Development of a reverse genetics system to generate a recombinant Ebola virus Makona expressing a green fluorescent protein

Abstract

Previous studies have demonstrated the potential application of reverse genetics technology in studying a broad range of aspects of viral biology, including gene regulation, protein function, cell entry, and pathogenesis. Here, we describe a highly efficient reverse genetics system used to generate recombinant Ebola virus (EBOV) based on a recent isolate from a human patient infected during the 2014-2015 outbreak in Western Africa. We also rescued a recombinant EBOV expressing a fluorescent reporter protein from a cleaved VP40 protein fusion. Using this virus and an inexpensive method to quantitate the expression of the foreign gene, we demonstrate its potential usefulness as a tool for screening antiviral compounds and measuring neutralizing antibodies.
Published by Elsevier Inc.

Full text

Brief Communication
Development of a reverse genetics system to generate a recombinant
Ebola virus Makona expressing a green fluorescent protein
$
César G. Albariño
n
, Lisa Wiggleton Guerrero, Michael K. Lo, Stuart T. Nichol,
Jonathan S. Towner
Centers for Disease Control and Prevention, Atlanta, GA, USA
article info
Article history:
Received 2 April 2015
Returned to author for revisions
21 April 2015
Accepted 10 June 2015
Available online 27 June 2015
abstract
Previous studies have demonstrated the potential application of reverse genetics technology in studying
a broad range of aspects of viral biology, including gene regulation, protein function, cell entry, and
pathogenesis. Here, we describe a highly efficient reverse genetics system used to generate recombinant
Ebola virus (EBOV) based on a recent isolate from a human patient infected during the 2014–2015
outbreak in Western Africa. We also rescued a recombinant EBOV expressing a fluorescent reporter
protein from a cleaved VP40 protein fusion. Using this virus and an inexpensive method to quantitate the
expression of the foreign gene, we demonstrate its potential usefulness as a tool for screening antiviral
compounds and measuring neutralizing antibodies.
Published by Elsevier Inc.
Introduction
The first outbreak of Ebola virus disease (EVD) caused by Ebola
virus (EBOV) was detected in 1976 in Zaire (now the Democratic
Republic of the Congo), and received significant international
attention due to its high case fatality rate (Johnson, 1978;
Johnson et al., 1977). During the early months of 2014, a large
EVD outbreak was detected in the Republic of Guinea (Baize et al.,
2014; Gatherer, 2014) and spread quickly to the neighboring
countries of Liberia and Sierra Leone (Dixon et al., 2014). To date,
this continuing outbreak has been the largest EVD outbreak ever
recorded, with over 23,000 cases and over 10,000 deaths in
Western Africa as of March 2015. A limited number of cases were
also recorded in Mali, Nigeria, Senegal, USA, Spain, Germany, and
the United Kingdom (Baize, 2015; CDC, 2015).
Several viruses of the Filoviridae family, including EBOV, Sudan
virus, Bundibugyo virus, Tai Forest virus, Marburg virus (MARV),
and Ravn virus, cause sporadic outbreaks of viral hemorrhagic
fevers (VHFs) with high case fatality rates in sub-Saharan Africa
(Albariño et al., 2013a; Feldmann et al., 2013; Hartman et al., 2010;
Leroy et al., 2011). Filoviruses are enveloped viruses that carry a
single-strand RNA genome with negative-sense polarity (Feldmann
et al., 2013). The filoviral genome is approximately 19 kb, and
encodes 7 genes, NP, VP35, VP40, VP30, VP24, and L, which are
transcribed in sequential order from the 3
0
end of the viral genome
and are separated by intergenic untranslated regions (Fig. 1A).
For more than a decade, reverse genetics technology has been a
useful tool for screening antiviral compounds and for studying
different aspects of filovirus biology, including virulence factors in
the viral genome, cell entry, mechanisms of transcription and
replication, and pathogenesis (Ebihara et al., 2005; Falzarano and
Feldmann, 2014; Hoenen and Feldmann, 2014; Hoenen et al., 2011;
Neumann et al., 2002; Theriault et al., 2005). Several reports also
describe modifying the genomes of EBOV and MARV by inserting
reporter genes, such as those coding for fluorescent proteins and
luciferase (Albariño et al., 2013b; Ebihara et al., 2007; Hoenen
et al., 2013; Schmidt et al., 2011; Schudt et al., 2013; Towner et al.,
2005; Uebelhoer et al., 2014).
In order to start studies on the biological characteristics of the
EBOV variant responsible for the current Western Africa outbreak,
we developed a reverse genetics system for an isolate (Makona)
that was obtained from a patient in 2014. Here, we report the
generation of recombinant viruses that would constitute an
appropriate tool for future studies on this particular EBOV variant.
Results and discussion
Comparisons of full-length genomes from EBOV samples iso-
lated from 1976 to 2014 show a high degree of sequence identity
(Fig. 1A). As expected, representative isolates of the Makona
variant from the 2014 outbreak in Western Africa, obtained from
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Virology
http://dx.doi.org/10.1016/j.virol.2015.06.013
0042-6822/Published by Elsevier Inc.
☆
The findings and conclusions in this report are those of the author(s) and do not
necessarily represent the official position of the Centers for Disease Control and
Prevention.
n
Corresponding author.
E-mail address: calbarino@cdc.gov (C.G. Albariño).
Virology 484 (2015) 259–264

patients in Liberia, Guinea, and Sierra Leone, showed over 99%
sequence identity with each other across the full length of the
genome (Baize et al., 2014; Gire et al., 2014). In contrast, these
isolates showed 97% sequence identity with the Mayinga isolate
of the Yambuku variant collected during the 1976 outbreak, and
with representative isolates of the Kikwit variant from 1995. Until
now, all published studies using reverse genetics technology with
EBOV have used only the historic Mayinga isolate (Groseth et al.,
2012; Hartman et al., 2008, 2006; Martinez et al., 2011; Neumann
et al., 2002; Volchkov et al., 2001). Despite the limited divergence
between the Makona and Yambuku variant isolates (Fig.1A: 97%
identities, 561 different nucleotides), concerns have been raised
about differences in pathogenicity, virulence, or transmission
associated with the 2014 variant (Dowall et al., 2014; Gire et al.,
2014; Stadler et al., 2014). For this reason, we developed a new
reverse genetics system based on an isolate from the current EVD
outbreak so that detailed studies on this 2014 variant or compar-
ison studies with previous variants could be performed.
After obtaining confirmation of the sequence identity of the
viral RNA termini by standard 5
0
and 3
0
RACE methods (data not
shown), we constructed a full-length recombinant clone (Fig. 1B)
based on the Makona variant isolate (Ebola virus/H.sapiens-wt/
LBR/2014/Makona-201403007; an isolate of the Makona variant
(GenBank accession #KP178538). Similarly to previously reported
constructs for EBOV and MARV (Albariño et al., 2013b; Enterlein
et al., 2006; Neumann et al., 2002; Volchkov et al., 2001), the viral
complementary (anti-genomic) sense genome was cloned into a
standard T7 transcription vector, flanked at the 3
0
end by the HDV
ribozyme and the T7 terminator.
Different cells lines, including 293T, BSRT7 and BHK, have been
traditionally used to rescue EBOV and MARV (Albariño et al.,
2013b; Neumann et al., 2002; Towner et al., 2005; Volchkov
et al., 2001). Since human liver cells are an important target of
EBOV infection, we decided to use Huh7 cells, a fully differentiated
human liver cell line, for viral rescue due to their high transfection
efficiency and the high titers obtained by propagating wild-type
filoviruses in these cells. In addition, Huh7 cells minimize selection
of variant viruses generated by reverse genetics (Tsuda et al.,
2015). To rescue the wild-type recombinant EBOV (rEBOV), we
co-transfected Huh7 cells with the full-length genomic plasmid,
with codon-optimized support plasmids expressing EBOV NP, L (or
inactive L; see below), VP35, and VP30, and with another plasmid
expressing a codon-optimized version of the T7 RNA polymerase
(Uebelhoer et al., 2014). For the negative control, we built a new
plasmid expressing an inactive form of the EBOV L polymerase
(L-inac), in which the GDN motif was removed from the L ORF.
Four days post transfection, we applied the clarified supernatants
from this transfection onto fresh monolayers of Huh7. We assessed
viral rescue 5 days later by performing an immunofluorescence
assay to detect EBOV antigens (Fig. 1C). As shown in Fig. 1D, the
rescue of rEBOV was successful in 11 of 11 replica wells, which
demonstrated the high efficiency of our new approach to
rescue rEBOV.
After confirming the successful rescue of rEBOV, we developed
a new recombinant virus expressing a reporter gene that could be
used to detect viral infection in live cells or to facilitate screening
antiviral agents against the 2014 EBOV variant. Historically, the
most common approach for introducing a reporter gene into the
filoviral genome has been to insert an additional transcription unit
into any of the 6 intergenic regions (Albariño et al., 2013b; Ebihara
et al., 2007; Hoenen et al., 2013; Schmidt et al., 2011; Schudt et al.,
2013; Towner et al., 2005; Uebelhoer et al., 2014). Unfortunately,
recombinant viruses generated using this approach exhibited
attenuated phenotypes in animal models (Ebihara et al., 2007)or
Fig. 1. (A) Sequence comparison. Full-length genomes of representative Ebola virus (EBOV) isolates compared at the nucleotide level. Sequence identity (%) is indicated in the
lower diagonal half, while the number of differing residues is indicated in the upper diagonal half. (B) Schematic representation of recombinant EBOV (rEBOV) genome.
Genome of recombinant EBOV in the viral complementary sense, with the 7 ORFs depicted in the 5
0
to 3
0
orientation. (C) Rescue of rEBOV. Wild-type rEBOV was rescued in
Huh7 cells using the full-length clone shown in B, T7 polymerase expression plasmid, and codon-optimized support plasmids that express NP, VP35, VP30, and L or inactive L
(L-inac). Supernatants from transfected cells were used to infect fresh monolayers of Huh7 cells. The infected cells were fixed 4 days post infection, and stained for
immunofluorescence assays (IFA) using a polyclonal rabbit anti-EBOV antibody followed by anti-rabbit Alexa-Fluor 594. (D) Efficiency of rescue. Rescue efficiency is shown as
the number of positive wells (pos)/total wells; average titer was assessed by standard TCID
50
assay.
C.G. Albariño et al. / Virology 484 (2015) 259–264260

reduced growth in interferon-competent cells (Albariño et al.,
2013b). An alternative strategy, used for non-segmented negative
strand viruses, has been to fuse the foreign gene to one of the viral
genes (Chambers and Takimoto, 2010; Duprex et al., 2002; Hoenen
et al., 2012; Lo et al., 2014; Schudt et al., 2013; Silin et al., 2007). A
particularly interesting report by Lo and colleagues describes the
successful generation of a fully functional recombinant Nipah virus
in which the reporter gene, green fluorescent protein (GFP), had
been fused to the matrix protein, M (Lo et al., 2014). In this
particular construct, M and GFP are released through the action of
self-cleaving peptides, such as F2A derived from foot and mouth
disease virus, or P2A derived from porcine teschovirus 2A (Kim
et al., 2011). We therefore decided to test this latest strategy by
fusing the modified green fluorescent protein, ZsGreen1 (ZsG), to
the self-cleaving peptide P2A and EBOV VP40.
To verify that VP40 is released from the protein fusion, ZsG/
P2A/VP40 was initially cloned into a standard expression vector
(Fig. 2A) and used to transfect Huh7 cells. As shown in Fig. 2B, the
fusion protein yields bright green fluorescence in transfected cells.
Moreover, protein analysis by western blotting showed that EBOV
VP40 is released from the fusion protein, although at lower
expression levels than wild-type VP40 (Fig. 2C).
Fig. 2. (A) VP40 fusion protein. Schematics of plasmids expressing VP40 or ZsG/P2A/VP40 fusion protein are shown. (B) ZsGreen (ZsG) expression. Huh7 cells were
transfected with pC-VP40 or pC-ZsG/VP40. Images shown were taken 3 days post transfection on an inverted fluorescent microscope using the GFP channel. (C) VP40
expression. Huh7 cells were transfected as above and harvested 3 days post transfection. Protein lysates were analyzed by western blotting using a Mab against EBOV-VP40
(lower panel). (D) Schematic representation of rEBOV genome expressing ZsG. Genome of rEBOV expressing ZsG fusion protein (rEBOV/ZsG) is shown in the viral
complementary sense. (E) Rescue of rEBOV/ZsG. rEBOV/ZsG was rescued using the same conditions as wild-type virus (see Fig. 1B). Fresh monolayers of Huh7 cells were
infected with transfection supernatants. ZsG expression was visualized on an inverted fluorescent microscope using the GFP channel (left panel), or via IFA as in Fig. 1D
(middle panel, and merged images panel at right). (F) Rescue efficiency. Efficiency of rescue and average titer of rEBOV/ZsG were assessed as in Fig. 1. (G) Growth kinetics of
wild-type and recombinant viruses. Huh7 cells were infected with wild-type EBOV Mayinga (EBOV-Z1976), or wild-type human isolate of the Makona variant (EBOV-L2014),
or with the 2 recombinant viruses, rEBOV or rEBOV/ZsG, with an MOI¼0.1. Growth kinetics were assessed by determining viral titers in cell supernatants using a standard
TCID
50
assay.
C.G. Albariño et al. / Virology 484 (2015) 259–264 261

After confirming that ZsG and EBOV VP40 can be expressed
from the fusion cassette, we modified our new full-length clone to
carry this cassette in the same locus as wild-type VP40 (Fig. 2D).
Using the conditions described above to rescue wild-type rEBOV,
we successfully generated rEBOV/ZsG in all 11 replica wells (Fig. 2E
and F). Both rEBOV and rEBOV/ZsG were successfully rescued with
100% efficiency (11 of 11 wells) in 2 successive rescue attempts.
As shown in Fig. 2E, rEBOV/ZsG recombinant virus expressed
ZsG and spread through the cell monolayer in a pattern similar to
that of wild-type rEBOV.
We compared the growth kinetics of the most relevant wild-
type viruses, EBOV Mayinga isolate and the human isolate of the
Makona variant, and of the 2 recombinant viruses, rEBOV and
rEBOV/ZsG. Interestingly, the wild-type Mayinga virus (EBOV-
Z1976) exhibited a slight growth advantage over the Makona
isolate (EBOV-L2014), while EBOV-L2014 and rEBOV exhibited
very similar growth characteristics (Fig. 2G). Moreover, rEBOV
grew slightly faster than rEBOV/ZsG (Fig.2G).
Quantifying the fluorescence signal from GFP- or RFP-
expressing viruses has an intrinsic limitation because it requi-
res expensive, high-content, sophisticated imaging techniques
(Panchal et al., 2010, 2012). To avoid this need, we sought to
validate an alternative method described by Lo et al. (2014), which
quantifies viral growth by measuring the fluorescence signal from
live cells infected with rEBOV/ZsG. In this experiment, we infected
Huh7 cells with rEBOV/ZsG at a low multiplicity of infection (MOI),
and captured daily micrographs to record the progress of infection
(Fig. 3A). Concurrently, we also measured the ZsG-specific signal
on a multi-mode microplate reader and removed sample aliquots
to determine viral titers over 5 days of infection. As shown in
Fig. 3B, the fluorescence signal of ZsG could be easily quantified in
a non-destructive way, and these data correlated with the spread
of virus shown in the captured images (Fig. 3A). Moreover, the
gradual increase of the ZsG signal also matched with increases in
viral titers (Fig. 3B).
Using this straightforward approach for quantifying the fluor-
escence signal, we conducted a proof of principle experiment
using rEBOV/ZsG as a rapid tool for screening antiviral compounds
and measuring neutralizing antibodies in sera collected from
convalescent rhesus monkeys. As shown in Fig. 3C, we used
rEBOV/ZsG virus in Huh7 cells to measure the antiviral effect of
6azaU, a known inhibitor of viral replication (Crance et al., 2003;
Morrey et al., 2002; Pyrc et al., 2006; Smee et al., 1987; Uebelhoer
et al., 2014). Consistent with our previous report (Uebelhoer et al.,
2014), 6azaU used at the maximum concentration tolerated with-
out toxicity significantly reduced ZsG signal: 94% and 97% signal
reduction at 62.5 μM and 125 μM concentrations of 6azaU,
respectively
As a second proof of principle experiment, we measured the
neutralizing effect of sera collected from convalescent rhesus
monkeys infected with EBOV Mayinga on rEBOV/ZsG (Fig. 3D). In
this experiment, we pre-incubated rEBOV/ZsG with different
dilutions of an IgGþor an IgGserum, and then used the treated
virus to infect Huh7 cells. As shown in Fig. 3D, the ZsG signal was
reduced by 98% after using a 1:400 dilution of the IgG þserum.
Conclusions
Here, we describe a highly efficient reverse genetics system to
generate a recombinant EBOV (rEBOV) based on a virus isolated
from a patient during the 2014–2015 Western Africa EVD out-
break. We also report the successful rescue of a recombinant EBOV
expressing a ZsG reporter protein that is initially fused to the EBOV
protein VP40, but is released by a cis-acting proteolytic cleavage
event. The fusion of this reporter gene did not adversely affect
Fig. 3. (A) Characterization of rEBOV/ZsG. Vero-E6 cells were infected with rEBOV/
ZsG viruses at MOI of 0.01, and pictures were taken as in Fig. 2C on indicated days
post infection (dpi). (B) Quantitation of ZsG expression and viral titers. Huh7 cells
were infected as indicated above, and fluorescence resulting from ZsG expression
was measured daily for 5 days using a multi-mode microplate reader (BioTek
Synergy). Fluorescence is reported as relative fluorescence units (RU). Viral titers
were determined as in Fig. 2G. (C) Effects of antiviral agent 6azaU. Huh7 cells
growing in 96-well plates were pre-treated with 6azaU at indicated concentrations
for 1 h, and then infected with rEBOV/ZsG at MOI of 0.1. Three days post infection,
ZsG expression (bars) was measured as indicated above. Mean and SEM of ZsG
expression from 4 wells are displayed (left Y-axis). Cytotoxicity was assayed by
measuring cellular ATP content (circles, right Y-axis) in uninfected cells treated
with 6azaU at indicated concentrations. (D) Serum neutralization assay. rEBOV/ZsG
was pre-incubated for 1 h with the indicated dilutions of an IgG-positive or control
rhesus monkey serum, and then used to infect Huh7 cells growing in 96-well
plates. Expression of ZsG was measured as indicated above; mean and SEM from
4 wells are depicted.
C.G. Albariño et al. / Virology 484 (2015) 259–264262

viral replication in vitro. Moreover, we show a straightforward
approach to quantify virus growth of rEBOV/ZsG in live infected
cells, and demonstrate that this virus can be used to screen
antiviral drugs and to measure the neutralizing effect of antibodies
in sera from convalescent animals. Finally, we consider that the
tools described here could have other potential applications, such
as testing the effects of novel mutations that could arise in nature,
specifically directed at the variant responsible for the largest
outbreak of EVD ever reported.
Materials and methods
Cell culture and biosafety
All work with recombinant viruses was performed in a biosaf-
ety level 4 (BSL-4) facility. Huh7 and Vero-E6 cells were propa-
gated in Dulbecco's modified Eagle's medium (DMEM, Life
Technologies, Grand Island, NY, USA) supplemented with 5% fetal
bovine serum (FBS) and penicillin–streptomycin (Pen–Strep).
Plasmid construction
(A) Support plasmids. The construction of EBOV support plas-
mids (pC-L, pC-NP, pC-V35, and pC-v30) has been described before
(Uebelhoer et al., 2014). Briefly, these clones were designed to
express rodent codon-optimized synthetic genes corresponding to
those of the EBOV Mayinga isolate. The original pC-L plasmid was
also modified to express an inactive form of the EBOV L polymer-
ase (L-inac) by removing the GDN motif from the L ORF. An
expression cassette containing ZsG/P2A/VP40 was made using a
previously described strategy (Lo et al., 2014). Briefly, ZsGreen1
(ZsG) ORF (Clontech, Mountain View, CA, USA) was fused to the
self-cleaving P2A peptide and to EBOV VP40, and cloned into the
standard Pol II expression vector pCAGGS (Niwa et al., 1991). (B)
Full-length clone. Viral RNA from Ebola virus/H.sapiens-wt/LBR/
2014/Makona-201403007 (GenBank accession KP178538) was
used as template to amplify by RT-PCR 2 overlapping fragments
of similar size spanning the full-length genome. These fragments
were gel-purified and used to assemble a full-length clone into the
T7 transcription vector. The final plasmid contained the full-length
anti-genome (viral complementary sense) preceded by the T7 RNA
polymerase promoter and followed by the hepatitis delta virus
ribozyme and T7 polymerase terminator. A spurious nucleotide
change in the GP-VP30 intergenic region was kept to differentiate
the recombinant viruses rEBOV and rEBOV/ZsG from the wild-
type virus.
The full-length clone was later modified by replacing the VP40
ORF with the ZsG/P2A/VP40 cassette described above. Both full-
length clones were sequenced to completion. Details regarding
construction strategies of support expression plasmids and plas-
mids encoding the full-length clone are available upon request.
Rescue of infectious viruses
Rescue of recombinant viruses was performed in Huh7 cells as
described previously (Albariño et al., 2013b). Briefly, a 70%
confluent monolayer of Huh7 cells grown in 12-well plates was
transfected with 1 μg pEBOV, 0.5 μg pC-L (wells #1–11) or pC-L-
inac (well #12), 0.5 μg pC-NP, 0.05 μg pC-VP35, 0.05 μg pC-VP30,
and 1 μg of codon-optimized pC-T7
Supernatants from transfected cells were harvested 4 days post
transfection, clarified by low-speed centrifugation, and passaged
twice in fresh monolayers of Huh7 cells. The rescue events were
confirmed by immunostaining Huh7 cell monolayers (also grown
in 12-well plates) infected with the first passage of the virus.
A 100% rescue efficiency (positive detection in 11 of 11 replica
wells) was obtained in 2 successive rescue experiments.
Both recombinant viruses were sequenced to completion, and
complete genomic sequences were deposited in GenBank (acces-
sion KR781608 and KR781609 for rEBOV and rEBOV/ZsG, respec-
tively). The viral genomic sequences were identical to those in the
full-length plasmids.
Virus titration and growth curves
To characterize the growth kinetics of wild-type and recombi-
nant viruses, 210
6
Huh7 cells were infected at MOI¼0.1. After
1 h of adsorption, cell monolayers were washed with PBS. Aliquots
of the supernatant were taken daily, and viral titers were deter-
mined by tissue culture infective dose 50 (TCID
50
) assay, as
described previously (Uebelhoer et al., 2014).
Protein expression
Expression of ZsG in transfected or infected live cells was
determined by direct UV microscopy using the GFP channel.
Expression of cleaved VP40 was detected by western blotting
using a monoclonal anti-EBOV-VP40 antibody followed by anti-
mouse HRP secondary antibody. Staining of TCID
50
plates was
done using a polyclonal rabbit anti-EBOV antibody followed by
anti-rabbit Alexa-Fluor 594 antibody.
Quantitation of ZsG
The protocol described by Lo et al. (2014) was used to
quantitate ZsG fluorescence in infected cells. Briefly, 410
4
Huh7 cells were seeded in 96-well flat-bottom black plates
(Corning) in 100 μL Fluorobrite medium (Life Technologies) per
well. The medium was removed on the following day, and cells
were pre-treated with 100 μL media containing various concen-
trations of 6azaU for 1 h. Virus was added in an additional 100 μL
media (final MOI¼0.1). Three days post infection, ZsG fluorescence
was measured in 4 replicates for each antiviral concentration using
a multi-mode microplate reader (BioTek Synergy) with a gain/
sensitivity preset of 90. Cell viability was determined by measur-
ing ATP content using CellTiter-Glo Luminescent Cell Viability
reagent (Promega) as previously described (Uebelhoer et al., 2014).
Serum neutralization
About 300 TCID
50
of rEBOV/ZsG were incubated for 1 h at 37 1C
with indicated dilutions of an IgGþor a control (IgG) serum
from rhesus monkeys, and then used to infect Huh7 cells growing
in 96-well flat-bottom black plates with Fluorobrite medium.
Three days post infection, ZsG fluorescence was measured in
4 replicates for each serum concentration, as described above.
Acknowledgments
We thank Marina L. Khristova for continuous excellent sequen-
cing support, and Tatyana Klimova for editing this manuscript. This
study was reviewed and approved by CDC's Institutional Biosafety
Committee.
References
Albariño, C.G., Shoemaker, T., Khristova, M.L., Wamala, J.F., Muyembe, J.J., Balinandi, S.,
Tumusiime, A., Campbell, S., Cannon, D., Gibbons, A., Bergeron, E., Bird, B., Dodd,
K., Spiropoulou, C., Erickson, B.R., Guerrero, L., Knust, B., Nichol, S.T., Rollin, P.E.,
Ströher, U., 2013a. Genomic analysis of filoviruses associated with four viral
C.G. Albariño et al. / Virology 484 (2015) 259–264 263

hemorrhagic fever outbreaks in Uganda and the Democratic Republic of the
Congo in 2012. Virology 442, 97–100.
Albariño, C.G., Uebelhoer, L.S., Vincent, J.P., Khristova, M.L., Chakrabarti, A.K.,
McElroy, A., Nichol, S.T., Towner, J.S., 2013b. Development of a reverse genetics
system to generate recombinant Marburg virus derived from a bat isolate.
Virology 446, 230–237.
Baize, S., 2015. Ebola virus in West Africa: new conquered territories and new risks-
or how I learned to stop worrying and (not) love Ebola virus. Curr. Opin. Virol.
10C, 70–76.
Baize, S., Pannetier, D., Oestereich, L., Rieger, T., Koivogui, L., Magassouba, N., Soropogui, B.,
Sow,M.S.,Keita,S.,DeClerck,H.,Tiffany,A.,Dominguez,G.,Loua,M.,Traore,A.,Kolie,
M.,Malano,E.R.,Heleze,E.,Bocquin,A.,Mely,S.,Raoul,H.,Caro,V.,Cadar,D.,Gabriel,
M., Pahlmann, M., Tappe, D., Schmidt-Chanasit, J., Impouma, B., Diallo, A.K., Formenty,
P., Van Herp, M., Günther, S., 2014. Emergence of Zaire Ebola virus disease in Guinea.
N.Engl.J.Med.371,1418–1425.
CDC, 2015. Update: Ebola virus disease epidemic –west Africa. MMWR Morb.
Mortal. Wkly. Rep. 64, 109–110.
Chambers, R., Takimoto, T., 2010. Trafficking of Sendai virus nucleocapsids is
mediated by intracellular vesicles. PLoS One 5, e10994.
Crance, J.M., Scaramozzino, N., Jouan, A., Garin, D., 2003. Interferon, ribavirin, 6-
azauridine and glycyrrhizin: antiviral compounds active against pathogenic
flaviviruses. Antiviral Res. 58, 73–79.
Dixon, M.G., Schafer, I.J., Centers for Disease, C., Prevention, 2014. Ebola viral
disease outbreak –West Africa. MMWR Morb. Mortal. Wkly. Rep. 63, 548–551.
Dowall, S.D., Matthews, D.A., Garcia-Dorival, I., Taylor, I., Kenny, J., Hertz-Fowler, C.,
Hall, N., Corbin-Lickfett, K., Empig, C., Schlunegger, K., Barr, J.N., Carroll, M.W.,
Hewson, R., Hiscox, J.A., 2014. Elucidating variations in the nucleotide sequence
of Ebola virus associated with increasing pathogenicity. Genome Biol. 15, 540.
Duprex, W.P., Collins, F.M., Rima, B.K., 2002. Modulating the function of the measles
virus RNA-dependent RNA polymerase by insertion of green fluorescent protein
into the open reading frame. J. Virol. 76, 7322–7328.
Ebihara, H., Groseth, A., Neumann, G., Kawaoka, Y., Feldmann, H., 2005. The role of
reverse genetics systems in studying viral hemorrhagic fevers. Thromb. Hae-
most. 94, 240–253.
Ebihara, H., Theriault, S., Neumann, G., Alimonti, J.B., Geisbert, J.B., Hensley, L.E.,
Groseth, A., Jones, S.M., Geisbert, T.W., Kawaoka, Y., Feldmann, H., 2007. In vitro
and in vivo characterization of recombinant Ebola viruses expressing enhanced
green fluorescent protein. J. Infect. Dis. 196 (Suppl. 2), S313–S322.
Enterlein, S., Volchkov, V., Weik, M., Kolesnikova, L., Volchkova, V., Klenk, H.D.,
Muhlberger, E., 2006. Rescue of recombinant Marburg virus from cDNA is
dependent on nucleocapsid protein VP30. J. Virol. 80, 1038–104 3.
Falzarano, D., Feldmann, H., 2014. Possible leap ahead in filovirus therapeutics. Cell
Res. 24, 647–648.
Feldmann, H., Sanchez, A., Geisbert, T.W., 2013. Filoviridae: marburg and Ebola
viruses. In: Knipe, D.M.a.H., P.M (Ed.), Fields Virology, 6th ed. Lippincott,
Williams and Wilkins, Philadelphia, pp. 923–956.
Gatherer, D., 2014. The 2014 Ebola virus disease outbreak in West Africa. J. Gen.
Virol. 95, 1619–16 24.
Gire, S.K., Goba, A., Andersen, K.G., Sealfon, R.S., Park, D.J., Kanneh, L., Jalloh, S.,
Momoh, M., Fullah, M., Dudas, G., Wohl, S., Moses, L.M., Yozwiak, N.L., Winnicki,
S., Matranga, C.B., Malboeuf, C.M., Qu, J., Gladden, A.D., Schaffner, S.F., Yang, X.,
Jiang, P.P., Nekoui, M., Colubri, A., Coomber, M.R., Fonnie, M., Moigboi, A.,
Gbakie, M., Kamara, F.K., Tucker, V., Konuwa, E., Saffa, S., Sellu, J., Jalloh, A.A.,
Kovoma, A., Koninga, J., Mustapha, I., Kargbo, K., Foday, M., Yillah, M., Kanneh, F.,
Robert, W., Massally, J.L., Chapman, S.B., Bochicchio, J., Murphy, C., Nusbaum, C.,
Young, S.,Birren, B.W., Grant, D.S., Scheiffelin, J.S., Lander, E.S., Happi, C., Gevao, S.
M., Gnirke, A., Rambaut, A., Garry, R.F., Khan, S.H., Sabeti, P.C., 2014. Genomic
surveillance elucidates Ebola virus origin and transmission during the 2014
outbreak. Science 345, 1369–1372.
Groseth, A., Marzi, A., Hoenen, T., Herwig, A., Gardner, D., Becker, S., Ebihara, H.,
Feldmann, H., 2012. The Ebola virus glycoprotein contributes to but is not
sufficient for virulence in vivo. PLoS Pathog. 8, e1002847.
Hartman, A.L., Bird, B.H., Towner, J.S., Antoniadou, Z.A., Zaki, S.R., Nichol, S.T., 2008.
Inhibition of IRF-3 activation by VP35 is critical for the high level of virulence of
Ebola virus. J. Virol. 82, 2699–2704.
Hartman, A.L., Dover, J.E., Towner, J.S., Nichol, S.T., 2006. Reverse genetic generation
of recombinant Zaire Ebola viruses containing disrupted IRF-3 inhibitory
domains results in attenuated virus growth in vitro and higher levels of IRF-3
activation without inhibiting viral transcription or replication. J. Virol. 80,
6430–6440.
Hartman, A.L., Towner, J.S., Nichol, S.T., 2010. Ebola and marburg hemorrhagic fever.
Clin. Lab. Med. 30, 161–177.
Hoenen, T., Feldmann, H., 2014. Reverse genetics systems as tools for the develop-
ment of novel therapies against filoviruses. Expert Rev. Anti-Infect. Ther. 12,
1253–1263.
Hoenen, T., Groseth, A., Callison, J., Takada, A., Feldmann, H., 2013. A novel Ebola
virus expressing luciferase allows for rapid and quantitative testing of anti-
virals. Antiviral Res. 99, 207–213.
Hoenen, T., Groseth, A., de Kok-Mercado, F., Kuhn, J.H., Wahl-Jensen, V., 2011.
Minigenomes, transcription and replication competent virus-like particles and
beyond: reverse genetics systems for filoviruses and other negative stranded
hemorrhagic fever viruses. Antiviral Res. 91, 195–208.
Hoenen, T., Shabman, R.S., Groseth, A., Herwig, A., Weber, M., Schudt, G., Dolnik, O.,
Basler, C.F., Becker, S., Feldmann, H., 2012. Inclusion bodies are a site of
ebolavirus replication. J. Virol. 86, 11779–11788.
Johnson, K.M., 1978. Ebola haemorrhagic fever in Zaire, 1976. Bull. World Health
Organ. 56, 271–293.
Johnson, K.M., Lange, J.V., Webb, P.A., Murphy, F.A., 1977. Isolation and partial
characterisation of a new virus causing acute haemorrhagic fever in Zaire.
Lancet 1, 569–571.
Kim, J.H., Lee, S.R., Li, L.H., Park, H.J., Park,J.H., Lee, K.Y., Kim, M.K., Shin, B.A., Choi, S.Y.,
2011. High cleavage efficiency of a 2A peptide derived from porcine teschovirus-
1 in human cell lines, zebrafish and mice. PLoS One 6, e18556.
Leroy, E.M., Gonzalez, J.P., Baize, S., 2011. Ebola and Marburg haemorrhagic fever
viruses: major scientific advances, but a relatively minor public health threat
for Africa. Clin. Microbiol. Infect. 17, 964–976.
Lo, M.K., Nichol, S.T., Spiropoulou, C.F., 2014. Evaluation of luciferase and GFP-
expressing Nipah viruses for rapid quantitative antiviral screening. Antiviral
Res. 106, 53–60.
Martinez, M.J., Volchkova, V.A., Raoul, H., Alazard-Dany, N., Reynard, O., Volchkov,
V.E, 2011. Role of VP30 phosphorylation in the Ebola virus replication cycle.
J. Infect. Dis. 204 (Suppl. 3), S934–S940.
Morrey, J.D., Smee, D.F., Sidwell, R.W., Tseng, C., 2002. Identification of active
antiviral compounds against a New York isolate of West Nile virus. Antiviral
Res. 55, 107–116.
Neumann, G., Feldmann, H., Watanabe, S., Lukashevich, I., Kawaoka, Y., 2002.
Reverse genetics demonstrates that proteolytic processing of the Ebola virus
glycoprotein is not essential for replication in cell culture. J. Virol. 76, 406–410.
Niwa, H., Yamamura, K., Miyazaki, J., 1991. Efficient selection for high-expression
transfectants with a novel eukaryotic vector. Gene 108, 193–199.
Panchal, R.G., Kota, K.P., Spurgers, K.B., Ruthel, G., Tran, J.P., Boltz, R.C., Bavari, S.,
2010. Development of high-content imaging assays for lethal viral pathogens.
J. Biomol. Screen. 15, 755–765.
Panchal, R.G., Reid, S.P., Tran, J.P., Bergeron, A.A., Wells, J., Kota, K.P., Aman, J., Bavari,
S., 2012. Identification of an antioxidant small-molecule with broad-spectrum
antiviral activity. Antiviral Res. 93, 23–29.
Pyrc, K., Bosch, B.J., Berkhout, B., Jebbink, M.F., Dijkman, R., Rottier, P., van der Hoek,
L., 2006. Inhibition of human coronavirus NL63 infection at early stages of the
replication cycle. Antimicrob. Agents Chemother. 50, 2000–2008.
Schmidt, K.M., Schumann, M., Olejnik, J., Krahling, V., Muhlberger, E., 2011.
Recombinant Marburg virus expressing EGFP allows rapid screening of virus
growth and real-time visualization of virus spread. J. Infect. Dis. 204 (Suppl. 3),
S861–S870.
Schudt, G., Kolesnikova, L., Dolnik, O., Sodeik, B., Becker, S., 2013. Live-cell imaging
of Marburg virus-infected cells uncovers actin-dependent transport of nucleo-
capsids over long distances. Proc. Natl. Acad. Sci. USA 110, 14402–14407.
Silin, D., Lyubomska, O., Ludlow, M., Duprex, W.P., Rima, B.K., 2007. Development of
a challenge-protective vaccine concept by modification of the viral RNA-
dependent RNA polymerase of canine distemper virus. J. Virol. 81,
13649–13658.
Smee, D.F., McKernan, P.A., Nord, L.D., Willis, R.C., Petrie, C.R., Riley, T.M., Revankar,
G.R., Robins, R.K., Smith, R.A., 1987. Novel pyrazolo[3,4-d]pyrimidine nucleoside
analog with broad-spectrum antiviral activity. Antimicrob. Agents Chemother.
31, 1535–1541.
Stadler, T., Kuhnert, D., Rasmussen, D.A., du Plessis, L., 2014. Insights into the early
epidemic spread of ebola in sierra leone provided by viral sequence data. PLoS Curr. 6,
Oct 6 . Edition 1. 10.1371/currents.outbreaks.02bc6d927ecee7bbd33532ec8ba6a25f.
Theriault, S., Groseth, A., Artsob, H., Feldmann, H., 2005. The role of reverse genetics
systems in determining filovirus pathogenicity. Arch. Virol. Suppl. 19, 157–177 .
Towner, J.S., Paragas, J., Dover, J.E., Gupta, M., Goldsmith, C.S., Huggins, J.W., Nichol,
S.T., 2005. Generation of eGFP expressing recombinant Zaire Ebola virus for
analysis of early pathogenesis events and high-throughput antiviral drug
screening. Virology 332, 20–27.
Tsuda, Y., Hoenen, T., Banadyga, L., Weisend, C., Ricklefs, S.M., Porcella, S.F., Ebihara,
H., 2015. An Improved reverse genetics system to overcome cell-type-
dependent Ebola virus genome plasticity. J. Infect. Dis., Mar 24. pii: jiu681.
[Epub ahead of print].
Uebelhoer, L.S., Albariño, C.G., McMullan, L.K., Chakrabarti, A.K., Vincent, J.P., Nichol,
S.T., Towner, J.S., 2014. High-throughput, luciferase-based reverse genetics
systems for identifying inhibitors of Marburg and Ebola viruses. Antiviral Res.
106, 86–94.
Volchkov, V.E., Volchkova, V.A., Muhlberger, E., Kolesnikova, L.V., Weik, M., Dolnik,O.,
Klenk, H.D., 2001. Recovery of infectious Ebola virus from complementary DNA:
RNA editing of the GP gene and viral cytotoxicity. Science 291, 1965–19 69.
C.G. Albariño et al. / Virology 484 (2015) 259–264264