molecules
ArticleUltrastructural Damages to H1N1 Influenza Virus Caused byVapor Essential Oils
Valentina Noemi Madia 1 , Walter Toscanelli 2 , Daniela De Vita 3, *, Marta De Angelis 2, * , Antonella Messore 1 ,Davide Ialongo 1 , Luigi Scipione 1 , Valeria Tudino 1 , Felicia Diodata D’Auria 2 , Roberto Di Santo 1 ,Stefania Garzoli 4 , Annarita Stringaro 5 , Marisa Colone 5 , Magda Marchetti 6 , Fabiana Superti 6 ,Lucia Nencioni 2 and Roberta Costi 1
1

2

3
4

5

6

*

Dipartimento di Chimica e Tecnologie del Farmaco, Istituto Pasteur-Fondazione Cenci Bolognetti,
Sapienza University of Rome, p.le Aldo Moro 5, I-00185 Rome, Italy;
valentinanoemi.madia@uniroma1.it (V.N.M.); antonella.messore@uniroma1.it (A.M.);
davide.ialongo@uniroma1.it (D.I.); luigi.scipione@uniroma1.it (L.S.); valeria.tudino@uniroma1.it (V.T.);
roberto.disanto@uniroma1.it (R.D.S.); roberta.costi@uniroma1.it (R.C.)
Department of Public Health and Infectious Diseases, Laboratory Affiliated to Istituto Pasteur-Fondazione
Cenci Bolognetti, Sapienza University of Rome, p.le Aldo Moro 5, I-00185 Rome, Italy;
walter.toscanelli@uniroma1.it (W.T.); felicia.dauria@uniroma1.it (F.D.D.); lucia.nencioni@uniroma1.it (L.N.)
Department of Environmental Biology, Sapienza University of Rome, p.le Aldo Moro 5, I-00185 Rome, Italy
Department of Drug Chemistry and Technologies, Sapienza University of Rome, p.le Aldo Moro 5,
I-00185 Rome, Italy; stefania.garzoli@uniroma1.it
National Center for Drug Research and Evaluation, Italian National Institute of Health, V.le Regina Elena, 299,
I-00161 Rome, Italy; annarita.stringaro@iss.it (A.S.); marisa.colone@iss.it (M.C.)
National Centre for Innovative Technologies in Public Health, Italian National Institute of Health,V.le Regina Elena, 299, I-00161 Rome, Italy; magda.marchetti@iss.it (M.M.); fabiana.superti@iss.it (F.S.)
Correspondence: daniela.devita@uniroma1.it (D.D.V.); marta.deangelis@uniroma1.it (M.D.A.);
Tel.: +39-06-4991-2788 (D.D.V.); Tel.: +39-06-4991-4608 (M.D.A.)

Citation: Madia, V.N.; Toscanelli, W.;
De Vita, D.; De Angelis, M.; Messore,
A.; Ialongo, D.; Scipione, L.; Tudino,V.; D’Auria, F.D.; Di Santo, R.; et al.
Ultrastructural Damages to H1N1
Influenza Virus Caused by Vapor
Essential Oils. Molecules 2022, 27,3718. https://doi.org/10.3390/
molecules27123718
Academic Editor: Asta Judzentiene
Received: 5 May 2022
Accepted: 6 June 2022
Published: 9 June 2022
Publisher’s Note: MDPI stays neutral
with regard to jurisdictional claims inpublished maps and institutional affil-

Abstract: Influenza viruses are transmitted from human to human via airborne droplets and canbe transferred through contaminated environmental surfaces. Some works have demonstrated theefficacy of essential oils (EOs) as antimicrobial and antiviral agents, but most of them examined theliquid phases, which are generally toxic for oral applications. In our study, we describe the antiviral
activity of Citrus bergamia, Melaleuca alternifolia, Illicium verum and Eucalyptus globulus vapor EOs
against influenza virus type A. In the vapor phase, C. bergamia and M. alternifolia strongly reduced viralcytopathic effect without exerting any cytotoxicity. The E. globulus vapor EO reduced viral infection by
78% with no cytotoxicity, while I. verum was not effective. Furthermore, we characterized the EOs andtheir vapor phase by the head-space gas chromatography–mass spectrometry technique, observing
that the major component found in each liquid EO is the same one of the corresponding vapor phases,
with the exception of M. alternifolia. To deepen the mechanism of action, the morphological integrityof virus particles was checked by negative staining transmission electron microscopy, showing thatthey interfere with the lipid bilayer of the viral envelope, leading to the decomposition of membranes.
We speculated that the most abundant components of the vapor EOs might directly interfere with
influenza virus envelope structures or mask viral structures important for early steps of viral infection.

iations.

Keywords: influenza A H1N1 virus; essential oil vapors; bergamot; Chinese star anise; tea tree oil;
eucalyptus; antivirals
Copyright: © 2022 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms andconditions of the Creative Commons
Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).

1. Introduction
Influenza viruses (Fam. Orthomyxoviridae) are etiological agents causing highly contagious acute respiratory syndromes and constituting a significant public health problem
worldwide [1]. The virus is mainly transmitted from human to human through airborne
droplets and may be transferred to a surface if it is touched by contaminated hands [2].

Molecules 2022, 27, 3718. https://doi.org/10.3390/molecules27123718

https://www.mdpi.com/journal/molecules

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For these reasons, viral spread may be controlled by the disinfection of objects and sanitization of indoor air, and cleaning and disinfecting are considered an essential part of
influenza control programs [3]. Indeed, it is well known that the microbial contaminationof surfaces and medical devices, for example, plays an important role in the transmissionof healthcare-associated pathogens [4]. Disinfection with chemical reagents is an importantelement of the biosecurity program against avian flu [5]. Even so, several issues are relatedwith the use of chemical disinfectants such as cytotoxicity, corrosion and environmental
toxicity. Phytochemicals, on the contrary, are an attractive source of environmental friendly,
relatively inexpensive and available disinfectants [4]. Among them, various essential oils
(EOs) from plants have been screened to assess their antimicrobial, antifungal, and antiviral
activities [6]. Essential oils are complex mixtures of volatile compounds, such as monoterpenes, sesquiterpenes and phenylpropenes [7,8], which have been investigated for a wide
range of properties [9–11].
Bergamot (Citrus × bergamia Risso & Poit.) belonging to Rutaceae family is classified
as a hybrid of lemon (Citrus limon (L.) Osbeck) and bitter orange (Citrus aurantium L.).
The plant is endemic in Calabria region (Italy) and is highly appreciated in the perfumery,
cosmetic and food industries [12].
Chinese star anise (Illicium verum Hook. f.) is a tree of Illiciaceae family well known
both for its EO used in liqueurs and the several medicinal properties, particularly in the
traditional Chinese medicine [13].
Melaleuca alternifolia (Maiden & Betche) Cheel, most commonly known as tea treeoil (TTO), and Eucalyptus globulus Labill. (eucalyptus) belonging to the Myrtaceae family,
are Australian endemic species. The leaves of both these plants are a source of EOs used
for millenia as antiseptics and with antimicrobial activity against several respiratory tract
pathogens, among others [14–17]. Interestingly, it was observed that bergamot, Chinese
star anise, TTO and eucalyptus EOs inhibit influenza type A (H1N1) virus, and they havealso been reported to be complementary and alternative treatment options for influenza
infections [16].
Several studies have reported the antiviral activity of EOs against influenza A virus(IAV) [18–26], even though these works were carried out with the liquid phases of theoils and their components. On the other hand, it was demonstrated that the vapor phaseof several EOs showed good antibacterial, antifungal and antiviral activity, sometimes
better than the corresponding liquid phase of the oil [27–30]. Still, a paucity of data isreported in the literature on the anti-influenza effect of EO vapors. Indeed, the work of
Vimalanathan and Hudson [31] is the only report describing the anti-influenza activity ofvapor EOs. The authors deepened the activity against influenza virus of some EOs such
as C. bergamia and E. globulus in their vapor phase without characterizing their chemicalcompositions. In the light of the above, we aimed at studying the activity of the vapors of
EOs previously reported for their anti-influenza properties in their liquid phases [16,32,33].
Therefore, we tested bergamot, Chinese star anise, TTO and eucalyptus vapor essential oils
for their anti-influenza activities. In addition, we carried out the phytochemical analysis of
EOs in both their liquid and vapor phases to elucidate their chemical compositions by thehead-space gas chromatography–mass spectrometry technique. This work is the first report
describing the anti-influenza activity of EO vapors phytochemically analyzed in their vapor
phase. Moreover, we deepened the mechanism of action of vapor EOs by checking themorphological integrity of virus particles through negative staining transmission electron
microscopy, showing that they interfere with the lipid bilayer of the viral envelope, leading
to the decomposition of membranes.
2. Results and Discussion
2.1. Headspace-Gas Chromatography/Mass Spectrometry (HS-GC/MS)
Essential oils were obtained from hydrodistillation with a yield of 0.9 and 4.7%.
Gas chromatographic analyses were carried out on the four EOs analyzed in this study
both in their liquid and vapor phases. We analyzed the C. bergamia EO (BEO), I. verum EO

Molecules 2022, 27, 3718

3 of 14(IV-EO), M. alternifolia (TTO), and E. globulus EO (EEO). The analysis of the liquid phaseof EOs allowed to compare their chemical composition with that of their corresponding
vapors.
Gas chromatographic analysis on liquid EOs led to the identification of 42 compounds
in total listed in Table 1. Limonene (31.9%) and linalyl acetate (31.4%) followed by linalool(17.0%) were the main compounds found in C. bergamia EO. Anethole (93.0%), terpinen4-ol (92.4%) and 1,8-cineole (93.7%) were the most abundant constituents in I. verum,
M. alternifolia and E. globulus EOs, respectively. In general, terpenoids prevailed oversesquiterpenoids in all investigated EOs and represented the family of compounds prevalent except for I. verum EO where anethole, a non-terpene compound, was the one withthe highest relative percentage. Results are in agreement with those reported in the literature [16,34–36].
Table 1. Chemical composition (percentage mean value ± standard deviation) of EOs.
N◦

Component 1

LRI 2

LRI 3

BEO 4 (%)

IV-EO 5 (%)

TTO 6 (%)

EEO 7 (%)

1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40

α-thujene
α-pinenesabinene
β-myrcene
β-pinene
α-phellandrene
α-terpinene
p-cymenelimonene
1,8-cineolecis-β-ocimene
β-terpinenetrans-β-ocimenetrans-sabinene hydrate
γ-terpinene
cis-sabinene hydrate
terpinolene
linalool
terpinen-4-ol
estragole
α-terpineol
carveol
cis-piperitol
p-anisaldehyde
linalyl acetate
anethole
α-citral
α-copaene
β-elemene
β-caryophyllene
α-bergamotene
aromadendrene
humulene
γ-muurolene
ledeneβ-bisabolene
α-farnesene
δ-cadinene
spathulenol
viridiflorol

920
941
973
981
988
1008
1013
1020
1021
1026
1035
1040
1044
1057
1059
1072
1079
1090
1164
1180
1185
1210
1219
1232
1259
1269
1291
1373
1410
1426
1435
1459
1470
1490
1499
1501
1510
1528
1573
1580

923
943
972
983
986
1005
1010
1016
1023
1025
1032
1036
1043
1053
1054
1069
1080
1092
1160
1177
1183
1201
1215
1229
1252
1260
1287
1370
1406
1424
1430
1460
1465
1486
1492
1501
1506
1530
1571
1583

0.3 ± 0.03
1.3 ± 0.02
1.5 ± 0.03
7.3 ± 0.02
0.7 ± 0.02
31.9 ± 0.06
0.5 ± 0.02
0.2 ± 0.02
7.5 ± 0.02
17.0 ± 0.03
tr
tr31.4 ± 0.02
0.1 ± 0.02
0.1 ± 0.02
0.1 ± 0.01-

0.3 ± 0.03
tr
0.2 ± 0.02
tr
0.1 ± 0.02
tr
0.2 ± 0.02
tr
1.0 ± 0.02
3.2 ± 0.02
0.2 ± 0.03
93.0 ± 0.07
0.7 ± 0.03
tr
tr
tr
tr
tr
-

0.2 ± 0.02
tr
0.1 ± 0.00
0.1 ± 0.00
tr
1.1 ± 0.02
0.3 ± 0.04
0.5 ± 0.02
2.7 ± 0.03
tr
92.4 ± 0.03
0.6 ± 0.02
tr
0.1 ± 0.00
tr
0.1 ± 0.00
0.4 ± 0.03
tr
0.2 ± 0.02
0.5 ± 0.02
0.4 ± 0.02
tr
0.1 ± 0.01

1.7 ± 0.02
0.6 ± 0.02
0.5 ± 0.02
1.7 ± 0.01
93.7 ± 0.02
1.6 ± 0.02
tr
tr
0.1 ± 0.00–-

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Table 1. Cont.
N◦

Component 1

LRI 2

LRI 3

BEO 4 (%)

IV-EO 5 (%)

TTO 6 (%)

EEO 7 (%)

41
42

globulol
cubenol
SUM
Terpenoids
Sesquiterpenoids
Other

1596
1635

1594
1631

99.9
68.3
0.2
31.4

98.9
1.8
0.7
96.4

0.1 ± 0.01
0.1 ± 0.01
100.0
98.0
1.9
0.1

99.9
99.9
-

1 The components are reported according to their elution order on apolar column; 2 Linear Retention Indicesmeasured on apolar column; 3 Linear Retention indices from literature; 4 Percentage mean values of C. bergamia
liquid phase components; 5 Percentage mean values of I. verum liquid phase components; 6 Percentage meanvalues of M. alternifolia liquid phase components; 7 Percentage mean values of E. globulus liquid phase components;- Not detected; tr: traces (mean value <0.1%).

The chemical composition of EOs in their vapor phase was described by the head-space
GC/MS technique. Twenty-three compounds were identified and reported in Table 2. The
volatile profile of EOs was dominated by terpenoids, while sesquiterpene compounds were
missing. Limonene (51.2%) and β-pinene (20.4%) were the major compounds in C. bergamia
EO. Anethole (49.1%) was the most abundant component even in the vapor phase of
I. verum EO followed by α-pinene as the second principal molecule (19.1%). γ-Terpinene(29.3%) and 1,8-cineole (89.8%) dominated in M. alternifolia and E. globulus EOs, respectively.
As observed by comparing Tables 1 and 2, the major component found in each EO in its
liquid phase is the same one present in the corresponding vapor phase, with the exceptionof M. alternifolia, where the main compound present in its liquid EO was the terpinen-4-ol,
while the γ-terpinene was the most abundant in the corresponding vapor. Interestingly, thechemical composition of the M. alternifolia and E. globulus vapor EOs has been previously
reported in the literature, and both analyses are in agreement with our results [37,38].
Table 2. Chemical composition of volatiles (percentage mean value ± standard deviation) of EOs.
N◦

Component 1

LRI 2

LRI 3

BEO 4 (%)

IV-EO 5 (%)

TTO 6 (%)

EEO 7 (%)

1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23

α-thujene
α-pinene
camphenesabinene
β-myrcene
β-pinene
α-phellandrene
α-terpinene
p-cymenelimonene
1,8-cineolecis-β-ocimene
β-terpinenetrans-β-ocimene
γ-terpinene
cis-sabinene-hydrate
terpinolene
linalool
terpinen-4-ol
estragole
α-terpineol
anethole
α-farnesene

920
941
950
974
981
988
1008
1013
1020
1021
1026
1035
1040
1044
1059
1072
1079
1090
1164
1180
1185
1269
1510

923
943
946
972
983
986
1005
1010
1016
1023
1025
1032
1036
1043
1054
1069
1080
1092
1160
1177
1183
1260
1506

1.5 ± 0.02
6.5 ± 0.02
0.3 ± 0.02
3.9 ± 0.03
20.4 ± 0.05
0.2 ± 0.01
1.1 ± 0.03
51.2 ± 0.03
0.7 ± 0.03
0.3 ± 0.04
9.2 ± 0.04
4.6 ± 0.02
0.1 ± 0.02-

0.4 ± 0.02
19.1 ± 0.06
0.3 ± 0.03
2.3 ± 0.04
5.9 ± 0.07
0.5 ± 0.03
3.8 ± 0.06
0.4 ± 0.05
0.7 ± 0.03
2.7 ± 0.03
5.0 ± 0.05
3.9 ± 0.05
5.5 ± 0.06
49.1 ± 0.05
0.3 ± 0.02

3.5 ± 0.04
10.0 ± 0.04
0.1 ± 0.00
0.3 ± 0.02
1.5 ± 0.04
1.7 ± 0.06
1.4 ± 0.01
18.1 ± 0.02
10.7 ± 0.04
8.3 ± 0.03
29.3 ± 0.03
4.6 ± 0.04
10.2 ± 0.05
0.3 ± 0.04-

5.6 ± 0.02
0.8 ± 0.00
0.6 ± 0.01
1.9 ± 0.04
89.8 ± 0.03
1.1 ± 0.02
tr
0.2 ± 0.01-

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Table 2. Cont.
N◦

Component 1

LRI 2

LRI 3

SUM
Terpenoids
Sesquiterpenoids
Other

BEO 4 (%)

IV-EO 5 (%)

TTO 6 (%)

EEO 7 (%)

100.0
100.0
-

99.9
45.0
0.3
54.6

100.0
100.0
-

100.0
100.0
-

1 The components are reported according to their elution order on apolar column; 2 Linear Retention Indicesmeasured on apolar column; 3 Linear Retention indices from the literature; 4 Percentage mean values of C. bergamia
vapor phase components; 5 Percentage mean values of I. verum vapor phase components; 6 Percentage meanvalues of M. alternifolia vapor phase components; 7 Percentage mean values of E. globulus vapor phase components;- Not detected; tr: traces (mean value <0.1%).

2.2. Antiviral Activity
As described in the methods, the virucidal activity was evaluated by exposing influenza A Puerto Rico 8/H1N1 virus (PR8) to vapor EOs for 30 min at 37 ◦ C in an incubator
at 5% CO2 . Following the exposure to vapor phases, the virus was used to infect cellmonolayers of Madin–Darby Canine Kidney (MDCK) cells. After 24 h infection, cells wereobserved under the microscope, and no cytopathic effect (CPE) was detectable in cells
infected with the virus exposed to vapor bergamot and TTO EOs compared to cells infectedwith untreated viral suspension (PR8 untreated) (Figure 1). Similarly, no viral titer wasquantified by Tissue Culture Infectious Dose 50% (TCID50 ), indicating a strong virucidal
activity of both the vapor EOs, which was reflected in a sharp reduction in viral infectivity.
We also analyzed two other oil vapors, deriving from eucalyptus and Chinese star aniseoils. As already described, PR8 was exposed to oil vapors for 30 min, and then, the virus
used to infect cell monolayers for 24 h. As shown in Figure 1, while the eucalyptus vapor
phase reduced CPE (viral titer measured by TCID50 was reduced by 78%: logTCID50 = 2.35
vs. logTCID50 = 1.68, PR8-untreated vs. -treated, respectively), Chinese star anise vapor
was not effective. In particular, the virus treated with this vapor phase produced someviscous oil droplets that aggregated on the cell monolayers. For this reason, we tried to
expose PR8 to the vapor phase of the main compound present (49.1%) in the Chinese staranise, anethole. Treatment with the anethole vapor phase of the viral suspension, at thesame condition described above, reduced the virus induced CPE (Figure 1), as confirmed
by TCID50 assay: ≈82% reduction, logTCID50 = 2.25 vs. log TCID50 = 1.5, PR8-untreated
vs. -treated, respectively. We can speculate that the most abundant components foundin each vapor EO (limonene and β-pinene in C. bergamia, γ-terpinene in M. alternifolia,
and 1,8-cineole in E. globulus) likely contribute to the vapor EOs efficacy against IAV.
This hypothesis could also be confirmed by previously reported results describing theanti-influenza activity of 1,8-cineole [39] and limonene [40].
To exclude that the observed antiviral effect of the EOs vapor phase was due to theircytotoxicity, cell monolayers were mock-infected with PBS treated with vapor EOs at thesame conditions described above. After 24 h, the cells observed by an optical microscope
showed no differences in morphology; the monolayers were confluent, and cells were
not detached, indicating that the vapor EOs did not exert any cytotoxic effect (Figure 2A).
Regarding the treatment with Chinese star anise, although the cell monolayer was stillintact, this vapor phase produced some vacuoles probably due to droplets released on thevapor phase.

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Figure 1. Cytopathic effect (CPE) induced by influenza virus treated with EOs vapor phases (PR8 + Bergamot; PR8 + TTO; PR8 + Eucalyptus; PR8 + Star Anise; PR8 + Anethole) or not treated (PR8untreated) on MDCK cell monolayers analyzed by an inverted optical microscope after 24 h from
infection. As control of infection, cells were mock-infected as described in methods.

To confirm that effective vapor EOs were not toxic for the cells, MTT assay was
per-formed on MDCK cell monolayers incubated with PBS treated with different vapor
phases as described above. As shown in Figure 2B, the percentage of cell proliferation wascomparable to cells not treated with vapor EOs and considered as control (100%).

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Figure 2. Cytotoxicity of EOs vapor phases. MDCK cell monolayers were treated with a mixture of
PBS + EOs vapors and compared to cells incubated with PBS untreated (control). (A) Cell monolayers
were observed by an inverted optical microscope or (B) evaluated by MTT assay. Cell viability was
expressed as percentage (%) by comparison to control cells (considered as 100%). The values represent
the mean ± S.D. of three technical replicates performed (n = 3).

To confirm that the virucidal activity observed for vapor EOs (especially bergamot
and TTO) led to reduced infectious ability, we chose to analyze viral hemagglutinin (HA)
protein expression on MDCK cell monolayers infected with the mixture of PR8 treatedor not with different vapor phases, by means of In Cell Western (ICW) assay (Figure 3,
left panel). The analysis of HA protein expression (green) on infected cell monolayers
indicated that HA was strongly reduced in cells infected with PR8 treated with bergamot
or TTO compared to cells infected with PR8 untreated. As control of the integrity of cellmonolayers, cells were confirmed by Cell Tag staining (red). In particular, the analysis ofRelative Fluorescence Unit (RFU, right panel) showed a significant inhibition of HA protein

Molecules 2022, 27, 3718

8 of 14expression (≈50% reduction) in cells infected with PR8 treated with bergamot vapor phasecompared to that of PR8 untreated-infected cells (considered 100%) (Figure 3, right panel).
As already observed in CPE and TCID50 assays, also, the mixture of PR8 plus TTO or
eucalyptus was able to reduce viral infectivity.

Figure 3. The expression of HA protein was analyzed on MDCK monolayers (left panel) by ICW
assay using LI-COR Image Studio Software to measure the Relative Fluorescence Units (RFU) (rightpanel). The graph represents the percentage (%) of RFU calculated in cells infected with PR8 treatedwith different vapor EOs compared to that of PR8 untreated-infected cells (considered 100%). The
values represent the mean ± S.D. of three technical replicates performed (n = 3). Statistical significanceof data vs. PR8 untreated-infected cells was defined as * p < 0.05 and ** p < 0.001.

2.3. Transmission Electron Microscopy (TEM) Imaging
It is well known that EOs, due to their lipophilic nature, can intercalate in the lipid
bilayer of the viral envelope [41]; thus, membrane fluidity is altered, the viral envelopes
are damaged and, in some cases, even broken [42]. As influenza viruses are envelopedby lipid membranes in which HA and neuraminidase (NA) viral surface glycoproteins
are inserted, it is possible that EO vapors may act by non-specific intercalation in the lipid
bilayer of the viral envelope, thus disorganizing the protein projections. To verify thishypothesis, the morphological integrity of virus particles was checked by negative staining
transmission electron microscopy (TEM).
The TEM images of the untreated IAV particles and the IAV particles after exposure tovapors of bergamot oil, TTO or anethole for 30 min are shown in Figure 4. Ultrastructural
analysis showed that oil vapors directly affect the virions. In particular, after exposure to
bergamot oil vapor, the IAV particles show a partially decomposed viral envelope, andexposure to the TTO vapor phase completely destroys the envelope, thus disorganizing
the viral tips. In particular, using TEM imaging, it was possible to visualize the preservation of the morphology in the control viral particles (Figure 4A) and, at the same time,
the structural changes in the viral particles following the treatments (Figure 4B–D). In particular, the particles exposed to vapors of bergamot, TTO or anethole appeared to expandin size.

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Figure 4. Negative staining of IAV particles. Control virions (A), bergamot (B), TTO (C) or anethole (D) vapor phases exposed virions.

As shown in Figure 4, the control virions display well-ordered surface tips (A); conversely, in the samples exposed to bergamot oil, the viral particles show a partially decomposed membrane and a disordered surface (B). In contrast, samples exposed to TTO werecompletely broken, and the tips appeared discontinuous (C). Although the virus particles
exposed to anethole (D) were larger than the untreated virus particles, they appeared morepreserved in morphology compared to the other treated samples (B, C).
In general, antiviral activity is classified into viral particle inactivation, adsorption
inhibition and growth inhibition. The antiviral activity of EOs, which are lipophilic bynature, is probably due to the penetration into viral membranes, easily leading to membrane
disintegration [39]. Basing on the results of the present study, it is very likely that there is adirect relationship between the lipophilic nature of the constituents of EOs and the viralenvelope disruption. This mechanism of action has been demonstrated in a previous study
on the herpes simplex virus type 1 (HSV-1), in which an ultrastructural alteration of the viralenvelope by EOs has been observed by electron microscopy [43]. It has been observed thatlimonene and β-pinene (the main constituents of vapor phase of C. bergamia) and anethole(the most abundant component even in the vapor phase of I. verum EO) exhibited highanti-HSV-1 activity by direct interaction with free virus particles [40,44,45]. Thus, we may
hypothesize that these compounds might directly interfere with influenza virion envelope
structures or mask viral structures important for early steps of viral infection. A similar

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behavior, but less potent, was observed for 1,8-cineole (the most abundant component in
the vapor phase of E. globulus EO) when added to HSV-infected cells prior to infection, evenif plaque formation was reduced also after penetration of the viruses into the host cells [19].
Another study demonstrated that eucalyptus essential oil had no effect on adenovirus,
a naked virus, confirming that EOs can affect viral infection by interfering with virion
envelope structures [46]. Moreover, it was observed that γ-terpinene, the main constituent
of vapor phase of M. alternifolia, shows only moderate antiviral effects when added tohost cells prior to infection or after the entry of HSV into cells, exhibiting high anti-HSV-1
activity by the direct inactivation of free virus particles [19].
3. Materials and Methods
3.1. Materials
Illicium verum fruits (production lot number 02220202) and bergamot fruits werepurchased from Salus in erbis (www.salusinerbis.it) and “Azienda Agricola Patea” (Brancaleone Marina, Italy), respectively. TTO (production lot number 192938014) and eucalyptus
EO (production lot number 192938005) were obtained commercially from Caesar & Loretz
GmbH (Hilden, Germany). Methanol was purchased from Sigma-Aldrich (Milano, Italy).
3.2. Gas Chromatography–Mass Spectrometry (GC–MS) Analysis
The analyses of the EOs were performed by a gas chromatograph with a flame ionization detector (FID) and coupled to a mass spectrometer (MS) Perkin Elmer Clarus 500 model(Waltham, MA, USA). The oven GC was equipped with a Varian Factor Four VF-1 capillarycolumn and helium was used as carrier gas at a flow rate of 1 mL/min. The operative
conditions followed [47] with some modifications.
Mass spectra were taken at 70 eV (EI) with a mass scan range of 40–500 m/z. The
identification of constituents was obtained by matching their mass spectra with thosestored in the Wiley and NIST 02 mass spectra libraries database. Furthermore, the Linear
Retention Indices (LRIs), with reference to the series of C8–C30 aliphatic hydrocarbons,
were calculated and compared with available retention data found in the literature. Relative
amounts were expressed as percentages obtained by peak area normalization from GC-FID
chromatograms without the use of an internal standard or correction factors. All analyses
were performed three times.
3.3. Head Space GC/MS Analysis
To obtain the chemical characterization of the EOs vapor phase, a Perkin-Elmer
Headspace Turbomatrix 40 (Waltham, MA, USA) autosampler connected to GC-MS wasused. The analytical conditions such as temperature and equilibration time were optimized
as previously reported [48]. For the sampling phase, about 1 mL of each EO was placedin 20 mL vials sealed with headspace PTFE-coated silicone rubber septa and caps. The
quantification of identified compounds was performed by GC-FID as described in theprevious section.
3.4. Cell Cultures
Madin–Darby canine kidney cells (MDCK) were acquired originally from ATCC
(American Type Culture Collection, Rockville, MD, USA) and were passaged in cell culture
flasks at 37 ◦ C in a 5% CO2 atmosphere in Minimum Essential Medium Eagle (MEM,
Sigma Aldrich, Milano, Italy) supplemented with 10% heat inactivated fetal bovine serum(FBS, Corning, NY, USA), glutamine 0.3 mg/mL, penicillin 100 U/mL and streptomycin
100 mg/mL.
3.5. Cell Toxicity Assays
The cytotoxicity of vapor EOs was studied after the observation of cell monolayers
under an inverted optical microscope and by MTT proliferation assay [49]. In the MTT
assay, MDCK cells were seeded in 96-well plates at a density of 2 × 104 cells/well in 100 µL

Molecules 2022, 27, 3718

11 of 14of complete MEM without phenol red for 24 h at 37 ◦ C. Subsequently, cell monolayers weretreated with PBS incubated with vapor phases and maintained at 37 ◦ C. After 24 h, 10 µL of
MTT solution (5 mg/mL) were added to each well for 3–4 h at 37 ◦ C. Each sample was thenacidified by adding 0.1 N HCl in isopropanol (100 µL/well) for 30 min under mild agitation
to ensure the dissolution of all formazan crystals. The absorbance of samples was read at
570 nm, using an automatic plate reader (Multiskan EX, Ascent Software, Thermo Fisher
Scientific, Waltham, MA, USA). The percentage (%) of cell proliferation in cells treated with
EOs vapor was compared to that of control cells treated with PBS at the same conditions(considered 100%).
3.6. TCID50 (Tissue Culture Infectious Dose 50%) and Cytopathic Effect Assays
The virucidal activity of the vapor phase of the different EOs was tested by TCID50 .
The exposure of pure virus to vapor phases was performed with a modified method alreadydescribed [31]. Briefly, 20 µL of human influenza virus A/Puerto Rico/8/34 H1N1 (PR8)
was dried for 2 h on the underside of the caps from sterile Eppendorf 1.5 mL tubes. EOs(250 µL) were added to each tube and the caps, containing dried virus film, were replacedon the tube. The exposure of dried virus to different EOs vapor phases lasted for 30 min at
37 ◦ C. Then, caps containing dried exposed film were removed from the tube, added to
new tubes and reconstituted in 1 mL PBS. Each mixture was serially diluted and added(20 µL) to MDCK cell monolayers plated at 2 × 104 cells/well in 96-well plates. Cells
infected with serial dilutions (until 10−4 ) of reconstituted dried virus (8 replicates/dilution)
were maintained at 37 ◦ C in a 5% CO2 atmosphere for 1 h. After viral challenge, theinoculum was removed, and cells were incubated in culture medium supplemented with
2% heat-inactivated FBS.
The Cytopathic Effect (CPE) induced by the infection was evaluated 24 h post infection
and TCID50 was calculated using the Reed–Muench method, as previously reported [50].
Dried virus, not exposed to any oil, was diluted in PBS and used as control. No viral
infectivity loss due to drying was detectable by TCID50 .
3.7. In-Cell Western (ICW) Assay
The viral protein expression was analyzed by ICW assay, as already described [11].
Briefly, MDCK confluent cell monolayers were infected with PR8 treated or not with EOs
vapor phases for 1 h at 37 ◦ C. After viral adsorption, cells were washed with PBS, supplemented with fresh medium plus 2% FBS and maintained for 24 h at 37 ◦ C. Cell monolayers
were then fixed with 3.7% formaldehyde for 20 min, permeabilized with 0.1% Triton X-100
and incubated with Odyssey blocking buffer (LI-COR Bioscience, Lincoln, NE, USA) for
60 min at room temperature. The cells were stained at 4 ◦ C overnight with primary antibodies against influenza HA protein. After incubation, three washes with PBS plus 0.1%
Tween 20 were performed, and then, the cells were stained with a mixture of fluorochromeconjugated secondary antibodies (fluorescence emission at 800 nm), properly diluted in
Odyssey blocking buffer and fluorochrome-conjugated Cell Tag (fluorescence emission at
700 nm), for 1 h at room temperature. Cell Tag was used as control of the integrity of cellmonolayer. Subsequently, three washes with PBS plus 0.1% Tween 20 were performed,
and plates were analyzed by the Odyssey infrared imaging system (LI-COR). Integrated
intensities of fluorescence were determined by the LI-COR Image Studio software, andthe relative fluorescence unit (RFU) was expressed as percentage compared to untreated
infected cells (100%).
3.8. Transmission Electron Microscopy (TEM) Imaging
For TEM visualization, after 30 min exposure to EOs vapors, unexposed (control)
or exposed viral particles were resuspended in PBS. A drop of each sample (10 µL) was
absorbed onto carbon-formvar-coated, 400-mesh EM copper grids. Negative staining was
carried out by utilizing 2% phosphotungstic acid (PTA) (pH 7.0) for 30 s, and samples
were examined using an EM 208 FEI transmission electron microscope operated at 80 kV.

Molecules 2022, 27, 3718

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Post-imaging manipulation was performed in Photoshop (Adobe, San Jose, CA, USA) andwas limited to image cropping and equal adjustments of image levels.
4. Conclusions
In conclusion, we analyzed the EOs and their vapor phase chemical compositions
by the head-space gas chromatography-mass spectrometry technique. Citrus bergamia
and M. alternifolia essential oil vapors showed interesting anti-influenza A virus activity
following 30 min exposure. Under these conditions, the vapors showed no measurable
adverse effect on epithelial cell monolayers. It was suggested from the results of the present
study that the essential oil inactivates directly the viral particles. Indeed, it was reported
that limonene, β-pinene, and anethole directly interact with free HSV-1 particles. Thus,
we may hypothesize that these compounds might directly interfere with influenza virion
envelope structures or mask viral structures important for early steps of viral replication.
A similar behavior, but less potent, was observed for 1,8-cineole (the most abundantcomponent in the vapor phase of E. globulus EO) when added to HSV-infected cells prior to
infection, even if plaque formation was reduced also after penetration of the viruses into thehost cells. Moreover, γ-terpinene, the main constituent of the vapor phase of M. alternifolia,
shows only moderate antiviral effects when added to host cells prior to infection or after
entry of HSV into cells, exhibiting high anti-HSV-1 activity by the direct inactivation offree virus particles. Furthermore, as both IAV and HSV-1 are surrounded by an envelope,these results could be useful for further studies on other enveloped viruses. In addition,
E. globulus vapor EO reduced viral infection by 78% with no cytotoxicity, while I. verum
was not effective. Therefore, we tested the most abundant component in the vapor phaseof I. verum EO, anethole, finding that it reduced viral infection by 82%. To deepen themechanism of action, the morphological integrity of virus particles was checked by negative
staining transmission electron microscopy, showing that, probably due to their lipophilic
nature, they penetrate the lipid bilayer of the viral envelope, damaging the membranes orleading to their complete destruction. These ultrastructural results support the use of EOs
as effective virucidal agents enveloped viruses.
Taken together, our data suggest that these EOs vapor phases possess effective antiinfluenza virus activity under conditions that did not adversely affect cultured epithelial
cells. The viral envelope appeared to be a major target. Thus, some of these oil vapors
could be potentially useful to reduce the viral infectivity and virus contamination of theenvironment.
Author Contributions: Conceptualization, D.D.V., L.S., R.C., R.D.S. and L.N.; methodology, V.N.M.,
D.D.V., M.D.A. and W.T.; validation, A.M., V.T., D.I., W.T. and M.D.A.; formal analysis, W.T., M.M.,
F.S. and L.N.; investigation, D.D.V., V.N.M., A.M., V.T., D.I., S.G., W.T., F.D.D., A.S., M.C., M.M., F.S.
and L.N.; resources and project administration, L.S., R.C., R.D.S. and L.N.; writing—original draft
preparation, V.N.M., D.D.V., M.D.A. and L.N.; writing—review and editing, V.N.M., D.D.V., A.M.,
S.G., R.C., F.S. and L.N.; visualization, V.N.M., D.D.V. and L.N.; supervision, D.D.V., L.S., R.C., R.D.S.
and L.N.; funding acquisition, R.C. and L.N. All authors have read and agreed to the publishedversion of the manuscript.
Funding: This work was partially supported by: Istituto Pasteur Italia Fondazione Cenci Bolognetti
Anna Tramontano 2019 grant (L.N.), “Sapienza” Ateneo grants (L.N. and R.C.), MIUR PRIN:
2017BMK8JR006 (LN).
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: Data is contained within the article.
Conflicts of Interest: The authors declare no conflict of interest.

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