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Evaluation of luciferase and GFP-expressing Nipah viruses for rapid
quantitative antiviral screening
Michael K. Lo
⇑
, Stuart T. Nichol, Christina F. Spiropoulou
⇑
Viral Special Pathogens Branch, Centers for Disease Control and Prevention, Atlanta, GA, United States
article info
Article history:
Received 8 February 2014
Revised 6 March 2014
Accepted 18 March 2014
Available online 27 March 2014
Keywords:
Henipavirus
Nipah virus
Antiviral screening
Luciferase
GFP
High-throughput screening
abstract
Nipah virus (NiV) outbreaks have occurred in Malaysia, India, and Bangladesh, and the virus continues to
cause annual outbreaks of fatal human encephalitis in Bangladesh due to spillover from its bat host res-
ervoir. Due to its high pathogenicity, its potential use for bio/agro-terrorism, and to the current lack of
approved therapeutics, NiV is designated as an overlap select agent requiring biosafety level-4 contain-
ment. Although the development of therapeutic monoclonal antibodies and soluble protein subunit vac-
cines have shown great promise, the paucity of effective antiviral drugs against NiV merits further
exploration of compound libraries using rapid quantitative antiviral assays. As a proof-of-concept study,
we evaluated the use of fluorescent and luminescent reporter NiVs for antiviral screening. We
constructed and rescued NiVs expressing either Renilla luciferase or green fluorescent protein, and
characterized their reporter signal kinetics in different cell types as well as in the presence of several
inhibitors. The 50% effective concentrations (EC
50
s) derived for inhibitors against both reporter viruses
are within range of EC
50
s derived from virus yield-based dose–response assays against wild-type NiV
(within 1Log
10
), thus demonstrating that both reporter NiVs can serve as robust antiviral screening tools.
Utilizing these live NiV-based reporter assays requires modest instrumentation, and circumvents the
time and labor-intensive steps associated with cytopathic effect or viral antigen-based assays. These
reporter NiVs will not only facilitate antiviral screening, but also the study of host cell components that
influence the virus life cycle.
Published by Elsevier B.V.
1. Introduction
Nipah virus (NiV) is a highly pathogenic paramyxovirus in the
genus Henipavirus of the subfamily Paramyxovirinae within the
family Paramyxoviridae (Rota and Lo, 2012). Humans are infected
upon spillover of NiV from its bat reservoir host or by person-to-
person transmission (Luby, 2013). Due to its high pathogenicity,
its potential use for bio/agro-terrorism, and to the current lack of
approved therapeutics, NiV is designated as an overlap select agent
requiring biosafety level-4 containment. Although several preven-
tative measures against exposure to bat saliva or urine have been
studied (Nahar et al., 2013), NiV continues to cause near-annual
outbreaks of fatal encephalitis in Bangladesh. The development
of therapeutic monoclonal antibodies and soluble subunit vaccines
against henipaviruses have shown great promise (Broder et al.,
2013), but screening available compound libraries for potentially
efficacious therapeutics against NiV remains a high priority for
investigation. Most high-throughput antiviral assays against NiV
were developed to screen for inhibitors of virus entry and/or cell-
to-cell fusion (Bossart et al., 2005; Porotto et al., 2009; Talekar
et al., 2012; Tamin et al., 2009), while the NiV minigenome assay
was used to screen for inhibitors of NiV replication (Freiberg
et al., 2008). A chemiluminescent immunodetection assay for live
henipavirus infection significantly streamlined antiviral screening
when compared with cytopathic effect (CPE) or immune plaque-
based assays, but had relatively limited signal-to-noise ratios
(<100), and also required cell fixation and multiple incubation
steps which are time and labor-intensive (Aljofan et al., 2008).
Reverse genetic systems for henipaviruses have enabled the gener-
ation of recombinant viruses expressing fluorescent or luminescent
proteins which allow for rapid detection of infection in vitro and
in vivo (Lo et al., 2012; Marsh et al., 2013; Yoneda et al., 2006).
We sought to determine whether NiVs expressing fluorescent or
luminescent reporters could facilitate rapid quantitative antiviral
screening. As a proof-of-concept study, we constructed and res-
cued NiVs expressing either Renilla luciferase (LUC2AM) or green
fluorescent protein (GFP2AM), characterized their reporter signal
http://dx.doi.org/10.1016/j.antiviral.2014.03.011
0166-3542/Published by Elsevier B.V.
⇑
Corresponding authors. Address: Centers for Disease Control and Prevention,
Viral Special Pathogens Branch, 1600 Clifton Road, Mailstop G-14, Atlanta, GA
30333, United States. Tel.:+1 4046390327 (M.K. Lo).
E-mail addresses: mko2@cdc.gov (M.K. Lo), ccs8@cdc.gov (C.F. Spiropoulou).
Antiviral Research 106 (2014) 53–60
Contents lists available at ScienceDirect
Antiviral Research
journal homepage: www.elsevier.com/locate/antiviral
Author's personal copy
kinetics in different human cell types, and tested them in parallel
against cellular inhibitors of pyrimidine biosynthesis as well as in-
nate immune agonists. We show that both reporter viruses can be
used in the 96-well format and have excellent signal-to-noise ra-
tios and Z
0
-factors. Furthermore, the 50% effective concentrations
(EC
50
s) derived for inhibitors tested against both reporter viruses
were not only within close range of one another (within 4-fold),
but also of EC
50
s derived from virus yield-based dose–response as-
says against wild-type NiV (within 1 Log
10
), thus demonstrating
that both reporter NiVs can serve as robust antiviral screening
tools.
2. Materials and methods
2.1. Cells
Vero (African green monkey kidney), A549 (human lung epithe-
lial carcinoma), HeLa (human cervical carcinoma), and HEK 293T
(human embryonic kidney) cells were maintained at 37 °C and
5% CO
2
in Dulbecco’s modified Eagle’s medium supplemented with
7.5% fetal bovine serum (FBS), 100 U/mL penicillin, 100
l
g/mL
streptomycin, and 2 mM
L
-glutamine. Baby hamster kidney cells
stably expressing T7 polymerase (BSRT7/5) (Buchholz et al.,
1999) were incubated at every third passage in the presence of
1 mg/mL G-418 (Geneticin).
2.2. Cloning and rescue of recombinant viruses
For LUC2AM, a modified NiV M gene fragment expressing the
Renilla luciferase (Promega) open reading frame (ORF) in-frame
with the NiV Matrix (M) gene ORF separated by the FMDV 2A pro-
tease cleavage peptide was constructed using a 4-step overlapping
PCR strategy as described for a DsRed-Express reporter NiV (RE-
D2AM) and inserted into a plasmid containing a full-length cDNA
copy of wild-type recombinant NiV genome using engineered SbfI
and SacII restriction sites (Lo et al., 2012). The total genome length
of LUC2AM was 19,254 nt.
The GFP2AM expressing the green fluorescence protein
Zsgreen1 (Clontech) ORF in-frame with the NiV M gene ORF sepa-
rated by the porcine teschovirus 2A (p2A) protease cleavage pep-
tide was constructed using a second-generation wild-type
recombinant NiV (rNiV) (Genbank sequence AF212302) devoid of
engineered restriction sites, assembled via 4 fragments into the
same plasmid backbone used for LUC2AM (Genscript). A synthe-
sized fragment containing the GFP, p2A, and NiV M coding se-
quence in-frame was cloned into WT using PacI restriction sites.
An additional 3 nucleotides ‘‘GGA’’ encoding the amino acid glycine
were inserted between the end of the Zsgreen1 ORF and the start of
the p2A cleavage peptide for the final genome length to obey the
‘‘Rule of six’’ at 19,014 nt (Kolakofsky et al., 1998). Both LUC2AM
and GFP2AM were rescued as previously described (Lo et al.,
2012). The genomes of the rescued viruses were fully sequenced
with no undesired mutations. Virus stock titers were determined
by median tissue culture infectious dose (TCID
50
) assay (see
below).
2.3. Infections with recombinant reporter viruses, reporter activity
measurements, cell type optimization, TCID
50
All infections in this study were performed in 96-well plates.
Cells were seeded in opaque black or white plates (Costar 3916,
3917) for fluorescence and luminescence-based assays respec-
tively. Initial time course experiments for rescued viruses were
performed by infecting 2 10
4
Vero cells/well at multiplicity of
infection (MOI) = 0.01 TCID
50
/cell of either virus for 2 h. Virus
inoculum was removed and replaced with 100
l
L Fluorobrite med-
ium (Life Technologies) supplemented with 2 mM
L
-glutamine and
10% FBS. At 24, 48, and 72 h post-infection (hpi), supernatants
were harvested for TCID
50
determination and replaced with
Fluorobrite medium. Fluorescence activity (gain = 65) was mea-
sured in a H1 Synergy plate reader (Biotek), while luminescence
activity (gain = 107) was measured in the same reader 10 min after
addition of Renilla-Glo substrate (Promega). Subsequent infections
were performed at MOI = 0.2 TCID
50
/cell unless stated otherwise.
Cytopathic effect-based TCID
50
assays were performed by infecting
Vero cells in 10-fold dilution series of samples (10
1
-10
8
) in a 96-
well plate format, infecting 9 wells per sample and dilution step.
Plates were read after 7 days to ensure a clear distinction between
infected and uninfected wells at the highest dilutions.
2.4. Fluorescence microscopy
210
4
HeLa cells were infected with LUC2AM or GFP2AM, and
at 24 hpi were fixed in 10% formalin for 15 min, permeablized with
PBS + 0.4% Triton-X for 5 min, washed with PBS + 0.1% Tween
(PBS-T), and blocked with PBS-T + 5% milk (PBS-T-M) for 30 min.
LUC2AM-infected cells were incubated with a mouse anti-NiV-N
monoclonal antibody 1A11C1 (Chiang et al., 2010) and a rabbit
anti-Renilla luciferase antibody (MBL) diluted 1:1000 and 1:200
respectively in PBS-T-M for 1 h at 37 °C, while GFP2AM-infected
cells were incubated with only 1A11C1 antibody. After 3 washes
with PBS-T, cells were incubated with anti-mouse DyLight 550
and anti-rabbit DyLight 488 antibodies (Bethyl Laboratories, Inc.)
diluted 1:1000 in PBS-T-M for 30 min at 37 °C. After 3 washes, fluo-
rescence images were taken on a Zeiss Axiovert 200 microscope at
40X magnification.
2.5. Antiviral compounds, assessment of cell viability (CC
50
) and 50%
effective concentrations (EC
50
)
The following compounds used in this study were dissolved in
dimethyl sulfoxide (DMSO): 6-azauridine acetate (Sigma), Pyra-
zofurin (TRC), and compound 09167 (Vitas-M Laboratory) (Yan
et al., 2013). The final DMSO concentration in Fluorobrite media
was 0.1%. Universal interferon (U-IFN) (Sigma) was dissolved in
sterile distilled H
2
O. The CellTiter-Glo assay (Promega) was used
to determine viability of uninfected 293T cells exposed to 3-fold
serial dilutions of the compounds for 48 h. Values were normalized
to DMSO controls according to % viability as follows: % viabil-
ity = [(specific valuereference value)/(DMSO control value ref-
erence value)] 100. Reference values were derived from control
wells without cells. DMSO control values (after subtraction of ref-
erence values) were set at 100% viability. 50% viability/cytotoxicity
(CC
50
) and 50% effective concentrations (EC
50
) were calculated
using four-parameter variable slope non-linear regression fitting
of mean values for three experiments. Therapeutic indices (TI)
were represented as CC
50
/EC
50
. In instances which CC
50
could not
be properly calculated due to non-convergence of dose response
curves, we designated the highest concentration of compound used
as the CC
50
.
2.6. Statistical analysis
Statistical analysis was performed using Prism 6 software
(GraphPad Software). Z
0
(separation band/dynamic range of the as-
say) values were calculated based on the following formula:
Z
0
=1[(3SD
C
+ 3SD
B
)/(mean
C
mean
B
)], where SD is the stan-
dard deviation, Cis the control, and Bis the background (Zhang
et al., 1999). The percent coefficient of variation (%CV) was calcu-
lated as follows: %CV = SD
C
/mean
C
100.
54 M.K. Lo et al. / Antiviral Research 106 (2014) 53–60
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3. Results and discussion
3.1. Construction and characterization of luciferase and GFP expressing
NiVs
To evaluate the use of luminescent reporters for antiviral
screening against NiV, we constructed a Renilla luciferase-
expressing NiV (LUC2AM) by employing previous methods used
to generate a red fluorescent protein-expressing NiV (RED2AM)
(Lo et al., 2012). We inserted the Renilla luciferase (LUC) gene into
the NiV matrix (M) gene 5
0
of the M ORF in the same reading frame,
separated by the coding sequence of the Foot and Mouth Disease
virus protease cleavage peptide (F2A) (Fig. 1A). We used the same
strategy to construct a second generation recombinant green fluo-
rescent protein(GFP)-expressing NiV (GFP2AM), except that we
used the more efficiently-cleaved porcine Teschovirus 2A cleavage
Fig. 1. Design and characterization of reporter Nipah (NiV) viruses. (A) Schematic of wild-type NiV genome alongside reporter NiVs expressing either luciferase (LUC2AM) or
green-fluorescent protein (GFP2AM) respective nucleotide (nt) lengths indicated. Black segments represent non-coding regions, while white segments indicate open reading
frames. Modified M gene coding regions for LUC2AM and GFP2AM are highlighted in yellow and green respectively. (B) Microscopic examination of HeLa cells infected with
LUC2AM and GFP2AM. LUC2AM-infected were stained with antibodies against NiV nucleoprotein (N) and anti-Renilla luciferase antibodies, while GFP2AM-infected cells
were stained only with N antibodies. (C) Time course analysis of growth kinetics and reporter activities of LUC2AM and GFP2AM alongside the growth kinetics of their
corresponding wild-type recombinant NiVs (WT, rNiV respectively). Vero cells were infected with each virus at an MOI of 0.01. At 1, 2, and 3 days post-infection, supernatants
titers were determined by TCID
50
, (left Y-axis) while relative light units (RLU) or relative fluorescence units (RFU) in the cells infected with reporter viruses were measured
(right Y-axis). Reporter activity levels at day 0 post-infection indicate background noise of the luminometer/fluorometer. Values represent means and standard deviations for
3 experiments.
M.K. Lo et al. / Antiviral Research 106 (2014) 53–60 55
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peptide (p2A) coding sequence instead of F2A (Kim et al., 2011)
(see Section 2). We rescued both LUC2AM and GFP2AM and con-
firmed expression of their respective reporter proteins by immuno-
fluorescence microscopy. The distribution of both LUC and GFP
were distinct from NiV nucleoprotein, with the luciferase localizing
exclusively to the cytoplasm, and with the GFP occupying both nu-
clear and cytoplasmic compartments (Fig. 1B). While GFP2AM
grew similarly in Vero cells (MOI = 0.01 TCID
50
/cell) compared
with the non-reporter recombinant (rNiV) (Fig. 1C, right) LUC2AM
had an attenuated growth phenotype when compared with its cor-
responding first generation non-reporter recombinant NiV (WT)
(Fig. 1C, left). This is consistent with previous observations that
the RED2AM virus was moderately attenuated compared with
WT (Lo et al., 2012). Furthermore our result is consistent with
Fig. 2. Assay optimization. (A) 293T cells yielded the highest reporter activity levels for both LUC2AM and GFP2AM reporter viruses. (B) 293T cells were infected at the
indicated multiplicity of infection (MOI) with LUC2AM or GFP2AM, and respective reporter values were measured at 48 hpi. (C) Time course analysis of growth kinetics and
reporter activities of LUC2AM and GFP2AM in 293T cells (MOI = 0.2 TCID
50
/cell). At 1, 2, and 3 days post-infection, supernatant titers were determined by TCID
50
(left Y-axis),
while respective reporter activities in the cells were measured (right Y-axis). Values represent means and standard deviations for 3 experiments.
56 M.K. Lo et al. / Antiviral Research 106 (2014) 53–60
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previous work showing that NiV M requires ubiquitination inside
the nucleus to become functional for budding (Wang et al.,
2010), since the comparatively less-efficient cleavage of F2A would
result in decreased amounts of functional NiV M for budding
new virus particles. In spite of this phenotype however, LUC2AM
generated increasing levels of LUC activity from 0 to 48 h post-
infection (hpi), and reached viral titers of over 10
6
TCID
50
/mL by
72 hpi (Fig. 1C, left panel). The fluorescence signal generated by
GFP2AM was detectable at 24 hpi, but increased by greater than
20-fold at 48 hpi, and continued to increase through 72 hpi. The
Fig. 3. Reporter NiVs as tools for antiviral testing. (A) Testing of 2 known inhibitors (6-azauridine acetate and pyrazofurin) of pyrimidine biosynthesis and (B) 2 innate
immune agonists (Universal interferon and compound 09167) against LUC2AM (left panels) and GFP2AM (right panels). 293T cells were infected with reporter NiVs
(MOI = 0.2 TCID
50
/cell) for 2 h, and then incubated with indicated compounds in 3-fold serial dilutions. Each concentration was assessed in three replicates. Reporter virus
activity was measured on the left Y-axis; % cell viability was measured on the right Y-axis. Values represent means and standard deviations of 3 experiments.
M.K. Lo et al. / Antiviral Research 106 (2014) 53–60 57
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accumulation of GFP signal over the course of infection corre-
sponded with the increase in GFP2AM viral titer (Fig. 1C, right
panel).
3.2. Optimization of cell type and infection conditions for LUC2AM and
GFP2AM-based assays
We then optimized both assays for antiviral testing. We tested
each virus against 3 common human cell lines (A549, HeLa, 293T)
alongside Vero cells to determine which would yield the strongest
reporter signal over the course of infection. For negative controls,
we assayed for reporter activity in each cell line infected with
wild-type recombinant NiV (rNiV). Infection with LUC2AM over
the course of 72 h (MOI = 0.1 TCID
50
/cell) showed that HeLa and
293T cells supported peak LUC activity to approximately equal or
greater levels than in Vero cells, while A549 only marginally sup-
ported LUC activity (Fig. 2A, left panel). In both Vero and HeLa cells,
LUC signal peaked at 24 hpi (4–5 10
4
relative light units) and
steadily decreased by over 10-fold by 72 hpi. In 293T cells how-
ever, the LUC signal was approximately equal to those of Vero
and HeLa cells at 24 hpi, but proceeded to double by 48 hpi
(810
4
RLU) before decreasing at 72 hpi to levels similar to
24 hpi. Infection with GFP2AM yielded similar results, showing
minimal NiV replication in A549 cells, and indicating 293T cells
as the optimal cell type which generated the highest GFP signal on-
ward from 48 hpi to 72 hpi (Fig. 2A right panel). In contrast to the
LUC2AM infections however, all 4 infected cell lines attained their
highest levels of GFP expression at 72 hpi. The different reporter
signal kinetics observed when comparing LUC2AM and GFP2AM
infections are likely a function of respective reporter protein mat-
uration rates and half-lives. Whereas LUC matures rapidly and has
a protein half-life of approximately 14 h in the cell cytoplasm
(Loening et al., 2006), GFP requires 8–12 h for maturation, and
has a protein half-life of approximately 26 h (Corish and
Tyler-Smith, 1999). Since the peak LUC signal in infected 293T cells
occurred at 48 hpi, and because of the minimal difference in GFP
signal between 48 hpi and 72 hpi, we chose 48 hpi as our assay
time point for both LUC2AM and GFP2AM.
To further optimize these reporter assays, we infected 293T
cells with either reporter virus at MOIs ranging from 0.05 to
0.4 TCID
50
/cell. Increasing the MOI for both LUC2AM and GFP2AM
led to corresponding increases in reporter activity (Fig. 2B). Since
the increase in MOI from 0.2 to 0.4 for both LUC2AM and GFP2AM
only marginally increased their respective reporter activity (less
than 2-fold), we used MOI = 0.2 TCID
50
/cell for both reporter
viruses for our antiviral assays. We performed growth curves for
both viruses in 293T cells by infecting at MOI = 0.2 TCID
50
/cell,
and observed increases in reporter signals corresponding with in-
creases in virus titers (Fig. 2C).
3.3. Testing antiviral inhibitors against LUC2AM and GFP2AM
Having optimized both fluorescent and luminescent reporter
assays in 293T cells, we assayed LUC2AM and GFP2AM against
two known inhibitors of NiV, 6-azauridine acetate and pyrazofurin.
Both of these compounds inhibit cellular pyrimidine biosynthesis
via blocking orotidine 5
0
-monophosphate decarboxylase (Bono
et al., 1964; Georges-Courbot et al., 2006; Gutowski et al., 1975).
Consistent with a prior study utilizing a CPE-based assay
(Georges-Courbot et al., 2006), 6-azauridine acetate and pyrazofu-
rin showed dose-dependent inhibition of LUC2AM and GFP2AM,
reducing reporter activity to background levels at the highest doses
(25–50
l
M) while having mild to minimal effects on cell viability
(Fig. 3A). The respective 50% effective concentrations (EC
50
) deter-
mined for each compound using the LUC2AM and GFP2AM were
within 3-fold of each other (Table 1).
Since several studies have demonstrated the sensitivity of
henipaviruses to type-1 interferon (IFN) signaling (Dhondt et al.,
2013; Escaffre et al., 2013; Virtue et al., 2011), we evaluated the
antiviral effect of universal interferon (U-IFN) against our reporter
viruses. Although increasing concentrations of U-IFN led to corre-
sponding decreases in respective reporter activity, even the highest
concentration (1000 IU) of U-IFN only reduced LUC and GFP activity
by approximately 4-fold and 3-fold respectively (Fig. 3B, top pan-
els). These results are consistent with the early expression of multi-
ple antagonists of IFN signaling by NiV (Kulkarni et al., 2009;
Rodriguez et al., 2002; Shaw et al., 2005). EC
50
values for U-IFN
against LUC2AM and GFP2AM were nearly identical (Table 1). In
addition to U-IFN we also tested compound 09167, a recently char-
acterized agonist of the innate antiviral response (Yan et al., 2013)
(Fig. 3B, lower panels). In contrast to U-IFN, treatment with com-
pound 09167 was able to completely ablate both LUC and GFP activ-
ity at the highest dose tested (12.5
l
M), indicating that IFN
signaling-independent components have crucial roles in blocking
NiV replication. Dose response curves for compound 09167 against
both LUC2AM and GFP2AM confirmed sub-micro molar EC
50
s,
which were consistent with EC
50
s previously obtained for 09167
against other paramyxoviruses (Yan et al., 2013)(Table 1). Further
studies of this compound’s effect on cell viability are warranted to
determine its suitability for antiviral testing in vivo.
3.4. Counter screening against wild-type NiV, evaluating reporter
assays for high-throughput screening
To confirm whether the dose–response curves and EC
50
values
obtained using these reporter viruses reflected actual inhibition
of NiV replication, we tested 6-azauridine acetate and compound
09167 against wild-type non-recombinant NiV via virus yield-
based dose–response assays (Fig. 4A). We observed dose-
dependent reductions in virus titers to below initial levels of input
virus at the two highest doses for each compound (>4 Log
10
TCID
50
/
mL reduction), suggesting complete ablation of viral replication at
these concentrations. EC
50
values calculated for 6-azauridine and
09167 against wild-type NiV were within 1 Log
10
of EC
50
values de-
rived for each compound against LUC2AM and GFP2AM, indicating
that inhibition of the reporter viruses paralleled inhibition of NiV
replication (Fig. 4B, Table 1). The moderately higher EC
50
s calcu-
lated from the reporter viruses suggest a lesser likelihood to detect
false positives in the context of high-throughput screening (HTS).
To quantitatively assess the suitability of these two viruses for
Table 1
50% effective concentrations (EC
50
) of compounds against reporter and wild-type NiVs (therapeutic indices in parentheses).
Inhibitor ID Units NiV-LUC2AM NiV-GFP2AM NiV-MY-99
6-Azauridine acetate
l
M 0.274 ± 0.0357 (P364) 0.553 ± 0.151 (P180) 0.120 ± 0.0445 (P833)
Pyrazofurin
l
M 0.432 ± 0.0871 (P115) 0.281 ± 0.0448 (P177) ND
09167
l
M 0.0723 ± 0.0123 (877) 0.254 ± 0.0905 (249) 0.0289 ± 0.0102 (2196)
U-IFN IU 10.96 ± 4.90 (P91) 10.2 ± 8.49 (P98) ND
ND - Not done.
58 M.K. Lo et al. / Antiviral Research 106 (2014) 53–60
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HTS, we used compound 09167 as a positive control for inhibition
and calculated Z
0
factor values, signal-to-noise ratios, and coeffi-
cients of variation for the 96-well plate format used throughout
this study. For both assays, these values were robust and well
within the acceptable range for HTS (Zhang et al., 1999)(Table 2).
4. Conclusions
The primary advantages to using viral reporter assays over viral
antigen-based assays are the significant reduction in assay turn-
around time and the minimal reagents required. Whereas a previ-
ously described henipavirus immunodetection assay required at
least 2 h of post-infection processing (fixing, blocking, antibody
staining) before the plate reading step, the LUC2AM assay required
just 10 min of reagent incubation before plate reading, while the
GFP2AM assay required even less time since no additional reagents
were required. The LUC2AM assay has several advantages over the
GFP2AM assay including better signal-to-noise ratios and less var-
iability between samples/wells. Although we established the LU-
C2AM assay to be read alongside the GFP2AM assay at 48 hpi,
the robust signal-to-noise ratio and Z
0
values for LUC2AM at even
24 hpi would allow for adaptation of the assay to span a shorter
infection period, whereas this would not be possible for GFP2AM
due to its longer maturation time (data not shown). The GFP2AM
assay has some advantages over LUC2AM assay in that GFP activity
can be measured multiple times, whereas LUC measurement is an
endpoint measurement. Furthermore, GFP2AM can be easily
adapted for high content imaging to simultaneously assess cell via-
bility and reporter activity, as well as for flow-cytometry-based
applications. In sum, we established reporter NiVs that will not
only facilitate antiviral screening, but also the study of cellular
components that influence viral replication.
Acknowledgements
We thank Mike Flint for helpful discussions and suggestions.
The findings and conclusions in this report are those of the authors
and do not necessarily represent those of the Centers for Disease
Control and Prevention.
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Fig. 4. Confirmatory counter-screening against wild-type NiV. (A) Virus titer-reduction assays. 293T cells were infected with wild-type NiV (MOI = 0.2 TCID
50
/cell) for 2 h,
and then incubated with 3-fold dilutions of 6-azauridine acetate (left panel) or compound 09167 (right panel) for 48 h. Supernatant virus titers were determined by TCID
50
titrations and plotted against compound concentration. Values represent means and standard deviations of 3 experiments. Virus titers were then (B) normalized to vehicle
(DMSO) controls. EC
50
s were calculated based on four-parameter variable-slope nonlinear regression models. Normalized data points represent means for three experiments.
Table 2
Comparison of Nipah LUC2AM and GFP2AM assays used in this study.
Plate format Target virus
a
Z
0
value
b
S/N ratio
c
%CV
96 wells NiV-LUC2AM 0.85 1.33E + 04 5%
96 wells NiV-GFP2AM 0.67 2.16E + 03 11%
a
293T cells were infected with either LUC2AM or GFP2AM viruses for 2 h before
treatment with host antiviral agonist compound 09167 (final concentration,
12.5 mM) or an equivalent amount of vehicle (DMSO). Relative luciferase/fluores-
cence units were determined at 48 hpi.
b
Z
0
factor (Zhang et al., 1999). Statistical analyses were based on means of four
independent experiments.
c
Ratio of signal to background noise.
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