Antiviral Research 147 (2017) 142–148

Contents lists available at ScienceDirect

Antiviral Research
journal homepage: www.elsevier.com/locate/antiviral

The susceptibility of circulating human influenza viruses to tizoxanide, the MARK

active metabolite of nitazoxanide
Danielle Tilmanisa, Carel van Baalenb, Ding Yuan Oha,c, Jean-Francois Rossignold,
Aeron C. Hurta,e,∗
a
WHO Collaborating Centre for Reference and Research on Influenza, VIDRL, Peter Doherty Institute for Infection and Immunity, Melbourne, VIC 3000, Australia
b
Viroclinics Biosciences B.V., Rotterdam, The Netherlands
c
Federation University, School of Applied and Biomedical Sciences, Gippsland VIC 3842, Australia
d
Romark Laboratories, L.C., Tampa, FL, USA
e
University of Melbourne, Department of Microbiology and Immunology, Parkville, VIC 3010, Australia

A R T I C L E I N F O A B S T R A C T

Keywords: Nitazoxanide is a thiazolide compound that was originally developed as an anti-parasitic agent, but has recently
Influenza been repurposed for the treatment of influenza virus infections. Thought to exert its anti-influenza activity via
Nitazoxanide the inhibition of hemagglutinin maturation and intracellular trafficking in infected cells, the effectiveness of
Antiviral activity nitazoxanide in treating patients with non-complicated influenza is currently being assessed in phase III clinical
Focus reduction assay
trials.
Susceptibility
Resistance
Here, we describe the susceptibility of 210 seasonal influenza viruses to tizoxanide, the active circulating
metabolite of nitazoxanide. An optimised cell culture-based focus reduction assay was used to determine the
susceptibility of A(H1N1)pdm09, A(H3N2), and influenza B viruses circulating in the southern hemisphere from
the period March 2014 to August 2016. Tizoxanide showed potent in vitro antiviral activity against all influenza
viruses tested, including neuraminidase inhibitor-resistant viruses, allowing the establishment of a baseline level
of susceptibility for each subtype. Median EC50 values ( ± IQR) of 0.48 μM (0.33–0.71), 0.62 μM (0.56–0.75),
0.66 μM (0.62–0.69), and 0.60 μM (0.51–0.67) were obtained for A(H1N1)pdm09, A(H3N2), B(Victoria lineage),
and B(Yamagata lineage) influenza viruses respectively. There was no significant difference in the median
baseline tizoxanide susceptibility for each influenza subtype tested. This is the first report on the susceptibility of
circulating viruses to tizoxanide. The focus reduction assay format described is sensitive, robust, and less la-
borious than traditional cell based antiviral assays, making it highly suitable for the surveillance of tizoxanide
susceptibility in circulating seasonal influenza viruses.

1. Introduction
Influenza remains a significant threat to global public health, and
causes high morbidity and mortality in immunocompromised, elderly,
and paediatric populations (Costantino and Vitale, 2016). While vac-
cination is the most common method for preventing influenza, the
protection it provides heavily depends on the antigenic match between
the vaccine strains and the circulating viruses, and the age and health
status of the recipient (Lambert and Fauci, 2010). Antiviral drugs
provide another option for the control of influenza, and can be used
either as short-term prophylaxis to prevent infection, or as a therapeutic
to reduce viral replication and duration of illness post-infection (Byrn
et al., 2015). Antivirals play a particularly important role in the
treatment of severely ill patients with influenza, and in the event of a
pandemic when pre-existing immunity is lacking and an appropriately
matched vaccine is unavailable (Naesens et al., 2016). Two classes of
licensed influenza antiviral drugs are currently available, the ada-
mantanes (M2 ion channel blockers), and the neuraminidase inhibitors
(NAIs). However, the adamantanes are no longer recommended for
influenza treatment because currently circulating influenza A viruses
are resistant to this class of antivirals (Hurt, 2014).
The two NAIs licensed in many countries, oseltamivir (the most
widely used NAI) and zanamivir, can reduce the duration of un-
complicated influenza illness when administered within 48 h of
symptom onset (Dobson et al., 2015). In addition, treatment with
oseltamivir has been shown to reduce rates of hospitalisation and

∗
Corresponding author. WHO Collaborating Centre for Reference and Research on Influenza, VIDRL, Peter Doherty Institute for Infection and Immunity, Melbourne, VIC 3000,
Australia.
E-mail address: Aeron.Hurt@influenzacentre.org (A.C. Hurt).

http://dx.doi.org/10.1016/j.antiviral.2017.10.002
Received 11 August 2017; Received in revised form 22 September 2017; Accepted 2 October 2017
Available online 03 October 2017
0166-3542/ © 2017 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY license (http://creativecommons.org/licenses/BY/4.0/).
D. Tilmanis et al.
mortality, (Fiore et al., 2011; Louie et al., 2012). Although the fre-
quency of resistance to the NAIs is low in currently circulating influenza
viruses (approximately 0.5%) (Hurt et al., 2016), the emergence and
rapid spread of oseltamivir-resistance in seasonal H1N1 viruses in
2007–2008 highlighted the limitations of this class of antiviral drug,
and is cause for concern should another NAI resistant strain become
prevalent. Furthermore, the therapeutic use of antivirals remains pro-
blematic when treating immunocompromised patients who typically
have prolonged virus shedding (Fiore et al., 2011), due to the increased
likelihood of resistance developing during extended courses of antiviral
treatment (Li et al., 2015). There is a need for new influenza antivirals
that are less likely to select for resistance and that are more effective in
treating influenza patients from high risk groups, particularly when
treatment is delayed by more than 48 h after symptom onset. Devel-
opment of influenza antivirals has accelerated in the last decade, and
several novel compounds that exert anti-influenza activity by targeting
either viral or host proteins are currently in various stages of devel-
opment (Koszalka et al., 2017). Among them is the thiazolide com-
pound nitazoxanide (NTZ).
NTZ was originally developed as an anti-protozoal drug, and is
currently licensed for the treatment of Cryptosporidium and Giardia in-
fections (Rossignol et al., 2009). In addition to anti-parasitic activity,
NTZ, via its active metabolite tizoxanide (TIZ), also exhibits anti-
bacterial activity against a range of anaerobic bacteria, as well as broad
spectrum in vitro antiviral activity against influenza, respiratory syn-
cytial virus, norovirus, rotavirus, and hepatitis B and C viruses
(Rossignol, 2014).
The broad spectrum antiviral activity of NTZ can be attributed to
the fact that it targets host cell mechanisms rather than the virus, al-
though the specific mode of action differs depending on the virus
(Rossignol, 2014). In the case of influenza, NTZ inhibits the function of
the endoplasmic reticulum (ER) protein ERp57, which is essential for
the maturation of the influenza virus hemagglutinin (HA), and post-
translational trafficking from the ER to the Golgi (Rossignol, 2017).
In vitro NTZ susceptibility has previously been determined for a
panel of eight reference influenza A viruses representing H1N1, H3N2,
H5N9, H7N1 subtypes, with reported IC50 values of 1.0–3.2 μM across
the panel (Belardo et al., 2015). NTZ also inhibited the growth of two
variant H3N2 influenza viruses with mean IC90 values of 1.09 μM and
0.86 μM (Sleeman et al., 2014). Rossignol et al. (2009), reported
equivalent antiviral activity for both NTZ and TIZ, and TIZ EC50 values
of 0.3–0.5 μg/mL (1.1–1.9 μM) for the three reference viruses, A/PR/8/
34 (H1N1), A/WSN/33 (H1N1) and A/Ck/It/9097/97 (H5N9). Fur-
thermore NTZ and TIZ also exhibited equal potency against three iso-
lates of canine influenza virus with EC50 values of 0.2 μM reported for
both compounds (Ashton et al., 2010). These results are not surprising
given that NTZ is readily hydrolysed to TIZ in aqueous buffers such as
cell culture medium (Broekhuysen et al., 2000).
NTZ has been effective in treating patients with uncomplicated in-
fluenza in a Phase IIb/III trial (Haffizulla et al., 2014). A placebo con-
trolled Phase III trial is currently underway to investigate treatment
with NTZ versus oseltamivir versus NTZ plus oseltamivir combination
in patients with uncomplicated influenza (ClinicalTrials.gov:
NCT01610245).
Although in vitro NTZ/TIZ susceptibility has been identified for a
small number of influenza reference viruses, data are not available for a
large number of seasonal influenza viruses. Here we describe the TIZ
susceptibility of 210 recently circulating seasonal influenza viruses,
including NAI-resistant viruses. This provides baseline TIZ suscept-
ibility data for A(H1N1)pdm09, A(H3N2), and B influenza viruses. In
addition, we describe in detail the development of a focus reduction
assay that is well suited to determining the TIZ susceptibility of a large
number of surveillance isolates.
143
Antiviral Research 147 (2017) 142–148
2. Materials and methods
2.1. Antiviral compounds
TIZ was supplied by Romark Laboratories, (Tampa, Florida, USA).
50 mM stocks were prepared in dimethyl sulfoxide (DMSO; Sigma,
USA), sterilised using 0.2 μm surfactant-free cellulose acetate (SFCA)
filters (ThermoFisher, USA), and aliquots stored at −20 °C. Working
solutions of TIZ were prepared from frozen stocks by further dilution in
DMSO, followed by a final dilution in cell culture media immediately
prior to assay setup.
2.2. Cells and viruses
Madin-Darby canine kidney (MDCK) cells were obtained from the
American Type Culture Collection (Manassas, VA, USA) and cultured in
MDCK cell growth medium (MGM) [Dulbecco's modified Eagles
medium (DMEM; Gibco, USA) supplemented with 10% foetal bovine
serum (FBS; Bovogen Biologicals, AUS), 100U/mL Penicillin-
Streptomycin, 20 μM HEPES, 0.06% Sodium Bicarbonate, 1x MEM non-
essential amino acids (NEAA), and 1x GlutaMAX, (Gibco, USA)].
MDCK-SIAT cells (MDCK cells modified to overexpress α 2,6-linked
sialic acids) were kindly provided by Dr. M. Matrosovich (Matrosovich
et al., 2003) and maintained in MDCK-SIAT cell growth medium (SGM)
[MGM supplemented with 1 mg/mL Geneticin (Gibco, USA)]. All cell
maintenance and cell-based in vitro assays were carried out in humi-
dified incubators with 5% CO2.
Influenza viruses were submitted to the WHO Collaborating Centre
for Reference and Research on Influenza, Melbourne, Australia, as part
of the WHO Global Influenza Surveillance and Response System
(GISRS). Viruses were selected for testing based on isolation between
2014 and 2016 and for breadth of regional distribution. The viruses
tested were collected from Australia (N = 99), New Zealand (N = 20),
Singapore (N = 17), Cambodia (N = 14), New Caledonia (N = 14), Fiji
(N = 11), Thailand (N = 6), Japan (N = 5), USA (N = 5), The
Philippines (N = 4), South Africa (N = 4), Sri Lanka (N = 4), China
(N = 3), Vietnam (N = 3), and Tonga (N = 1). Six seasonal viruses
were used for assay development, A/Perth/265/2009 and A/Osaka/
180/2009 (A(H1N1)pdm09), A/New Caledonia/104/2014 and A/
Newcastle/22/2014 (A(H3N2)), B/Brisbane/46/2015 (B-Victoria
lineage) and B/Sydney/39/2015 (B-Yamagata lineage). A(H1N1)
pdm09, B-Yamagata lineage, and B-Victoria lineage viruses were pro-
pagated in MDCK cells, while A(H3N2) viruses were propagated in
MDCK-SIAT cells. Virus dilutions for use in subsequent assays were
prepared in maintenance medium (MM)[MGM without FBS, supple-
mented with 2 μg/mL Amphotericin B (Gibco, USA), 1 μg/mL TPCK-
treated trypsin (Worthington, USA), and 0.5% DMSO].
2.3. Cytotoxicity assays
The cytotoxicity of TIZ was determined in MDCK and MDCK-SIAT
cells to identify a suitable non-toxic concentration range for use in the
susceptibility assay. MDCK cells (3.5 × 104 cells/well, 100 μl/well),
and MDCK-SIAT cells (2.5 × 104 cells/well, 100 μl/well), were seeded
into flat-bottom 96-well plates (Corning, USA) in MGM and SGM re-
spectively, and incubated overnight at 37 °C. Confluent cell monolayers
were washed with phosphate buffered saline (PBS; Sigma, USA), and
100 μl of a 2-fold serial dilution of TIZ (0.4–100 μM) in MM was added
to cells. The final DMSO concentration in each well was 0.5%. Due to
the low solubility of TIZ in aqueous solutions, concentrations greater
than 100 μM were not tested. DMSO at 0.5% in MM was added to cell
only control wells. Cytotoxicity was assessed using the CellTiterGlo cell
viability assay (CTG; Promega, USA), and the MTT (3-(4,5-di-
methylthiazol-2-yl)- 2,5-diphenyltetrazolium bromide; MP
Biomedicals, USA) assay, following incubation of plates for 24 h at
35 °C. The CTG assay was performed as per manufacturer's instructions,
D. Tilmanis et al.
and luminescence was measured using a FLUOstar Optima luminometer
(BMG Labtech, Germany). The procedure for the MTT assay was based
on previously described methods (Mosmann, 1983). Briefly, cell
monolayers were incubated for 4 h in the presence of 1 mg/mL MTT at
37 °C. The supernatant was removed and 200 μl isopropanol (Sigma,
USA) added to dissolve the purple formazan that was produced. Ab-
sorbance was measured at 570 nm using a Multiskan Ascent plate
reader (Thermo Fisher, USA). The 50% cytotoxic concentration for TIZ
(CC50; drug concentration that reduced the cell viability by 50% com-
pared to the cell only control) was determined using nonlinear regres-
sion analysis (GraphPad Prism, USA) for both cytotoxicity readouts.
2.4. Titration of virus
To determine the appropriate viral input for the focus reduction
assay, a virus titration was performed for each virus. MDCK and MDCK-
SIAT cells were seeded in 96-well plates overnight as previously de-
scribed (Section 2.3). Half-log dilutions of each virus were prepared in
MM in a 96 well U-bottom plate. Cell monolayers were washed with
PBS and 50 μl of each virus dilution was added to the plates, followed
by incubation at 35 °C for 90 min. Mock-infected cells in MM served as
a cell-only control. After incubation, the virus/mock inoculum was
removed. Monolayers were then washed with PBS to remove unbound
virus and overlaid with 100 μl infection medium [IM; equal parts 3.2%
carboxymethyl cellulose (CMC; Sigma Aldrich, USA) and 2x DMEM
[Sigma Aldrich (USA), containing 20 μM HEPES (Gibco, USA), 100 U/
mL Penicillin-Streptomycin (Gibco, USA), 0.06% Sodium Bicarbonate
(Gibco, USA), and 1 μg/mL L-tosylamido 2-phenylethyl chloromethyl
ketone (TPCK)-treated trypsin (Worthington, USA)] and 0.5% DMSO.
Plates were then incubated at 35 °C for either 8, 16, 24 or 30 h, after
which IM was removed, and cells were fixed and immunostained using
methods described previously (van Baalen et al., 2017) with some
minor modifications. Briefly, the IM was removed and the cells were
fixed for 15 min at room temperature (RT) with 100 μl 10% formalin
(Sigma Aldrich, USA). After removing the fixative and washing the cell
monolayers with 150 μl PBS, then 100 μl 0.5% Triton X-100 (Sigma
Aldrich, USA) in PBS was added to all wells and the plates were in-
cubated for 10 min at RT. The plates were then washed three times with
0.05% Tween 20 (Sigma Aldrich, USA) in PBS, and the cells were in-
cubated for 1 h with a mouse anti-influenza monoclonal antibody
against influenza A virus nucleoprotein (Millipore, USA,
Cat#MAB8251) or against influenza B virus nucleoprotein (Millipore,
USA, Cat#MAB8661), diluted 1:5000 in blocking solution (2% skim
milk powder in PBS). After repeating the washing step, cells were in-
cubated for 1 h with goat anti-mouse HRP-conjugated secondary anti-
body (BioRad, USA) diluted 1:1000 in blocking solution. The washing
step was repeated, and the infected cells were stained with 50 μl True
Blue peroxidase substrate (KPL, USA) for 10 min in the dark. The plates
were washed with Milli-Q water three times and allowed to dry com-
pletely. Plate images were captured and the number of focus forming
units (FFU) per well quantified using an Immunospot analyser and
Biospot software (CTL Immunospot, USA). The parameters used for
counting FFU were set up to reflect one focus unit to represent one
individual virus infected cell (Table S1 Supplementary material). Virus
dilutions that resulted in approximately 1000 FFU/well were used in
subsequent focus reduction assays.
2.5. Focus reduction assay
The susceptibility of influenza viruses to TIZ was determined by
measuring the reduction in FFU in the presence of the drug. Confluent
monolayers of MDCK or MDCK-SIAT cells in 96 well plates (seeded
overnight as described previously), were washed with PBS and 50 μl of
virus diluted to approximately 1000 FFU/well as determined pre-
viously, was added to each well of the culture plates. 50 μL of MM was
added to cell control wells. After incubation for 90 min at 35 °C the
144
Antiviral Research 147 (2017) 142–148
virus/mock inoculum was removed. Cell monolayers were washed once
with PBS, and overlaid with 2-fold serial dilutions of TIZ (0.08–10 μM)
in 100 μL IM. IM +0.5% DMSO was added to virus control and cell
control wells, and the plates were incubated at 35 °C for 8, 16, 24 or
30 h. Plates were then immunostained as described in Section 2.4, and
the number of foci/well determined as described above. Each virus was
tested against each TIZ concentration in duplicate wells and a mean
number of FFU calculated for each pair. Using the mean FFU, the per-
centage inhibition of FFU was calculated by formula 100-[(X-CC)/(VC-
CC) x100], where X = FFU in test wells (virus + drug at each con-
centration), CC= FFU in cell control wells (no virus, to account for
background), and VC=FFU in virus control wells (no drug).
The 50% effective concentration of TIZ for each virus (EC50; drug
concentration that inhibited FFU formation by 50% compared to the
cell only control) was determined using non-linear regression analysis
(GraphPad Prism, USA).
2.6. Statistical methods
Linear regression analysis, unpaired student's t-tests, and Kruskall
Wallis tests were performed using GraphPad Prism (USA) (P < 0.05
was considered statistically significant). Statistical plots were con-
structed using R version 3.1.2 (R Foundation for Statistical Computing,
Austria). To evaluate assay reproducibility, focus reduction assays were
performed with replicate (N = 48) wells of positive and negative
controls ( ± virus) on a 96-well plate and Z factors were calculated
using the equation outlined in Zhang et al. (1999).
3. Results
3.1. Cytotoxicity of TIZ
To determine the maximum non-toxic concentration for use in the
susceptibility assay, the effect of TIZ on the viability of MDCK and
MDCK-SIAT cells was determined using the CellTiterGlo assay and MTT
assay over a 24 h incubation period. In both cell lines the CC50 of TIZ
was higher in the MTT assay readout compared to the CellTiterGlo
assay (Table 1). The CC50 of TIZ was > 100 μM and 61.2 ± 15.1 μM in
the MTT assay in MDCK and MDCK-SIAT cells respectively, compared
to 77.1 ± 17.9 μM and 47.8 ± 18.9 μM for MDCK and MDCK-SIAT
cells using CellTiterGlo. To avoid underestimating cytotoxicity of TIZ,
the results from the CellTiterGlo cytotoxicity assays were used to select
a maximum test concentration of 10 μM for use in all further experi-
ments. At this concentration and below, the viability of both cell lines
was ≥80%.
3.2. Determination of optimal assay conditions
The appearance of foci, reproducibility, and EC50 values for TIZ
were examined at 8, 16, 24, and 30 h post infection (hpi) in both MDCK
and MDCK-SIAT cells, using six representative viruses [2x A(H1N1)
pdm09, 2x A(H3N2), 2x influenza B viruses]. Foci produced at 8 hpi for
all subtypes were low in intensity and fewer in number in comparison
to other time points (Fig. 1). In addition, 2-fold variations in foci
number were observed in duplicate wells for some viruses at 8 hpi,
Table 1
Cytotoxicity of TIZ in MDCK and SIAT1-MDCK cells.
Cell line TIZ CC50 (μM)a
CellTiterGlo MTT
MDCK 77.1 ± 17.9 > 100
MDCK-SIAT 47.8 ± 18.9 61.2 ± 15.1
a
CC50 values are presented as the mean ± SD of six separate experiments.
D. Tilmanis et al. Antiviral Research 147 (2017) 142–148

Fig. 1. Examples of foci formation in virus control wells after 8, 16,

24 and 30 h post infection in MDCK cells and MDCK-SIAT cells for
A(H1N1)pdm09, A(H3N2) and B influenza viruses. (Figure com-
posed of images captured across multiple assay plates).

Fig. 2. Effect of assay duration on assay reproducibility (A), and TIZ susceptibility (B), for A(H1N1)pdm09 (A/Perth/265/2009), A(H3N2) (A/New Caledonia/104/2014) and B (B/
Brisbane/46/2015) influenza viruses. Each virus was tested in a single experiment at each condition. One representative virus for each subtype/type is shown. Please refer to
Supplementary Material Fig. S1 for results for an additional three viruses.

145
D. Tilmanis et al.
therefore this time point was not analysed further. Examination of Z
factors for all time points indicated that the assay was more re-
producible (i.e. Z factors were closer to 1) when outside wells were
omitted from the analysis (Fig. 2A). Therefore only Z factors calculated
in the absence of the outside wells were used to select assay duration. A
Z factor of > 0.5 is considered a requirement for an assay to deliver
reproducible and accurate results. Z-factors > 0.5 were observed at 16
and 24 h for A(H1N1)pdm09 viruses and A(H3N2) viruses, and 16, 24,
and 30 h for influenza B viruses using both cell lines. For influenza A
viruses, EC50 values dropped markedly from 16 to 24 h, suggesting that
a 24 h assay duration is required to achieve a complete antiviral effect
(Fig. 2B). For influenza B viruses, the EC50 values were similar at 16 h,
24 h, and 30 h (Fig. 2B). For all subtypes/types, comparable Z factors
were obtained in each cell line, and while marginally lower EC50 values
were obtained in MDCK-SIAT cells compared to MDCK cells, either cell
line is considered suitable for performing the susceptibility assay.
Taking into account these findings, and the convenience of choosing a
single protocol that was suitable for testing all types/subtypes, all fur-
ther virus titrations and susceptibility assays were carried out using a
duration of 24 h with MDCK-SIAT cells. In order to assess inter-assay
variability, the same six representative viruses were tested for TIZ
susceptibility in an additional 3–6 assays using these optimised condi-
tions, and the mean EC50 and standard deviation was calculated for
each virus (Table S2 supplementary material). Due to the small stan-
dard deviations of the mean EC50 for each virus, the inter-assay varia-
bility was considered minimal and therefore allowed testing of each test
virus in a single assay.
The viral inoculum is an important parameter, and had to be high
enough to produce a statistically significant number of foci, yet not too
great to compromise drug sensitivity. As a result of initial titration
experiments, the virus dilution that yielded approximately 1000 foci
per well was used as the virus input. A plot of the number of foci versus
virus dilution produced a sigmoidal curve, and for all viruses, 1000 foci
fell in the lower linear region indicative of suitable sensitivity.
However, we investigated the significance of small variations in virus
input and the subsequent impact on the final EC50 result. The range of
FFU in the virus control well that resulted in comparable and accurate
TIZ EC50 values was tested for four representative viruses from different
subtypes/lineages. Comparable EC50 values were obtained for each
virus when the FFU/well was in the range of 200–2500 (Fig. S2 sup-
plementary material). Therefore, while 1000 foci/well was the target
virus input, the assay was considered valid if the FFU/well of the virus
control was in the range of 200–2500.
3.3. TIZ susceptibility of circulating seasonal influenza viruses
The in vitro TIZ susceptibility of 210 seasonal influenza viruses (54x
A(H1N1)pdm09, 53x A(H3N2), 56x B/Yamagata lineage and 47x B/
Victoria lineage) circulating from the period March 2014 to August
2016, was assessed in a 24 h assay in MDCK-SIAT cells. TIZ showed
potent activity against all influenza viruses tested (Fig. 3), with median
EC50 values of 0.48 μM, 0.62 μM, 0.66 μM, and 0.60 μM for A(H1N1)
pdm09, A(H3N2), B(Victoria lineage), and B(Yamagata lineage) influ-
enza viruses respectively. There was no significant difference between
the median values for each subtype (P > 0.05), although a larger
range of EC50 values was observed for the influenza A virus subtypes
[0.1–1.23 μM for A(H1N1)pdm09 viruses and 0.22–1.14 μM for
A(H3N2 influenza viruses] than for both B lineage viruses
(0.34–0.88 μM and 0.25–0.92 μM for B/Victoria lineage and B/Yama-
gata lineage viruses respectively).
3.4. TIZ susceptibility of neuraminidase inhibitor-resistant influenza viruses
A panel of fourteen circulating influenza viruses with reduced sus-
ceptibility to NAIs was also assessed for susceptibility to TIZ. All NAI-
resistant viruses tested were susceptible to TIZ (Table 2), and
146
Antiviral Research 147 (2017) 142–148
Fig. 3. Distribution of TIZ EC50 values for A(H1N1)pdm09, A(H3N2), B/Victoria lineage,
and B/Yamagata lineage influenza viruses circulating from March 2014 to August 2016.
Viruses were tested using a 24 h focus reduction assay in MDCK-SIAT cells.
furthermore there was no significant difference between mean TIZ EC50
of NAI-sensitive viruses and NAI-resistant viruses, 0.61 μM and 0.62 μM
respectively (P > 0.05).
4. Discussion
Ongoing surveillance of the antiviral susceptibility of circulating
viruses to currently available influenza antivirals, the adamantanes and
NAIs, has been important for the detection of emerging resistant
viruses, identification of the genetic mutations that confer resistance,
and ultimately for guiding therapeutic options for patients requiring
treatment (Hurt et al., 2016; Kossyvakis et al., 2017; Takashita et al.,
2015). With the potential licensure of several new influenza ther-
apeutics, including nitazoxanide, cell-based susceptibility assays that
are amenable to screening a large number of viruses are required for
surveillance purposes. Traditionally, cell-based yield reduction assays
have been used extensively for the assessment of influenza antivirals,
including nitazoxanide (Ashton et al., 2010; Belardo et al., 2015; Byrn
et al., 2015; Jones et al., 2016; Perwitasari et al., 2014; Sleeman et al.,
2014). However this assay format is very laborious and time con-
suming, and is therefore unsuitable for screening a large number of
samples. Focus reduction assays have recently become a useful method
for the antigenic characterisation of influenza viruses, particularly
A(H3N2) viruses that have lost the ability to agglutinate red blood cells
(Lin et al., 2015; Terletskaia-Ladwig et al., 2013; van Baalen et al.,
2017). Here, we have utilised the virus infectivity detection method
recently developed for antigenic characterisation assays (van Baalen
et al., 2017), to assess the TIZ susceptibility of recently circulating
seasonal influenza viruses and NAI-resistant viruses. The assay method
enabled rapid and direct determination of the reduction of virus in-
fectivity in the presence of TIZ, and was amenable to the analysis of a
large number of viruses. TIZ was selected for use in the assay, rather
than NTZ, as it is the active metabolite form, however the use of NTZ in
this assay generated highly similar results (data not shown). As is ty-
pical with surveillance testing of circulating viruses for NAI suscept-
ibility (Takashita et al., 2015), each virus was tested in a single assay
with repeat confirmatory testing necessary if the EC50 value was outside
the typical range. In addition, this assay format could be utilised for the
determination of the susceptibility of influenza viruses to other antiviral
drugs.
The potent activity of TIZ against 210 circulating A(H1N1)pdm09,
A(H3N2), B (Victoria lineage), and B (Yamagata lineage) influenza
viruses shown in the present study, was similar to previous in vitro data
which report EC50 values for laboratory reference viruses of these
D. Tilmanis et al. Antiviral Research 147 (2017) 142–148

Table 2
Susceptibility of NAI resistant viruses to TIZ.

Virus Designation Subtype NA mutation NAI susceptibility of virus to:

Tizoxanide EC50 (μM)b Oseltamivira Zanamivira Peramivira Laninamivira

A/Sydney/185/2015 A(H1N1)pdm09 H275Y 0.31 HRI NI HRI NI

A/Singapore/TT1275/2015 A(H1N1)pdm09 H275Y 0.34 HRI NI HRI NI
A/Sydney/158/2016 A(H1N1)pdm09 H275Y 0.32 HRI NI HRI NI
A/Victoria/2500/2016 A(H1N1)pdm09 H275Y 0.55 HRI NI HRI NI
A/Malaysia/2/2014 A(H1N1)pdm09 I223R 1.00 RI NI NI NI
A/Victoria/1031/2010 A(H3N2) E119V 0.38 RI NI NI NI
A/Victoria/1035/2010 A(H3N2) E119V 0.47 RI NI NI NI
A/Victoria/92/2012 A(H3N2) E119V 0.72 RI NI NI NI
A/Malaysia/22/2013 A(H3N2) Q136K 0.78 NI RI NI NI
A/Sydney/236/2014 A(H3N2) Q136K 0.45 NI RI NI NI
B/South Australia/2/2015 B D197N 0.79 RI NI RI NI
B/Canberra/1/2015 B D197N 0.92 RI NI RI NI
B/South Australia/5/2015 B D197N 0.79 RI NI RI NI
B/Perth/136/2015 B H273Y 0.80 RI NI HRI NI

a
NI = normal inhibition (Influenza A:within 10 fold of the median IC50; Influenza B: within 5-fold of the median IC50) ; RI = reduced inhibition (Influenza A:10–100 fold the median
IC50; Influenza B: 5–50 fold the median IC50); HRI = highly reduced inhibition (Influenza A: > 100 fold the median IC50; Influenza B: > 50 fold the median IC50). NAI susceptibility was
determined in a fluorometric neuraminidase inhibition assay.
b
EC50 values were determined using the focus reduction assay.

subtypes/types ranging from 1.1 μM to 5.6 μM (0.3–1.5 μg/mL) References

(Rossignol, 2014). In contrast to the reduced oseltamivir-susceptibility
of circulating influenza B viruses compared to influenza A viruses
(Farrukee et al., 2015), there was no significant difference in the
median TIZ EC50 values for influenza A and influenza B viruses. How-
ever, there was a greater range of EC50 values for influenza A virus
subtypes than for influenza B viruses, although the reason for this is
currently unknown.
Historically, the majority of FDA- approved drugs used to treat
human virus infections have targeted viral enzymes (de Chassey et al.,
2014). While a number of compounds that directly target specific in-
fluenza viral proteins are currently in pre-clinical and clinical devel-
opment, several host targeted compounds are also being investigated
(Koszalka et al., 2017). These compounds, such as nitazoxanide, act on
host cellular functions that are essential for virus replication. Because
the emergence of viruses resistant to host cell targeted antivirals is less
likely compared to virus targeted compounds (Loregian et al., 2014),
drugs acting on host cellular functions will be of particular importance
for treatment of viruses such as influenza which have a high rate of
mutation.
While preliminary reports indicate that the likelihood of selecting
TIZ resistant virus is low (Belardo et al., 2011), further studies to in-
vestigate the propensity of influenza viruses developing resistance to
TIZ in vitro, in vivo, and in clinical settings will be important. The
methods described here provide a means for the surveillance of influ-
enza virus susceptibility to TIZ, and furthermore may be used to test
other influenza antiviral drugs under development that require cell
based assays.
Acknowledgements
This work was funded by a grant from Romark Laboratories (Tampa,
Florida, USA). We thank Vivian Leung from the WHO Collaborating
Centre for Reference and Research on Influenza (Melbourne, Australia)
for her assistance with statistical analyses and production of figures.
The Melbourne WHO Collaborating Centre for Reference and Research
on Influenza is supported by the Australian Government Department of
Health.
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://dx.
doi.org/10.1016/j.antiviral.2017.10.002.
Ashton, L.V., Callan, R.L., Rao, S., Landolt, G.A., 2010. Vitro susceptibility of canine in-
fluenza A (H3N8) virus to nitazoxanide and tizoxanide. In: Veterinary Medicine
International 2010, , 891010.
Belardo, G., Cenciarelli, O., La Frazia, S., Rossignol, J.F., Santoro, M.G., 2015. Synergistic
effect of nitazoxanide with neuraminidase inhibitors against influenza a viruses in
vitro. Antimicrob. Agents Chemother. 59, 1061–1069.
Belardo, G., La Frazia, S., Cenciarelli, O., Carta, S., Rossignol, J.F., Santoro, M.G., 2011.
Nitazoxanide, a novel potential anti-influenza drug, acting in synergism with neur-
aminidase inhibitors. In: 49th Infectious Disease Society of America Annual Meeting,
Boston, Massachusetts USA, Paper 31075.
Broekhuysen, J., Stockis, A., Lins, R.L., De Graeve, J., Rossignol, J.F., 2000. Nitazoxanide:
pharmacokinetics and metabolism in man. Int. J. Clin. Pharmacol. Ther. 38, 387–394.
Byrn, R.A., Jones, S.M., Bennett, H.B., Bral, C., Clark, M.P., Jacobs, M.D., Kwong, A.D.,
Ledeboer, M.W., Leeman, J.R., McNeil, C.F., Murcko, M.A., Nezami, A., Perola, E.,
Rijnbrand, R., Saxena, K., Tsai, A.W., Zhou, Y., Charifson, P.S., 2015. Preclinical
activity of VX-787, a first-in-class, orally bioavailable inhibitor of the influenza virus
polymerase PB2 subunit. Antimicrob. Agents Chemother. 59, 1569–1582.
Costantino, C., Vitale, F., 2016. Influenza vaccination in high-risk groups: a revision of
existing guidelines and rationale for an evidence-based preventive strategy. J. Prev.
Med. Hyg. 57, E13–E18.
de Chassey, B., Meyniel-Schicklin, L., Vonderscher, J., Andre, P., Lotteau, V., 2014. Virus-
host interactomics: new insights and opportunities for antiviral drug discovery.
Genome Med. 6.
Dobson, J., Whitley, R.J., Pocock, S., Monto, A.S., 2015. Articles: oseltamivir treatment
for influenza in adults: a meta-analysis of randomised controlled trials. Lancet 385,
1729–1737.
Farrukee, R., Leang, S.-K., Butler, J., Lee, R.T.C., Maurer-Stroh, S., Tilmanis, D., Sullivan,
S., Mosse, J., Barr, I.G., Hurt, A.C., 2015. Influenza viruses with B/Yamagata- and B/
Victoria-like neuraminidases are differentially affected by mutations that alter anti-
viral susceptibility. J. Antimicrob. Chemother. (JAC) 70, 2004–2012.
Fiore, A.E., Fry, A., Shay, D., Gubareva, L., Bresee, J.S., Uyeki, T.M., 2011. Antiviral
agents for the treatment and chemoprophylaxis of influenza: recommendations of the
advisory committee on immunization practices (ACIP). Centers Dis. Control Prev.
1–27.
Haffizulla, J., Hartman, A., Hoppers, M., Resnick, H., Samudrala, S., Ginocchio, C.,
Bardin, M., Rossignol, J.F., Group, U.S.N.I.C.S., 2014. Effect of nitazoxanide in adults
and adolescents with acute uncomplicated influenza: a double-blind, randomised,
placebo-controlled, phase 2b/3 trial. Lancet. Infect. Dis. 14, 609–618.
Hurt, A.C., 2014. The epidemiology and spread of drug resistant human influenza viruses.
Curr. Opin. Virol. 8, 22–29.
Hurt, A.C., Besselaar, T.G., Daniels, R.S., Ermetal, B., Fry, A., Gubareva, L., Huang, W.,
Lackenby, A., Lee, R.T.C., Lo, J., Maurer-Stroh, S., Nguyen, H.T., Pereyaslov, D.,
Rebelo-de-Andrade, H., Siqueira, M.M., Takashita, E., Tashiro, M., Tilmanis, D.,
Wang, D., Zhang, W., Meijer, A., 2016. Global update on the susceptibility of human
influenza viruses to neuraminidase inhibitors, 2014–2015. Antivir. Res. 132,
178–185.
Jones, J.C., Marathe, B.M., Lerner, C., Kreis, L., Gasser, R., Pascua, P.N.Q., Najera, I.,
Govorkova, E.A., 2016. A novel endonuclease inhibitor exhibits broad-spectrum anti-
influenza virus activity in vitro. Antimicrob. Agents Chemother. 60, 5504–5514.
Kossyvakis, A., Mentis, A.F.A., Tryfinopoulou, K., Pogka, V., Kalliaropoulos, A., Antalis,
E., Lytras, T., Meijer, A., Tsiodras, S., Karakitsos, P., Mentis, A.F., 2017. Antiviral
susceptibility profile of influenza A viruses keep an eye on immunocompromised
patients under prolonged treatment. Eur. J. Clin. Microbiol. Infect. Dis. 361–371.
Koszalka, P., Tilmanis, D., Hurt, A.C., 2017. Influenza antivirals currently in late-phase

147
D. Tilmanis et al.
clinical trial. Influenza Other Respir. Viruses 240–246.
Lambert, L.C., Fauci, A.S., 2010. Influenza vaccines for the future. N. Engl. J. Med. 363,
2036–2044.
Li, T.C.M., Chan, M.C.W., Lee, N., 2015. Clinical implications of antiviral resistance in
influenza. Viruses 7, 4929–4944.
Lin, Y., Gu, Y., Wharton, S.A., Whittaker, L., Gregory, V., Li, X., Metin, S., Cattle, N.,
Daniels, R.S., Hay, A.J., McCauley, J.W., 2015. Optimisation of a micro-neutralisation
assay and its application in antigenic characterisation of influenza viruses. Influenza
Other Respir. Viruses 331–340.
Loregian, A., Mercorelli, B., Nannetti, G., Compagnin, C., Palù, G., 2014. Antiviral stra-
tegies against influenza virus: towards new therapeutic approaches. Cell. Mol. life Sci.
71, 3659–3683.
Louie, J.K., Yang, S., Acosta, M., Yen, C., Samuel, M.C., Schechter, R., Guevara, H., Uyeki,
T.M., 2012. Treatment with neuraminidase inhibitors for critically ill patients with
influenza A (H1N1) pdm09. Clin. Infect. Dis. 1198–1204.
Matrosovich, M., Matrosovich, T., Carr, J., Roberts, N.A., Klenk, H.D., 2003.
Overexpression of the alpha-2,6-sialyltransferase in MDCK cells increases influenza
virus sensitivity to neuraminidase inhibitors. J. Virol. 77, 8418–8425.
Mosmann, T., 1983. Rapid colorimetric assay for cellular growth and survival: application
to proliferation and cytotoxicity assays. J. Immunol. Methods 65, 55–63.
Naesens, L., Stevaert, A., Vanderlinden, E., 2016. Antiviral therapies on the horizon for
influenza. Curr. Opin. Pharmacol. 30, 106–115.
Perwitasari, O., Johnson, S., Yan, X., Howerth, E., Shacham, S., Landesman, Y., Baloglu,
E., McCauley, D., Tamir, S., Tompkins, S.M., 2014. Verdinexor, a novel selective
inhibitor of nuclear export, reduces influenza a virus replication in vitro and in vivo.
J. Virol. 88, 10228–10243.
148
Antiviral Research 147 (2017) 142–148
Rossignol, J.-F., 2014. Review: nitazoxanide: A first-in-class broad-spectrum antiviral
agent. Antivir. Res. 110, 94–103.
Rossignol, J.F., 2017. Nitazoxanide: a first-in-class indirect-acting antiviral for treatment
of respiratory virus infections. In: Fifth Isirv-Antiviral Group Conference Abstract
S10–S17 P58, Shanghai, China.
Rossignol, J.F., La Frazia, S., Chiappa, L., Ciucci, A., Santoro, M.G., 2009. Thiazolides, a
new class of anti-influenza molecules targeting viral hemagglutinin at the post-
translational level. J. Biol. Chem. 284, 29798–29808.
Sleeman, K., Mishin, V.P., Guo, Z., Garten, R.J., Balish, A., Fry, A.M., Villanueva, J.,
Stevens, J., Gubareva, L.V., 2014. Antiviral susceptibility of variant influenza
A(H3N2)v viruses isolated in the United States from 2011 to 2013. Antimicrob.
Agents Chemother. 58, 2045–2051.
Takashita, E., Meijer, A., Lackenby, A., Gubareva, L., Rebelo-de-Andrade, H., Besselaar,
T., Fry, A., Gregory, V., Leang, S.-K., Huang, W., Lo, J., Pereyaslov, D., Siqueira,
M.M., Wang, D., Mak, G.C., Zhang, W., Daniels, R.S., Hurt, A.C., Tashiro, M., 2015.
Global update on the susceptibility of human influenza viruses to neuraminidase
inhibitors, 2013–2014. Antivir. Res. 117, 27–38.
Terletskaia-Ladwig, E., Meier, S., Enders, M., 2013. Improved high-throughput virus
neutralisation assay for antibody estimation against pandemic and seasonal influenza
strains from 2009 to 2011. J. Virol. Methods 189, 341–347.
van Baalen, C.A., Jeeninga, R.E., Penders, G.H.W.M., van Gent, B., van Beek, R.,
Koopmans, M.P.G., Rimmelzwaan, G.F., 2017. ViroSpot microneutralization assay for
antigenic characterization of human influenza viruses. Vaccine 35, 46–52.
Zhang, J.H., Chung, T.D.Y., Oldenburg, K.R., 1999. A simple statistical parameter for use
in evaluation and validation of high throughput screening assays. J. Biomol. Screen 4,
67–73.