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Aerosolized pathogens are a leading cause of respiratory infection and transmission. Currently used protective measures pose potential risk of primary/secondary infection and transmission. Here, we report the development of a universal, reusable virus deactivation system by functionalization of the main fibrous filtration unit of surgical mask with sodium chloride salt. The salt coating on the fiber surface dissolves upon exposure to virus aerosols and recrystallizes during drying, destroying the pathogens. When tested with tightly sealed sides, salt-coated filters showed remarkably higher filtration efficiency than conventional mask filtration layer, and 100% survival rate was observed in mice infected with virus penetrated through salt-coated filters. Viruses captured on salt-coated filters exhibited rapid infectivity loss compared to gradual decrease on bare filters. Salt-coated filters proved highly effective in deactivating influenza viruses regardless of subtypes and following storage in harsh environmental conditions. Our results can be applied in obtaining a broad-spectrum, airborne pathogen prevention device in preparation for epidemic and pandemic of respiratory diseases.
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Scientific RepoRts | 7:39956 | DOI: 10.1038/srep39956
Universal and reusable virus
deactivation system for respiratory
Fu-Shi Quan1,*, Ilaria Rubino2,*, Su-Hwa Lee3, Brendan Koch2 & Hyo-Jick Choi2
Aerosolized pathogens are a leading cause of respiratory infection and transmission. Currently used
protective measures pose potential risk of primary/secondary infection and transmission. Here, we
report the development of a universal, reusable virus deactivation system by functionalization of
the main brous ltration unit of surgical mask with sodium chloride salt. The salt coating on the
ber surface dissolves upon exposure to virus aerosols and recrystallizes during drying, destroying
the pathogens. When tested with tightly sealed sides, salt-coated lters showed remarkably higher
ltration eciency than conventional mask ltration layer, and 100% survival rate was observed in
mice infected with virus penetrated through salt-coated lters. Viruses captured on salt-coated lters
exhibited rapid infectivity loss compared to gradual decrease on bare lters. Salt-coated lters proved
highly eective in deactivating inuenza viruses regardless of subtypes and following storage in harsh
environmental conditions. Our results can be applied in obtaining a broad-spectrum, airborne pathogen
prevention device in preparation for epidemic and pandemic of respiratory diseases.
Aerosols take a prominent role in airborne transmission of respiratory diseases. Droplets with aerodynamic size
(da) < 10 μ m and 10 < da < 100 μ m are known to infect the alveolar regions and upper respiratory tract, respec-
tively1,2. Notably, aerosols can also be a route of infection in diseases that, contrary to for instance inuenza, do
not specically target the respiratory tract, as it could be the case of Ebola virus3. While vaccination can greatly
reduce morbidity and mortality, during a pandemic or epidemic new vaccines matching the specic strain would
be available, at the earliest, six months aer the initial outbreak. Additionally, following development of an eec-
tive viral vaccine, several potential problems would remain, such as limited supply due to insucient production
capacity and time-consuming manufacturing processes. As a result, individuals close to the point of an outbreak
would be in imminent danger of exposure to infectious diseases during the non-vaccine period. In the absence
of vaccination, respirators and masks can be worn to prevent transmission of airborne pathogenic aerosols and
control diseases, such as inuenza4.
e main alternative, the N95 respirator, requires training prior to use, must be expertly tted to address the
risk of faceseal leakage at the face-mask interface, and must be disposed of as biohazard5. Due to these factors,
the use of N95 respirators on a large scale is impractical and expensive during an epidemic or pandemic. Past
experiences of severe acute respiratory syndrome (SARS), H1N1 swine u in 2009, and Middle East respira-
tory syndrome (MERS) indicate that surgical masks have been most widely adopted by the public as personal
protective measure, despite controversy on their eectiveness6–9. Currently, among other factors, ltration in
respirators and masks depends on lter characteristics, including ber diameter, packing density, charge of bers
and lter thickness, as well as particle properties, such as diameter, density and velocity10–14. However, in the lack
of a system to deactivate the collected pathogens, safety concerns naturally arise about secondary infection and
contamination from virus-laden lter media during utilization and disposal. Furthermore, since re-sterilization is
not possible without causing damage, respirators and masks are recommended for single use only9,15,16. Scientic
eorts have been focused on treatment of lters with materials possessing well-known antimicrobial properties,
such as iodine, chlorine and metals17–25, although with limited eectiveness against virus aerosols26–28. erefore,
a key challenge is the development of an easy-to-use, universal virus negation system, which is reusable without
1Department of Medical Zoology, Kyung Hee University School of Medicine, Seoul, 130-701, Korea. 2Department
of Chemical and Materials Engineering, University of Alberta, Edmonton, AB T6G 1H9, Canada. 3Department of
Biomedical Science, Graduate School, Kyung Hee University, Seoul, 130-701, Korea. *These authors contributed
equally to this work. Correspondence and requests for materials should be addressed to H.J.C. (email: hyojick@
Received: 04 August 2016
Accepted: 30 November 2016
Published: 04 January 2017
Scientific RepoRts | 7:39956 | DOI: 10.1038/srep39956
reprocessing and capable of deactivating pathogens, thereby reducing potential risk of secondary infection and
Here, we report a simple but ecient virus inactivation system exploiting the naturally occurring salt recrys-
tallization. Our strategy is to modify the surface of the brous ltration layer within masks with a continuous
salt lm for virus deactivation via two successive processes: i) salt is locally dissolved by the viral aerosols and ii)
supersaturation is followed by evaporation-induced salt recrystallization. Consequently, viruses are exposed to
increasingly higher concentrations of saline solution during drying and physically damaged by recrystallization.
Preparation and characterization of salt-functionalized lters. To demonstrate the concept of virus
deactivation system based on salt recrystallization, the middle layer of three-ply surgical mask, polypropylene
(PP) microber lter, was coated with NaCl salt as an active virus negation unit (see SupplementaryFig.S1 for
bare PP lter). e coating formulations contained surfactant to enhance wetting of saline solution on the sur-
face of hydrophobic PP bers. Bare PP lters (abbreviated as Filterbare) were pre-wet to contain about 600 μ L of
coating solution (abbreviated as Filterwet). e amount of NaCl salt (Wsalt in mg/cm2) coated on the lter per unit
area, considering that the lters thickness is constant, was easily controlled by changing the coating solution
volume (Vsalt in μ L) during drying of pre-wet lter (radius: 3 cm, Wsalt = 3.011 + 0.013 × V s at, n = 7) (Fig.S2).
Scanning electron microscopy (SEM) and energy dispersive X-ray (EDX) mapping analysis showed the for-
mation of homogeneous NaCl coating during drying, as also conrmed by X-ray diraction (XRD) (Fig.1a,b
and SupplementaryFig.S3). Both the formation of NaCl coating on PP bers and presence of surfactant in the
coating formulation appeared to alter the lter surface properties from hydrophobic (bare lter; contact angle,
c = 133.0 ± 4.7°) to completely hydrophilic (salt-coated lter; θ
c ~ 0°, n = 10) (Fig.1c and SupplementaryFig.S4).
Hydrophilic nature of salt coating can greatly improve adhesion of viral aerosols to PP fibers compared to
Filterbare, as seen in Raman microscope images (Fig.1d and SupplementaryFig.S5).
Figure 1. Mask with salt-coated lter for prevention and deactivation of airborne pathogens. (a) SEM
image of Filterwet+600μL (top le) and EDX mapping images of Na (red), Cl (green), and NaCl (combination of
Na and Cl mapping images), showing the formation of NaCl coating, as also conrmed by XRD spectra (b)
of Filterbare, Filterwet, Filterwet+100μL, Filterwet+300μL, Filterwet+600μL, Filterwet+900μL and Filterwet+1200μL (labelled as
Bare, wet, wet+ 100 μ L, wet+ 300 μ L, wet+ 600 μ L, wet+ 900 μ L and wet+ 1200 μ L, respectively; miller indices
corresponding to NaCl crystal are shown at the top of XRD spectra for each position). (c) Optical microscope
images for contact angle measurements using 3 μ L DI water droplets on (i) Filterbare and (ii) Filterwet+600μL
(n = 10). (d) Microscope images of aerosol on (i) Filterbare and (ii) Filterwet+600μL (n = 10).
Scientific RepoRts | 7:39956 | DOI: 10.1038/srep39956
Filtration eciency against viral aerosols and protective ecacy in vivo. Filtration eciency of
salt-coated lters was tested against aerosols with volumetric mean diameter (VMD) of 2.5–4 μ m containing
H1N1 pandemic inuenza virus (A/California/04/2009, abbreviated as CA/09) at dierent pressure conditions
(see Fig.2a for transmission electron microscope (TEM) image of H1N1 virus). Interestingly, as shown in Fig.2b,
Filterbare did not exhibit any signicant level of resistance against penetration of virus under our experimental
conditions (i.e., 0% ltration eciency). Conversely, salt-coated lters showed substantially increasing ltra-
tion eciency with pressure and amount of coated salt. In particular, in the case of Filterwet+600μL, ltration e-
ciency varied from 43 to 70%, with increasing pressure from 3 to 17 kPa, and Filterwet+1200μL exhibited persistent,
high-level eciency (~85%) (one-way ANOVA, P = 0.85).
To probe the eects of ltration eciency on protective ecacy, in vivo experiments were performed using
mice intranasally (IN) infected with penetrated dosages of H1N1 virus under breathing pressure (~10 kPa)29.
As shown in Fig.2c, similarly to negative control groups (mice infected with lethal dose of virus stock and
aerosolized virus), mice exposed to a dose penetrated through the bare lter showed rapid body weight loss,
followed by death within 10 days aer infection, in good agreement with the observed 0% ltration eciency
(Fig.2b). In contrast, mice groups exposed to virus derived from salt-coated lters resulted in 100% survival rate
(Fig.2d). Furthermore, lungs of mice from negative control groups exhibited severe lung infection 4 days aer
challenge (Fig.2e). Conversely, mice groups exposed to virus derived from salt-coated lters showed signicantly
lower levels of lung viral titers (t-test, P < 0.005). is is consistent with lower levels of inammatory cytokines,
interferon-γ (IFN-γ ), from salt-coated lter groups compared to negative control and bare lter groups (t-test,
P < 0.001) (Fig.2f).
Deactivation of virus on salt-functionalized lters. Inuenza virus stability tests were performed to
investigate the eects of salt coating. e same amount of recovered viruses from the PP bers was used, and, in
the case of bare lters, viral aerosols exposure was conducted in the absence of pressure due to 100% penetra-
tion of viral aerosols. Unlike bare lters (Fig.S6a(i)), formation of micron-sized NaCl phase represents a typical
feature of salt-coated lters due to recrystallization of NaCl salt, following local dissolution upon aerosols expo-
sure (SEM images in Fig.S6a, ii to iv, and EDX mapping in Fig.S6b). In contrast to 8% HA activity loss of virus
adsorbed onto Filterbare, salt-coated lters exhibited almost complete HA activity loss within 5 min of incubation
(Fig.3a). Such dramatic virus destabilization on salt-coated lters is further supported by negligible levels of
viral titers compared to Filterbare with incubation time (t-test, P < 0.001) (Fig.3b). It is also noted that virus titers
exhibited signicant decrease with increase of incubation time and amount of coated salt (ANOVA general lin-
ear model, P < 0.001). TEM analysis showed that inuenza virus on Filterbare experiences morphological change
into non-spherical shape during aerosol drying (Fig.3c(i)). Notably, inuenza virus was severely damaged on
salt-coated lters even at 5 min of incubation (Fig.3c(ii)). From microscopic analysis, aerosol drying time was
about 3 min, indicating that destruction of virus observed at 5 min is associated with drying-induced salt crystal-
lization. Physical damage of virus due to crystallization was similarly reported as a major destabilizing factor of
inactivated inuenza virus30,31. Lower levels of native uorescence and nile red uorescence from virus recovered
from salt-coated lters accounted for more severe conformational change of antigenic proteins and destabili-
zation of viral envelope, respectively, consistent with TEM analysis (t-test, P < 0.001) (Fig.3d). In parallel, we
investigated the separate eect of salt concentration increase on virus stability during the aerosol drying process,
irrespective of crystal growth. As displayed in SupplementaryFig.S7, the materials collected in suspension from
Filterwet+600μL induced visible morphological transformation of the virus (SupplementaryFig.S7b) compared to
suspension of Filterbare (SupplementaryFig.S7a). is can be attributed to the high salt/surfactant concentra-
tion and osmotic pressure, which have been well-known to destabilize proteins and viruses31–33. erefore, the
marked virus destabilization on salt-coated PP bers can be explained by the combined eects of salt concentra-
tion increase during drying and evaporation-induced salt crystallization.
To verify in vitro virus stability on the lters, an in vivo study was performed by infecting mice with virus
incubated for 60 min on PP lters. As shown in Fig.3e, Filterbare group exhibited 5% body weight loss at day 9
post-infection, reaching a body weight lower than that of salt-coated lter groups by 10–15%. us, signicantly
higher lung virus titers in the negative control group were observed in contrast to no detectable titers in the
salt-coated lter groups (Fig.3f).
Strain-nonspecic virus deactivation and eects of storage under harsh environmental conditions
on salt coating stability. Broad-spectrum protection of salt-coated lters against multiple subtypes of
viral aerosols was evaluated by investigating both lethal infectivity by penetrated virus in vivo and infectivity by
virus collected on lters during ltration in vitro using A/Puerto Rico/08/1934 (PR/34 H1N1) and A/Vietnam/
1203/2004 (VN/04 H5N1). Similarly to CA/09 H1N1, 100% of mice survived viral infection (PR/34 and VN/04),
with no evidence of weight loss, due to higher ltration eciency of salt-coated lter than that of bare lter
(Fig.4a). is is supported by no signicant level of viral titer in the lung. In addition, as shown in Fig.4b,
salt-coated PP lters destroyed adsorbed inuenza viruses irrespective of both subtypes and amount of coated
e stability of salt coating on PP bers was tested under harsh environmental conditions. Incubation at 37 °C
and 70% relative humidity (RH) for 1 day did not cause any signicant dierence in ltration eciency (t-test,
P = 0.718) (SupplementaryFig.S8). As a result, all mice infected with dosage of penetrated virus through the lter
stored at high temperature and RH displayed 100% survival with 7% of body weight loss (Fig.4c,d). Even aer
15 days of incubation, salts remained to coat PP bers (Fig.4e, and SupplementaryFig.S9a,b), despite change in
grain orientation due to recrystallization (Fig.4f, and SupplementaryFig.S10a,b).
Scientific RepoRts | 7:39956 | DOI: 10.1038/srep39956
Figure 2. Filtration eciency of salt-coated lters. (a) TEM image of CA/09 H1N1 inuenza virus.
(b) Pressure-dependent ltration eciency (n = 8–10, mean ± standard deviation (SD)). (cf) Eects of
ltration eciency on protective ecacy in vivo. Body weight change of mice aer infection with the dosages
of penetrated virus (n = 12, mean ± SD) (c), survival rates (mean; 100% means that all mice in the group
survived as penetrated dosages were lower than lethal dose) (d), lung virus titers (n = 4, mean ± SD) (e), and
lung inammatory cytokine (interferon-γ (IFN-γ )) assay (n = 11, mean ± SD) (f). Legends: lters are labelled
as in Fig.1b.
Scientific RepoRts | 7:39956 | DOI: 10.1038/srep39956
Figure 3. Inactivation of virus adsorbed on salt-coated lters. (a,b) HA activity (a) and virus titer
(b) displaying the eects of incubation time on the remaining activity of virus (n = 4–8, mean ± SD). (c) TEM
images of viruses reconstituted, aer incubation for 5 and 60 min, from (i) Filterbare and (ii) Filterwet+600μL.
(d) Native uorescence/nile red uorescence of viruses incubated for 60 min (n = 12, mean ± SD). (e,f) Body
weight change of mice aer infection with virus recovered from lters aer incubation for 60 min (n = 12,
mean ± SD) (e), and lung virus titers (n = 6, mean ± SD) (f). Asterisk (*): below detection limit. Legends: lters
are labelled as in Fig.1b.
Scientific RepoRts | 7:39956 | DOI: 10.1038/srep39956
Figure 4. Strain- and environment-dependent performance of salt-coated lters. (a) Body weight change
of mice infected with penetrated PR/34 H1N1 and VN/04 H5N1 viruses through Filterwet+600μL (n = 12,
mean ± SD). (b) Virus titers of recovered viruses from bare and salt-coated lters (n = 4, mean ± SD; data
for Filterwet, Filterwet+600μL and Filterwet+1200μL are overlapped). (c,d) Body weight change (c) and survival rate
(d) of mice infected with dosage of penetrated virus through Filterwet+600μL before and aer exposure to harsh
environmental conditions (37 °C and 70% RH) for 1 day (lled square and open square overlap in (d)). (e) EDX
mapping image of NaCl-coated Filterwet+600μL aer incubation for 15 days at 37 °C and 70% RH (combination of
Na (red) and Cl (green) mapping images). (f) XRD spectra of Filterwet+600μL before and aer incubation at 37 °C
70% for 1 day and 15 days. Legends: lters are labelled as in Fig.1b.
Scientific RepoRts | 7:39956 | DOI: 10.1038/srep39956
Development of a universally applicable, low-cost, and ecient mechanism for virus negation is regarded as a
major challenge in public health against general airborne biological threats. is led us to propose a new con-
cept of personal/public preventive and control measures using salt-recrystallization against pathogenic aerosols
based on two hypotheses. e salt-coating can enhance adsorption of virus on the lter bers and inactivate
virus by the increase of osmotic pressure followed by the crystallization of salts. As shown in Fig.2b, salt-coated
lters exhibited signicantly higher levels of ltration eciency than bare lters. Notably, the bacterial ltration
eciency (BFE) reported by the mask manufacturer is 99%. e dierent value of ltration eciency for bare
lters obtained under our experimental conditions may be partially due to the use of aerosols with dierent bio-
logical origins. e FDA-recognized ASTM F2101 – 14 standard for evaluation of BFE exposes surgical masks
to Staphylococcus aureus aerosols, by employing S. aureus ATCC 653834, which has an average diameter of about
1 μm. In this study, ltration eciency was calculated following exposure of bare and salt-coated lters to inu-
enza virus, which exhibits a smaller diameter than that of S. aureus by one order of magnitude. Additionally,
whereas during BFE evaluation all three layers of surgical masks are used, in this work ltration eciency refers
to mask lters (middle layer). It is worth noting that the conditions for BFE standard evaluation (such as ow
rate and time of application of ow) do not coincide with the experimental procedure we used for measurement
of the ltration eciency, which may further contribute to the dierent result. e enhanced ltration eciency
of salt-coated lters against inuenza virus aerosols as compared to bare lters can be explained by the observed
wetting of aerosols, favoring greater adhesion to salt-coated lters. Furthermore, the signicant improvement in
ltration eciency resulted in complete protection of mice against lethal inuenza aerosols, which demonstrates
the high level of protection provided by salt-coated lters, outperforming currently used bare lters.
Rapid loss of HA activity and viral infectivity on salt-coated lters can be explained by physical destruction of
virus during recrystallization of coated salts. When the salt-coated lter is exposed to virus aerosols, salt crystals
below the aerosol droplet dissolve to increase osmotic pressure to virus. Due to evaporation, the salt concentra-
tion of the droplet signicantly increases and reaches the solubility limit, leading to recrystallization of salt. As a
consequence, virus particles are exposed to increasing osmotic pressure during the drying process and are phys-
ically damaged by crystallization. As shown in Fig.3e,f, the superior advantage of physically destroying the virus
adsorbed to the salt-coated PP lters through natural salt crystallization process was further conrmed in vivo.
According to previous reports, hyperosmotic stress (> 541 mOsm) and crystallization induce membrane pertur-
bation with irreversible deformation of the viral envelope and structural virus damage, respectively, resulting in
infectivity loss of virus30,31. erefore, our data support that the extensive level of infectivity loss associated with
a salt recrystallization process caused by physical contact between virus aerosols and salt coating can be used in
developing virus negation systems that are reusable without reprocessing.
Similarly to CA/09 H1N1 aerosols, increased protection in vivo due to higher ltration eciency of salt-coated
lters compared to bare lters and deactivation of virus on salt-coated lters were observed following exposure
to PR/34 H1N1 and VN/04 H5N1 (Fig.4a,b). is suggests that salt-coated lters prevent virus penetration
and destroy virus attached to the lter in a non-specic way. Furthermore, the performance of salt-coated l-
ters was not degraded by storage at 37 °C and 70% RH, demonstrating that salt recrystallization-based lters
can ensure protection even under harsh environmental conditions. Notably, for demonstration of the concept
of salt-recrystallization based virus deactivation system, NaCl salt was used, which has a critical RH of 75% at
30 °C35. However, salts with higher critical RH can be easily used, such as ammonium sulfate, potassium chloride
and potassium sulfate, which have critical RH of 80%, 84% and 96.3% at 30 °C, respectively35. is suggests that
salt-coated lters may be developed for specic environmental conditions.
In conclusion, we demonstrated that the developed salt-recrystallization based ltration system provides high
ltration eciency and successfully deactivates multiple subtypes of adsorbed viruses. Moreover, we have shown
that stability of the salt coating is not compromised by high temperature and humidity, which suggests safe use
and long-term storage/reuse at such environmental conditions. Although our tests are based on exposure to dif-
ferent types of inuenza virus, the signicance of these results for personal and public protective measures may
be generally extended to enveloped respiratory viruses where infection and transmission can occur by aerosol.
Our salt-coated lter unit can promise the development of long-term stable, versatile airborne pathogen negation
system, without safety concerns. In fact, the destruction mechanism of viruses solely depends on the simple, yet
robust naturally occurring salt recrystallization process, combining the destabilizing eects of salt crystal growth
and concentration increase during drying of aerosols. is idea can be easily applied to a wide range of existing
technologies to obtain low-cost, universal personal and public means of protection against airborne pathogens,
such as masks and air lters in hospitals. erefore, we believe that salt-recrystallization based virus deactivation
system can contribute to global health by providing a more reliable means of preventing transmission and infec-
tion of pandemic or epidemic diseases and bioterrorism.
Bare and salt-coated lter samples preparation. e commercial surgical masks had a three-ply
structure. e middle layer is the lter media, whereas the inner and outer layers provide support and protect
the lter against wear and tear. e metal nose clips and elastic ear loops were removed and circular samples
(radius: 3 cm) were cut from the masks. e PP lters (middle layer) were isolated by removing the inner and
outer protective layers (bare lters, Filterbare). e coating solution was prepared by dissolving sodium chloride
(NaCl; Sigma Aldrich, St. Louis, MO) in ltered DI water (0.22 μ m pore size; Corning, Tewksbury, MA) under
stirring at 400 rpm and 90 °C, followed by the addition of Tween 20 (Fisher Scientic) to a nal concentration of
29.03 w/v% of NaCl and 1 v/v% of Tween 20. To obtain the salt-coated lters, the mask bare PP lters were pre-wet
to contain approximately 600 μ L of coating solution by incubating overnight at room temperature. Any remain-
ing dry areas were removed by applying gentle strokes with tweezers to the lters while immersed in the coating
Scientific RepoRts | 7:39956 | DOI: 10.1038/srep39956
solution. Subsequently, the lters were deposited in the desired volume of coating solution (0, 100, 300, 600,
900 and 1200 μL, of which corresponding membranes are abbreviated as Filterwet, Filterwet+100μL, Filterwet+300μL,
Filterwet+600μL, Filterwet+900μL, and Filterwet+1200μL, respectively) on petri dishes (60 × 15 mm; Fisher Scientic) to
control the amount of NaCl per unit area and dried in an oven (Isotemp Incubator, Fisher Scientic) at 37 °C
for 1 day.
Inuenza virus preparation. Inuenza viruses A/California/04/2009 (CA/09, H1N1), A/Puerto Rico/8/34
(PR/34, H1N1) and A/Vietnam/1203/2004 (VN/04, H5N1) were grown in 10-day old embryonated hen eggs, in
which H5N1 virus was derived by reverse genetics from HPAI A/Vietnam/1203/200436. Inuenza viruses were
puried from allantoic uid using discontinuous sucrose gradient (15%, 30% and 60%) layers following the pre-
viously reported procedure37.
Aerosols exposure to lters. For experiments involving aerosols exposure, an aerosol chamber (L × W ×
H = 145 × 145 × 150 mm; Emka Inc., Middletown, PA) was used (Fig.S11). It has a connection to the vacuum
line and a circular aperture in the top wall (diameter: 22 mm) to exactly accommodate the cylindrical part (diam-
eter: 20 mm, height; 20 mm) of the nebulizer unit that is below the aerosol generator (Aeroneb Lab Nebulizer
System; Aerogen, Galway, Ireland). Bleach was used as trap between the chamber and the vacuum pump (Welch
2522C-10, 22 L/min; Niles, IL). e lters were placed on top of the chamber aperture and the nebulizer unit
was inserted, ensuring the tight seal of the lters against the side of the aperture. 5 μ L of virus stock were added
to the nebulizer unit, aerosols (VMD 2.5–4 μ m from manufacturer specications) were generated for 30 sec and
subsequently the desired vacuum level (3, 10 or 17 kPa) was applied, by manual control, three times in 1 sec cycles.
Notably, in the case of bare lters, pressure was only applied for ltration eciency tests.
For all assays and analysis, suspensions of the lters were prepared as follows, unless otherwise indicated. To
reconstitute virus adsorbed onto lters, virus-laden lters were immersed in 400 μ L of sterilized DI water for
about 5 min, and then removed aer vortexing from the suspension. e virus suspension was centrifuged at
19,800 g and 4 °C for 10 min (Centrifuge 5810 R, Eppendorf, Hauppauge, NY), followed by resuspension of pellets
in 70 μ L of DI water to eliminate any interference from materials in supernatant during assays.
Filtration eciency tests. e lters were exposed to the virus aerosols at 3, 10 and 17 kPa and suspen-
sions of the lters were obtained, as described above. e ltration eciency was calculated as the ratio of the
amount of virus (i.e., total proteins measured from the virus) reconstituted from the lter to that from the virus
in the exposure aerosols. e concentration of virus in aerosols was determined by generating viral aerosols into
a 15 mL centrifuge tube, containing 1 mL of DI water. Aer vortexing, virus concentrations (i.e., total protein
concentration) were measured with bicinchoninic acid assay (BCA protein assay kit; ermo Fischer scientic,
Waltham, IL) with bovine serum albumin as a standard. In the case of virus reconstituted from salt-coated lters,
virus-laden lter suspension was replaced with DI water prior to BCA assay.
In vivo infection tests. Lethal infectivity of inuenza viruses (CA/09 H1N1) was examined in 8 week old
female inbred BALB/c mice (Nara Biotech; Seoul, Korea) by using the intranasal route. For bare and salt-coated
lters, 12 mice per group were infected with individual penetration dosage of inuenza virus through each lter.
e penetration dosage of the virus through the lters (Filterbare, Filterwet, Filterwet+600μL, and Filterwet+1200μL) was
calculated from the ltration eciency at 10 kPa (near breathing pressure) using the relationship: penetration dos-
age = virus dosage in lethal aerosol × penetration eciency (%)/100, where penetration eciency (%) = 100
ltration eciency (%). To examine the eects of the aerosolization process on the viral infectivity change, two
mice groups were infected with a lethal dose of virus before and aer aerosol formation, which served as negative
control groups. Body weight changes and survival rate of mice were monitored daily for 15 days. Mice with body
weight loss greater than 25% were euthanized. All animal protocols were approved by the Kyung Hee University
(KHU) Institutional Animal Care and Use Committee (IACUC). All animal experiments and husbandry involved
in this work were conducted under the approved protocols and guidelines of KHU IACUC. KHU IACUC oper-
ates under National Veterinary Research and Quarantine Service (NVRQS), and animal welfare law and regula-
tions of the WOAH-OIE (World organization for animal health).
To test strain-dependent lethal infection behavior, mice (12 per group) were infected with the penetrated dos-
age of viral aerosols (PR/34 H1N1 and VN/04 H5N1 viruses) through Filterwet+600μL at 10 kPa. Time-dependent
body weight change was monitored in the same manner described above.
Lung viral titer and lung inammatory cytokine assays after infection. On day 4 aer infection 6
mice of each group were sacriced for the collection of lung samples. Lung virus titers were measured on six-well
plates containing conuent MDCK cell monolayers. Inammatory cytokines (IFN-γ ) were determined using BD
OptEIA mouse IFN-γ ELISA kit (BD Biosciences, San Jose, CA) following the manufacturer’s procedure.
Test of viral infectivity change on lters. To investigate the eects of salt-coating on viral infectivity
loss, lethal inuenza aerosols were exposed to four dierent types of lters (Filterbare, Filterwet, Filterwet+600μL, and
Filterwet+1200μL). Since Filterbare exhibited almost complete penetration upon pressure application, aerosols were
exposed to the bare lter in the absence of pressure and samples were carefully handled to prevent mechanical
agitation. To measure time-dependent stability change of virus, virus-laden lters were incubated at ambient
conditions for 0, 5, 15, and 60 min aer aerosol exposure, and suspended in DI water to reconstitute virus at each
time point. In vitro stability of virus was characterized by measuring hemagglutinin activity (HA) and virus titers
at the same concentration as lethal dose30. e conformational stability of antigenic proteins was characterized
by measuring intrinsic uorescence using 0.1 mg/mL of virus suspension38. To investigate morphological change
of virus, lipid stability of viral wall was characterized by nile red uorescence (Sigma Aldrich), a uorescent lipid
Scientific RepoRts | 7:39956 | DOI: 10.1038/srep39956
stain, following manufacturers protocol39. A decrease in uorescence intensity can be used to examine the level
of disintegration of the virus. Both intrinsic and nile red uorescence were measured by using a uorimeter (LB
50B; PerkinElmer, Waltham, MA). Intensity changes of uorescent spectra were compared relative to those of a
control from virus stock.
To test infectivity dierence observed from in vitro ndings, in vivo study was performed for the virus recon-
stituted from the lters (Filterbare, Filterwet, Filterwet+600μL, and Filterwet+1200μL) aer incubation for 60 min at RT
(aerosol exposure at 10 kPa, except for Filterbare). 12 mice per group were infected with a lethal dose of virus
collected from each type of lter. Body weight change and lung virus titers were measured as described above.
Eects of environmental conditions on the performance of salt-coated lter. Salt-coated lters
(Filterwet, Filterwet+600μL, and Filterwet+1200μL) were stored at 37 °C, 70% RH in an incubator (Maru Max; Rcom,
Gyeonggi-do, South Korea) for 15 days. Every day, the lters were collected and incubated at ambient conditions
for 5 min. At 1-day incubation, ltration eciency was measured at 10 kPa from Filterwet+600μL, followed by in vivo
infection test. Lethal infectivity between two dierent lter groups (before and aer incubation at 37 °C, 70% RH)
was compared by measuring body weight change and survival rate of mice aer exposure to lethal CA/09 H1N1
aerosols. XRD analysis was performed to salt-coated lters incubated for 1 and 15 days, and SEM/EDX mapping
analysis for 15-day incubated samples.
Contact angle measurements and imaging of aerosols. e bare and salt-coated lters were xed
with carbon tape (Ted Pella, Inc., Redding, CA) to a metal, at substrate and 3 μ L of DI water were added on the
surface of the lters. e contact angles were measured from images collected with an optical microscope (10×
lens, Motic SMZ-140; Motic, Richmond, Canada) at RT. Images of aerosols on lter bers were obtained using a
dispersive Raman microscope (Nicolet Almega XR; Fisher Scientic).
Aerosol drying time on lters. e bare and salt-coated lters were xed with carbon tape to a metal, at
substrate and exposed to aerosols generated from 5 μ L of Sulforhodamine B Dye solution (1 mM, Sigma-Aldrich).
Aerosol drying time was determined with timer by observation with optical microscope.
Electron microscopy analysis. For virus stability tests, bare and salt-coated lters were exposed to CA/09
H1N1 aerosols and, aer 5 and 60 min incubation, virus was recovered by suspension of the lters, as described
above. To study the eects of the coating formulation during aerosol drying independently from crystal growth,
bare and salt-coated lters were immersed in DI water and removed aer 60 min. Subsequently, virus was incu-
bated in the obtained suspension for 60 min. Additionally, the virus suspension was centrifuged at 19,800 g and
4 °C for 10 min to collect the samples and suspend them in DI water. For TEM analysis (200 kV, JEOL JEM 2100;
JEOL, Peabody, MA), samples were deposited on copper grid (Electron Microscopy Sciences, Hateld, PA) and
negatively stained with solution comprised of phosphotungstic acid hydrate (1.5 w/v%, pH = 7.0; Sigma-Aldrich,
Oakville, Canada).
To identify the morphology of salt-coated lters and recrystallized salts, SEM/EDX analysis was performed
for bare and salt-coated lters aer coating with 10 nm thick gold layer. Scanning electron microscopy analysis
(Hitachi S-3000N; Hitachi, Toronto, Canada) was operated in secondary electron mode at 20 kV and EDX analy-
sis was obtained with EDX detector (Oxford Instruments, Concord, MA).
XRD analysis. To conrm the formation of crystalline NaCl coating during drying process and its stability
during storage at 37 °C and 70% RH, XRD analysis (BRU-1098; Bruker, Billerica, MA) was performed at dierent
coating conditions. Filters (1 × 1 cm) were mounted on a slide glass for XRD analysis (θ –2θ mode) using a CuKα
Statistical analysis. To compare multiple conditions, Students t-test, One-way analysis of variance
(ANOVA), and general linear model were used (Minitab release 14; Minitab, State College, PA). P value of less
than 0.05 was considered to be signicant.
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is research was nancially supported by startup funds from University of Alberta (H.J.C.), and grants from
National Research Foundation of Korea (NRF) (NRF-2014R1A2A2A01004899) and Ministry of Health &
Welfare, Republic of Korea (HI15C2928).
Author Contributions
H.J.C. conceived and designed the experiments. F.S.Q., I.R., S.H.L., B.K., and H.J.C. performed the experiments.
F.S.Q., I.R., S.H.L., B.K., and H.J.C. analyzed the data. I.R. and H.J.C. wrote the manuscript. F.S.Q. and B.K. edited
the manuscript.
Additional Information
Supplementary information accompanies this paper at
Competing nancial interests: e authors declare no competing nancial interests.
How to cite this article: Quan, F.-S. et al. Universal and reusable virus deactivation system for respiratory
protection. Sci. Rep. 7, 39956; doi: 10.1038/srep39956 (2017).
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Supplementary resource (1)

... They found that within 5 min, the H1N1 in uenza virus is inactivated when the coating layer wetted with the virus -laden aerosols locally dissolves, subsequently evaporates, and recrystallizes. They concluded that these physicochemical processes damage the viral capsid, leading to viral inactivation (Quan et al. 2017). They further reported that NaCl can functionalize inert membranes, causing the e cient capture and inactivation of airborne pathogens (Rubino et al. 2020). ...
... A salt solution containing 29.03% w/v NaCl in demineralized water (29.03 g/100 ml) and 1% Tween 20 (Merck Sigma Aldrich, Darmstadt, Germany) was used as the starting concentration (Quan et al. 2017). A ve-fold dilution in demineralized water was also prepared. ...
... The TEER values measured at 24 h post-infection were signi cantly lower in the epithelial inserts that received the viral input exposed to the salt-coated material than in the control inserts (viral control and noncoated In conclusion, the in vitro bioassay using human lung epithelia proved to be suitable for the assessment of antiviral coating effective against SARS-CoV-2 and could be used to test other types of antiviral face masks in the future. The antiviral effect of salt coatings previously reported for the in uenza virus A H1N1 (Quan et al. 2017) and H3N2 (Schorderet Weber et al. 2022) is also con rmed for SARS-CoV-2. The survival of virus particles was signi cantly reduced after contact with salt-coated materials. ...
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In the Covid-19 pandemic, caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), face masks have become a very important safety measure against the main route of transmission of the virus: droplets and aerosols. Concerns that masks contaminated with SARS-CoV-2 infectious particles could be a risk for self-contamination have emerged early in the pandemic as well as solutions to mitigate this risk. The coating of masks with sodium chloride, an anti-viral and non-hazardous to health chemical, could be an option for reusable masks. To assess the antiviral properties of salt coatings deposited onto common fabrics by spraying and dipping, the present study established an in vitro bioassay using three-dimensional airway epithelial cell cultures and SARS-CoV-2 virus. Virus particles were given directly on salt-coated material, collected, and added to the cell cultures. Infectious virus particles were measured by plaque forming unit assay and in parallel viral genome copies were quantified over time. Relative to noncoated material, the sodium chloride coating significantly reduced virus replication, confirming the effectiveness of the method to prevent fomite contamination with SARS-CoV-2. In addition, the lung epithelia bioassay proved to be suitable for future evaluation of novel antiviral coatings.
... Five spray and three dip coating conditions were defined. Spray deposition with the salt formulation containing Tween-20 as wetting agent 20 was performed, one piece of fabric per condition, using a spray device whose valve aperture was changed according to the arbitrary stroke units 1, 3, 5, and 10, labeled Spr S1, Spr S3, Spr S5, and Spr S10, respectively. The spray formulation was fivefold diluted for an additional sample with stroke unit 3 (Spr S3 Dil5 ×). ...
... The present study extended the findings of Quan et al. 20 on material that has good particle filtration properties, that can be washed and integrated into reusable face masks 10 . In addition, we used salt coating techniques applicable in a household setting. ...
... The antiviral effect of a salt coating appears to be a consequence of virus capsid disruption from local salt dissolution followed by recrystallization 20,30 . This effect may vary not only with the amount of salt in the coating, but also with the distribution and size of the salt crystals on the fabrics, which in turn depends on various saltdeposition parameters and techniques. ...
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During the coronavirus disease (COVID-19) pandemic, wearing face masks in public spaces became mandatory in most countries. The risk of self-contamination when handling face masks, which was one of the earliest concerns, can be mitigated by adding antiviral coatings to the masks. In the present study, we evaluated the antiviral effectiveness of sodium chloride deposited on a fabric suitable for the manufacturing of reusable cloth masks using techniques adapted to the home environment. We tested eight coating conditions, involving both spraying and dipping methods and three salt dilutions. Influenza A H3N2 virus particles were incubated directly on the salt-coated materials, collected, and added to human 3D airway epithelial cultures. Live virus replication in the epithelia was quantified over time in collected apical washes. Relative to the non-coated material, salt deposits at or above 4.3 mg/cm² markedly reduced viral replication. However, even for larger quantities of salt, the effectiveness of the coating remained dependent on the crystal size and distribution, which in turn depended on the coating technique. These findings confirm the suitability of salt coating as antiviral protection on cloth masks, but also emphasize that particular attention should be paid to the coating protocol when developing consumer solutions.
... Overall concerns in many of the lab studies were that decontamination processes were not tested in a true healthcare setting, suggesting further research would be needed before implementing changes in medical facilities. Thirteen of these studies (26%) did not list limitations [31,[33][34][35]38,39,53,57,60,61,73,75,77]. ...
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During the start of the COVID-19 pandemic, shortages of personal protective equipment (PPE) necessitated unprecedented and non-validated approaches to conserve PPE at healthcare facilities, especially in high income countries where single-use disposable PPE was ubiquitous. Our team conducted a systematic literature review to evaluate historic approaches for conserving single-use PPE, expecting that lower-income countries or developing contexts may already be uniquely conserving PPE. However, of the 50 included studies, only 3 originated from middle-income countries and none originated from low-income countries. Data from the included studies suggest PPE remained effective with extended use and with multiple or repeated use in clinical settings, as long as donning and doffing were performed in a standard manner. Multiple decontamination techniques were effective in disinfecting single use PPE for repeated use. These findings can inform healthcare facilities and providers in establishing protocols for safe conservation of PPE supplies and updating existing protocols to improve sustainability and overall resilience. Future studies should evaluate conservation practices in low-resource settings during non-pandemic times to develop strategies for more sustainable and resilient healthcare worldwide.
... In the first approach, partly discussed in earlier section, the mask material is functionalized or additional material having novel property is incorporated so as to deactivate the pathogens. One of the strategies is to functionalize fibrous filtration unit of mask by salts such as sodium chloride (Quan et al., 2017). In this experiment, salt coating on the fiber surface dissolved when exposed to virus aerosols. ...
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Respiratory protection devices such as face masks and respirators minimize the transmission of infectious diseases by providing a physical barrier to respiratory virus particles. The level of protection from a face mask and respirator depends on the nature of filter material, the size of infectious particle, breathing and environmental conditions, facial seal, and user compliance. The ongoing COVID‒19 pandemic has resulted in the global shortage of surgical face mask and respirator. In such a situation, significant global populations have either reused the single‒use face mask and respirator or used a substandard face mask fabricated from locally available materials. At the same time, researchers are actively exploring filter materials having novel functionalities such as antimicrobial, enhanced charge holding, and heat regulating properties to design potentially better face mask. In this work, we reviewed research papers and guidelines published primarily in last decade focusing on, (a) virus filtering efficiency, (b) impact of type of filter material on filtering efficiency, (c) emerging technologies in mask design, and (d) decontamination approaches. Finally, we provide future prospective on the need of novel filter materials and improved design.
... Many experts recommend reducing the Covid-19 through a healthy, clean lifestyle and using masks. Someone who is required to be in a public place (market, public transportation, and hospital) is always recommended to wear a mask to minimize the transmission of the virus into the respiratory tract (Quan et al., 2017). ...
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The corona virus or SARS-CoV2 is a pathogenic virus that has become a pandemic and a threat in almost all countries in the world. SARS-CoV2 belongs to the Coronaviridae family with particle sizes varying around 60 nm - 140 nm. Various regulations and prevention have been designed to reduce the impact of Covid-19 by SARS-CoV2. Several technologies and studies have been developed to form nanofiber woven membranes. Cellulose nanofibers and chitin/chitosan nanofibers have been studied and are known to have nanometer-sized structures smaller than SARS-CoV2. Chitin/chitosan has been investigated to have antiviral properties, especially corona virus. Cellulose nanofibers, and chitin/chitosan nanofibers has the potential to be developed for Covid-19 virus nanofiltration masks. Various active agents (nanosilver, nanogold, CuO, etc.) have been known to have antiviral and/or antibacterial properties so that they can be used as nanofillers to enhance the performance and effectiveness of nanofibers based masks against SARS-CoV2.
... A number of candidate salt antimicrobials were identified in a small proportion of products. The antimicrobial ability of salts has long been known and they mainly function by desiccation or by physical damage to cells during recrystallization (e.g., Quan et al. 2017). The most prominent among the candidate antimicrobials in the present work was chlorhexidine gluconate, which has recently been evaluated in Canada and proposed for addition to Schedule 1 List of Toxic Substances under CEPA, under the category of "chlorhexidine and its salts". ...
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In response to the coronavirus (COVID-19) pandemic there has been an increased need for personal and environmental decontamination to aid in curbing transmission of the SARS-CoV-2 virus. Products used for this purpose include sanitizers for hands and disinfectants for surfaces. The active chemical ingredients used in these products, termed antimicrobials, can enter waste streams after application and may be emerging as more prominent environmental contaminants. Even prior to COVID-19, there was recognized need to examine their implications for aquatic biota, which is now made more pressing due to their exaggerated use in response to the pandemic. Our objectives were to identify current antimicrobial active ingredients, quantify their increased use, and determine which may be candidates for further consideration as possible aquatic contaminants. By consulting multiple sources of publicly available information in Canada, we identified current-use antimicrobials from the lists of sanitizers and surface disinfectants approved for use against SARS-CoV-2 by Health Canada and the drug registration database. To estimate the use of sanitizers and disinfectants, we evaluated import quantities and grocery store retail sales of related compounds and products (Statistics Canada) and both lines of evidence supported increased use trends. The list of identified antimicrobials was refined to include only candidates with potential to reach aquatic ecosystems, and information on their environmental concentrations and toxicity to aquatic biota were reviewed. Candidate antimicrobials (n=32) fell into four main categories: quaternary ammonium compounds (QACs), phenols, acids and salts. Benzalkonium chloride, a QAC, was the most prominent active ingredient used in both non-alcohol-based hand sanitizers and surface disinfectants. Four QACs followed in prevalence and the next most used antimicrobial was triclosan (hand sanitizers only), an established and regulated environmental contaminant. Little information was found on environmental concentrations of other candidates, suggesting that the majority would fall into the category of emerging contaminants if they enter aquatic systems. Several were classified as acutely or chronically toxic to aquatic biota (Globally Harmonised System) and thus we recommend empirical research begin focusing on environmental monitoring of all candidate antimicrobials as a critical next step, with detection method development first where needed.
The global pandemic of COVID-19 and emerging antimicrobial drug resistance highlights the need for sustainable technology that enables more preparedness and active control measures. It is thus important to have a reliable solution to avert the present situations as well as preserve nature for habitable life in the future. One time use of PPE kits is promoting the accumulation of nondegradable waste, which may pose an unforeseen challenge in the future. We have developed a biocompatible, biodegradable, and nonirritating nanoemulsion coating for textiles. The study focused on coating cotton fabric to functionalize it with broad spectrum antimicrobial, antibiofilm, and anti-SARS-CoV-2 activity. The nanoemulsion comprises spherical particles of chitosan, oleic acid, and eugenol that are cross-linked to fibers. The nanoemulsion caused complete destruction of pathogens even for the most rigid biofilms formed by drug resistant Staphylococcus aureus, Pseudomonas aeruginosa, and Candida albicans on the surface of the coated fabric. The secondary coat with beeswax imparts super hydrophobicity and 20 wash cycle resistance and leads to enhanced barrier properties with superior particulate filtration, bacterial filtration, and viral penetration efficiency as compared to an N95 respirator. The coated fabric qualifies as per standard parameters like breathability, flammability, splash resistance, and filtration efficiency for submicrometer particles, bacteria, and viruses. The scaleup and bulk manufacturing of the coating technology on fabric masks complied with standards. The consumer feedback rated the coated mask with high scores in breathability and comfortability as compared to an N95. The strategy promises to provide a long-term sustainable model compared to single use masks and PPE that will remain a nondegradable burden on the ecosystem for years to come.
The outbreak of COVID-19 provided a warning sign for society worldwide: that is, we urgently need to explore effective strategies for combating unpredictable viral pandemics. Protective textiles such as surgery masks have played an important role in the mitigation of the COVID-19 pandemic, while revealing serious challenges in terms of supply, cross-infection risk, and environmental pollution. In this context, textiles with an antivirus functionality have attracted increasing attention, and many innovative proposals with exciting commercial possibilities have been reported over the past three years. In this review, we illustrate the progress of textile filtration for pandemics and summarize the recent development of antiviral textiles for personal protective purposes by cataloging them into three classes: metal-based, carbon-based, and polymer-based materials. We focused on the preparation routes of emerging antiviral textiles, providing a forward-looking perspective on their opportunities and challenges, to evaluate their efficacy, scale up their manufacturing processes, and expand their high-volume applications. Based on this review, we conclude that ideal antiviral textiles are characterized by a high filtration efficiency, reliable antiviral effect, long storage life, and recyclability. The expected manufacturing processes should be economically feasible, scalable, and quickly responsive.
Purpose Given the COVID-19 Pandemic outbreak and the role of medical textiles for protection, this study aims to identify the leading research foci on using textile materials for personal protection in pandemic situations. Design/methodology/approach A systematic review and systemic analysis of the literature on the subject were performed using the process knowledge development – constructivist (ProKnow-C) methodology. Findings A bibliographic portfolio with 16 relevant studies was obtained. This portfolio represents the main focus of this research field, including the main filtration mechanisms, ways of disinfecting N95 respirators and proposed methods to evaluate the filtration efficiency of different materials with potential for mask development. Originality/value To the best of the authors’ knowledge, this is the first time the ProKnow-C methodology was used in the textile field. Thus, future studies can benefit from using the Proknow-C for selecting and analyzing relevant textile studies following a systematic approach.
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COVİD-19 hızlı bulaşma oranı ve yüzeylerde uzun süre kalabilmesi nedeni ile kısa sürede küresel bir salgına dönüştü. Hızla yayılımı özellikle belediyecilik hizmetlerinden olan Atık Yönetiminde sorunlara nedeni oldu. Tek kullanımlık maske, eldiven, gözlük gibi koruyucu ekipmanlar ve hastane yoğun bakımlarından çıkan enfekte atık miktarlarında ciddi artışlar meydana geldi. Bu durum mevcut atık bertaraf sistemleri üzerinde baskı oluşturdu. Ulusal ve uluslararası genelgeler yayınlanarak salgının yayılımı engellenmeye çalışıldı. Bu süreçte biyolojik parçalanabilir plastikler, çevresel ve ekonomik olarak uygun bertaraf yöntemleri ve uygun yönetim planlarının oluşturulmasına yönelik sistem optimizasyon çalışmaları ile veri üretme, saklama ve işleme konusunda yenilikçi çalışmalar ortaya çıktı. Bu çalışma da salgın döneminde Katı Atık Yönetiminde ortaya çıkan sorunlar, çevresel ve ekonomik etkileri, sorunların çözümüne yönelik yapılan yasal düzenlemeler, alınan tedbirler ile önerilen uygun bertaraf yöntemleri incelenmiştir. Artan nüfus ve iklim değişikliğinin uzun süreli etkileri dikkate alındığında bu salgın ilk değil, son olmayacaktır. Bu nedenle mevcut çalışmalar ışığında öneriler derlenmiştir.
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Early detection of Ebola virus (EBOV) infection is essential to halting transmission and adjudicating appropriate treatment. However, current methods rely on viral identification, and this approach can misdiagnose presymptomatic and asymptomatic individuals. In contrast, disease-driven alterations in the host transcriptome can be exploited for pathogen-specific diagnostic biomarkers. Here, we present for the first time EBOV-induced changes in circulating miRNA populations of nonhuman primates (NHPs) and humans. We retrospectively profiled longitudinally-collected plasma samples from rhesus macaques challenged via intramuscular and aerosol routes and found 36 miRNAs differentially present in both groups. Comparison of miRNA abundances to viral loads uncovered 15 highly correlated miRNAs common to EBOV-infected NHPs and humans. As proof of principle, we developed an eight-miRNA classifier that correctly categorized infection status in 64/74 (86%) human and NHP samples. The classifier identified acute infections in 27/29 (93.1%) samples and in 6/12 (50%) presymptomatic NHPs. These findings showed applicability of NHP-derived miRNAs to a human cohort, and with additional research the resulting classifiers could impact the current capability to diagnose presymptomatic and asymptomatic EBOV infections.
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Enveloped virus vaccines can be damaged by high osmotic strength solutions, such as those used to protect the vaccine antigen during drying, which contain high concentrations of sugars. We therefore studied shrinkage and activity loss of whole inactivated influenza virus in hyperosmotic solutions and used those findings to improve vaccine coating of microneedle patches for influenza vaccination. Using stopped-flow light scattering analysis, we found that the virus underwent an initial shrinkage on the order of 10% by volume within 5 s upon exposure to a hyperosmotic stress difference of 217 milliosmolarity. During this shrinkage, the virus envelope had very low osmotic water permeability (1 - 6×10-4 cm s-1) and high Arrhenius activation energy (Ea = 15.0 kcal mol-1), indicating that the water molecules diffused through the viral lipid membranes. After a quasi-stable state of approximately 20 s to 2 min, depending on the species and hypertonic osmotic strength difference of disaccharides, there was a second phase of viral shrinkage. At the highest osmotic strengths, this led to an undulating light scattering profile that appeared to be related to perturbation of the viral envelope resulting in loss of virus activity, as determined by in vitro hemagglutination measurements and in vivo immunogenicity studies in mice. Addition of carboxymethyl cellulose effectively prevented vaccine activity loss in vitro and in vivo, believed to be due to increasing the viscosity of concentrated sugar solution and thereby reducing osmotic stress during coating of microneedles. These results suggest that hyperosmotic solutions can cause biphasic shrinkage of whole inactivated influenza virus which can damage vaccine activity at high osmotic strength and that addition of a viscosity enhancer to the vaccine coating solution can prevent osmotically driven damage and thereby enable preparation of stable microneedle coating formulations for vaccination.
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Infectious micro-organisms may be transmitted by a variety of routes, and some may be spread by more than one route. Respiratory and facial protection is required for those organisms that are usually transmitted via the droplet/airborne route, or when airborne particles have been artificially created, such as during ‘aerosol-generating procedures’. A range of personal protective equipment that provides different degrees of facial and respiratory protection is available. It is apparent from the recent experiences with severe acute respiratory syndrome and pandemic (H1N1) 2009 influenza that healthcare workers may have difficulty in choosing the correct type of facial and respiratory protection in any given clinical situation. To address this issue, the Scientific Development Committee of the Healthcare Infection Society established a short-life working group to develop guidance. The guidance is based upon a review of the literature, which is published separately, and expert consensus.
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Oral immunization using whole inactivated influenza virus vaccine promises an efficient vaccination strategy. While oral vaccination was hampered by harsh gastric environment, a systematic understanding about vaccine destabilization mechanisms was not performed. Here, we investigated the separate and combined effects of temperature, retention time, pH, and osmotic stress on the stability of influenza vaccine by monitoring the time-dependent morphological change using stopped-flow light scattering. When exposed to osmotic stress, clustering of vaccine particles was enhanced in an acidic medium (pH 2.0) at ≥25°C. Fluorescence spectroscopic studies showed that hyper-osmotic stress at pH 2.0 and 37°C caused a considerable increase in conformational change of antigenic proteins compared to that in acidic iso-osmotic medium. A structural integrity of membrane was destroyed upon exposure to hyper-osmotic stress, leading to irreversible morphological change, as observed by undulation in stopped-flow light scattering intensity and transmission electron microscopy. Consistent with these analyses, hemagglutination activity decreased more significantly with an increasing magnitude of hyper-osmotic stress than in the presence of the hypo- and iso-osmotic stresses. This study shows that the magnitude and direction of the osmotic gradient has a substantial impact on the stability of orally administrated influenza vaccine.
We report that the dye nile red, 9-diethylamino-5H-benzo[alpha]phenoxazine-5-one, is an excellent vital stain for the detection of intracellular lipid droplets by fluorescence microscopy and flow cytofluorometry. The specificity of the dye for lipid droplets was assessed on cultured aortic smooth muscle cells and on cultured peritoneal macrophages that were incubated with acetylated low density lipoprotein to induce cytoplasmic lipid overloading. Better selectivity for cytoplasmic lipid droplets was obtained when the cells were viewed for yellow-gold fluorescence (excitation, 450-500 nm; emission, greater than 528 nm) rather than red fluorescence (excitation, 515-560 nm; emission, greater than 590 nm). Nile red-stained, lipid droplet-filled macrophages exhibited greater fluorescence intensity than did nile red-stained control macrophages, and the two cell populations could be differentiated and analyzed by flow cytofluorometry. Such analyses could be performed with either yellow-gold or red fluorescence, but when few lipid droplets per cell were present, the yellow-gold fluorescence was more discriminating. Nile red exhibits properties of a near-ideal lysochrome. It is strongly fluorescent, but only in the presence of a hydrophobic environment. The dye is very soluble in the lipids it is intended to show, and it does not interact with any tissue constituent except by solution. Nile red can be applied to cells in an aqueous medium, and it does not dissolve the lipids it is supposed to reveal.
Anionic and cationic N-halamine polyelectrolytes were synthesized, characterized and then immobilized onto melt-blown polypropylene fabrics having two different basis-weights. The coatings were rendered biocidal upon exposure to dilute sodium hypochlorite solution. The effect of single and multilayer deposition of the polyelectrolytes on the surfaces was investigated in terms of chlorine loadings, rechargeabilities, antimicrobial efficacies, and air permeabilities. It was found that all of the coatings provided remarkable biocidal efficacies with about six log reductions of bacteria within two min of contact time on filters having higher basis-weight, whereas slower inactivation was observed for lower-basis weight filters due to diminished surface areas and numbers of active halogen atoms. The antimicrobial coatings reduced the air permeabilities of the filters somewhat; however, the air permeabilities of the coated swatches were comparable to those of most protective textiles.
Infectious micro-organisms may be transmitted by a variety of routes. This is dependent on the particular pathogen and includes bloodborne, droplet, airborne, and contact transmission. Some micro-organisms are spread by more than one route. Respiratory and facial protection is required for those organisms which are usually transmitted via the droplet and/or airborne routes or when airborne particles have been created during 'aerosol-generating procedures'. This article presents a critical review of the recently published literature in this area that was undertaken by Health Protection Scotland and the Healthcare Infection Society and which informed the development of guidance on the use of respiratory and facial protection equipment by healthcare workers.
Crosslinked polyacrylamide (PAM) was durably immobilized onto polypropylene (PP) by forming a surface thermoplastic semi-interpenetrating network (IPN). Upon conversion of the immobilized amide to N-halamine, the PP substrate was imparted with durable and potent antibacterial activity. The successful modification was limited to one side of the PP fabric only as confirmed by XPS. After the surface modification, surface morphology of the fiber remained relatively unchanged, with only a slight increase in fiber diameter. When the immobilization percentage (IP) was less than 2.5%, a decrease of less than 6.6% in air permeability of the fabric was found. The tensile strength of the fabrics after the modification was well retained and even showed significant improvement as the IP exceeded 2.5%. After a sharp decrease in the first 4 regeneration cycles, the amount of N-halamine on the chlorinated polyacrylamide modified PP fabric leveled off up to 25 regeneration cycles. Even after 25 regeneration cycles, the chlorinated PAM–PP–D fabric was still able to result in 100% reduction of HA-MRSA in 30min contact.
Immunization using a microneedle patch coated with vaccine offers the promise of simplified vaccination logistics and increased vaccine immunogenicity. This study examined the stability of influenza vaccine during the microneedle coating process, with a focus on the role of coating formulation excipients. Thick, uniform coatings were obtained using coating formulations containing a viscosity enhancer and surfactant, but these formulations retained little functional vaccine hemagglutinin (HA) activity after coating. Vaccine coating in a trehalose-only formulation retained about 40 - 50% of vaccine activity, which is a significant improvement. The partial viral activity loss observed in the trehalose-only formulation was hypothesized to come from osmotic pressure-induced vaccine destabilization. We found that inclusion of a viscosity enhancer, carboxymethyl cellulose, overcame this effect and retained full vaccine activity on both washed and plasma-cleaned titanium surfaces. The addition of polymeric surfactant, Lutrol® micro 68, to the trehalose formulation generated phase transformations of the vaccine coating, such as crystallization and phase separation, which was correlated to additional vaccine activity loss, especially when coating on hydrophilic, plasma-cleaned titanium. Again, the addition of a viscosity enhancer suppressed the surfactant-induced phase transformations during drying, which was confirmed by in vivo assessment of antibody response and survival rate after immunization in mice. We conclude that trehalose and a viscosity enhancer are beneficial coating excipients, but the inclusion of surfactant is detrimental to vaccine stability.