the paper.
Funding: This work was supported by the
Program for Innovative Research Team in IMPLAD
(PIRTI). The funders had no role in study design,
data collection and analysis, decision to publish, or
preparation of the manuscript.
Competing Interests: The authors have declared
that no competing interests exist.
PLOS ONE | DOI:10.1371/journal.pone.0113604 December 1, 2014 1/17
in aged population, children and young persons, about one per 1000 or higher in
infants, persons at age 65 (approx. 20% of deaths) and older as well as persons
with chronic medical conditions [2].
Influenza virus, which is an enveloped, negative-strand RNA virus with a
segmented RNA genome, is characterized by frequent mutations - antigenic drifts
(minor antigenic change, both A and B) and antigenic shifts (major antigenic
change, only A). To combat the virus, a number of treatments are currently
available. Vaccines, such as cell-based whole-virion inactivated vaccines and dose-
sparing adjuvants [3,4,5], can provide prophylactic protection by stimulating the
production of antibodies to viral strains. However, these vaccines generally have
lower efficacy in the most susceptible populations, such as the elderly over 61
years of age and children less than 11 years of age [6]. Antiviral agents can be used
in either a therapeutic or a prophylactic mode. These antiviral agents include M2
ion channel blockers (amantadine and rimantadine) and neuraminidase
inhibitors (oseltamivir, zanamivir, and peraivir) [7,8]. However, the potential
usefulness of M2 ion channel blockers is limited due to their lack of activity
against influenza B virus, the global distribution of amantadine-resistant influenza
A viruses, and the occurrence of neurological side effects [9]. The neuraminidase
inhibitor resistance is also mounting with continued use. Oseltamivir-resistant
mutants in A/H3N2- and A/H5N1-infected patients receiving this drug have been
isolated [10,11], and oseltamivir-resistant A/H1N1 strains have worldwide spread
(during the period 2007 to 2009). Therefore, novel antiviral agents are in urgent
need to prepare for future influenza epidemics and pandemics.
Natural products can be candidates to be identified as new generations of
antivirals [12]. The medicinal use of mushrooms has a very long tradition in the
Asian countries, and their use in the Western hemisphere has been slightly
increasing since the last decades [13,14,15,16]. Antiviral effects are described not
only for whole extracts of mushrooms [17] but also for their isolated compounds
[18,19]. The antiviral activity could be caused directly by inhibition of viral
enzymes, synthesis of viral nucleic acids, or adsorption and uptake of viruses into
mammalian cells. These direct antiviral effects are exhibited especially by smaller
molecules. Indirect antiviral effects are the result of the immunostimulating
activity of polysaccharides or other complex molecules [20]. Cryptoporus volvatus
belongs to Eumycota,Cryptoporus [21], and grows in certain areas in China. Its
fruiting body has been used for asthma and bronchitis back to the 15
th
century
a.d. when the record of Cryptoporus volvatus appeared in ‘‘Materia Medica of
Yunnan’’ [22]. Chemical analysis of Cryptoporus volvatus reveals that it contains
many physiological activators, such as polysaccharose, amino acid, volatile oil,
and cryptoporic acids etc. [23]. Aqueous extract from the fruiting body of
Cryptoporus volvatus has been reported to have anti-tumor, anti-allergy, anti-
inflammation, and immunomodulatory activities [24,25,26].
We previously showed that the aqueous extract from the fruiting body of
Cryptoporus volvatus has potential antiviral effects against porcine reproductive and
respiratory syndrome virus (PRRSV) infection in vivo and in vitro [27]. In the
present study, we investigated whether aqueous extract from the fruiting body of
Cryptoporus volvatus Inhibits Virus Replication
PLOS ONE | DOI:10.1371/journal.pone.0113604 December 1, 2014 2/17
Cryptoporus volvatus has the ability to inhibit influenza virus infection. We first
examined its potential to inhibit different influenza virus strain replication in vitro,
and then determined if the extract could protect mice from lethal challenge with
2009 pandemic H1N1 influenza virus. Our results showed that the extract from
Cryptoporus volvatus inhibited influenza virus infections in vitro through targeting
an early stage in the replication cycle, very likely the virus entry into host cells, and
the release of the virus from cells. More importantly, Cryptoporus volvatus efficiently
inhibited 2009 pandemic H1N1 influenza virus in vivo. These results implicate that
the aqueous extract from the fruiting body of Cryptoporus volvatus has the potential
to be an antiviral therapeutics against influenza virus infection.
Materials and Methods
Ethics statement
All animal research was approved by the Beijing Association for Science and
Technology (approval ID SYXK (Beijing) 2007–0023) and complied with the
guidelines of Beijing Laboratory Animal Welfare and Ethics of the Beijing
Administration Committee of Laboratory Animals.
Cells and viruses
Madin-Darby Canine Kidney (MDCK) cells were maintained in DMEM
supplemented with 10% FBS and penicillin/streptomycin.
The pandemic H1N1/2009 influenza virus, A/Beijing/7/2009(H1N1/09), was
isolated from a young patient with an influenza-like illness in December 2009 [28].
Influenza A viruses A/WSN/33(H1N1), A/Jiangxi/262/05(H3N2), and H1N1/09
were grown and titrated on MDCK cells and then stored at 280˚C. Briefly, virus was
serially diluted 10-fold in DMEM to infect MDCK cells in 96-well plates. Influenza
virus infection was determined 36 h post infection using immunofluorescent
staining for the virus NP protein. Virus titer was determined using Reed-Muench
method, and expressed as tissue culture infective dose 50% (TCID
50
).
Indirect immunofluorescence assay
Cells were fixed with cold methanol-acetone (1:1) for 10 min at 4˚C, washed with
phosphate-buffered saline (PBS), and then blocked with 5% normal goat serum for
30minatroomtemperature.Afterblocking,cellswerestainedwithmouse
monoclonal antibody AA5H (Abcam, Hong Kong) against influenza A virus NP.
Cells were then washed and incubated with FITC-conjugated goat anti-mouse IgG
(H+L) (1:2000, Jackson ImmunoResearch) for 60 min at 37 ˚C. After three washes in
PBS, cells were counter-stained with DAPI and examined by fluorescence microscopy.
Cryptoporus volvatus Inhibits Virus Replication
PLOS ONE | DOI:10.1371/journal.pone.0113604 December 1, 2014 3/17
Preparation of the Cryptoporus volvatus extract
The Cryptoporus volvatus was purchased from Yunnan Province, China. The dry
fruiting body of Cryptoporus volvatus was crushed by grinder and soaked in
distilled water (1 g dry fruiting body in 20 ml H
2
O) overnight at 4˚
C, and then
centrifuged at 8000–10,000 g for 30 min. The supernatant was harvested and
freeze-dried, and then stored at 280˚C until use. When used, the freeze-dried
powder was re-dissolved with normal saline or culture medium and filtered with
0.22 mm filters. The final concentration was determined by the weight of the dry
fruiting body and the volume of solvent finally used,
Cell viability assay
The MTT [3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazo-lium bromide]
assay was used to examine the effect of the Cryptoporus volvatus extract on cell
viability. MDCK cells in 96-well plates were treated with sequential dilutions of
the extract or normal saline in a total of 100 ml growth medium for 48 h. And
then, 20 ml of freshly made 5 mg/ml MTT solution was added to each well, and
the cells were incubated at 37˚C for another 5 h before the medium was replaced
with 200 ml DMSO to dissolve the crystals. The plates were further incubated at
37˚C for 5 min to dissolve any air bubbles before the MTT signal was measured at
an absorbance of 550 nm. The 50% cytotoxic concentrations (CC
50
) were
analyzed by GraphPad Prism (GraphPad Software, San Diego, CA).
Inhibition of virus infection assay
Confluent MDCK cell monolayers in 96-well plate were inoculated with the different
influenza virus (multiplicity of infection [MOI] 50.1) in the presence of different
concentrations of the Cryptoporus volvatus extract and 2 mg/ml TPCK-treated
trypsin. Twenty-four hours later, the supernatant was collected for virus titration and
cells were fixed for indirect immunofluorescence assay. The 50% effective
concentration (EC
50
) was determined using a 4 parameter, nonlinear regression of
dose response inhibition by plotting log (inhibitor(concentration) vs. viral titer
(variable slope) using GraphPad Prism (GraphPad Software, San Diego, CA).
Time-of-addition experiment
Confluent MDCK cell monolayers in 96-well plate were inoculated with influenza
virus A/WSN at an MOI of 1 at 4˚C for 2 h and then shifted to 37˚
C (this time point
was set up as 0 h). And the extract was added at 21h,0h,1h,2h,4h,6hor8h
p.i.. At 9 h p.i., the supernatants were collected for virus titration.
Cryptoporus volvatus Inhibits Virus Replication
PLOS ONE | DOI:10.1371/journal.pone.0113604 December 1, 2014 4/17
Determination of direct virion inactivation activity of the
Cryptoporus volvatus extract
Influenza virus A/WSN of 10
5
TCID
50
was incubated with different concentra-
tions of the Cryptoporus volvatus extract for 1 h or 3 h at 37˚
C. Following the
treatment, viral infectivity was determined on MDCK cells.
Virus attachment assay
MDCK cells were incubated with different concentrations of the Cryptoporus
volvatus extract and A/WSN (MOI of 2) at 4˚
C for 2 h. After cells were washed 6
times with cold PBS, cell lysates were prepared by rapid freeze-thaw. Virus titer
was determined as described above.
Virus entry assay
MDCK cells in 6-well plates were infected with influenza virus at an MOI of 10 at
4˚
C for 1 h. The inoculum was aspirated, and the cell monolayer was washed three
times with cold PBS, replaced with fresh DMEM medium containing different
concentration of the extract, and switched to 37˚C. At 1 h post switching to 37˚
C,
cells were washed twice with acidic PBS-HCl (pH1.3) to remove any un-
internalized viral particles on cell surface, followed by washing twice with PBS.
Intracellular virus was analyzed using quantitative RT-PCR. Total cellular RNAs
were extracted using Trizol Reagent (Invitrogen) and reverse transcription (RT)
was conducted using oligonucleotides specific for vRNA (59-AGCAAAAGCAGG-
39). A (GAPDH)-specific primer (59- GAAGATGGTGATGGGATTTC-39) was
also included in the RT reaction mixture. The quantitative real-time PCR was
carried out with a 20 ml reaction mixture containing primers specific for influenza
virus M1 gene (59-ACAGATTGCTGACTCCCAGC-39and
59-TCTCATCGCCTGCACCATTT-39) or for GAPDH RNA
(59-GAAGGTGAAGGTCGGAGTC-39and 59-GAAGATGGTGATGGGATTTC-
39) by using SYBR green DNA dye (TAKARA) following the manufacturer’s
introductions.
Virus Release Assay
MDCK cells were infected with A/WSN (MOI50.1). At 10 h p.i., cells were
washed 3 times with PBS and replaced with fresh medium containing different
concentrations of the Cryptoporus volvatus extract or BFA (1 mg/ml). At 0.5 h and
1 h following medium replacement, supernatants were collected and cells were
lysed by rapid freeze-thaw on dry ice-ethanol and a 37 ˚C water bath. Influenza
virus RNA copies in the supernatants (extracellular virus) and cell lysates
(intracellular virus) were then quantified using quantitative real-time PCR as
described above.
Cryptoporus volvatus Inhibits Virus Replication
PLOS ONE | DOI:10.1371/journal.pone.0113604 December 1, 2014 5/17
Treatment studies in mice
Six-week-old female BALB/c mice (Vital River Laboratories [VRL], China) were
used in five treatment groups, and mice were anaesthetized with Zoletil
(tiletamine-zolazepam; Virbac S.A., Carros, France; 20 mg/g body weight) before
treatment. Two groups of mice were intranasally inoculated with 10
3.5
pfu BJ09
H1N1 virus and then treated with the Cryptoporus volvatus extract (16.5 mg
extract/g body weight or 50 mg extract/g body weight, respectively). Treatment
was given twice daily for 8 days post virus inoculation (dpi) with half of the
dosage via intragastric administration and the other half via thigh muscle
injection. One group of mice was administered with the extract (50 mg extract/g
body weight) without virus inoculation. One group of mice inoculated with BJ09
H1N1 virus was provided twice daily with normal saline, and one group of mice
was just provided with normal saline. At 1, 4, and 7 dpi, three mice in BJ09/
normal saline, or BJ09/50 mg/g treatment groups were euthanized and the lungs
were collected for virus detection and titration. Also, at 4 dpi, lung sections from
three mice in BJ09/normal saline, BJ09/16.5 mg/g, or BJ09/50 mg/g treatment
group were collected for immunohistological analysis. Five mice in each group
were monitored daily for 14 days for clinical signs post inoculation. Any mouse
that lost .25% of its pre-inoculation body weight was euthanized.
Histopathological analyses
The lung sections were fixed in 10% (wt/vol) buffered formalin. Formalin-fixed
tissues were then embedded in paraffin wax, sectioned into 5-mm slices, and
mounted on glass slides. Tissues were stained with hematoxylin and eosin (H&E)
for light microscopy, or stained for viral NP by immunohistochemistry (IHC)
with a mouse monoclonal antibody as previously described [29]. Lung pathology
was scored on a scale of 0 to 4 as previously described [29,30]. Briefly, four easily
identifiable pathological processes were chosen to be scored on a scale of 0–4:
alveolar and interstitial edema; haemorrhage; margination; and infiltration of
inflammatory cells and formation of bronchiolitis. A score of 0 represented
normal lungs; a score of 1 represents mild (,25% lung involvement); a score of 2
represents moderate (25–50% lung involvement); a score of 3 represents severe
(50–75% lung involvement); and a score of 4 represents the severest (.75% lung
involvement). The results of histopathological changes were expressed as mean
+SD (five lung sections from each mouse, and 3 mice per group).
Statistical analysis
Results were analyzed using One-way ANOVA except for the animal experiment,
which was analyzed using Student’s t test. Differences were considered to be
statistically significant if the Pvalue is less than 0.05. *P,0.05;**P,0.01.
Cryptoporus volvatus Inhibits Virus Replication
PLOS ONE | DOI:10.1371/journal.pone.0113604 December 1, 2014 6/17
Results
Cryptoporus volvatus extract inhibits influenza virus infection
in vitro
To evaluate the therapeutic potential of the Cryptoporus volvatus extract, we first
investigated whether the extract could inhibit influenza virus replication in vitro.
Madin-Daby canine kidney (MDCK) cells were inoculated with A/Beijing/7/2009
H1N1 (H1N1/09) influenza virus and then treated with the extract at different
concentrations. Twenty-four hours post infection, infected cells were directly
observed under microscope following immunofluorescence staining of the virus
(Fig. 1A) and the virus quantity in supernatants for each treatment was measured
(Fig. 1B). Our results showed that the Cryptoporus volvatus extract treatment
induced a significant dose-dependent reduction of infected cells and suppressed
the propagation of the H1N1/09 virus to a low level (about 7-fold and 30-fold
reduction when treated with 2.5 mg/ml, and 5 mg/ml extract, respectively). To
further verify its anti-influenza virus activity, we examined whether the
Cryptoporus volvatus extract could inhibit different influenza virus strain
replication in MDCK cells. As illustrated in Figure 1C, the Cryptoporus volvatus
extract also inhibited seasonal influenza virus A/Jiangxi/262/05(H3N2) and
laboratory-adapted A/WSN/33 H1N1 (A/WSN) infections. Virus titer was
decreased about 60-fold for H3N2 when the extract was at 5 mg/ml. For A/WSN,
the suppression even reached to 10
4
fold with the extract at the concentration of
5 mg/ml. The extract inhibited influenza virus infection with 50% effective
concentration (EC50) values of 0.45 mg/ml for H1N1/09 strain, 1.21 mg/ml for
H3N2 strain, and 0.37 mg/ml for A/WSN strain.
To exclude the possibility that nonspecific toxicity induced by the extract could
affect influenza virus replication, we evaluated MDCK cell viability under various
concentrations of the Cryptoporus volvatus extract using the MTT assay (Fig. 1D).
Forty-eight hours following treatment, the cells cultured in medium containing
50 mg/ml Cryptoporus volvatus retained approximately relative viability of 100%
compared with control. And the relative viability of MDCK cells was reduced to
less than 10% when the Cryptoporus volvatus extract concentration in medium was
400 mg/ml. The 50% cytotoxic concentration (CC50) of the Cryptoporus volvatus
extract for MDCK cells was 148 mg/ml, which greatly exceeded its EC50. The
therapeutic index (CC50/EC50) in MDCK cells was 328 for H1N1/09 virus
strain,68 for H3N2 virus strain, and 400 for A/WSN virus strain.
These results suggested that the Cryptoporus volvatus extract could inhibit
influenza virus infection in vitro.
Cryptoporus volvatus extract acts at early and late stages in the
replication cycle.
To characterize the specific step(s) of the influenza virus life cycle that is inhibited
by the Cryptoporus volvatus extract, we examined the time course of their
inhibitory effects. MDCK cells were infected with A/WSN virus at an MOI of 1
Cryptoporus volvatus Inhibits Virus Replication
PLOS ONE | DOI:10.1371/journal.pone.0113604 December 1, 2014 7/17
and, then treated with the extract at various time points post infection from -1 h
to 8 h p.i.. We then measured the titer of infectious viral particles released into the
supernatant at 9 h p.i. [31]. As shown in Figure 2A, when the Cryptoporus
volvatus extract was added at 21 h p.i., virus production was strongly blocked by
a factor of about 3000 folds, while the extract displayed partial antiviral effect
when added at 1 h p.i. or later to 8 h p.i., suggesting that the Cryptoporus volvatus
extract is able to inhibit earlier and late stages in the virus replication cycle.
However, for ribavirin which inhibits virus RNA synthesis, virus production was
continuously inhibited between 1 h and 8 h p.i. as a function of compound
addition. To verify whether the Cryptoporus volvatus extract can directly inactivate
the virus infectivity, we incubated A/WSN (H1N1) virus with the Cryptoporus
volvatus extract at 37˚C for 1 h or 3 h. As shown in Fig. 2B, incubation of A/WSN
Figure 1. Cryptoporus volvatus extract inhibits influenza virus infection in vitro.(A and B) The Cryptoporus volvatus extract represses H1N1/BJ09
replication in MDCK cells. MDCK cells were infected with H1N1/BJ09 at an MOI of 0.1, and then treated with the Cryptoporus volvatus extract at various
concentrations or the control normal saline. At 24 h p.i., cells were fixed and analyzed by IFA using antibody against virus NP protein (A), and virus yield in
the supernatants was also quantified (B). Cultures treated with normal saline were set up as control (0 mg/ml). (C) Cryptoporus volvatus extract potently
inhibits both A/Jiangxi (H3N2) and A/WSN (H1N1) replication in MDCK cells. A similar virus inhibition assay was performed with MDCK cells infected with A/
Jiangxi (H3N2) or A/WSN (H1N1) at an MOI of 0.1 in the presence of the Cryptoporus volvatus extract at various concentrations or the control normal saline.
(D) Determination of cytotoxicity of the Cryptoporus volvatus extract by MTT assay. MDCK cells were incubated with various concentrations of the
Cryptoporus volvatus extract or the control normal saline for 48 h prior to the MTT assay. Data are representative of three independent experiments (mean
¡SEM). Statistical significance was analyzed by One-way ANOVA. *P,0.05;**P,0.01.
doi:10.1371/journal.pone.0113604.g001
Cryptoporus volvatus Inhibits Virus Replication
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(H1N1) virus with the Cryptoporus volvatus extract had no effect on viral
infectivity, suggesting that the inhibitory effect of the Cryptoporus volvatus extract
is not due to its direct inactivation of H1N1 virion particles.
Cryptoporus volvatus extract blocks influenza virus entry into cells
Early events of influenza virus infection cycle include virus attachment and cell
entry. Thus, we investigated whether the Cryptoporus volvatus extract could block
virus attachment or entry into host cells.
We first determined the role of the Cryptoporus volvatus extract in virus
attachment. As shown in Fig. 3A, the Cryptoporus volvatus extract did not affect
the quantity of infectious virus particles that can attach to host membranes. To
test whether the Cryptoporus volvatus extract acts at the internalization stage of
infection, we used an assay described before [32]. Influenza viruses were allowed
to bind MDCK cells at 4˚C for 1 h. Then the inoculum was replaced with fresh
DMEM medium containing the Cryptoporus volvatus extract, and the cells were
transferred to 37˚C. Cell-attached viruses started to enter cells through
endocytosis when the temperature was shifted from 4 ˚Cto37
˚C. At 1 h post
switching to 37˚C, cells were washed with acidic PBS-HCl (pH51.3) to remove
any un-internalized viral particles on the cell surface, and intracellular virus was
analyzed using Real-Time PCR for the M1 gene. As illustrated in Fig. 3B,
intracellular uptake of influenza virus was dose-dependently blocked by the
Cryptoporus volvatus extract, and only about 20% virus entry into cells compared
of control was detected when the extract was at 5 mg/ml.
Figure 2. Cryptoporus volvatus extract acts at an early and late stages in the replication cycle. (A) Time
course analysis of the extract inhibitory effects on influenza A virus replication. MDCK cells were infected with
A/WSN (H1N1) at an MOI of 1, and then at different time points, treated with the normal saline control, extract
(5 mg/ml), or ribavirin (20 mm). Virus titer at 9 hpi was determined. (B) The Cryptoporus volvatus extract did
not inactivate influenza virus directly. Incubating the A/WSN (H1N1) virus with different concentrations of the
extract at 37˚C for 1 h or 3 h, then virus titer was determined in MDCK cells. Data are representative of three
independent experiments (mean ¡SEM). Statistical significance was analyzed by One-way ANOVA.
*P,0.05;**P,0.01.
doi:10.1371/journal.pone.0113604.g002
Cryptoporus volvatus Inhibits Virus Replication
PLOS ONE | DOI:10.1371/journal.pone.0113604 December 1, 2014 9/17
Cryptoporus volvatus extract inhibits the release of influenza virus
particles
To investigate whether the Cryptoporus volvatus extract inhibits virus release, we
used an assay described previously [31] to quantify viruses that are either in cells
or released into the supernatants. We first infected MDCK cells with A/WSN
(MOI50.1), and at 10 hours post infection, cells were then extensively washed
with PBS and replaced with fresh medium containing different concentrations of
the extract or BFA (1 mg/ml), a known inhibitor of protein transport [33]. Viral
RNA copies that were either in cells or released into the supernatants were then
quantified at 0.5 h and 1 h following treatments. At each time point, comparable
amounts of intracellular viral RNAs were found in either extract or BFA-treated
samples (Fig. 3C). In contrast, the relative copies of released viral RNA in
Figure 3. Cryptoporus volvatus extract inhibits influenza virus entry into and release from MDCK cells
but not attachment. (A) Effects of the Cryptoporus volvatus extract on virus attachment. MDCK cells were
incubated with various concentrations of the Cryptoporus volvatus extract and A/WSN (MOI of 2) at 4˚C for
2 h. After cells were washed 6 times with PBS to remove unattached virus, cell lysates were prepared by rapid
freeze thaw. Virus titer was determined in MDCK cells. (B) Inhibition of influenza virus entry by the
Cryptoporus volvatus extract. MDCK cells were incubated with A/WSN (MOI of 10) at 4˚C for 2 h. The
inoculum was then aspirated, and cell monolayer was washed with cold PBS, replaced with fresh DMEM
medium containing various concentrations of the extract or normal saline control, and switched the
temperature to 37˚C. At 1 h post switching to 37˚C, cells were washed twice with acidic PBS-HCl (pH1.3) to
remove any un-internalized viral particles on the cell surface, followed by washing twice with PBS. Intracelluar
virus was analyzed by quantified M1 gene using Real-Time RT-PCR and the quantity of virus in the control
group was set up at 100%. Determination of viral RNA intracellular (C) or released to the supernatants (D).
MDCK cells were infected with A/WSN (MOI of 0.1) for 10 h. After washing with PBS, cells were treated with
either Cryptoporus volvatus extract or BFA (a known inhibitor of protein transport) for 0.5 or 1 h, and then
copies of viral RNA in the supernatants and in the cells were determined using quantitative real-time RT-PCR
assay. The relative quantities of viral RNA compared to the control at 0.5 h post treatment (set up as 1) were
shown. Results shown are the averages from three independent experiments (mean ¡SEM). Statistical
significance was analyzed by One-way ANOVA. *P,0.05;**P,0.01.
doi:10.1371/journal.pone.0113604.g003
Cryptoporus volvatus Inhibits Virus Replication
PLOS ONE | DOI:10.1371/journal.pone.0113604 December 1, 2014 10 / 17
supernatants were significantly decreased by 70% when treated with the extract at
the concentration of 5 mg/ml compared to the control. And the extract at the
concentration of 2.5 mg/ml also potently blocked virus release by about 50%
compared to the control at 1 h following treatment (Fig. 3D). There was no
significant effects observed when the extract was at 1 mg/ml. It should be noted
that we analyzed the released viral RNA, which cannot exactly represent the
released virus particles. Nevertheless, our data suggest that the extract block
influenza virus particle release.
Cryptoporus volvatus extract inhibits H1N1/09 influenza virus
infection in vivo
Previous study demonstrated that H1N1/09 virus was restricted to the respiratory
systems of mice and replicated most efficiently in lungs [28]. To examine the
ability of the Cryptoporus volvatus extract to inhibit H1N1/09 virus replication in
mice, we collected the lungs of three mice in two of the three treatment groups to
quantify H1N1/09 virus at day 1, 4, and 7 post infection (Fig. 4A). H1N1/09 virus
replicated efficiently in the lungs of mice from saline treatment group with a titer
of 10
5.27
TCID
50
/g. In comparison, the viral burdens in high-dosage drug
treatment group were about 4- fold lower than that in no treatment control group
at 4 days post infection (P,0.01). Similar results were also observed at 7 days post
infection. These findings indicated that the Cryptoporus volvatus extract treatment
was capable of suppressing H1N1/09 virus replication in mice. We further
investigated influenza virus distribution in lungs by IHC (Fig. 4B). In virus-
infected mice treated with normal saline, the bronchiolar wall was ulcerated and
there was extensive and intense viral antigen staining in bronchiolar epithelium,
which was sloughing into the lumen. There was also viral antigen staining in the
alveolar and staining of cells in the peribronchiolar inflammation areas. The level
of influenza virus-positive cells in the extract-treated group, especially in the high
concentration group, was lower than that in the lungs of normal saline-treated
mice, and viral antigen staining appeared to be limited to the bronchial
epithelium and minimal in the interstitial epithelium (alveolar septa). These
results suggest that the extract could decrease influenza virus distribution in the
lungs.
We also examined the ability of the Cryptoporus volvatus extract to prevent
virus infection- induced pulmonary lesions at day 4 following H1N1/09 infection.
As shown in Fig 4C and 4D, H&E staining showed that H1N1/09 virus caused
severe interstitial pneumonia and bronchopneumonia characterized by serious
dropout of bronchial mucosa and extensive infiltration of inflammatory cells and
red blood cells in bronchia and alveolus (Fig. 4C and D, BJ09/normal saline). In
comparison, less severe pneumonia was observed in animals treated with low-
dosage Cryptoporus volvatus extract (Fig. 4C and D, BJ09/16.5 mg extract/g body),
while pathology observed in animals of high-dosage treatment group was milder
(Fig. 4C and D, BJ09/50 mg extract/g body).
Cryptoporus volvatus Inhibits Virus Replication
PLOS ONE | DOI:10.1371/journal.pone.0113604 December 1, 2014 11 / 1 7
Cryptoporus volvatus extract protects mice from virus challenge
H1N1/09 influenza virus could cause mice lethal disease. To determine whether
the Cryptoporus volvatus extract can protect mice from H1N1/09 influenza virus
lethal challenge, we treated H1N1/09 infected mice with low-dose or high dose of
the Cryptoporus volvatus extract for 8 days. By day 4 p.i., most of the mice infected
with H1N1/09 virus in saline treatment group showed severe clinical signs of
respiratory disease, including labored respirations and respiratory distress. All five
mice died from 7 to 8 day p.i (Fig. 5A). Mice exhibited unable response to exterior
stimuli, polypnea, and labored respirations before death. The mortality of infected
mice was reduced following low-dosage Cryptoporus volvatus extract treatment (2/
5, 40%). However, mice still showed obvious clinical signs, including decreased
activity, huddling, hunched posture, and ruffled fur. Strikingly, high-dosage of the
Figure 4. Cryptoporus volvatus extract inhibits H1N1 influenza virus replication in vivo, and reduces severity of pathological changes. (A) Virus
replication in lung of mice. Titers of virus recovered from the supernatant of homogenized lung at day 1, 4, and 7 p.i. are shown. (B) Immunohistochemistry
analysis of the lung of influenza virus-infected mice treated with normal saline or the extract. Scale bars, 100 mm. (C) Lung histological grading of virus-
infected mice treated with normal saline or the extract (5 sections from each lung, and 3 mice per group). (D) Representative of histopathological changes in
H&E stained lung tissues from mice sacrificed at day 4 p.i.. normal saline (negative control group); 50 mg/g (extract control group), no histopathology lesion;
BJ09/normal saline (virus infection control group): severe desquamation and droplet of bronchial mucosa (q), massive immune cell and red blood cell
infiltrates around bronchi and blood vessels (red arrow), and inflammatory cells within alveolar spaces (g); BJ09/16.5 mg/g (virus infection and treated with
low-dosage extract group): a small number of immune cell infiltrates around bronchi and blood vessels (red arrow), and alveolar wall thickened (*); BJ09/
50 mg/g (virus infection and treated with high-dosage extract group): a small number of immune cell infiltrates around bronchi and blood vessels (red arrow),
and mild desquamation of bronchial mucosa (q). (Scale bar: 50 mm). Data are presented as mean ¡SD. Statistical significance was analyzed by Student’s
t test;*P,0.05;**P,0.01.
doi:10.1371/journal.pone.0113604.g004
Cryptoporus volvatus Inhibits Virus Replication
PLOS ONE | DOI:10.1371/journal.pone.0113604 December 1, 2014 12 / 17
Cryptoporus volvatus extract treatment prevented all 5 mice from death. Mice in
this group did not show obvious clinical signs except for slight weight loss
(Fig. 5B). Mice in normal saline control and extract control groups did not
display any clinical signs during the course of the experiment. Taken together,
these data suggested that the Cryptoporus volvatus extract could inhibit H1N1/09
influenza virus replication, and protect mice from H1N1 influenza virus infection.
Discussion
Influenza viruses are still the cause of significant morbidity and mortality, posing
a serious health threat during seasonal outbreaks as well as periodic pandemics,
although influenza vaccines and two classes of anti-influenza virus drugs are
available. Thus, there is an urgent need to develop new regimens.
Chinese herbal medicines are a unique source of medical complexity and
diversity, and they have been exploited extensively in pursuit of new antiviral
agents [12]. Cryptoporus volvatus has a long medical use history for treating
asthma and bronchitis in China [22]. We previously reported that the aqueous
extract from the fruiting body of Cryptoporus volvatus has the potential to inhibit
porcine reproductive and respiratory syndrome virus (PRRSV) infection in vivo
and in vitro. In this study, we found that the aqueous extract from the fruiting
body of Cryptoporus volvatus also had broad and robust activity against influenza
virus infection. Our data demonstrated that the extract could inhibit different
influenza virus strain infection. And most importantly, we showed that the extract
completely protected mice from lethal challenge with H1N1/09 influenza virus in
a mouse model when we treated the mice with a high dose of the extract.
We first investigated the potential of the Cryptoporus volvatus extract to inhibit
influenza virus infection in vitro, and its toxicity on cells. We showed that the
CC
50
of the Cryptoporus volvatus extract for MDCK cells was 148 mg/ml, which
greatly exceeded its EC
50
(0.45 mg/ml for H1N1/09 strain, 1.21 mg/ml for H3N2
strain and 0.37 mg/ml for A/WSN strain). The therapeutic index (CC
50
/EC
50
)in
MDCK cells was 328 for H1N1/09 influenza virus, 68 for H3N2 virus, and 400 for
A/WSN virus strain.
Then we investigated on which step(s) of influenza virus life cycle the extract
exerted its effect to inhibit virus infection. The inhibitory effect of the Cryptoporus
volvatus extract is not due to its direct inactivation of H1N1 virion particles, as its
incubation with A/WSN (H1N1) virus at 37˚C for 1 h or 3 h had no effect on viral
infectivity (Fig. 2B). In the time-of-addition experiments, the extract lost partial
anti-influenza virus activity when added at 1 h p.i. and still had the comparable
inhibition activity when added at 8 h p.i, indicating that its target is situated at
both the earlier and late stages of virus replication. Earlier event during influenza
virus infection is virus entry into cells, which is consisted of virus binding to cells
and internalization, virus uncoating, release of the vRNP in the cytoplasm, and
importing of the vRNP into the nucleus [34]. Our virus binding experiments at
4˚
C provided evidence that the extract did not have inhibitory effect on receptor-
Cryptoporus volvatus Inhibits Virus Replication
PLOS ONE | DOI:10.1371/journal.pone.0113604 December 1, 2014 13 / 17
mediated virus binding. However, using time of addition experiment, we
demonstrated that cellular uptake of the virus was significantly blocked by the
extract. Entry process is attractive as targets to block infection efficiently as it is the
first essential step for virus replication. The acute nature of influenza virus
infection and the accompanying inflammatory disease also make an intervention
strategy by blocking the early viral entry process particularly favorable [35]. This is
consistent with our previous study that the Cryptoporus volvatus extract blocks
PRRSV entry into cells. Both viruses use similar entry routes and so it is
conceivable that the extract maybe target cellular factor(s) which is essential for
virus entry. However, as the extract is a mixture of many chemical compounds, we
could not exclude the possibility of targeting viral proteins that mediate virus
entry into cells, e.g. the receptor binding protein haemagglutinin (HA). Further
studies are needed to illustrate the mechanism by which the extract inhibits virus
entry.
More importantly, in the animal study, we found that the extract could
completely protected mice from lethal challenge with H1N1/2009 influenza virus.
To make sure that the dose we used in mouse study did not cause much direct
damage to the mice, the highest dose we used was 50 mg/g body weight. Indeed,
the extract alone at this dose did not cause obvious weight loss and lung lesions
compared to the saline/no virus control. To our surprise, the extract at high dose
(50 mg/g body weight) not only reduced virus loads in lungs of mice, but also
protected mice from lethal challenge with H1N1 influenza virus. These results
Figure 5. Characterization of Cryptoporus volvatus extract efficacy in a mouse model of H1N1/09
influenza virus infection. (A) Survival rate and (B) Weight changes of mice. The dashed line indicates 75%
of initial body weight, and data are presented as mean ¡SD. Mice infected with or without H1N1/09 influenza
virus were treated with the extract or with saline. Each group contained five BALB/c mice. Body weight and
survival status were checked daily. Mice were euthanized upon the loss of 25% of their initial body weight.
normal saline (no virus as negative control group); 50 mg/g (no virus as extract control group); BJ09/normal
saline (virus infection (10
3.5
pfu) control group); BJ09/16.5 mg/g (virus infection (10
3.5
pfu) and treated with
low-dosage extract group); BJ09/50 mg/g (virus infection (10
3.5
pfu) and treated with high-dosage extract
group). Data are presented as mean ¡SD.
doi:10.1371/journal.pone.0113604.g005
Cryptoporus volvatus Inhibits Virus Replication
PLOS ONE | DOI:10.1371/journal.pone.0113604 December 1, 2014 14 / 17
suggest that the Cryptoporus volvatus extract has the potential to be used to treat
H1N1 influenza virus infected mice. However, we could not exclude the fact that
the H1N1 influenza virus infected mice treated with high dose of the extract still
lost some weight compared to saline/no virus controls, even though these mice
survived. Even though we showed that Cryptoporus volvatus could significantly
inhibit H1N1 influenza virus replication in lungs of mice, the reduction of virus
titre was minimal. We propose that there might be other mechanisms existed for
Cryptoporus volvatus to protect mice from H1N1 influenza virus lethal challenge,
e.g. suppressing inflammation. Indeed, Cryptoporus volvatus is used to treat
bronchitis [22]. Thus, more works remain for us to do. Aqueous extract from the
fruiting body of Cryptoporus volvatus is a crude extract, which includes many
components. The antiviral effects of the extract might result from a mixture of
active compounds rather than from a single chemical entity. The efficacy of
Traditional Chinese Medicine (TCM) is a characteristic of a complex mixture of
chemical compounds present in the various herbs. The concept of combinatorial
medicines has been exemplified by the drug cocktail used in the treatment of
acquired immunodeficiency syndrome [36]. However, in order to develop new
generation of antiviral agents, it is necessary to isolate and purify the active
compounds in the aqueous extract from the fruiting body of Cryptoporus volvatus.
Obviously, more work remains for us to do to identify molecules in the
Cryptoporus volvatus extract.
Conclusions
In conclusion, our findings reveal that the aqueous extract of Cryptoporus volvatus
exhibits antiviral activity against influenza A virus in vitro and in vivo, and has the
potential to be developed into a new antiviral agent. Further studies are in
progress to identify the molecules that are responsible for the inhibitions of virus
replication.
Acknowledgments
We thank Ye Shen for her help during virus preparation and Junchi Wang for his
help during the Cryptoporus Volvatus extract preparation.
Author Contributions
Conceived and designed the experiments: LG YS LC. Performed the experiments:
LG YS. Analyzed the data: LG LC. Contributed reagents/materials/analysis tools:
JL GS XS JS. Wrote the paper: LG LC.
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