Archives of Virology (2018) 163:2099–2109
https://doi.org/10.1007/s00705-018-3830-x

ORIGINAL ARTICLE

Functional growth inhibition of influenza A and B viruses by liquidand powder components of leaves from the subtropical plant Melia
azedarach L.
Kuniaki Nerome1 · Kazufumi Shimizu2 · Shiori Zukeran1 · Yasuhiro Igarashi3 · Kazumichi Kuroda2 · Shigeo Sugita4 ·Toshikatsu Shibata5 · Yasuhiko Ito6 · Reiko Nerome1
Received: 25 January 2018 / Accepted: 21 February 2018 / Published online: 9 April 2018
© The Author(s) 2018

Abstract
We evaluated the anti-influenza-virus effects of Melia components and discuss the utility of these components. The effects ofleaf components of Melia azedarach L. on viruses were examined, and plaque inhibition tests were performed. The in vivo
efficacy of M. azedarach L. was tested in a mouse model. Leaf components of Melia azedarach L. markedly inhibited thegrowth of various influenza viruses. In an initial screening, multiplication and haemagglutination (HA) activities of H1N1,
H3N2, H5, and B influenza viruses were inactivated by the liquid extract of leaves of M. azedarach L. (MLE). Furthermore,plaque inhibition titres of H1N1, H3N2, and B influenza viruses treated with MLE ranged from ­103.7 to ­104.2. MLE possessed high plaque-inhibitory activity against pandemic avian H5N1, H7N9, and H9N2 vaccine candidate strains, with aplaque inhibition titre of more than ­104.2. Notably, the buoyant density decreased from 1.175 to 1.137 g/cm3, and spikelessparticles appeared. We identified four anti-influenza virus substances: pheophorbide b, pheophorbide a, pyropheophorbidea, and pheophytin a. Photomorphogenesis inside the envelope may lead to removal of HA and neuraminidase spikes fromviruses. Thus, MLE could efficiently remove floating influenza virus in the air space without toxicity. Consistent with thisfinding, intranasal administration of MLE in mice significantly decreased the occurrence of pneumonia. Additionally, leaf
powder of Melia (MLP) inactivated influenza viruses and viruses in the intestines of chickens. MLE and MLP may haveapplications as novel, safe biological disinfectants for use in humans and poultry.

Handling Editor: Hans Dieter Klenk.
* Kuniaki Nerome
rnerome_ibr@train.ocn.ne.jp
1

The Institute of Biological Resources, 893‑2, Nakayama,
Nago‑shi, Okinawa 905‑0004, Japan

2

Division of Microbiology, Nihon University Schoolof Medicine, 30‑1, Oyaguchi‑kamicho, Itabashi‑ku,
Tokyo 173‑8610, Japan

3

Biotechnology Research Center and Departmentof Biotechnology, Faculty of Engineering, Toyama
Prefectural University, 5180 Kurokawa, Imizu‑shi,Toyama 939‑0398, Japan

4

Equine Research Institute, Japan Racing Association, 1400‑4,
Shiba, Shimotsuke‑shi, Tochigi 329‑0412, Japan

5

Division of Gastroenterology and Hepatology, Nihon
University School of Medicine, 30‑1, Oyaguchi‑kamicho,
Itabashi‑ku, Tokyo 173‑8610, Japan

6

Department of Biomedical Sciences (Graduate School),College of Life and Health Sciences, Chubu University,
1200, Matsumoto‑cho, Kasugai, Aichi 487‑6501, Japan

Introduction
As an important respiratory disease, influenza may have thegreatest impact of all known highly communicable diseases
worldwide. However, in contrast to bacterial diseases, forwhich various antibiotics have been developed over many
decades, the most effective approach for the treatment of viralcommunicable diseases is establishment of vaccine strategies
rather than antiviral drugs. Indeed, to prevent virus infection and related complications caused by epidemic influenza
viruses, e.g. H1N1, H3N2, and B influenza viruses, trivalentvaccines have been established to combat A (H1N1) pdm 09,
H3N2 subtype, and B viruses [1]. Despite major vaccination
efforts, antigenic variation occurring in epidemic seasons
frequently reduces the efficacy of seasonal inactivated or live
vaccines in Japan and the United States of America (USA) [2,3]. For example, although 100% of children in primary andjunior high schools in the southwestern region of Japan are
vaccinated, large outbreaks have occurred in these schools,
indicating that influenza virus variants quite different from the13Vol.:(0123456789)

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vaccine strains are prevalent in this area during the spring season [2]. Moreover, in the USA, reduced vaccine efficacy wasconfirmed during the 2015–2016 season due to the antigenic
gap between the A (H1N1) pdm vaccine strain and epidemic
viruses. Importantly, the major reason for the lack of efficacy
during the 2016–2017 season was that the vaccine strains were
not altered from those in the 2015–2016 season [3].
After the appearance of the H7N9 virus in southern China
and subsequent detection in humans, rapid vaccine development was required [4–8]. Accordingly, several vaccines havebeen developed, such as virus-like particle (VLP) vaccines [9]and DNA-based vaccines [10]. However, despite the use ofnovel vaccine strategies in humans and poultry, frequent antigenic drift or shift occurs, allowing viruses to evade the effectsof vaccines. Thus, it is necessary to design new strategies toovercome the limitations of currently available vaccines. For
example, recently developed neuraminidase inhibitors haveplayed an important role in alleviating the symptoms of influenza infection; however, preventive efficacy has not yet beenachieved [11]. Based on this information, novel and highly
effective anti-influenza drugs are urgently needed.
As an alternative preventative strategy, researchers may
consider the use of novel compounds. For example, somechemical and physicochemical compounds targeting H5N1
influenza have been reported [12, 13]. Povidone-iodine-based
compounds have been shown to have antiviral and therapeutic activities when administered via intravenous inoculation[14]. Additionally, disodium cromoglycate may also inactivate influenza viruses in the lungs [15]. Effective, safe disinfectants that are mildly disruptive to influenza should be used
to improve quality of life. Zhao et al. [16] reported usefulantiviral compounds derived from the Melia tree. Melia compounds inhibit herpes simplex virus, flaviviruses, and Mycobacterium tuberculosis [17–20]. Additionally, Melia-derived
natural resources extracted with C­ O2, ethanol, or water havebeen widely used in folk medicine [21].
In our studies, we also found antiviral substances insubtropical plants, including Melia azedarach L., which
is distributed in the Okinawan area of Japan. Our researchshowed that Melia extracts exhibit a variety of anti-influenza activities. Here, we evaluated the anti-influenza-virus
effects of Melia components and discuss the utility of thesecomponents.

Materials and methods
Preparation of leaf powder and its liquid extractfrom M. azedarach L.
Leaves of M. azedarach L. were provided by the forestry
department of Nago City, Okinawa, Japan, dried at 65 °C
overnight, and powdered in a machine used to pulverise grain

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(MKCA6-25; Masuko Co. Ltd., Saitama, Japan). To prepare
the liquid extract (MLE), samples were suspended in ninevolumes of distilled water, heated at 115 °C for 35 min, andcentrifuged at 2,900 xg for 30 min in a Kubota 7780 centrifuge(Kubota, Tokyo, Japan) fitted with a Kubota AG-1K rotor. The
resulting supernatant was mixed with an equal volume of ethanol (Wako, Tokyo, Japan), incubated overnight at 4 °C, andcentrifuged at 2,900 xg for 20 min. Ethanol was then removedby evaporation, and the dried material was resuspended in an
equal volume of distilled water and filtered through a 10-kDa
membrane (Pellicon 2 Mini Filters; Millipore Corporation,
Germany). The resuspended sample was adjusted to contain10.5 mg Melia extract/mL and was thereafter considered tohave an activity of 1 U in subsequent examinations. To obtainleaf powder (MLP), dried leaves from the Melia tree were
powdered as described above and dried at 37 °C for a few days.

Cells and viruses
Madin-Darby canine kidney (MDCK) cells were grown inminimum essential medium containing 5–10% foetal calf
serum. A/PR/8/34 (H1N1) (PR-8), A/Okinawa/248/2009(H1N1) pdm (Okinawa), A/Moscow/1/00 (H3N2) (Moscow), B/Yamagata/16/88 (Yamagata), and A/duck/Singapore-Q/F119-3/97 (H5N3) (Singapore) strains of influenza
virus were propagated in MDCK cells or 10-day-old embryonated chicken eggs. Except for the Okinawa strain, all of
the above strains were obtained from the Non-Profit Organization of Biomedical Science Association (Tokyo, Japan);the Okinawa strain was kindly provided by the Okinawa
Prefectural Institute of Health and Environment. In addition, the following low pathogenic avian influenza (vaccineseed) viruses were used, which were kindly provided byand imported (Import Permit No.: 29 douken 322, issued
on 12 June, 2017 by Ministry of Agriculture, Forestry and
Fisheries, Japan) from Dr. R. G. Webster, St. Jude Children
Research Hospital, Memphis, TN, USA: RG-A/Barn Swallow/Hong Kong/1161/2010-A/PR/8/34 H5N1 [R] (6+2):(Swallow HK H5), RG-A/Anhui/2013-A/PR/8/34 H7N9 [R](6+2): (Anhui H7), and A/Hong Kong/308/2014 -A/PR/8/34
H9N2 [R] (6+2): (HK H9).

Plaque assay
The infectivity of the challenge influenza viruses was
adjusted to 200 PFU/mL, and 100 μL of each virus was
mixed with an equal volume of MLE. Mixtures were incubated at 35 °C for 30 min and spread on 30-mm culture
plates containing monolayers of MDCK cells. Subsequently,
cells were overlaid with 3 mL of 1% agar containing 15 μg/
mL acetylated trypsin from bovine pancreas (Sigma-Aldrich,Germany). After 3 days at 35 °C in a 5% ­CO2 incubator, theagar of the overlaid media was removed, and cells were fixedInhibition of influenza by Melia azedarach extracts

at room temperature for 1 h with 1 mL of 3.7% formaldehydein phosphate-buffered saline (PBS), rinsed with water, andstained with 1 mL of methylene blue tetrahydrate (Wako)
in distilled water. Finally, plates were rinsed with water anddried at room temperature, and plaques were counted.

Growth inhibition and HA inactivation tests
Growth inhibition was examined on the basis of the cytopathic effect (CPE). A 0.1-mL aliquot of 10-fold-diluted
challenge viruses was mixed with 0.9 mL of MLE sample,
and the mixtures were incubated at 37 °C for 60 min. The
mixtures were then inoculated onto monolayers of MDCK
cells, which were incubated at 37 °C for 30 min.

HA testViruses were two-fold serially diluted in 50 μL of PBS in
96-well U-bottom plates, and 50 μL of 0.5% (v/v) chicken
erythrocytes in PBS was added and mixed. After 30 minof incubation, HA titres were determined based on the last
dilution showing complete hemagglutination, as described
previously [13].

Physicochemical and morphologicalcharacterisation of viruses treated with Melia
extract
A/PR-8 virus was purified by centrifugation at 142,190 ×g
for 4 h along a 10–50% (w/w) sucrose density gradientand treated with concentrated Melia extract for 120 min at
37 °C. Samples were then stained with 2% phosphotungstic
acid and examined by electron microscopy (H500; Hitachi,
Japan). For comparison, untreated virus was purified along a
10–50% (w/w) sucrose density gradient by centrifugation at
142,190×g for 4 h in an SW 50.1 rotor, and buoyant density
was determined by centrifugation at 142,190×g for 4 h alongthe 10–50% (w/w) sucrose density gradient. Morphologicalobservation of the virus particles was performed using a
Hitachi H7000 electron microscope, as described previously[9].

Identification of active components in Melia extract
Dried Melia leaves (1 kg) were extracted with 5 L of distilledwater, filtered through a 10-kDa membrane (Pellicon 2 Mini
Filter; Millipore Corporation), dried, and further extracted
for 1 h with 1 L of 1:1 C­ HCl3-MeOH (Wako). For chemical
analysis, approximately 200 L of this preparation was dried
and resuspended. The aqueous layer, which exhibited activity, was collected and dried, yielding 1.46 g of dark solid.
The extract was fractionated by silica gel column chromatography ­(CHCl3/MeOH = 1:0, 20:1, 10:1, 4:1, 2:1, 1:1, or 0:1,

2101v/v), and antiviral activity against PR-8 influenza virus was
detected in fractions CM4:1 (730 mg) and CM2:1 (168 mg).
CM4:1 (50 mg) was fractionated by high-performance liquid
chromatography (HPLC) along a gradient of MeCN-0.1%­HCO2H (74% MeCN for 0–20 min, 74–100% for 20–25 min,and 100% for 25–36 min) to yield components A (2.6 mg),
B (0.5 mg), C (1.0 mg), and D (0.2 mg). Based on nuclear
magnetic resonance (AVANCE 500; Bruker Daltonics K.K.,
Japan), mass spectrometry (MicroTOF Focus; Bruker Daltonics K.K.), and UV spectrometry (U-3210; Hitachi), thecomponents were identified as pheophorbide a, pheophorbide b, pyropheophorbide a, and pheophytin a, respectively.

Safety of Melia extract
Samples of Melia extract were filtered through ultramembrane filters (Millipore Corporation) with molecular weightcut offs of 10, 30, 50, and 100 kDa and sterilised using a
200-μm filter. In vitro toxicity was assessed based on cytopathic morphological changes (CPEs) in MDCK cells. In
vivo mortality was evaluated in 4-week-old ddY specificpathogen-free (SPF) mice (n = 5) purchased from JapanSLC (Shizuoka, Japan). Mice were orally administered with
0.5 mL of sample, and mortality was examined daily for
7 days. Mouse and chicken experiments were approved andperformed in accordance with the Fundamental Rules for
Animal Experiments and the Guidelines for Animal Experiments Performed at The Institute of Biological Resources
published by the Animal Welfare and Animal Care Committee including the Animal Ethics Committee of the Instituteof Biological Resources, Okinawa, Japan.

Virus clearance from air space
Samples of PR-8 (H1N1) virus were adjusted to 2 × 107 PFU/
mL, and 5 mL was sprayed for 10 min into a 96-L closed
chamber. Subsequently, a sample of Melia extract was also
sprayed into the same chamber for another 10 min. Virus
particles were then recovered into the MEM by aspirator andinfectivity was measured by the PFU method, as described
previously.

Inactivation of virus infectivity by Melia powder(MLP)
We prepared three stainless steel plates (560 cm2), whichwere covered (0.015 g/cm2) with MLP, bamboo powder (Bam), or wood shavings (Wood) and placed into
the 96-L closed chamber. Then, 5 mL of Singapore virus(5 × 106 PFU/mL) was sprayed onto the plate for 30 min,using a nebulizer kept in the chamber. After infection, wecollected the infected powder sample into a tube from theplate. Subsequently, 9 mL of PBS was added to 1 g of the

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sample and kept at room temperature for 30 min to extract
the reaction fluid. The same procedure was repeated forthe remaining two sample plates. Prior to plaque formation
tests, the three test specimens were filtered through a 0.2-μm
membrane filter.

Virus clearance test in the intestines
Different concentrations of Melia powder were mixed withchicken bait at ratios ranging from 0.1 to 10%, and theireffects in chickens were investigated. In this test, 10-folddiluted A/Singapore virus was also orally inoculated, andvirus titres were examined in the chicken droppings for the
next 2 days.

In vivo antiviral activity
Female SPF ddY mice (4 weeks of age) were purchasedfrom Japan SLC Co. Ltd. (Shizuoka, Japan). Mice wereadministered Melia extract or PBS intranasally and infectedwith PR-8 virus at an infectivity of 10–20 PFU (n = 5
mice per group) using a 100 μL plastic pipette tip. Mice
were sacrificed on the indicated days after infection, andpneumonia was scored on a scale of 0–5 after dissection,
depending on the number of infected lobes in the lung.

Results
Growth inhibition of influenza viruses by MLE
First, we screened subtropical plants collected in the
Okinawan area of Japan for anti-influenza activity. Our
results showed that MLE inactivated the HA activity ofhuman and avian influenza viruses tested. Moreover, as
seen in Table 1, four human and three avian strains ofinfluenza virus showed markedly plaque reduction following treatment with MLE. For example, 50% plaque inhibition titres of four human influenza virus ranged from 1­ 03.7
and ­1 04.2. We also examined growth inhibition of threeavian vaccine candidate strains with the use of 10-folddiluted MLE samples. It was quite evident that 50% plaqueinhibition titres of avian viruses were 1­ 02.6 (Anhui H7),
­103.6 (HK H 9), and ­103.7 PFU/mL (Swallow HK H 5).

Physicochemical and morphological analysesof growth inhibition mechanisms
Since the HA activities of human and avian viruses were inactivated by MLE treatment, highly purified A/PR-8 virus wastreated with MLE for 120 min, and the mixture was subjected
to 10–50% (w/w) sucrose density gradient centrifugation at

Table 1  Screening of the anti-influenza virus activity of MLE
Sample treatment
Test viruses
HA subtypes
H1N1
H3N2

H5N3

Strains
1. Human strains
A/PR/8/34
A/Moscow/1/00
B/Yamagata/16/88
2. Avian strain
A/duck/singapore-Q/F119-3/97 (H5N3)

PBS

MLE

HA titre

HA titre

256
64
128

‒*
8
8

128

‒*

Inactivation efficacy against plaque infectivity
HA Subtypes

H1N1
H1N1
H3N2

H5N1
H7N9
H9N2
*: HA titre less than 32

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Strains
1. Human strains
A/PR/8/34
A/Okinawa/248/2009
A/Moscow/1/00
B/Yamagata/16/88
2. Avian strains
RG-A/Barn Swallow/HK/1161/2010-A/PR/8/34
RG-A/Anhui/1/2013-A/PR/8/34
RG-A/HK/308/2014-A/PR/8/34

50% plaqueInhibition titre/ml ­(log10)
4.0
4.2
3.8
3.7
3.7
2.6
3.6

Inhibition of influenza by Melia azedarach extracts

35,000 rpm for 4 h using a SW 501 swing rotor. Notably, twodifferent virus bands were detected in two centrifugation tubes(Fig. 1(a)). The two target virus samples were correctly analysed
under a clear linear sucrose concentration, showing two different peaks (Fig. 1(a)). The first peak was detected in untreatedvirus specimens (Fig. 1(a)). This was different from that of theMLE-treated virus sample, in which the protein peak was present in lighter fractions 9–10; however, HA activity was notdetermined in these fractions (Fig. 1(a)). Moreover, the buoyant
density of MLE-treated virus was decreased from 1.175 g/cm3
of normal virions to 1.137 g/cm3 (Fig. 1(a)). Conversely, protein
and HA peaks of normal and MLE-treated viruses were present
in fraction 7 (Fig. 1(a)). As shown in Fig. 1(b), typical normal

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virions of influenza virus were surrounded with dense projections (two pictures on the left). In contrast, all spikes on thesurface of MLE-treated virus were completely removed fromvirus particles (two pictures on the right).

Identification of chemical structuresof anti‑influenza components from Melia specimens
Crushed leaves were collected by centrifugation andextracted with a mixture of chloroform and methanol. The
lower chloroform layer was concentrated to give a crude
extract, which was further fractionated by silica gel column
chromatography (Fig. 2(a)). The active fraction was thenFig. 1  Buoyant density analysis and electron microscopic
examination of MLE-treatedand untreated influenza
viruses. (a) Estimation of
the buoyant density of A/
PR-8 (H1N1) influenza virus
particles treated with or without
MLE. Samples were subjected
to 10–50% sucrose (w/w)
density gradient centrifugation at 142,190 xg for 4 h andseparated into 21 fractions.
Examination of the linearity ofthe sucrose concentration andconfirmation of virus bands.
Fractionation patterns of MLEtreated virus and fractionation
patterns of untreated normal
virus. (b) Electron microscopic
examination of virus particles
treated with and without MLE.
Untreated viruses were two
pictures on the left and MLEtreated viruses were two on the
right

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Fig. 2  Chemical analysis of
MLE and antiviral activityof purified components. (a)Identification, purification, andstructure of MLE components.
(b) Antiviral activity of eachpurified component, alone or in
combination, against PR-8 virus

subjected to HPLC fractionation, and the antiviral activity of each fraction was evaluated (Fig. 2(b)). Structure
analysis of the most active fraction revealed that the major
active component was pheophorbide a, a degraded productof chlorophyll. Three more components, pheophorbide b,

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pyropheophorbide a, and pheophytin, were also identified
as active components on the basis of mass spectrometry andUV spectral data (Fig. 2(a)). As a result, the antiviral activityof the mixture of pheophorbide b, pyropheophorbide a, andpheophytin a showed enhanced antiviral activity (Fig. 2(b)).

Inhibition of influenza by Melia azedarach extracts

Fig. 3  Spraying and powder treatment efficacy of MLE and MLPagainst virus inactivation. (a) Plaque titration of the three sprayedsamples. Distilled water-sprayed virus (left), 50-fold-diluted MLEsprayed samples (centre) and 100-fold-diluted MLE-sprayed sample(right). (b) Virus inactivation efficacy in the air space with MLE
spray. (c) Virus inactivation efficacy of MLE powder (MLP), Bamboo

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and Wood powder. Challenge viruses possessing 5 × 106 PFU infectivity were mixed with MLP on the plates and allowed to stand at room
temperature. After 30 min their infectivity were measured. (d) Virus
infectivity in the chicken intestine, which ate MLP including bait. (e)Safety test MLE toxicity in MDCK cells and mice

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Virus clearance efficacy of MLE in the closed air
space
Next, we investigated virus clearance or removal efficacy
in a closed air space with MLE. In this experiment, viruses(2 × 107 PFU) were sprayed for 10 min in a revelation chamber in a safety cabinet (90 L air space), and 10 min later,
distilled water (control) or MLE test samples were subsequently sprayed for 10 min. 1­ 03.81 PFU/mL of test virus wasobserved in the distilled water control (Fig. 3(a), left). In
contrast, the above active viruses were completely inactivated or removed by administration of 50-fold-diluted
MLE (Fig. 3(a), center), whereas ­102.39 PFU/mL of virus
remained in the air space in samples treated with 100-folddiluted MLE (Fig. 3(a), right). These results were summarized in Fig. 3(b) together with the complete disappearanceof MLE + virus sample. The finding shown in Fig. 3(a)(b),indicates Melia samples have the potential for virus clearance in a closed air space.

Efficacy of MLP in influenza virus inactivation
To evaluate the virus inactivation efficacy of MLP, Singapore virus sprayed onto bamboo powder (Bam), wood savings (Wood), or MLP on the stainless steel plates was used
to simulate the ground at poultry farms. The Bam and Woodtest specimens showed high titres similar to virus control(PBS) titre of ­105.2 PFU/mL (Fig. 3(c)). In contrast, the MLP
test specimen showed complete inactivation (Fig. 3(c)). Furthermore, in a chicken model infected with the low pathogenic Singapore virus, high virus titres of approximately
6,000 ­(103.78) PFU/mL were observed in droppings, andvirus titres gradually decreased as the concentration of
MLP was increased from 0.1% to 10% in the bait (Fig. 3(d)).
These experiments demonstrate that MLP markedly reduced
virus titres in the intestine of chickens.

Safety analysis of the MLE sample
To apply MLE in the treatment of human disease, a minimum safety requirement must be met. As shown in Fig. 3(d),partially purified MLE did not show any CPE in MDCK
cells or mortality in mice.

Prevention of experimental pneumonia in micefollowing MLE treatment
Finally, we examined the efficacy of MLE in the preventionof mouse pneumonia. As shown in Fig. 4(a), body weightsof mice treated with PBS decreased from 3 days after infection, whereas mice treated with MLE (Fig. 4(b)) retained
their body weight until 5 days after infection. Moreover,13

K. Nerome et al.
Fig. 4  Preventive efficacy of MLE against influenza-related pneumonia in mice. (a)–(c) Mean body weights in mice pretreated with
PBS (a) or MLE (b) or prior to infection, mice treated with MLE
containing PR-8 virus (MLE + virus) (c). (d) Macropathologicalcomparison of pneumonia occurrence in mice treated with or without
MLE. Morphological and colour changes in the dorsal and abdominal sides of the lung. (e) Mean pneumonia score in the lungs of threemouse groups treated with MLE, MLE-virus mixture (MLE + V), andPBS. (f) Survival rates in the three mouse groups treated with PBS(control), MLE, and MLE + V samples

mice treated with a virus-MLE mixture showed a constantincrease in body weight (Fig. 4(c)). Consistent with thesefindings, the occurrence of pneumonia in these three mousegroups differed (Fig. 4(d)). For example, four of the mouse
lungs obtained from MLE-pretreated mice showed pinkshading (Fig. 4(d)). In contrast, most lungs obtained from
PBS control mice showed a strong black colour, indicating severe pneumonia (Fig. 4(d)). This conclusive evidence
was further evaluated based on the degree of pneumonia inmouse lungs (Fig. 4(e)). Scores for pneumonia occurrencein MLE-treated, MLE+virus-treated, and PBS-treated mice
were 1.5, 0.5, and 5, respectively. Consistent with the above
results, MLE-treated mice showed higher preventive efficacy(80% survival; Fig. 4(f)), which was apparently different
from that of PBS-treated mice (40% survival) in the absenceof the MLE component (Fig. 4(f)). In contrast, the survival
rate in mice treated with the MLE-virus mixture was 100%.
These experiments suggest that the MLE and MLP components could prevent or alleviate the damage attributable toinfluenza virus infection.

Discussion
In this study, we found that the growth of influenza A and
B viruses was markedly influenced by the presence of MLE
and/or MLP. Additionally, the effects of MLE and MLP on
growth inhibition could overcome the risk of antigenic differences in types and subtypes of all orthomyxovirus family
members. Based on these findings, MLE may be used tosupplement inactivated or live influenza vaccines, which are
greatly influenced by the frequent occurrence of antigenic
drift or limited antigenic shift.
Influenza viruses spread from human to human throughdirect droplets within a short distance of 1–2 m. However,the presence of other important transmission routes should
be considered. For example, transmission may occur throughcoughing and sneezing, during which many virus particles
are scattered in the air space and human environment; thetransmission of these aerosols may play important roles in
the rapid spread of influenza [22].

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In 1997, a commercial jet airplane experienced engine
trouble and was unable to take off for approximately 4 h. A
woman infected with influenza virus had boarded the airplane and suffered from a violent acute onset of influenza
infection, resulting in frequent coughing [23]. In this case,
Moser et al. investigated the infection rate in all passengers and the number of hours they stayed on the airplane indetail. They showed that the infection rate for 5 passengers
who stayed for less than 1 hour, 9 passengers who stayedfor 1–3 hours, and 29 passengers who stayed for more than
3 hours were 54%, 56%, and 86%, respectively [23], suggesting that most passengers were infected through an aerosol
route originating from the single patient [23]. Thus, many
influenza cases, such as those occurring in families, schools,
companies, or the general social population, may be attributable to aerosol infection, although droplet infection is also
an important transmission route.
When considering the above complicated transmissionof aerosol influenza viruses in the living air space, countermeasures against influenza, including removal or killing offloating viruses outside the body, are essential. As describedabove, MLE samples could efficiently remove or kill floating
viruses in a closed air space, and through this mode of virus
inactivation, infection could be prevented in some individuals. Thus, these results may be important for decreasing the
spread of the virus through social populations.
Based on our findings, we suggest that MLE may haveapplications in viral prevention. Indeed, Zhao et al. reviewed
a large number of dimonoids isolated from Melia trees andshowed that some compounds had antiviral effects on herpes
simplex virus-1 and vesicular stomatitis virus [16].
In summary, our findings show that MLE could obviously
remove or kill the floating virus in the air space, leading todecreased influenza infection. Moreover, MLP could inactivate or remove influenza viruses distributed on the groundat poultry farms or in the intestines of chickens, suggesting
reduced rates of avian influenza virus infection at poultry
farms or even in nature. Thus, Melia-derived compounds
may be established as strategic countermeasures combinablewith influenza vaccines and antiviral drugs. Vaccines andanti-influenza drugs are important measures against viral
infection. In contrast, measures for the treatment of influenza virus using biological resources such as Melia may act
outside the living body or cells, suggesting another effective
strategy for treating humans and animals. Further studies areneeded to assess whether such an approach may be efficacious when combined with appropriate vaccines.
Acknowledgements The authors thank Mr. S. Mukai and Ms. S. Matsuda (Institute of Biological Resources, Okinawa) for their skilful technical help and general support.
Funding This work was partially supported by a grant from the
Okinawa New Industry Creation Project for fiscal year 2011 and 201213

K. Nerome et al.
by the Okinawa Industry Promotion Public Corporation trusted fromthe Okinawa prefectural government.

Compliance with ethical standards
Conflict of interest The authors declare that they have no conflict ofinterest.
Data availability statement All data generated or analysed during thisstudy are included in this published article.
Open Access This article is distributed under the terms of the Creative
Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate
credit to the original author(s) and the source, provide a link to theCreative Commons license, and indicate if changes were made.

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