ArticlePDF Available

D, L-lysine acetylsalicylate + glycine Impairs Coronavirus Replication

Authors:

Abstract and Figures

Coronaviruses (CoV) belong to the large family Coronaviridae within the order of Nidovirales. Among them, several human pathogenic strains (HCoV) are known to mainly cause respiratory diseases. While most strains contribute to common cold-like illnesses others lead to severe infections. Most prominent representatives are SARS-CoV and MERS-CoV, which can lead to fatal infections with around 10% and 39% mortality, respectively. This resulted in 8098 casualties in the 2002/2003 SARS-CoV outbreak and in 1806 documented human infections (September 2016) during the recent ongoing MERS-CoV outbreak in Saudi Arabia. Currently patients receive treatment focusing on the symptoms connected to the disease rather than addressing the virus as the cause. Therefore, additional treatment options are urgently needed which would ideally be widely available and show a broad affectivity against different human CoVs. Here we show that D, L-lysine acetylsalicylate + glycine sold as " Asprin i.v. 500mg® " (LASAG), which is an approved drug inter alia in the treatment of acute pain, migraine and fever, impairs propagation of different CoV including the highly-pathogenic MERS-CoV in vitro. We demonstrate that the LASAG-dependent impact on virus-induced NF-κB activity coincides with (i) reduced viral titres, (ii) decreased viral protein accumulation and viral RNA synthesis and (iii) impaired formation of viral replication transcription complexes.
Content may be subject to copyright.
D, L-lysine acetylsalicylate + glycine Impairs Coronavirus Replication
Christin Müller, Nadja Karl, John Ziebuhr and Stephan Pleschka*
Institute of Medical Virology, Justus Liebig University Giessen, Schubertstr 81, 35392 Giessen, Germany
*Corresponding author: Stephan Pleschka, Institute of Medical Virology, Schubertstr. 81, 35392 Giessen, Germany, Tel: 0049 (0)641-99-47750; Fax: 0049
(0)641-99-41209; E-mail: stephan.pleschka@viro.med.uni-giessen.de
Received date: December 05, 2016, Accepted date: December 20, 2016, Published date: December 30, 2016
Copyright: © 2016 Müller C, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted
use, distribution, and reproduction in any medium, provided the original author and source are credited.
Abstract
Coronaviruses (CoV) belong to the large family Coronaviridae within the order of Nidovirales. Among them,
several human pathogenic strains (HCoV) are known to mainly cause respiratory diseases. While most strains
contribute to common cold-like illnesses others lead to severe infections. Most prominent representatives are SARS-
CoV and MERS-CoV, which can lead to fatal infections with around 10% and 39% mortality, respectively. This
resulted in 8098 casualties in the 2002/2003 SARS-CoV outbreak and in 1806 documented human infections
(September 2016) during the recent on-going MERS-CoV outbreak in Saudi Arabia. Currently patients receive
treatment focusing on the symptoms connected to the disease rather than addressing the virus as the cause.
Therefore, additional treatment options are urgently needed which would ideally be widely available and show a
broad affectivity against different human CoVs. Here we show that D, L-lysine acetylsalicylate + glycine sold as
“Asprin i.v. 500mg®” (LASAG), which is an approved drug inter alia in the treatment of acute pain, migraine and
fever, impairs propagation of different CoV including the highly-pathogenic MERS-CoV in vitro. We demonstrate that
the LASAG-dependent impact on virus-induced NF-κB activity coincides with (i) reduced viral titres, (ii) decreased
viral protein accumulation and viral RNA synthesis and (iii) impaired formation of viral replication transcription
complexes.
Keywords: Coronavirus replication; D, L-lysine acetylsalicylate
+glycine; NF-κB inhibition; Replication/Transcription Complexes
Introduction
Coronaviruses (CoV) are enveloped, positive-strand RNA viruses
belonging to the family of
Coronaviridae
[1,2] and are known to infect
mammals and birds. Prior to 2003 it was believed that circulating
human coronaviruses (HCoV), such as HCoV-OC43 [3,4] and
HCoV-229E [5], cause low-pathogenic infections of the upper
respiratory tract and were not recognized as a signicant threat to
human health. is changed, aer the emerging Severe Acute
Respiratory Syndrome-Coronavirus (SARS-CoV) caused an epidemic
outbreak in 2002 in Asia, Canada, the U.S. and Europe [6], which
resulted in 8098 laboratory-conrmed cases, including 774 deaths
(average mortality rate 10%) [6-9]. In 2013, a new human pathogenic
CoV with zoonotic origin was discovered in Saudi Arabia: the Middle
East Respiratory Syndrome coronavirus (MERS-CoV) causing SARS-
like symptoms including lethal pneumonia [10] and renal dysfunction
up to complete failure [11-13]. Since September 2012 1806 conrmed
cases have been reported of whom 643 died [14].
Against both highly pathogenic emerging viruses no specic anti-
viral treatment or vaccination is currently available and treatment of
infected individuals presently only aims at relieving symptoms [15,16].
In cell culture, Type 1 Interferon (IFN-1) inhibits MERS-CoV as well
as SARS-CoV replication [17-19]. IFN-1 is also used as potential
treatment of other +ssRNA viruses [20], but beside high costs it shows
massive side eects. Several other drugs were also shown to impair
MERS-CoV replication in cell culture including cyclosporine,
chloroquine, chlorpromazine, loperamide and lopinavir [21-23].
Whether these drugs will be useful in future treatment of HCoV
infections remains to be investigated.
Targeting CoV proteins [24,25] is always prone to lead to the
emergence of resistant variants/escape-mutants due to the high
mutation rate of RNA viruses [26] including CoV [27]. As all viruses
depend on their host cell for their replication, alternative anti-viral
strategies target host functions or factors. is approach has a low
potential to lead to viral resistance and provides the possibility of
broad-spectrum anti-viral applications.
For many years acetylsalicylic acid (ASA) has been therapeutically
used as an analgesic, anti-pyretic, anti-rheumatic and also as a non-
steroidal anti-inammatory drug (NSIAD). It can also slow down the
progression of Alzheimer’s disease and prevent colon cancer [28-30].
However, ASA is only soluble to a limited extent and the rate of bio-
absorption is thus limited. By using salts of ASA with basic amino
acids, the dissolution rate of the active compound itself can be
increased and high blood concentrations of the active compound can
be achieved [31]. D, L-lysine Acetylsalicylate (LASA) is the water
soluble salt of ASA and the amino acid lysine and due to its improved
solubility in water (compared to ASA) it is faster acting and can not
only be applied orally, but also intravenously. A further disadvantage of
the o-acetylsalicylate was their inadequate stability. e addition of
glycine in the production process of D, L-lysine acetylsalicylated +
glycine (LASAG) resulted in a stable active compound complex of salts
of o-acetylsalicylic acid with basic amino acids and glycine [31].
ASA as well as other salicylates are able to block the activation of
NF-κB [32,33] via inhibition of IKK2 in a low millimolar range [34].
e transcription factor NF-κB is essential for cell responses to
infection with various pathogens. It controls expression of a variety of
anti-viral cytokines and it regulates apoptotic gene expression.
erefore, it is considered to be a main mediator of the anti-viral cell
response to viral infection [35]. Besides the wide usage of ASA in pain
therapy, it was shown that ASA-mediated inhibition of the NF-κB-
Journal of Antivirals &
Antiretrovirals Müller et al., J Antivir Antiretrovir 2016, 8:4
DOI:10.4172/jaa.1000151
Research Article Open Access
J Antivir Antiretrovir
ISSN:1948-5964 ,an open access journal Volume 8(4): 142-150 (2016) - 142
J
o
u
r
n
a
l
o
f
A
n
t
i
v
i
r
a
l
s
&
A
n
t
i
r
e
t
r
o
v
i
r
a
l
s
ISSN: 1948-5964
dependent induction of TRAIL and Fas/FasL, reduces inuenza virus
propagation [36-38]. ASA also displays anti-viral activity against
cytomegalovirus [39] and human rhinoviruses [40].
As SARS-CoV was shown to activate NF-κB in the lung tissue of
infected mice and NF-κB-inhibition (using CAPE, resveratrol,
BAY11-7082 or parthenolide) improved survival rates of SARS-CoV-
infected mice [41], we speculated that NF-κB activity might be
important for ecient CoV propagation. We therefore investigated
whether NF-κB inhibition via the clinically tested and approved
LASAG could negatively aect CoV replication. is hypothesis was
analysed by LASAG treatment of cells infected with the low pathogenic
human strain HCoV-229E as well as the high pathogenic MERS-CoV.
Our results could demonstrate that LASAG interferes with virus-
induced NF-κB activation, the formation of viral replication
transcription complexes, viral RNA synthesis and viral protein
accumulation resulting in an overall impaired CoV propagation.
Materials and Methods
Cells and viruses
Huh7 cells (human hepatocellular carcinoma cells) were maintained
in complete Dulbecco’s modied Eagles medium (DMEM, Gibco Life
Technologies, UK) supplemented with 10% foetal calf serum (FCS)
and antibiotics (100 U/ml penicillin, 0.1 mg/ml streptomycin (P/S)).
Conuent cells were infected with human coronavirus 229E
(HCoV-229E, strain collection of the Institute of Medical Virology,
Giessen, Germany) and MERS-CoV (EMC/2012, kindly provided by
Christian Drosten, Bonn, Germany) at the indicated multiplicity of
infection (MOI). Aer 1h the inoculum was aspirated, and the cells
were incubated with complete DMEM at 33°C (HCoV-229E) or 37°C
(MERS-CoV).
PBMC isolation
Peripheral blood mononuclear cells (PBMCs) were isolated using
the standard Ficoll-Paque gradient centrifugation and maintained in
completed DMEM. Briey, human blood, diluted 1:2 in PBS, was
layered carefully on top of Ficoll-Paque PLUS solution (GE Healthcare
Life Science; USA) and centrifuged for 30 min at room temperature
(RT) at 400 x g without brake. Aerwards the PBMCs containing
Plasma-Ficoll interface was collected, the cells were washed twice with
PBS (for 10 min at 640 ×g followed by 10 min at 400 ×g) and
resuspended in complete RPMI 1640 medium (Gibco Life
Technologies, UK) with Glutamax (Gibco Life Technologies, UK)
supplemented with P/S and 10% FCS and directly seeded in 24 or 96
well plates.
Inhibitors
D, L-lysine acetylsalicylate + glycine (LASAG, C15H22N2O6, 326.3 g/
mol) was obtained as Asprin i.v. 500mg®” (Bayer Vital GmbH,
Germany) and dissolved in dH2O to provide a stock concentration of 1
M. BAY11-7082 (Selleckchem, USA) was solved in DMSO at a stock
concentration of 50 mM and stored at -20°C until further usage.
Lornoxicam (Selleckchem, USA) was solved in DMSO for stock
concentration of 20 mM.
Virus titration
Virus titers were determined by focus forming assay [42] as
previously described [43]. Briey, infected Huh7 cells in 96-well plates
were xed and permeabilized (4% paraformaldehyde (PFA, Roth,
Germany), and 1 % Triton X-100 (Roth, Germany) in PBS++) and kept
at 4°C for 1h. ereaer, the solution was discarded and cells were
washed 3x with PBS++/0.05% Tween-20 (Roth, Germany). Aerwards,
cells were incubated with a mouse anti-coronavirus nucleoprotein
mAb (Ingenasa, Spain) primary antibody diluted 1:1,000 in PBS
containing 3% BSA (PAN Biotech, Germany) for 1h at RT. e cells
were washed again and then incubated with a goat anti-mouse HRP-
antibody (Santa Cruz, USA) secondary antibody diluted 1:1,000 in PBS
containing 3% BSA, for 1h at RT. Aer additional washing the cells
were incubated with 40 μl AEC (3-Amino-9-ethylcarbazole) staining
solution (Santa Cruz, USA). Following incubation for 40 min at 37°C,
the substrate solution was removed and cells were washed 2x with
dH2O to remove salts. To detect and quantify foci, the plates were
scanned with a resolution of 1200 dpi using an Epson Perfection V500
Photo scanner (Epson, Japan) and analysed using Photoshop soware
(Adobe, USA). Results represent the averages from three biological
replicates.
Cell viability (CC50)
To determine the median cytotoxic concentration of the compounds
at which 50% of the cells are still viable (CC50), Huh7 cells or PBMCs
were grown in 96 well micro plates. Growth medium was replaced with
DMEM containing dierent, indicated concentrations of LASAG
solved in dH20 or BAY11-7082 or Lornoxicam both solved in DMSO
and were further incubated under conditions of infection for the
indicated time periods. Subsequently, an MTT assay was performed as
previously described [37].
To determine the CC50, the MTT values were calculated in
percentage (% viability = (100/MTT value of untreated sample) x MTT
value of inhibitor treated sample) with the control set as 100 %.
Antiviral activity (EC50)
To determine the eective concentration at which virus titres are
reduced by 50% (EC50), Huh7 were infected with MOI 0.1 in 100 μl for
1h at 33°C (HCoV-229E) or 37°C (MERS-CoV). Aer removing the
inoculum, cells were incubated directly with 500 µl complete DMEM
containing dierent inhibitor concentrations. Samples of the
supernatants were collected at the indicated time points post infection
(p.i.) and the amount of infectious virus particles was determined by
focus forming assay.
In order to dene the EC50 the viral titre of the untreated virus-
infected control was set at 100% and the titres of LASAG-,
BAY11-7082- and Lornoxicam-treated samples were calculated in
relation to it.
Western blot
Cells were infected in a time course experiment and treated with the
indicated concentrations of LASAG or le untreated. Aer cell lysis,
proteins were separated by 12% SDS-PAGE and blotted onto
nitrocellulose membrane (Amersham, UK) as previously described
[44]. Membranes were incubated with the respective primary antibody
(mouse anti-nucleocapsid protein mAb [Ingenasa, Spain], mouse anti-
phospho-IκB-α antibody (Cell Signaling, USA] or rabbit anti-actin
Citation: Müller C, Karl N, Ziebuhr J, Pleschka S (2016) D, L-lysine acetylsalicylate + glycine Impairs Coronavirus Replication. J Antivir
Antiretrovir 8: 142-150. doi:10.4172/jaa.1000151
J Antivir Antiretrovir
ISSN:1948-5964 ,an open access journal Volume 8(4): 142-150 (2016) - 143
antibody [abcam, USA]) in PBS containing 3% BSA). Aer washing
the membranes three times with TBS/T (20 mM Tris-HCl, pH 7.6, 140
mM NaCl, 0.05 % Tween 20), they were further incubated with
Infrared IRDye-conjugated anti-mouse and anti-rabbit monoclonal
secondary antibodies (Li-Cor, Germany) diluted 1:10,000 in PBS
containing 3% BSA and analyzed via Li-Cor Odyssey (Li-Cor,
Germany). Western Blots were further graphically analysed by
measuring the intensity of the protein bands in regard to the loading
control of four dierent experiments for nucleocapsid protein
reduction or two independent experiments for IkB reduction.
Nothern blot
Huh7 cells were infected with HCoV-229E (MOI=3) and treated
with LASAG (20, 10, 5 mM) or le untreated. Total RNA was isolated
using Trizol (Invitrogen, Germany) 24 h p.i. and 5 µg total RNA was
separated in a denaturing gel (1% agarose, 6 % formaldehyde, 1x
MOPS) and transferred onto a positively charged nitrocellulose
membrane via Vacuum Blot. Northern Blot analysis was done using a
32P-labled probe specic for the HCoV-229E genome (nt 29297 to
27273) as described previously [45].
Immunouorescence
Huh7 cells were infected with HCoV-229E (MOI=3) and treated
with LASAG (20 mM) or le untreated. 24 h p.i. the cells were xed
with ice-cold methanol and stained with mouse anti-dsRNA mAb (J2,
English & Scientic Consulting Bt., Hungary) and rabbit anti-
HCoV-229E nsp8 mAb (both diluted 1:100 in PBS containing 3%
BSA). For detection Alexa Fluor 594 goat anti-mouse IgG and Alexa
Fluor 488 F (ab’) 2 fragment of goat anti-rabbit IgG (Invitrogen, USA)
both diluted 1:500 in PBS containing 3% BSA were used. Images were
acquired using a confocal laser-scanning microscope (Leica SP05
CLSM, Leica, Germany).
Analysis of NF-κB activation
NF-κB activation was measured as previously described [37] with
the commercial available TransAM kit (Active motif, USA) according
to the manufacturers instruction (n=4). In our study, HCoV-229E-
infected (MOI=3) Huh7 cells that were either treated with LASAG (20
mM) or le untreated were lysed at 4 or 12 h p.i.. Relative NF-κB
activation was calculated as fold induction compared to mock-infected
control cells.
Temporal LASAG treatment
To determine the eect of LASAG-treatment on the early and the
late phase of the CoV replication cycle, Huh7 cells were infected with
HCoV-229E (MOI=3) and treated with LASAG (20 mM) either from
3-6 h p.i. or from 9 -12 h p.i.. For this the cells were incubated in
regular media, which was replaced with LASAG-containing media for
the indicated times. 12 h p.i. the supernatant was collected and pooled
and the virus titer was determined via focus forming assay.
Statistical analysis
e results correspond to the mean ± SD of the indicated
experiments. e statistical signicance of dierences between the
indicated groups was tested using the unpaired, two-tailed Student’s t-
test with a threshold of p:*<0.05; **<0.005; and ***<0.0005.
Results
LASAG reduces HCoV-229E and MERS-CoV titres in cell
culture as well as in primary human cells at non-toxic
concentrations
Based on improved survival rates of SARS-CoV-infected mice,
reduced lung pathology following NF-κB inhibition [41] and the fact,
that ASA can block NF-κb activation [31,32] we aimed to elucidate
whether LASAG (Figure 1) might have a negative eect on CoV
propagation
in vitro
.
Figure 1: Structural formula of D,L-lysine acetylsalicylate glycine.
Treatment was performed with commercial Aspirin® i.v.
containing the active compound acetylsalicylic acid (ASA). D,L-
lysine acetylsalicylate glycine (LASAG) is a salt of ASA and the two
amino acids glycine and lysine. Aer dissolving in water, LASAG
dissociates readily into ASA and the two amino acids glycine and
lysine.
In a rst set of experiments the cell viability was investigated via
MTT assay aer 24 and 48 h incubation for Huh7 cells and aer 24 h
incubation for PBMCs, applying dierent amounts of LASAG. For this
we determined the cytotoxic concentration 50 (CC50) of LASAG for
Huh7 cells, which are regularly used for CoV propagation [46,47] as
20.43 mM and 58.73 mM at 48 and 24 h, respectively (Figures 2A and
2B). Furthermore, we determined the CC50 of LASAG for human
peripheral blood mononuclear cells (PBMC) at 37.95 mM at 24 h
(Figure 2C). PBMCs can be infected with HCoV-229E [48] and served
as a primary human cell system.
Next, we analysed whether LASAG aects the replication of the
human strain HCoV-229E as well as of MERS-CoV. erefor we
treated HCoV-229E- and MERS-CoV-infected Huh7 cells (MOI=0.1)
with LASAG at dierent concentrations and analysed the viral titres
via focus forming assay at 48 h and 24 h post infection (p.i.). e
results indicate an eective concentration 50 (EC50) of 1.31 mM for
HCoV-229E resulting in a selectivity index (SI: CC50/IC50) of 15.6 as
well as an EC50 of 3.69 mM for MERS-CoV, leading to an SI of 15.9,
similar to that of HCoV-229E (Figures 2D and 2E). e EC50 for
HCoV-229E-infected PBMCs at 24 h was 6.71 mM resulting in an SI of
5.7 (Figure 2F).
Citation: Müller C, Karl N, Ziebuhr J, Pleschka S (2016) D, L-lysine acetylsalicylate + glycine Impairs Coronavirus Replication. J Antivir
Antiretrovir 8: 142-150. doi:10.4172/jaa.1000151
J Antivir Antiretrovir
ISSN:1948-5964 ,an open access journal Volume 8(4): 142-150 (2016) - 144
Figure 2: LASAG impairs coronaviral replication. To investigate the eect of LASAG on coronaviral replication we determined the CC50 (A, B,
C) and the EC50 (D, E, F) of LASAG. Huh7 cells were treated with the indicated concentrations of LASAG for 48 h (A, D) and 24 h (B, E), and
PBMC were treated for 24 h (C,F). EC50 concentrations were determined for HCoV-229E on Huh7 cells (D) and PBMC (F). For MERS-CoV
the EC50 was determined aer 24 h on sHuh7 cells (E).
ese results indicate that LASAG can eectively reduce titres of
HCoV-229E and MERS-CoV with similar SI values in Huh7 cells and,
to a lesser extent, HCoV-229E titers in primary human PBMCs in a
dose-dependent manner at non-toxic concentrations. Nevertheless,
even though the SI values are distinct, increased SI values for both cell
systems, would be advantageous for a therapeutic approach.
LASAG reduces viral protein accumulation and viral RNA
synthesis of HCoV-229E
e reduction of viral titres following LASAG treatment led us to
speculate that LASAG might impair the intra-cellular replication of
CoV. In order to elucidate which step of the viral life cycle is aected by
the treatment of LASAG, we rst determined viral protein
accumulation via Western Blot and also analysed viral RNA synthesis
via Northern Blot of HCoV-229E infected Huh7 cells treated for 24 h
p.i. at non-toxic LASAG concentrations of 20, 10 and 5 mM.
As shown in Figures 3A and 3B we found that treatment with 20
mM LASAG led to a strong reduction of the viral nucleocapsid (N)
protein as well as viral RNA amount (Figure 3C). e eect on viral
protein accumulation appears to be specic, as the quantity of cellular
actin (loading control) was not aected.
Citation: Müller C, Karl N, Ziebuhr J, Pleschka S (2016) D, L-lysine acetylsalicylate + glycine Impairs Coronavirus Replication. J Antivir
Antiretrovir 8: 142-150. doi:10.4172/jaa.1000151
J Antivir Antiretrovir
ISSN:1948-5964 ,an open access journal Volume 8(4): 142-150 (2016) - 145
Figure 3: LASAG reduces coronaviral RNA and protein
accumulation. Huh7 were infected with HCoV-229E and treated
with the indicated LASAG concentrations. 24 h p.i. the amount of
viral N protein and actin as loading control in infected and un-
infected (mock) cells were detected by Western Blot using mouse
anti-nucleocapsid protein mAb and rabbit anti-actin antibody (A)
and quantied (B). e amount of viral RNAs was detected by
Northern Blot analysis (C).
Compared to the strong reduction of the HCoV-229E titre
(EC50=1.31 mM) 48 h p.i. by LASAG the eects of 5 and 10 mM
LASAG on viral protein/RNA production are less evident.
Nevertheless, it should be considered that these assays (Western Blot/
Northern Blot) were performed 24 h p.i.. is was done, as the CC50
for LASAG at 48 h was 20.43 mM, whereas it was 58.73 mM for 24 h
(Figure 2), indicating that a LASAG concentration of 20 mM is less
toxic at 24 h. erefore, the eect of the lower LASAG concentrations
(5, 10 mM) on viral protein/RNA production might be less evident at
this earlier time point. Also, the MOI to determine the EC50 was only
0.1, whereas the MOI to analyse viral protein/RNA production was 3.
erefore, the eect of the LASAG concentrations used to analyse the
eect on viral protein/RNA production might be weaker. Furthermore,
it should be considered that these assays are generally less sensitive
than the direct virus titration used to determine the EC50, which could
also explain the discrepancy.
Taken together, the results indicate that the reduction in the viral
titre observed under LASAG treatment could be related to the negative
eect of LASAG on the viral protein and RNA production, which
might therefore account for the reduction of infectious progeny virions
produced.
LASAG treatment results in reduction of replication
transcription complexes
e impaired viral protein accumulation and viral RNA synthesis
led us to assume that the mode of action exerted by LASAG on CoV
propagation impairs a step of the viral life cycle before viral RNA/
protein synthesis starts. CoVs replicate and transcribe their genome
via so called replication/ transcription complexes (RTCs), anchored in
virus-induced membrane alterations that consist of double membrane
vesicles (DMVs) [49].
Figure 4: e abundance of coronaviral RTCs is reduced under
LASAG treatment. HCoV-229E-infected Huh7 cells +/- LASAG (20
mM) were analysed 24 h p.i. using confocal microscopy. As a
marker for viral RTCs, the CoV replication intermediate dsRNA
(green) and the viral non-structural protein nsp8 (red) were
detected using target specic antibodies. e nuclei were stained
with DAPI (blue). Co-localization of dsRNA and nsp8 is shown in
the merge (yellow). Open boxes (marked in white) indicate the
magnied areas shown underneath the respective panel.
We therefore assessed whether LASAG may impact the formation of
these host cell-derived DMVs. To this point, Huh7 cells were infected
with HCoV-229E (MOI=3) and analysed 24 h p.i. for the appearance of
RTCs. As markers in our immunouorescence studies (Figure 4) we
assessed the presence of dsRNA, a viral replication intermediate,
indicating the site of viral genome replication [50,51] and the viral
Citation: Müller C, Karl N, Ziebuhr J, Pleschka S (2016) D, L-lysine acetylsalicylate + glycine Impairs Coronavirus Replication. J Antivir
Antiretrovir 8: 142-150. doi:10.4172/jaa.1000151
J Antivir Antiretrovir
ISSN:1948-5964 ,an open access journal Volume 8(4): 142-150 (2016) - 146
nsp8 protein as an integral part of the viral RTCs [52]. In infected,
untreated cells the characteristic peri-nuclear immunouorescence
pattern for RTCs marked by nsp8 and dsRNA [53], which was absent
in the mock-infected cells (Figure 4, mock), was clearly visible (Figure
4, infected). In infected and LASAG-treated cells the amount of cells
showing an nsp8/dsRNA signal (RTCs in DMVs) was decreased,
compared to the untreated, infected cells (Figure 4, infected+20 mM
LASAG). is result demonstrates that 24 h p.i. LASAG-treatment
severely aects the formation of viral RTCs and/or DMVs and it is
tempting to speculate that this is the cause for the decreased
production of viral proteins and RNA. It should be noted that LASAG
might also aect a step in the viral life cycle before RTC formation,
which needs to be investigated.
HCoV-229E infection triggers NF-κB activation in the early
phase of infection, which is decreased by LASAG
Activation of the transcription factor NF-κB is one of the hallmarks
of host cell response to invasion by dierent pathogens. e
phosphorylation of the NF-κB inhibitor IκBα leads to its degradation
and release of NF-κB [54], which then translocates into the nucleus to
transactivate responsive genes [55] related to host defence mechanisms
[56].
NF-κB inhibitors have been studied as potential therapeutic drugs
in anti-viral therapy [57]. Previously, we and others [36-38,58]
demonstrated that NF-κB activity is essential for ecient inuenza
virus propagation, as inhibition of NF-κB activation results in impaired
nuclear RNP export and therefore in reduced virus titers.
For coronavirus infections there are diverting reports of NF-κB
activation [41,59-66] as well as NF-κB inhibition during CoV infection
[67,68]. To assess whether HCoV-229E infection also activates NF-κB
in Huh 7 cells we determined the level of NF-κB activation in
HCoV-229E-infected Huh7 cells via TransAM assay and Western Blot
analysis. We found that HCoV-229E infection leads to a markedly
reduced amount of IκBα at 2 h p.i., which later increases again in the
virus-infected cells (Figure 5A). When analysing the activation of NF-
κB we found an early (4 h p.i.) activation (Figure 5B, le part),
coinciding with decreased amounts of phospho-IκBα early in the
infection (Figure 5A). According to the increased amounts of IκBα at
later time points of the infection, the activation of NF-kB was found to
be lower at 12 h p.i. compared to 4 h p.i. (Figure 5B, right part).
As mentioned before, ASA is known to block NF-κB activation.
erefore we tested whether LASAG treatment also has an eect on
HCoV-229E-induced NF-κB activation. e treatment of HCoV-229E-
infected cells with 20 mM LASAG resulted in a signicant 1.5 fold
reduction of NF-κB activation at early (4 h p.i.) and late (12 h p.i.) time
points of infection (Figure 5B).
Furthermore, the addition of LASAG in the early stage of infection
(3-6 h p.i.), when NF-κB activation was most prominent and viral
RTCs are being formed, resulted in a reduction of virus titres. In
contrast, the addition of LASAG in later stages of viral life cycle (9-12 h
p.i.) resulted in less pronounced titre reduction (Figure 5C). In
summary HCoV-229E infection activates NF-κB in the early stages of
viral life cycle and the LASAG-dependent reduction of virus-induced
NF-κB activation coincides with reduced viral titers.
Figure 5: Virus-induced NF-κB activation is aected by LASAG
treatment. e total amount of IκBα in HCoV-229E-infected Huh7
cells was analysed at the indicated time points p.i. by Western Blot
using mouse anti-phospho-IκB-α antibody (A upper panel) and
quantied (A lower panel). HCoV-229E-infected Huh7 cells (+/-)
LASAG-treatment (20mM) were analysed for the activation of NF-
κB at the indicated time points p.i. via detection of the amount of
activated NF-κB (B). HCoV-229E-infected Huh7 cells were treated
with LASAG (20mM) for the indicated time frames p.i. or le
untreated (control) and virus titres (FFU/ml) were determined 12 h
p.i. (C).
NF-κB plays a pivotal role in HCoV-229E infection
ASA is a multi-target compound [69] not only blocking NF-κB
activation, but also interfering with several other cellular factors, such
as cyclooxygenase 1/2 (COX1/2) a main enzyme in the synthesis of
inammation mediators [70,71] or AMPK/mTOR [72].
To highlight the role of NF-κB activity in CoV infection, we
elucidated the eect of the NF-κB-specic inhibitor (BAY11-7082) on
HCoV-229E replication. HCoV-229E-infected (MOI=0.5) Huh7 cells
were treated with the indicated concentrations of BAY11-7082 and
virus titres were analysed 24 h p.i.. Addition of BAY11-7082 at non-
toxic concentration of 50 µM (Figure 6A) resulted in a reduction of
viral titres by about 4 log10 (Figure 6B). is result supports our
assumption that inhibition of NF-κB activity by LASAG impairs virus
replication. Beside NF-κB inhibition, one of the major activities of
ASA is the inhibition of the cellular COX1/2. We further investigated
whether this activity might (also) be responsible for the anti-viral eect
Citation: Müller C, Karl N, Ziebuhr J, Pleschka S (2016) D, L-lysine acetylsalicylate + glycine Impairs Coronavirus Replication. J Antivir
Antiretrovir 8: 142-150. doi:10.4172/jaa.1000151
J Antivir Antiretrovir
ISSN:1948-5964 ,an open access journal Volume 8(4): 142-150 (2016) - 147
of LASAG against CoV. For this we employed Lornoxicam, a non-
steroidal COX1/2 inhibitor, which is used as an anti-inammatory
drug to treat pain, osteoarthritis, and rheumatoid arthritis [73,74].
However, at the indicated non-toxic concentrations (Figure 6C)
Lornoxicam had no eect on HCoV-229E propagation in infected
Huh7 cells (MOI=0.5) analysed 24 h p.i (Figure 6D). is indicates that
the inhibitory eect of ASA on COX1/2 activity does not seem to be
important for HCoV-229E propagation.
Figure 6: Eect of BAY11-7082 and Lornoxicam on HCoV-229E
replication at non-toxic concentrations. Huh7 cells were treated
with the indicated concentrations of BAY11-7082 and Lornoxicam
for 24h and cell viability was determined via MTT assay (A, C).
HCoV-229E-infected Huh7 cells were treated with the indicated
concentration of BAY11-7082 and Lornoxicam and virus titres were
determined 24 h p.i. (B, D).
Discussion
Among the CoV several human pathogenic strains can cause
common cold-like illnesses, while others as SARS-CoV and MERS-
CoV lead to severe infections. Currently, no treatment focusing on the
virus as the cause is available. Here we show that LASAG, which is an
approved drug, impairs propagation of HCoV-229E and of the highly
pathogenic MERS-CoV
in vitro
. Our results demonstrate that
inhibition of virus-induced NF-κB activity early in the viral replication
cycle via LASAG coincides with (i) reduced viral titres, (ii) decreased
viral protein accumulation and viral RNA synthesis and (iii) impaired
formation of viral replication transcription complexes.
It should be mentioned that upon NF-κB inhibition (BAY11-7082)
DeDiego et al. did not observe any virus titre reduction of the beta-
coronavirus SARS-CoV [41] which was adapted to murine cell lines.
However, it cannot be excluded that this system of a mouse-adapted
SARS-CoV in a murine cell line might not reect the situation of
human CoV or wild type SARS-CoV infection of human cells.
Despite the possibility that other cellular factors, which are also
targeted by LASAG might aect CoV propagation, the results obtained
with the NF-κB inhibitor (BAY11-7082) and the COX1/2 inhibitor
(Lornoxicam) further support the notion that NF-κB inhibition is a
likely reason for the anti-CoV eect of LASAG. To elucidate the NF-
κB-specic eect, further analysis will be needed. It should be noted
that similar results were obtained for the treatment of inuenza virus
(IV) infection with ASA. Here, the COX inhibitor Indometacin also
had no eect on the virus titer in cell culture [38].
Even though cell viability is not impaired by the applied LASAG
concentrations, the anti-coronaviral action of LASAG in cell culture
lies in a millimolar range, likewise to the anti-viral action of ASA
against IV [38]. To achieve a 20 mM LASAG concentration in the
blood, 6.53 g/L would be needed, which is toxic [75]. e Cmax in the
blood aer applying 500 mg ASA i.v. is 54.25 mg/L and 4.84 mg/L aer
oral application [76]. Nevertheless, treatment of patients with a CoV-
caused severe acute respiratory syndrome via inhalation might allow
achieving locally eective LASAG concentrations. Results from a
clinical study investigating the eectiveness of inhaled lysine-
acetylsalicylate in the treatment of asthma showed, that patients that
received a dosage of 720 mg of inhaled LASAG twice a day over a two-
week period did not experience any signicant side eects [77]. Also, a
dose escalation study of inhaled LASAG in humans for the clinical
development of an antiviral treatment of IV infections demonstrated
that inhalative doses up to 750 mg LASAG were safe and well tolerated
without serious adverse events [78]. Furthermore, administration of
aerosolic ASA via intubation directly into the trachea resulted in
increased survival rates of mice infected with a lethal dose of IV [38].
In light of the fact that currently no approved anti-viral treatment
against severe CoV-infections exist, it should be mentioned that
LASAG (i) is widely available and it is tested and approved for humans,
(ii) it targets cellular functions, (iii) is so far not known to target CoV
functions, which reduces the chance that resistant virus variants
emerge and (iv) adjacent to its direct anti-viral property, patients could
also benet from the eects of LASAG on infection-related symptoms
based on the analgetic- and anti-inammatory characteristics of
LASAG.
In conclusion, we were able to demonstrate that LASAG inhibits
virus-induced NF-κB activity, which might be connected to the anti-
viral eect against CoV, including the impaired formation of RTCs
and/or DMVs in CoV-infected cells, leading to reduced viral RNA
production and consequently decreased production of viral proteins,
resulting in an overall diminished virus titre.
Acknowledgments
We want to thank C. Drosten, Bonn, Germany, for providing
MERS-CoV EMC/2012. is work was funded in part by the German
Centre for Infection Research (DZIF), partner site Giessen, Germany
(TTU Emerging Infections to S.P. and J.Z.), and the DFG-funded
Collaborative Research Centre 1021 "RNA viruses: RNA metabolism,
host response and pathogenesis" (SFB1021; projects A01 and C01 to
J.Z. and S.P., respectively). Furthermore the work was supported in
part by a fund of the Activaero GmbH (acquired by Vectura). e
funders had no role in study design, data collection and analysis,
decision to publish, or preparation of the manuscript.
Disclosure Statement
e authors have no conict of interest.
References
1. de Groot RJ, Baker SC, Baric R, Enjuanes L, Gorbalenya AE, et al. (2012)
Family Coronaviridae. p 806-828.
2. Masters PS, Perlman S (2013) Coronaviridae. Philadelphia, p 825-858.
Citation: Müller C, Karl N, Ziebuhr J, Pleschka S (2016) D, L-lysine acetylsalicylate + glycine Impairs Coronavirus Replication. J Antivir
Antiretrovir 8: 142-150. doi:10.4172/jaa.1000151
J Antivir Antiretrovir
ISSN:1948-5964 ,an open access journal Volume 8(4): 142-150 (2016) - 148
3. McIntosh K, Dees JH, Becker WB, Kapikian AZ, Chanock RM (1967)
Recovery in tracheal organ cultures of novel viruses from patients with
respiratory disease. Proc Natl Acad Sci U S A 57: 933-940.
4. Tyrrell DA, Bynoe ML (1965) Cultivation of a Novel Type of Common-
Cold Virus in Organ Cultures. Br Med J 1: 1467-1470.
5. Hamre D, Procknow JJ (1966) A new virus isolated from the human
respiratory tract. Proc Soc Exp Biol Med 121: 190-193.
6. Kuiken T, Fouchier RA, Schutten M, Rimmelzwaan GF, van Amerongen
G, et al. (2003) Newly discovered coronavirus as the primary cause of
severe acute respiratory syndrome. Lancet 362: 263-270.
7. Ksiazek TG, Erdman D, Goldsmith CS, Zaki SR, Peret T, et al. (2003) A
novel coronavirus associated with severe acute respiratory syndrome. N
Engl J Med 348: 1953-1966.
8. Drosten C, Gunther S, Preiser W, van der Werf S, Brodt HR, et al. (2003)
Identication of a novel coronavirus in patients with severe acute
respiratory syndrome. N Engl J Med 348:1967-1976.
9. Peiris JSM, Lai ST, Poon LLM, Guan Y, Yam LYC, et al. (2003)
Coronavirus as a possible cause of severe acute respiratory syndrome.
Lancet 361: 1319-1325.
10. Zaki AM, van Boheemen S, Bestebroer TM, Osterhaus AD, Fouchier RA
(2012) Isolation of a novel coronavirus from a man with pneumonia in
Saudi Arabia. N Engl J Med 367: 1814-1820.
11. Arabi YM, Ari AA, Balkhy HH, Najm H, Aldawood AS, et al. (2014)
Clinical course and outcomes of critically ill patients with Middle East
respiratory syndrome coronavirus infection. Ann Intern Med 160:
389-397.
12. Al-Abdallat MM, Payne DC, Alqasrawi S, Rha B, Tohme RA, et al. (2014)
Hospital-Associated Outbreak of Middle East Respiratory Syndrome
Coronavirus: A Serologic, Epidemiologic, and Clinical Description. Clin
Infect Dis 59: 1225-1233.
13. Guery B, Poissy J, Mansouf L, Sejourne C, Ettahar N, et al. (2013) Clinical
features and viral diagnosis of two cases of infection with Middle East
Respiratory Syndrome coronavirus: a report of nosocomial transmission.
Lancet 381: 2265-2272.
14. WHO (2015) Middle East respiratory syndrome coronavirus (MERS
CoV)-Saudi Arabia.
15. (2012) MERS in the Arabian Peninsula. CDC.
16. (2016) Prevention & Treatment CDC.
17. Cinatl J, Morgenstern B, Bauer G, Chandra P, Rabenau H, et al. (2003)
Treatment of SARS with human interferons. Lancet 362: 293-294.
18. Zielecki F, Weber M, Eickmann M, Spiegelberg L, Zaki AM, et al. (2013)
Human cell tropism and innate immune system interactions of human
respiratory coronavirus EMC compared to those of severe acute
respiratory syndrome coronavirus. J Virol 87: 5300-5304.
19. Chan RW, Chan MC, Agnihothram S, Chan LL, Kuok DI, et al. (2013)
Tropism of and innate immune responses to the novel human
betacoronavirus lineage C virus in human ex vivo respiratory organ
cultures. J Virol 87: 6604-6614.
20. Samuel CE (2001) Antiviral actions of interferons. Clin Microbiol Rev 14:
778-809.
21. de Wilde AH, Raj VS, Oudshoorn D, Bestebroer TM, van Nieuwkoop S,
et al. (2013) MERS-coronavirus replication induces severe in vitro
cytopathology and is strongly inhibited by cyclosporin A or interferon-
alpha treatment. J Gen Virol 94: 1749-1760.
22. de Wilde AH, Jochmans D, Posthuma CC, Zevenhoven-Dobbe JC, van
Nieuwkoop S, et al. (2014) Screening of an FDA-approved compound
library identies four small-molecule inhibitors of Middle East
respiratory syndrome coronavirus replication in cell culture. Antimicrob
Agents Chemother 58: 4875-4884.
23. Dyall J, Coleman CM, Hart BJ, Venkataraman T, Holbrook MR, et al.
(2014) Repurposing of clinically developed drugs for treatment of Middle
East respiratory syndrome coronavirus infection. Antimicrob Agents
Chemother 58: 4885-4893.
24. Ziebuhr J, Snijder EJ, Gorbalenya AE (2000) Virus-encoded proteinases
and proteolytic processing in the Nidovirales. J Gen Virol 81: 853-879.
25. Yang H, Xie W, Xue X, Yang K, Ma J, et al. (2005) Design of wide-
spectrum inhibitors targeting coronavirus main proteases. PLoS Biol 3:
e324.
26. Steinhauer DA, Holland JJ (1986) Direct Method for Quantitation of
Extreme Polymerase Error Frequencies at Selected Single Base Sites in
Viral-Rna. J Virol 57: 219-228.
27. Zhao Z, Li H, Wu X, Zhong Y, Zhang K, et al. (2004) Moderate mutation
rate in the SARS coronavirus genome and its implications. BMC Evol Biol
4: 21.
28. Weissmann G (1991) Aspirin. Sci Am 264: 84-90.
29. Weiss HA, Forman D (1996) Aspirin, non-steroidal anti-inammatory
drugs and protection from colorectal cancer: a review of the
epidemiological evidence. Scand J Gastroenterol Suppl 220: 137-141.
30. Stewart WF, Kawas C, Corrada M, Metter EJ (1997) Risk of Alzheimer's
disease and duration of NSAID use. Neurology 48: 626-632.
31. Franckowiak G, Ledwoch W, Schweinheim E, Hayauchi Y (2011) Stable
active compound complex of salts of o-acetylsalicylic acid with basic
amino acids and glycine. USA.
32. Kopp E, Ghosh S (1994) Inhibition of NF-kappa B by sodium salicylate
and aspirin. Science 265: 956-959.
33. Grilli M, Pizzi M, Memo M, Spano P (1996) Neuroprotection by aspirin
and sodium salicylate through blockade of NF-kappaB activation. Science
274: 1383-1385.
34. Yin MJ, Yamamoto Y , Gaynor RB (1998) e anti-inammatory agents
aspirin and salicylate inhibit the activity of I(kappa)B kinase-beta. Nature
396: 77-80.
35. Chu WM, Ostertag D, Li ZW, Chang L, Chen Y, et al. (1999) JNK2 and
IKKbeta are required for activating the innate response to viral infection.
Immunity 11: 721-731.
36. Wurzer WJ, Ehrhardt C, Pleschka S, Berberich-Siebelt F, Wol T, et al.
(2004) NF-kappaB-dependent induction of tumor necrosis factor-related
apoptosis-inducing ligand (TRAIL) and Fas/FasL is crucial for ecient
inuenza virus propagation. J Biol Chem 279: 30931-30937.
37. Pinto R, Herold S, Cakarova L, Hoegner K, Lohmeyer J, et al. (2011)
Inhibition of inuenza virus-induced NF-kappaB and Raf/MEK/ERK
activation can reduce both virus titers and cytokine expression
simultaneously in vitro and in vivo. Antiviral Res 92: 45-56.
38. Mazur I, Wurzer WJ, Ehrhardt C, Pleschka S, Puthavathana P, et al.
(2007) Acetylsalicylic acid (ASA) blocks inuenza virus propagation via
its NF-kappaB-inhibiting activity. Cell Microbiol 9: 1683-1694.
39. Speir V, Yu ZX, Ferrans VJ, Huang ES, Epstein SE (1998) Aspirin
attenuates cytomegalovirus infectivity and gene expression mediated by
cyclooxygenase-2 in coronary artery smooth muscle cells. Circ Res 83:
210-216.
40. Glatthaar-Saalmuller B, Mair KH, Saalmuller A (2016) Antiviral activity
of aspirin against RNA viruses of the respiratory tract-an in vitro study.
Inuenza Other Respir Viruses 11: 85-92.
41. DeDiego ML, Nieto-Torres JL, Regla-Nava JA, Jimenez-Guardeno JM,
Fernandez-Delgado R, et al. (2014) Inhibition of NF-kappaB-mediated
inammation in severe acute respiratory syndrome coronavirus-infected
mice increases survival. J Virol 88: 913-924.
42. Matrosovich M, Matrosovich T, Garten W, Klenk HD (2006) New low-
viscosity overlay medium for viral plaque assays. J Virol 3.
43. Ma W, Brenner D, Wang Z, Dauber B, Ehrhardt C, et al. (2010) e NS
segment of an H5N1 highly pathogenic avian inuenza virus (HPAIV) is
sucient to alter replication eciency, cell tropism, and host range of an
H7N1 HPAIV. J Virol 84: 2122-2133.
44. Marjuki H, Alam MI, Ehrhardt C, Wagner R, Planz O, et al. (2006)
Membrane accumulation of inuenza A virus hemagglutinin triggers
nuclear export of the viral genome via protein kinase Calpha-mediated
activation of ERK signaling. J Biol Chem 281: 16707-16715.
45. iel V, Ivanov KA, Putics A, Hertzig T, Schelle B, et al. (2003)
Mechanisms and enzymes involved in SARS coronavirus genome
expression. J Gen Virol 84: 2305-2315.
Citation: Müller C, Karl N, Ziebuhr J, Pleschka S (2016) D, L-lysine acetylsalicylate + glycine Impairs Coronavirus Replication. J Antivir
Antiretrovir 8: 142-150. doi:10.4172/jaa.1000151
J Antivir Antiretrovir
ISSN:1948-5964 ,an open access journal Volume 8(4): 142-150 (2016) - 149
46. Chan JF, Chan KH, Choi GK, To KK, Tse H, et al. (2013) Dierential cell
line susceptibility to the emerging novel human betacoronavirus 2c EMC/
2012: implications for disease pathogenesis and clinical manifestation. J
Infect Dis 207: 1743-1752.
47. Tang BS, Chan KH, Cheng VC, Woo PC, Lau SK, et al. (2005)
Comparative host gene transcription by microarray analysis early aer
infection of the Huh7 cell line by severe acute respiratory syndrome
coronavirus and human coronavirus 229E. J Virol 79: 6180-6193.
48. Desforges M, Miletti TC, Gagnon M, Talbot PJ (2007) Activation of
human monocytes aer infection by human coronavirus 229E. Virus Res
130: 228-240.
49. V'Kovski P, Al-Mulla H, iel V, Neuman BW (2015) New insights on the
role of paired membrane structures in coronavirus replication. Virus Res
202: 33-40.
50. Schonborn J, Oberstrass J, Breyel E, Tittgen J, Schumacher J et al. (1991)
Monoclonal antibodies to double-stranded RNA as probes of RNA
structure in crude nucleic acid extracts. Nucleic Acids Res 19: 2993-3000.
51. Weber F, Wagner V, Rasmussen SB, Hartmann R, Paludan SR (2006)
Double-stranded RNA is produced by positive-strand RNA viruses and
DNA viruses but not in detectable amounts by negative-strand RNA
viruses. J Virol 80: 5059-5064.
52. Imbert I, Guillemot JC, Bourhis JM, Bussetta C, Coutard B, et al. (2006) A
second, non-canonical RNA-dependent RNA polymerase in SARS
coronavirus. EMBO J 25: 4933-4942.
53. Lundin A, Dijkman R, Bergstrom T, N Kann, B Adamiak, et al. (2014)
Targeting membrane-bound viral RNA synthesis reveals potent inhibition
of diverse coronaviruses including the middle East respiratory syndrome
virus. PLoS Pathog 10: e1004166.
54. Israel A (2010) e IKK complex, a central regulator of NF-kappaB
activation. Cold Spring Harb Perspect Biol 2: a000158.
55. Schmitz ML, Mattioli I, Buss H, Kracht M (2004) NF-kappaB: a
multifaceted transcription factor regulated at several levels.
Chembiochem 5: 1348-1358.
56. Hiscott J, Kwon H, Genin P (2001) Hostile takeovers: viral appropriation
of the NF-kappaB pathway. J Clin Invest 107: 143-151.
57. Gilmore TD, Herscovitch M (2006) Inhibitors of NF-kappaB signaling:
785 and counting. Oncogene 25: 6887-6899.
58. Nimmerjahn F, Dudziak D, Dirmeier U, Hobom G, Riedel A, et al. (2004)
Active NF-kappaB signalling is a prerequisite for inuenza virus
infection. J Gen Virol 85: 2347-2356.
59. Li J, Liu Y, Zhang X (2010) Murine coronavirus induces type I interferon
in oligodendrocytes through recognition by RIG-I and MDA5. J Virol 84:
6472-6482.
60. Cao L, Ge X, Gao Y, Herrler G, Ren Y, et al. (2015) Porcine epidemic
diarrhea virus inhibits dsRNA-induced interferon-beta production in
porcine intestinal epithelial cells by blockade of the RIG-I-mediated
pathway. Virol J 12: 127.
61. Cao L, Ge X, Gao Y, Ren Y, Ren X, et al. (2015) Porcine epidemic diarrhea
virus infection induces NF-kappaB activation through the TLR2, TLR3
and TLR9 pathways in porcine intestinal epithelial cells. J Gen Virol 96:
1757-1767.
62. Zhang X, Wu K, Wang D, Yue X, Song D, et al. (2007) Nucleocapsid
protein of SARS-CoV activates interleukin-6 expression through cellular
transcription factor NF-kappaB. Virology 365: 324-335.
63. Liao QJ, Ye LB, Timani KA, Zeng YC, She YL, et al. (2005) Activation of
NF-kappaB by the full-length nucleocapsid protein of the SARS
coronavirus. Acta Biochim Biophys Sin (Shanghai) 37: 607-612.
64. Yan X, Hao Q, Mu Y, Timani KA, Ye L, et al. (2006) Nucleocapsid protein
of SARS-CoV activates the expression of cyclooxygenase-2 by binding
directly to regulatory elements for nuclear factor-kappa B and CCAAT/
enhancer binding protein. Int J Biochem Cell Biol 38: 1417-1428.
65. Dosch SF, Mahajan SD, Collins AR (2009) SARS coronavirus spike
protein-induced innate immune response occurs via activation of the NF-
kappaB pathway in human monocyte macrophages in vitro. Virus Res
142: 19-27.
66. Kanzawa N, Nishigaki K, Hayashi T, Ishii Y, Furukawa S, et al. (2006)
Augmentation of chemokine production by severe acute respiratory
syndrome coronavirus 3a/X1 and 7a/X4 proteins through NF-kappaB
activation. FEBS Lett 580: 6807-6812.
67. Kopecky-Bromberg SA, Martinez-Sobrido L, Frieman M, Baric RA,
Palese P (2007) Severe acute respiratory syndrome coronavirus open
reading frame (ORF) 3b, ORF 6, and nucleocapsid proteins function as
interferon antagonists. J Virol 81: 548-557.
68. Frieman M, Ratia K, Johnston RE, Mesecar AD, Baric RS (2009) Severe
acute respiratory syndrome coronavirus papain-like protease ubiquitin-
like domain and catalytic domain regulate antagonism of IRF3 and NF-
kappaB signaling. J Virol 83: 6689-6705.
69. Pillinger MH, Capodici C, Rosenthal P, Kheterpal N, Han S, et al. (1998)
Modes of action of aspirin-like drugs: salicylates inhibit erk activation
and integrin-dependent neutrophil adhesion. Proc Natl Acad Sci U S A
95: 14540-14545.
70. Patrono C, Baigent C, Hirsh J, Roth G (2008) Antiplatelet drugs:
American College of Chest Physicians Evidence-Based Clinical Practice
Guidelines (8th Edition). Chest 133: 199S-233S.
71. Clarke RJ, Mayo G, Price P, FitzGerald GA (1991) Suppression of
thromboxane A2 but not of systemic prostacyclin by controlled-release
aspirin. N Engl J Med 325: 1137-1141.
72. Din FV, Valanciute A, Houde VP, Zibrova D, Green KA, et al. (2012)
Aspirin inhibits mTOR signaling, activates AMP-activated protein kinase,
and induces autophagy in colorectal cancer cells. Gastroenterology 142:
1504-1515.
73. Kidd B, Frenzel W (1996) A multicenter, randomized, double blind study
comparing lornoxicam with diclofenac in osteoarthritis. J Rheumatol 23:
1605-1611.
74. Balfour JA, Fitton A, Barradell LB (1996) Lornoxicam. A review of its
pharmacology and therapeutic potential in the management of painful
and inammatory conditions. Drugs 51: 639-657.
75. Temple AR (1981) Acute and chronic eects of aspirin toxicity and their
treatment. Arch Intern Med 141: 364-369.
76. Nagelschmitz J, Blunck M, Kraetzschmar J, Ludwig M, Wensing G, et al.
(2014) Pharmacokinetics and pharmacodynamics of acetylsalicylic acid
aer intravenous and oral administration to healthy volunteers. Clin
Pharmacol 6: 51-59.
77. Bianco S, Vaghi A, Robuschi M, Reni RM, Pieroni MG, et al. (1995)
Steroid-sparing eect of inhaled lysine acetylsalicylate and furosemide in
high-dose beclomethasone-dependent asthma. J Allergy Clin Immunol
95: 937-943.
78. Nagelschmitz J, Scheerans C, Kraetzschmar J, von Degenfeld G, Müllinger
B, et al. (2015) First-In-Man Dose Escalation Study of Aspirin® Inhaled
For e Clinical Development of A New Antiviral Treatment of Resistant
Inuenza.
Citation: Müller C, Karl N, Ziebuhr J, Pleschka S (2016) D, L-lysine acetylsalicylate + glycine Impairs Coronavirus Replication. J Antivir
Antiretrovir 8: 142-150. doi:10.4172/jaa.1000151
J Antivir Antiretrovir
ISSN:1948-5964 ,an open access journal Volume 8(4): 142-150 (2016) - 150
... 86 LASAG dissociates readily after topical administration and the pharmacodynamic properties of LASAG are similar to aspirin. 88 LASAG was effective at inhibiting coronavirus replication in an in vitro cell system infected with either a low (human corona virous (HCoV)-229E) or high pathogenic strain (Middle East Respiratory Syndrome (MERS)-CoV). 87 Virus-induced NF-κB activity was attenuated early in the viral replication cycle by LASAG in highly pathogenic avian influenza virus strains leading to reduced viral titers, decreased viral protein accumulation, viral ribonucleic acid (RNA) synthesis, and impaired formation of viral replication transcription complexes. ...
... 87 Virus-induced NF-κB activity was attenuated early in the viral replication cycle by LASAG in highly pathogenic avian influenza virus strains leading to reduced viral titers, decreased viral protein accumulation, viral ribonucleic acid (RNA) synthesis, and impaired formation of viral replication transcription complexes. 88 Nebulized LASAG three times a day to achieve a total daily alveolar dose of 133.5 mg in 24 patients afflicted with severe influenza was found to be associated with faster alleviation of symptoms versus controls. 85 LASAG is currently available and approved for intravenous administration in Germany. ...
... 85 An inhaled nanoparticle aspirin formulation that can be administered as a dry powder inhaler to enhance the speed of platelet inhibition is under development. 88 In a recent pilot, Phase 1, open-label, single dose-escalation study, the inhalation formulation of aspirin compared to chewing and swallowing soluble aspirin formulation is associated with earlier drug exposure (2-4 minutes versus 30 minutes, respectively) and earlier greater inhibition of arachidonic acid-induced platelet inhibition (2 minutes versus 40 minutes, respectively). 89 Treatment with the above two inhaled formulations of aspirin may attenuate serious symptoms in COVID-19 during initial acute conditions. ...
Article
Full-text available
Introduction: Pharmacologic therapy options for COVID-19 should include antiviral, anti-inflammatory, and anticoagulant agents. With the limited effectiveness, currently available virus-directed therapies may have a substantial impact on global health due to continued reports of mutant variants affecting repeated waves of COVID-19 around the world. Methods: We searched articles pertaining to aspirin, COVID-19, acute lung injury and pharmacology in PubMed and provide a comprehensive appraisal of potential use of aspirin in the management of patients with COVID-19. The scope of this article is to provide an overview of the rationale and currently available clinical evidence that supports aspirin as an effective therapeutic option in COVID-19. Results: Experimental and clinical evidence are available for the potential use of aspirin in patients with COVID-19. Discussion: Aspirin targets the intracellular signaling pathway that is essential for viral replication, and resultant inflammatory responses, hypercoagulability, and platelet activation. With these multiple benefits, aspirin can be a credible adjunctive therapeutic option for the treatment of COVID-19. In addition, inhaled formulation with its rapid effects may enhance direct delivery to the lung, which is the key organ damaged in COVID-19 during the critical initial course of the disease, whereas the 150-325 mg/day can be used for long-term treatment to prevent thrombotic event occurrences. Being economical and widely available, aspirin can be exploited globally, particularly in underserved communities and remote areas of the world to combat the ongoing COVID-19 pandemic.
... 6 Aspirin was previously reported as capable in reducing RNA synthesis and replication of human coronavirus-299E (CoV-229E) and Middle East Respiratory Syndrome (MERS)-CoV in one in vitro study. 7 Remarkably, the use of aspirin in COVID-19 is still controversial due to conflicting results. Chow et al. showed the use of aspirin was associated with improved outcome among hospitalized patients with COVID-19, 8 while Yuan et al. and Sahai et al. reported otherwise. ...
... Anti-inflammatory effect Non-selective inhibitor of cyclooxygenase (COX-1 and COX-2) enzymes 34 Inducing overactivation Heme oxygenase-1 35 Modulation of immune system and inhibition of viral replication and/or entry Reducing reactive oxygen species (ROS) production and nuclear factor kappa beta (NF-κB) activation 7,26 Stimulating over-expression of ubiquitin-protein ligase E6A, adenylosuccinate lyase, and nibrin 25 Enhancing proteosomal degradation of claudin-1 36 Reducing virus affinity to CCAAT/ enhancer-binding protein-beta (C/ EBP-β) 37 Enhancing the expression and activity of Cu/Zn superoxide dismutase (SOD1) 38 Inhibiting of prostaglandin-E2 (PGE2) activity in macrophages and upregulating of interferon type I (IFN-I) production 39 Dose dependent anti-viral activity with unknown molecular mechanism 6 D-L lysine acetylsalicylate interferes NF-κB activation 7 Anti-thrombotic effect Inhibiting the production of Thromboxane-A2 22,34, Acetylation of proteins (e.g., fibrinogen) involved in the coagulation cascade 33 with COVID-19 could not be drawn definitively. ...
... Anti-inflammatory effect Non-selective inhibitor of cyclooxygenase (COX-1 and COX-2) enzymes 34 Inducing overactivation Heme oxygenase-1 35 Modulation of immune system and inhibition of viral replication and/or entry Reducing reactive oxygen species (ROS) production and nuclear factor kappa beta (NF-κB) activation 7,26 Stimulating over-expression of ubiquitin-protein ligase E6A, adenylosuccinate lyase, and nibrin 25 Enhancing proteosomal degradation of claudin-1 36 Reducing virus affinity to CCAAT/ enhancer-binding protein-beta (C/ EBP-β) 37 Enhancing the expression and activity of Cu/Zn superoxide dismutase (SOD1) 38 Inhibiting of prostaglandin-E2 (PGE2) activity in macrophages and upregulating of interferon type I (IFN-I) production 39 Dose dependent anti-viral activity with unknown molecular mechanism 6 D-L lysine acetylsalicylate interferes NF-κB activation 7 Anti-thrombotic effect Inhibiting the production of Thromboxane-A2 22,34, Acetylation of proteins (e.g., fibrinogen) involved in the coagulation cascade 33 with COVID-19 could not be drawn definitively. ...
Article
Full-text available
Background Repurposing the use of aspirin to treat hospitalized patients with COVID-19 is a sensible approach. However, several previous studies showed conflicting results. This meta-analysis was aimed to assess the effect of aspirin on the outcome in patients with COVID-19. Methods Systematic search using relevant keywords was carried out via several electronic databases until 21 February 2021. Research studies on adults COVID-19 patients with documentation on the use of aspirin and reported our outcomes of interest were included in the analysis. Our main outcome of interest was all types of mortality, while the incidence of thrombosis and bleeding were considered as secondary outcomes. Estimated risk estimates of the included studies were then pooled using DerSimonian-Laird random-effect models regardless heterogeneity. Results Seven studies with a total of 34,415 patients were included in this systematic review and meta-analysis. The use of aspirin was associated with a reduced risk of mortality (RR 0.56, 95% CI 0.38–0.81, P = 0.002; I²: 68%, P = 0.005). Sensitivity analysis by differentiating in-hospital (active aspirin prescription) and pre-hospital use of aspirin could significantly reduce the heterogeneity (I²: 1%, P = 0.4). Only one study reported the incidence of major bleeding between aspirin and non-aspirin users (6.1% vs. 7.6%, P = 0.61). The association between the use of aspirin and the incidence of thrombosis were contradictory in two studies. Conclusion The use of aspirin was significantly associated with a reduced risk of mortality among patients with COVID-19. Due to limited studies, the effect of aspirin on the incidence of thrombosis and bleeding in patients with COVID-19 could not be drawn definitively.
... First, its analgesic and antipyretic effects may help alleviate the specific symptoms of COVID-19. Second, it may exert antiinflammatory, antithrombotic and antiviral effects (229)(230)(231)(232)(233), which may be useful in preventing pathophysiological processes of severe clinical manifestations involved in COVID-19. Much solid evidence from in vitro and experimental models support that aspirin reduces the synthesis and replication of several RNA-encapsulated viruses (including human CoV-229E and MERS-coronavirus) in infected cells, thereby reducing viral titers and virulence (229). ...
... Second, it may exert antiinflammatory, antithrombotic and antiviral effects (229)(230)(231)(232)(233), which may be useful in preventing pathophysiological processes of severe clinical manifestations involved in COVID-19. Much solid evidence from in vitro and experimental models support that aspirin reduces the synthesis and replication of several RNA-encapsulated viruses (including human CoV-229E and MERS-coronavirus) in infected cells, thereby reducing viral titers and virulence (229). Moreover, excess Ang II signaling in COVID-19 may activate the STING pathway (234), which promotes hypercoagulation through the secretion of interferon-b and tissue factors by monocyte-macrophages. Aspirin has been found to directly inhibit the STING pathway (235), thereby reducing tissue factor procoagulant activities. ...
Article
Full-text available
The potential relationship between diabetes and COVID-19 has been evaluated. However, new knowledge is rapidly emerging. In this study, we systematically reviewed the relationship between viral cell surface receptors (ACE2, AXL, CD147, DC-SIGN, L-SIGN and DPP4) and SARS-CoV-2 infection risk, and emphasized the implications of ACE2 on SARS-CoV-2 infection and COVID-19 pathogenesis. Besides, we updated on the two-way interactions between diabetes and COVID-19, as well as the treatment options for COVID-19 comorbid patients from the perspective of ACE2. The efficacies of various clinical chemotherapeutic options, including anti-diabetic drugs, renin-angiotensin-aldosterone system inhibitors, lipid-lowering drugs, anticoagulants, and glucocorticoids for COVID-19 positive diabetic patients were discussed. Moreover, we reviewed the significance of two different forms of ACE2 (mACE2 and sACE2) and gender on COVID-19 susceptibility and severity. This review summarizes COVID-19 pathophysiology and the best strategies for clinical management of diabetes patients with COVID-19.
... 27 Because it has antiviral activity related to its ability to inhibit the coronavirus-induced nuclear factor kappa B pathway based on in silico docking analysis and in vitro cell culture study. 28 This efficacy of ASA has also been demonstrated in non-COVID patients. So that; A more recent meta-analysis of ten cohort studies enrolling 689 897 patients with sepsis revealed that ASA, administered either before or after sepsis, reduced ICU or hospital mortality. ...
Article
Full-text available
We aimed to investigate association between mean platelet volume (MVP), platelet distribution width (PDW) and red cell distribution width (RDW) and mortality in patients with COVID-19 and find out in which patients the use of acetylsalicylic acid (ASA) affects the prognosis due to the effect of MPV on thromboxan A2. A total of 5142 patients were divided into those followed in the intensive care unit (ICU) and those followed in the ward. Patient medical records were examined retrospectively. ROC analysis showed that the area under curve (AUC) values were 0.714, 0.750, 0.843 for MPV, RDW and D-Dimer, the cutoff value was 10.45fl, 43.65fl, 500.2 ng/mL respectively. (all P < .001). Survival analysis showed that patients with MPV >10.45 f/l and D-Dimer >500.2 ng/mL, treatment with ASA had lower in-hospital and 180-day mortality than patients without ASA in ICU patients (HR = 0.773; 95% CI = 0.595-0.992; P = .048, HR = 0.763; 95% CI = 0.590-0.987; P = .036). Administration of low-dose ASA in addition to anti-coagulant according to MPV and D-dimer levels reduces mortality.
... Тези молекули инхибират тромбоцитната агрегация и формирането на артериален тромб. Аспиринът инхибира активирания от SARS-CoV-2 нуклеарен фактор kappa B път, докато дипиридамолът има директно противовирусно действие [20,27]. ...
Article
Full-text available
COVID-19 infection is characterized with hyperstimulated infl ammatory response that affects lungs, cytokine storm and acute respiratory distress syndrome. Thrombotic complications are the leading reason for death in COVID-19 patients. Those of them with previous cardio-vascular diseases or risk factors – obesity, arterial hypertension, diabetes mellitus, advanced age are with higher risk for worse clinical outcome. Coagulopathy as well as thrombocytopathy and endothelial dysfunction are signifi cant pathophysiological factors for the severe clinical course of the infection. Beside anticoagulation therapy, targetеd strategies regarding thrombocytopathy and endothelial dysfunction are necessary for the treatment of patients with COVID-19 infection.
... In addition to the well-established anti-inflammatory and antithrombotic properties [103], aspirin is effective in reducing replication, propagation, and infectivity of several DNA and RNA viruses, including different human coronavirus (such as the human CoV-229E and the MERS-CoV) [104,105]. ...
Article
Full-text available
Coronavirus disease 2019 (COVID-19) is a pandemic syndrome caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. SARS-CoV-2 infection induces a process of inflammation and thrombosis supported by an altered platelet activation state. This platelet activation is peculiar being characterized by the formation of platelet-leukocytes rather than platelet–platelet aggregates and by an increased procoagulant potential supported by elevated levels of TF positive platelets and microvesicles. Therapeutic strategies targeting, beyond systemic inflammation (i.e. with tocilizumab, an anti interleukin-6 receptor), this state of platelet activation might therefore be beneficial. Among the antithrombotic drugs proposed as candidates to treat patients with SARS-CoV-2 infection, antiplatelet drugs, such as aspirin are showing promising results.
... On the other hand, same can also assist SARS coronaviruses for avoiding ribosomal slippage and producing more asymptomatic cases. Coronavirus replication in vitro gets inhibited after supplementation of 'D, L-lysine acetylsalicylate and glycine' (Muller et al. 2016). These two studies invite further investigations to understand the evolution of molecular mechanisms for coronavirus replication strategies and their relation with the prevalence of asymptomatic carriers. ...
Article
Full-text available
Analytical observations (in silico) indicate molecular features of SARS-Cov2 genome that potentially explains the high prevalence of asymptomatic cases in Covid-19 pandemic. We observed that the virus maintains a low preference for ‘GGG’ codon for glycine (3%) in its genome. We also observed multiple putative introns of 26–44 nucleotide (nt) length in the genomic region between the coding regions of Nsp10 and RPol in the viral ORF1ab, like several other beta-coronaviruses of similar infectivity levels. It appears that the virus employs a dual strategy to ensure unhindered replication within the host. One of the strategies employ a (− )1 frameshift translation event through programmed ribosomal slippage at the ribosomal slippage site in the ORF1ab. The alternate strategy relies on intron excision to generate a read through frame. The presence of ‘GGG’ in this conserved ribosomal slippage site ensures adequate tRNA in cytoplasm to match the codon, implying no additional frameshift translation due to ribosomal stalling. With fewer replication events, viral load remains low and resulting in asymptomatic cases. We suggest that this strategy is the primary reason for the prevalence of asymptomatic cases in the disease, enabling the virus to spread rapidly.
Article
The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causing the coronavirus disease-19 (COVID-19) is still challenging healthcare systems and societies worldwide. While vaccines are available, therapeutic strategies are developing and need to be adapted to each patient. Many clinical approaches focus on the repurposing of approved therapeutics against other diseases. However, the efficacy of these compounds on viral infection or even harmful secondary effects in the context of SARS-CoV-2 infection are sparsely investigated. Similarly, adverse effects of commonly used therapeutics against lifestyle diseases have not been studied in detail. Using mono cell culture systems and a more complex chip model, we investigated the effects of the acetylsalicylic acid (ASA) salt D,L-lysine-acetylsalicylate + glycine (LASAG) on SARS-CoV-2 infection in vitro. ASA is commonly known as Aspirin® and is one of the most frequently used medications worldwide. Our data indicate an inhibitory effect of LASAG on SARS-CoV-2 replication and SARS-CoV-2-induced expression of pro-inflammatory cytokines and coagulation factors. Remarkably, our data point to an additive effect of the combination of LASAG and the antiviral acting drug remdesivir on SARS-CoV-2 replication in vitro.
Article
Full-text available
Severe Acute Respiratory Syndrome-Coronavirus-2 (SARS-CoV-2) can induce inflammatory and thrombotic complications of pulmonary district (interstitial pneumonia), sometimes evolving toward acute respiratory failure. In adults, Acetylsalicylic Acid (ASA) is widely employed at low doses for primary and secondary prevention of cardiovascular diseases (CVD). Apart their anti-thrombotic effect, low ASA doses also exert an anti-inflammatory action. So, when these are assumed for CVD prevention, could prevent both inflammatory reaction and pro-coagulant tendency of Coronavirus-2019 (COVID-19) infection. In addition, some patients receiving ASA are simultaneously treated with Statins, to correct dyslipidemia. But, for their pleiotropic effects, Statins can also be useful to antagonize pulmonary thrombo-inflammation induced by COVID-19. Thus ASA, with or without Statins, employed for CVD prevention, could be useful to avoid or minimize inflammatory reaction and thrombotic complications of COVID-19. But, further studies performed in a wide range are requested to validate this hypothesis.
Article
Full-text available
Aim: Aspirin (Acetylsalicylic acid) has been used for more than 115 years in medicine. Research exists to show that aspirin has antiviral effects in vitro e.g. by blocking influenza virus propagation via NF-κB inhibition when used at high concentrations and short-term incubation steps. The aim of this study was to confirm the antiviral activity of aspirin against influenza virus and further elucidate the activity of aspirin against other respiratory viruses. Methods: Tests to detect antiviral activity were performed using plaque-reduction assays. Aspirin was administered to the virus-infected cell cultures one hour after infection. Prior to these assays, the non-cytotoxic concentrations of aspirin on cells used for propagation of the respective viruses were determined. Results: Aspirin was found to be highly effective against influenza A H1N1 virus. The antiviral activity against further respiratory RNA viruses was less distinct. Respiratory syncytial virus was minimally inhibited. However, the activity of aspirin against rhinoviruses was more pronounced. Aspirin demonstrated antiviral activity against all human rhinoviruses (HRV) but the effect on members of the "major group" viruses, namely HRV14 and HRV39, was greater than on those of the "minor group", HRV1A and HRV2. Conclusions: These data demonstrate a specific antiviral activity of aspirin against influenza A virus and HRV. The mode of action against rhinoviruses is still unknown and requires further investigation, as does the possibility of aspirin being effective in vivo to treat the common cold. This article is protected by copyright. All rights reserved.
Article
Full-text available
The lack of optimal porcine cell lines has severely impeded the study and progress in elucidation of porcine epidemic diarrhea virus (PEDV) pathogenesis. Vero cell, an African green monkey kidney cell line, was often used to isolate and propagate PEDV. Nonetheless, the target cells of PEDV in vivo are intestinal epithelial cells, during infection, intestinal epithelia would be damaged and resulted in digestive disorders. The immune functions of porcine epithelial cells and interactions with other immune cell populations display a number of differences compared to other species. Type I interferon (IFN) plays an important role in antiviral immune response. Limited reports showed that PEDV could inhibit type I interferon production. In this study, porcine small intestinal epithelial cells (IECs), the target cells of PEDV, were used as the infection model in vitro to identify the possible molecular mechanisms of PEDV-inhibition IFN-β production. PEDV not only failed to induce IFN-β expression, but also inhibited dsRNA-mediated IFN-β production in IECs. As the key IFN-β transcription factors, we found that dsRNA-induced activation of IFN regulatory factor 3 (IRF-3) was inhibited after PEDV infection, but not nuclear factor-kappaB (NF-κB). To identify the mechanism of PEDV intervention with dsRNA-mediated IFN-β expression more accurately, the role of individual molecules of RIG-I signaling pathway were investigated. In the upstream of IRF-3, TANK-binding kinase 1 (TBK1)-or inhibitor of κB kinase-ε (IKKε)-mediated IFN-β production was not blocked by PEDV, while RIG-I-and its adapter molecule IFN-β promoter stimulator 1 (IPS-1)-mediated IFN-β production were completely inhibited after PEDV infection. Taken together, our data demonstrated for the first time that PEDV infection of its target cell line, IECs, inhibited dsRNA-mediated IFN-β production by blocking the activation of IPS-1 in RIG-I-mediated pathway. Our studies offered new visions in understanding of the interaction between PEDV and host innate immune system.
Article
Full-text available
Coronaviruses can cause respiratory and enteric disease in a wide variety of human and animal hosts. The 2003 outbreak of severe acute respiratory syndrome (SARS) first demonstrated the potentially lethal consequences of zoonotic coronavirus infections in humans. In 2012, a similar previously unknown coronavirus emerged, Middle East respiratory syndrome coronavirus (MERS-CoV), thus far causing over 650 laboratory-confirmed infections, with an unexplained steep rise in the number of cases being recorded over recent months. The human MERS fatality rate of similar to 30% is alarmingly high, even though many deaths were associated with underlying medical conditions. Registered therapeutics for the treatment of coronavirus infections are not available. Moreover, the pace of drug development and registration for human use is generally incompatible with strategies to combat emerging infectious diseases. Therefore, we have screened a library of 348 FDA-approved drugs for anti-MERS-CoV activity in cell culture. If such compounds proved sufficiently potent, their efficacy might be directly assessed in MERS patients. We identified four compounds (chloroquine, chlorpromazine, loperamide, and lopinavir) inhibiting MERS-CoV replication in the low-micromolar range (50% effective concentrations [EC(50)s], 3 to 8 mu M). Moreover, these compounds also inhibit the replication of SARS coronavirus and human coronavirus 229E. Although their protective activity (alone or in combination) remains to be assessed in animal models, our findings may offer a starting point for treatment of patients infected with zoonotic coronaviruses like MERS-CoV. Although they may not necessarily reduce viral replication to very low levels, a moderate viral load reduction may create a window during which to mount a protective immune response.
Article
Full-text available
In September 2012, a case of novel coronavirus (CoV) infection was reported in Saudi Arabia. It is caused by a CoV called Middle East Respiratory Syndrome (MERS). Eight countries have reported the virus so far, with most of the reported cases from Saudi Arabia. Fatality rate is about 44% and most people who have been confirmed to have MERS-CoV developed severe acute respiratory illness. There is very limited information on transmission, severity and clinical impact with only small number of cases reported so far. However, the virus has not shown to spread in sustained way in communities. There is no available vaccine or proven treatment for this novel virus; however, there are several treatment protocols under trail. Healthcare is provided to infected individuals by alleviating symptoms and treating its complications; nevertheless the situation is still evolving.
Article
Porcine epidemic diarrhea virus (PEDV) is a coronavirus that induces persistent diarrhea in swine, resulting in severe economic losses in swine-producing countries. Insights into the interplay between PEDV infection and innate immune system are necessary for understanding the associated mechanism of pathogenesis. The transcription factor nuclear factor kappa B (NF-κB) plays an important role in regulating host immune responses. Here, we elucidated for the first time the potential mechanism of PEDV-mediated NF-κB activation in porcine small intestinal epithelial cells (IECs). During PEDV infection, NF-κB p65 was found to translocate from the cytoplasm to the nucleus, and PEDV-dependent NF-κB activity was associated with viral dose and active replication. Using small interfering RNAs to screen different mRNA components of the Toll-like receptor (TLR) or RIG-I-like receptor signaling pathways, we demonstrated that TLR2, TLR3, and TLR9 contribute to NF-κB activation in response to PEDV infection, but not RIG-I. By screening PEDV structural proteins for their abilities to induce NF-κB activities, we found that PEDV nucleocapsid protein (N) could activate NF-κB and that the central region of N was essential for NF-κB activation. Furthermore, TLR2 was involved in PEDV N-induced NF-κB activation in IECs. Collectively, these findings provide new avenues of investigation into the molecular mechanisms of NF-κB activation induced by PEDV infection.