key: cord-0798773-9dsrzfsa
authors: Machado, Rafael R. G.; Glaser, Talita; Araujo, Danielle B.; Petiz, Lyvia Lintzmaier; Oliveira, Danielle B. L.; Durigon, Giuliana S.; Leal, Alessandra L.; Pinho, João Renato R.; Ferreira, Luis Carlos S.; Ulrich, Henning; Durigon, Edison L.; Guzzo, Cristiane R.
title: Hypertonic saline solution inhibits SARS-CoV-2 in vitro assay
date: 2020-08-06
journal: bioRxiv
DOI: 10.1101/2020.08.04.235549
sha: 88ec6e033489067bbc22e1c0f5c43a4f78f845f6
doc_id: 798773
cord_uid: 9dsrzfsa

We are facing an unprecedented global health crisis caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). At this date more than 680 thousand people have died due to coronavirus disease 2019 (COVID-19). Unfortunately, until now no effective treatment to combat the virus and vaccine are available. We performed experiments to test if hypertonic saline solution is able to inhibit virus replication in vitro. Our data shows that 260 mM NaCl (1.5%) inhibits 100% SARS-CoV-2 replication in Vero cells. Furthermore, our results suggest that the virus replication inhibition is due to an intracellular mechanism and not due to the dissociation between spike SARS-CoV-2 protein and its human receptor angiotensin-converting enzyme 2 interaction. NaCl depolarizes the plasma membrane supposedly associated with the inhibition of the SARS-CoV-2 life cycle. This observation could lead to simple, safe and low cost interventions at various stages of COVID-19 treatment, improving the prognosis of infected patients, thereby mitigating the social and economic costs of the pandemic.

The world is facing a pandemic situation due to the pathogenic SARS-COV-2 (Severe Acute Respiratory Syndrome Coronavirus 2) 1,2 , a zoonotic virus, that spread from China to many countries around the world in less than 3 months. So far there were more than 17 million confirmed patients in 213 countries, and more than 680,000 deaths due to COVID-19 disease, according to WHO data as of July 26, 2020 (https://covid19.who.int). Few medical procedures are available, which could reduce viral load, decreasing patient hospitalization time, virus dissemination and the number of patients with severe symptoms. SARS-CoV-2 uses a surface glycosylated spike (S) protein to bind human angiotensin-converting enzyme 2 (ACE-2) 3 , CD147 4 and sialic acid receptors 5 and to develop coronavirus disease . In the case of SARS-CoV, that shares 76% of identity with SARS-CoV-2 S protein, it has been suggested that the virus may also bind to host cells through DC-SIGN or L-SIGN receptors 6, 7 . SARS-CoV-2 and ACE-2 interaction is essential to mediate virus infection 8 . The S protein has two main subunits, the S1 subunit involved in binding to the host cellular receptor, and the S2 subunit that mediates fusion of the viral and cellular membranes. Initially, the homotrimeric S protein via its receptor binding domain (RBD) located at the S1 subunit, binds to human ACE-2 (hACE-2), causing a conformational change in the S1 and S2 subunits leading to the fusion of virus membrane with the host cell membrane 9, 10 .

The SARS-CoV-2 S protein structure has been recently solved in the apo form 11 and in complex with ACE-2 protein 12 . Interestingly, the interface between the RBD of S protein with hACE-2 is highly polar, involving charged residues located mainly at the hACE-2 interface and polar residues at the RBD interface 12 . These ionic interactions could in principle be affected by increasing the ionic strength and could be used to inhibit the interaction of the virus with the host cell. Moreover, the potential antiviral activity, in vitro, of sodium chloride (NaCl) has been already studied for RNA viruses, such as mengovirus 13 , respiratory syncytial virus, influenza A virus, human coronavirus 229E and coxsackievirus B3, and also for DNA virus, like herpes simplex virus-1 and murine gammaherpesvirus 68 14 . Furthermore, a randomised controlled clinical trial studying the effectiveness of the treatment for viral upper respiratory tract infection, using hypertonic saline nasal irrigation and gargle (HSNIG) 15 , observed a decrease in the duration of illness, over-the-counter medications use, transmission within household contacts and viral shedding 15 .

However, the molecular details of the antiviral NaCl activity remains unresolved.

In order to test our initial hypothesis, we performed different assays to determine if different concentrations of NaCl affect the SARS-CoV-2 replication, an RNA enveloped virus, when cultured in monkey kidney epithelial cells (Vero CCL-81), and we also evaluated the effect of NaCl in the membrane potential.

We performed experiments to determine if hypertonic saline solution is able to inhibit SARS-CoV-2 replication in vitro. We measured the effects of increasing concentrations of NaCl (135, 160, 185, 210, 235, 260 and 285 mM equivalent to 0.8, 0.9, 1.1, 1.2, 1.4, 1.5, 1.7%, respectively) on cell viability and their antiviral activity on SARS-CoV-2. Efficacies were evaluated by quantification of viral copy numbers in the cell supernatant via quantitative real-time RT-PCR (RT-qPCR) and confirmed with visualization of cytopathic effect by optical microscopy at 72 hours post infection (h.p.i.). Our data shows that 210 mM NaCl (1.2%) was sufficient to inhibit the virus replication by 90% (Fig. 1a) , achieving 100% of inhibition on 260mM (1.5%). Next, to determine which stage of virus replication was affected by the NaCl, we evaluated if viral inhibition was a direct effect of NaCl on the virus particles, for that SARS-CoV-2 was pre-incubated with media and increasing concentrations of NaCl for 1 hour before absorption. Virus pre-exposure to NaCl did not affect viral replication at any concentration of NaCl tested (Fig. 1a , VPI curve). The same pattern of absence of inhibition was observed when we treated the cells one hour before infection, analyzing the adsorption process, which consists of the interaction of virus with cell receptors (Fig. 1a, AD curve) . On the other hand, significant inhibition of viral replication (up to 50%) was seen when as little as 160 mM of NaCl was available during virus replication alone (Fig. 1a , p = 0.032, PI curve) or full-time, during adsorption and replication (Fig. 1a , p = 0.030, FT curve). There was no statistically significant difference between post-infection (PI) and adsorption plus post-infection (FT) treatments (p = 0.985). These data together suggest that SARS-CoV-2 inhibition in the presence of NaCl was an intracellular mechanism and was not due to the dissociation of spike SARS-CoV-2 and human angiotensin-converting enzyme 2 (ACE-2) complexes.

The antiviral activity of NaCl was not caused by cytotoxicity in the Vero cell line, as determined by the AlamarBlue™ Cell Viability Reagent (Fig. 1b) and LDH assay (Fig. 1c) . Furthermore, we performed a cell death experiment to confirm the integrity of cells after being exposed to NaCl in the same conditions performed with the presence of the virus. We observed less than 8% of death at all concentrations tested (Fig. 1d) . Related to NaCl cytotoxicity, we observed less than 20% of cytotoxicity (cell viability up to 80%) at all concentrations tested (Fig 1b, 1c and 1d) . 

Additionally, we conducted experiments to measure the hyperosmotic stress effect on cell membrane. Vero cells were incubated with different NaCl concentrations (135, 160, 185, 210, 235, 260 and 285 mM equivalent to 0.8, 0.9, 1.1, 1.2, 1.4, 1.5, 1.7%, respectively), and cell membrane potential was measured at 1, 24 and 72h after incubation (Fig. 2a) . Results are indicated as relative fluorescence units (RFU), whose increased values refers to membrane depolarization. Cell membranes showed a rapid depolarization upon stimulation with increasing NaCl concentration (Fig. 2b) . Depolarization was dose-dependent, with statistically difference starting at 160 mM of NaCl. The results of the 24 hours after incubation showed that the depolarization of the membrane continues to increase with time, since the values are greater than those at the time of 1 hour. Membrane potential tended to return to control values at 72h, indicating that Vero cells were partially able to recover their resting membrane potential. one-way ANOVA (*p ≤ 0.05). The increasing NaCl concentration caused an immediate membrane depolarization that was maximal after 24h and tends to return to control values after 72h of treatment. b: The increasing NaCl concentration causes an immediate membrane depolarization in NaCl concentrations above 160 mM. The depolarization for cells treated with up to 210 mM NaCl tends to restore to the resting point after 70 seconds.

Our data show the efficiency of hypertonic NaCl solutions in blocking SARS-CoV-2 replication in Vero cells. This data suggests that hypertonic solution can be used as a prophylaxis and an alternative treatment for COVID-19 patients.

Nevertheless, clinical trials must be done to prove the efficacy of the treatment in humans. In order to understand the reason to explain the virus inhibition by NaCl, we performed membrane potential assays that also show clearly a direct relation with membrane depolarization. The SARS-CoV-2 inhibition assays were performed using monkey kidney cells and as lung epithelial cells, may be heavily infected by SARS-CoV-2 16 and are capable of undergoing endo-and exocytosis, being therefore a good model cell to study the SARS-CoV-2 life cycle. Vesicle formation is important for kidney and lung cells as responses to alteration of osmosis, regulated by aquaporin channels 17 , as well as for vesicle transfer between cells, which has been suggested to contribute to lung disease and possibly to the propagation of the SARS-CoV-2 virus into neighboring cells 18 .

We postulate here the importance of a hyperosmotic response by high extracellular salt concentration on the expression of aquaporin channels, which would limit endocytosis and thereby inhibit entrance of SARS-CoV-2 into cells.

Endocytic, clathrin-independent, lipid-raft involving virus entry has been described for SARS-CoV 19 , supporting our proposed mechanism of endocytosis participation in SARS-CoV-2 entry. During SARS-CoV-2 infection, pulmonary edema and cytokine storm are responsible for the most lethal scenarios 20 . The increased amount of liquid blocking the airway at the alveoli causes extreme injury, and a scar fibrotic tissue substitutes the lesioned tissue (Fig. 3a) . Along the embryonic pulmonary development, the organ is full of liquid, which is removed mainly by the action of sodium channels such as epithelial sodium (Na + ) channels (ENaC) that are sensitive to amiloride [21] [22] [23] [24] . These ENaC are expressed mainly by epithelial cells of many organs, such as kidney, lungs, colon, skin, and by some neurons in the brain 21 . In adulthood ENaC usually controls the concentration of Na + ions in the extracellular environment. Therefore, the channels are extremely important for the organism water balance, by controlling the reabsorption of Na + in kidneys, sweat and colon. In this way, the activity of ENaC can influence blood pressure and the renin-angiotensin system, as well as the thickness and dryness of the airway 21 . Interestingly, this channel is also involved in the taste sensation and its improper functioning could be related to loss of taste 21 .

The data we obtained about the membrane potential points to a possible explanation about the inhibition mechanism of SARS-CoV-2 infection in Vero cells by extracellular increased concentration of NaCl. Various pulmonary virus infections impair the activity of ENaC, and the increased activation state of these channels can significantly decrease the influenza A infection [25] [26] [27] . Additionally, the spike and envelope small membrane protein (E) of SARS-CoV can directly decrease the inward current of ENaC as well as decrease its expression through the PKC signaling pathway 28 . Impairment of ENac activity itself would be enough to explain the pulmonary edema in the alveoli, since its inhibition would increase the amount of Na + in the airway, and water would flow by osmosis 29 .

Membrane potential has been shown to be important for some virus entry, and in the case of human Rhinovirus Type 2 infection the membrane hyperpolarization enhances infection 30 . Inhibition of membrane depolarization is part of viral lung infection strategy, as shown for SARS-CoV 21 and respiratory syncytial virus 31 , whose cell infection inhibits the entry of Na + into the cell. While the ENac is voltage-insensitive, the gradient between high and low Na + (out-and inside of the cell) forces the entry of Na + , being an unfavorable condition for SARS-CoV-2 infection.

Our results show that the increase of NaCl concentration causes an immediate membrane depolarization, probably through the activation of an extracellular Na + sensitive channel, called Na X

. The activation of Na x leads to Na + inward flow, causing the depolarization, which in turn can open the voltage gated Ca 2+ channels Cav2.1 that are expressed at the apical membrane of epithelial cells 33 . Moreover, Na x activation upregulates prostasin (protease) release into the extracellular space activating then ENaC by cleaving the extracellular loop of the γ ENaC subunit [34] [35] [36] . Following Na + influx via ENaC, the cells also present downstream mRNA synthesis elevation of inflammatory mediators 36 . In the same line, Na x expression blockade can improve scarring 36 . Therefore, extracellular Na + imbalance through ENac inefficiency by SARS-CoV-2 infection may explain the formation of pulmonary fibrosis due to virus infection We observed mainly two patterns of depolarization: (1) lower doses of NaCl concentration triggered depolarization and after 180s the cell membrane reestablished the resting state, causing a timid inhibition of the virus release, while (2) doses higher than 210 mM could trigger a high amplitude depolarization, in which the cells needed periods longer than 24h to recover the resting state, causing over 90% inhibition of the virus. In the first situation, we hypothesize that activation of Ca 2+ sensitive K + channels and Na + /K + ATPase pump could recover the steady state, while in the second situation, we hypothesize that the over stimulation of Na x , ENaC and Ca2.1, would overwhelm the cell, consume the ATP production, thus stressing the mitochondria. Two types of Ca 2+ -dependent K + channels, small (SK) and big (BK) conductance, can be responsible for Ca 2+ -activated outward K + currents. While SK channels are expressed especially in the central nervous system and heart, being responsible for the afterhyperpolarization of excitable cells, BK channels are broadly expressed in mammalian cells. These two K + channel subtypes differ mainly in their single channel conductance, which is in the order of 10 pS for SK channels and 150-250 pS for BK channels 37 . This large K + conductance makes BK channels important for cell membrane repolarization, since BK channels are activated by membrane depolarization and/or high cytosolic Ca 2+ concentration ([Ca 2+ ] i ).

Literature reports that BK channels are activated in Vero cells during hypotonic stress, where cells are submitted to a 45% less NaCl environment, in which BK channels were activated by Ca 2+ released by intracellular stores 38 . Here, membrane potential assays showed that Vero cells were able to cope with high NaCl concentrations and recover its resting membrane potential, probably due to activation of BK channels, which would activate either by the observed depolarization, or by the Ca 2+ inward flow that probably occurred.

Our results showed that SARS-CoV-2 inhibition effects occurs at the onset of hypertonic stress, since the viral inhibition did not change when Vero cells had been only pre-treated with NaCl (Fig 1a, AD curve) . Mitochondrial membrane potential can also be determinant for antiviral cell responses. In human embryonic kidney (HEK) and mouse embryonic kidney (MEF) cells, the application of mitochondrial uncoupler CCCP, which dissipates the transmembrane potential through the increase of membrane permeability, caused an impaired antiviral response by the cell 39 . This did not occur for the F 1 F O ATP synthase inhibitor oligomycin B, indicating that the diminished antiviral effect observed was associated with mitochondrial membrane depolarization, but not with impaired ATP synthesis alone. Our hypothesis shown in Fig. 3 b is that the overflow of Na + in Vero cells, followed by increased [Ca 2+ ] i , would cause an extreme low energy state (high ADP/ATP ratio), leading to impaired virus replication. Another possible effect is that the increased concentration of intracellular Na + that causes membrane depolarization results in a decreased intracellular K + concentration due to restoring the membrane potential. Thus, the imbalance of the intracellular K + concentration can affect the functioning of different potassium channels that are important for the life cycle of some viruses, as in the case of HIV 40 .

It is known that hyperosmotic stress induced by only 30 mM above isotonic concentration, resulting in 150 mM NaCl, dissipates mitochondrial membrane potential within minutes, whereas the addition of 100 mM NaCl (220 mM NaCl final concentration) causes instant mitochondrial depolarization in Vero cells, with no detectable cytochrome c release to the cytoplasm 41 . In addition, high intracellular Na + concentrations stimulate ion active transport through Na + /K + ATPase, and an hyperosmotic stress caused by NaCl (and not sorbitol or mannitol) causes an increase of the pump activity, since elevated chloride levels specifically increases the expression of the Na + /K + ATPase γ -subunit 42 .

In our study, mitochondrial depolarization combined with the elevated activity of this ATP-consuming pump probably resulted in an extremely low energetic state, with increased ADP/ATP ratio, which would compromise viral replication. Besides the energetic state, mitochondria are associated with other cellular functions that can be interesting for viral infection. In A549 cells, a cell line often used as a model of type II pulmonary epithelial cells, the SARS-CoV ORF-9b protein localizes to mitochondria and reduces the levels of DRP1, a GTPase that regulates mitochondrial fission. This resulted in enhanced fusion and elongated mitochondria compared to control cells, which led to an impaired innate immune system signaling, necessary for viral replication 43 44 . In virus-host interactions, viruses can use different calcium-binding proteins of different cell compartments, including cytoplasm and endoplasmic reticulum, such as calmodulin, proteins of the S100 family and calnexin, and a disturbance in these metabolic processes would be detrimental for viral replication.

Since NaCl decreases viral replication by more than 90%, probably due to a mechanism associated with the effect of membrane depolarization, our results suggest that tests on humans should be carried out to validate the effectiveness of hypertonic solution treatment in patients with COVID-19. This treatment could be effective for either those, who are hospitalized, or as prophylaxis treatment for nonhospitalized cases. It is worth to mention that this procedure is already used to improve lung function in cystic fibrosis patients 45 and nebulized 3% hypertonic saline treatment for infants with moderate to severe bronchiolitis is safe without adverse events such as bronchospasm, cough or wheezing aggravation [46] [47] [48] . However, this safe protocol has not, to our knowledge, been used to treat COVID-19 patients. The 

In summary, hypertonic saline solution inhibits SARS-CoV-2 virus replication in Vero cells due to perturbation in one or several steps of the virus intracellular cycle. NaCl is known to drive type I interferon signalling in macrophages, and that elevated concentrations of NaCl enhance the production of HOCl in non-myeloid cells 14, 50 . Hypertonic saline solution decreased Respiratory syncytial virus infection and pro-inflammatory response such as IL-6 and IL-8 release in cultures of human respiratory epithelial lines 51 . Therefore, the complete molecular mechanism underlying the observed phenomenon may be more complex, involving several factors that together result in the inhibition of viral infection. Therefore, further assays must be done to elucidate the real mechanism of virus inhibition by high concentrations of NaCl. Hypothesis for a possible mechanism involving the effect of NaCl in the inhibition of SARS-CoV-2 replication in Vero cells. Hypertonic saline solution causes membrane depolarization and an overflow of Na + in cells, followed by increased cytosolic Ca 2+ causing low energy state (high ADP/ATP ratio), leading to impaired virus replication..

Hyperosmotic extracellular NaCl concentrations activate Na x channels, a sodiumsensitive (but not voltage-sensitive) channel, which is critically involved in body-fluid homeostasis. High cytoplasmic Na + can recruit epithelial sodium (Na + ) channels (ENaC), further increasing intracellular Na + concentration, causing cell membrane to depolarize and to open voltage-gated calcium channels Cav2.1. The membrane depolarization and inward Ca 2+ current would cause impairment in mitochondrial function, which combined to Na + /K + ATPase high activity -due to cell attempt to restore rest membrane potential -results in increased ADP/ATP ratio. This low energetic state would be detrimental for viral replication. Since cells with time do restore the rest membrane potential, large conductance K + channels (BK channels), that are both calcium and voltage-gated, would cause an outward K + current, reestablishing rest membrane potential. Thus, the imbalance of the intracellular K + concentration may also affect the functioning of different potassium channels that may be important for the virus life cycle. The illustration was created with the webbased tool BioRender (https://biorender.com). Nucleic acid extraction and quantitative real-time RT-PCR (RT-qPCR). In order to perform the quantification of SARS-CoV-2 viral load, the extraction of total nucleic acid (RNA and DNA) from the collected cell culture supernatant was carried out, using the semi-automated NucliSENS® easyMag® platform (BioMerieux, Lyon, France), following the manufacturer's' instructions. The quantification of viral RNA was done using the AgPath-ID One-Step RT-PCR Kit (Applied Biosystems, Weiterstadt, Germany) on an ABI 7500 SDS real-time PCR machine (Applied Biosystems) using a reference published sequence of primers and probe for E gene 56 . Analysis of membrane potential variation by microfluorimetry. Changes of the membrane potential of Vero cells exposed to increasing concentrations of NaCl was determined by plate microfluorimetry recordings with the FlexStation III microplate reader and the FLIPR Membrane Potential Assay Kit (Molecular Devices Corp., Sunnyvale, CA) following the manufactures' instructions, as previously described 59 .

The kit provides results in good correlation with those obtained in patch-clamping assays. Recordings of the fluorescence intensity of 5x10 4 

A novel coronavirus from patients with pneumonia in China

A pneumonia outbreak associated with a new coronavirus of probable bat origin

Functional assessment of cell entry and receptor usage for SARS-CoV-2 and other lineage B betacoronaviruses

SARS-CoV-2 invades host cells via a novel route: CD147-spike protein

In-Silico evidence for two receptors based strategy of SARS-CoV-2

CD209L (L-SIGN) is a receptor for severe acute respiratory syndrome coronavirus

pH-dependent entry of severe acute respiratory syndrome coronavirus is mediated by the spike glycoprotein and enhanced by dendritic cell transfer through DC-SIGN

SARS-CoV-2 Cell Entry Depends on ACE2 and TMPRSS2 and Is Blocked by a Clinically Proven Protease Inhibitor

Structure, Function, and Evolution of Coronavirus Spike Proteins

The coronavirus spike protein is a class I virus fusion protein: structural and functional characterization of the fusion core complex

Cryo-EM structure of the 2019-nCoV spike in the prefusion conformation

Structure of the SARS-CoV-2 spike receptor-binding domain bound to the ACE2 receptor

Effect of several inorganic salts on infectivity of Mengo virus

Antiviral innate immune response in non-myeloid cells is augmented by chloride ions via an increase in intracellular hypochlorous acid levels

A pilot, open labelled, randomised controlled trial of hypertonic saline nasal irrigation and gargling for the common cold

Long Term Culture of Human Kidney Proximal Tubule Epithelial Cells Maintains Lineage Functions and Serves as an Ex vivo Model for Coronavirus Associated Kidney Injury

Targeting the Trafficking of Kidney Water Channels for Therapeutic Benefit

Extracellular vesicles: novel communicators in lung diseases

SARS coronavirus entry into host cells through a novel clathrin-and caveolae-independent endocytic pathway

The pathogenesis and treatment of the 'Cytokine Storm'' in COVID-19

SARS-CoV proteins decrease levels and activity of human ENaC via activation of distinct PKC isoforms

Early death due to defective neonatal lung liquid clearance in alpha-ENaC-deficient mice

Invited review: biophysical properties of sodium channels in lung alveolar epithelial cells

Sodium channels in alveolar epithelial cells: molecular characterization, biophysical properties, and physiological significance

Modulation of influenza virus replication by alteration of sodium ion transport and protein kinase C activity

Influenza virus inhibits amiloride-sensitive Na+ channels in respiratory epithelia

Influenza influences ion channels

SARS-CoV proteins decrease levels and activity of human ENaC via activation of distinct PKC isoforms

Viral dependence on cellular ion channels -an emerging anti-viral target?

Human Rhinovirus Type 2 Uncoating at the Plasma Membrane Is Not Affected by a pH Gradient but Is Affected by the Membrane Potential

Respiratory syncytial virus inhibits lung epithelial Na+ channels by upregulating inducible nitric-oxide synthase

Sodium channel Nax is a regulator in epithelial sodium homeostasis

Quantitative localization of Cav2.1 (P/Q-type) voltage-dependent calcium channels in Purkinje cells: somatodendritic gradient and distinct somatic 20

coclustering with calcium-activated potassium channels

Urinary serine proteases and activation of ENaC in kidney--implications for physiological renal salt handling and hypertensive disorders with albuminuria

Epithelial Na+ channels are fully activated by furin-and prostasindependent release of an inhibitory peptide from the gamma-subunit

Na(+) homeostasis by epithelial Na(+) channel (ENaC) and Nax channel (Nax): cooperation of ENaC and Nax

Endothelium as target for large-conductance calcium-activated potassium channel openers

Purinergic activation of BK channels in clonal kidney cells (Vero cells)

Mitochondrial membrane potential is required for MAVS-mediated antiviral signaling

G protein-coupled and ATP-sensitive inwardly rectifying potassium ion channels are essential for HIV entry

Hypertonic shock inhibits growth factor receptor signaling, induces caspase-3 activation, and causes reversible fragmentation of the mitochondrial network

Chloride, not sodium, stimulates expression of the γ subunit of Na/K-ATPase and activates JNK in response to hypertonicity in mouse IMCD3 cells

SARS-Coronavirus Open Reading Frame-9b Suppresses Innate Immunity by Targeting Mitochondria and the MAVS/TRAF3/TRAF6 Signalosome

Viral calciomics: Interplays between Ca2+ and virus

A controlled trial of long-term inhaled hypertonic saline in patients with cystic fibrosis

Nebulised hypertonic saline solution for acute bronchiolitis in infants

Nebulized hypertonic saline treatment in hospitalized children with moderate to severe viral bronchiolitis

Saline nasal irrigation for upper respiratory conditions

Hypertonic saline nasal irrigations and gargling should be considered as a treatment option for COVID-19

Elevated sodium chloride drives type I interferon signaling in macrophages and increases antiviral resistance

Effect of Hypertonic Saline Solution on cultures of human respiratory epithelium infected by respiratory syncytial virus

SARS-CoV-2 isolation from the first reported patients in Brazil and establishment of a coordinated task network

Laboratory biosafety guidance related to the novel coronavirus ( 2019-nCoV )

Tests for in vitro cytotoxicity

A flow cytometric method using Hoechst 33342 and propidium iodide for simultaneous cell cycle analysis and apoptosis determination in unfixed cells

Detection of 2019 novel coronavirus (2019-nCoV) by real-time RT-PCR

Identification of a novel coronavirus in patients with severe acute respiratory syndrome

Remdesivir and chloroquine effectively inhibit the recently emerged novel coronavirus (2019-nCoV) in vitro

ATP and spontaneous calcium oscillations control neural stem cell fate determination in Huntington's disease: a novel approach for cell clock research