Transgenic expression of full-length 2′,5′-oligoadenylate synthetase 1b confers to
BALB/c mice resistance against West Nile virus-induced encephalitis
Dominique Simon-Chazottesa,d, Marie-Pascale Frenkielb, Xavier Montagutellia,d, Jean-Louis Guénetc,
Philippe Desprèsb, Jean-Jacques Panthiera,d,⁎
aMouse Functional Genetics, Institut Pasteur, Paris, France
bFlavivirus–Host Molecular Interactions, Institut Pasteur, Paris, France
cDepartment of Developmental Biology, Institut Pasteur, Paris, France
dCentre National de la Recherche Scientifique, Unité de Recherche Associée 2578, Paris, France
a b s t r a c ta r t i c l e i n f o
Received 4 March 2011
Accepted 31 May 2011
Available online 17 June 2011
Susceptibility of inbred strains to infection with West Nile virus (WNV) has been genetically associated with
an arginine-to-a nonsense codon substitution at position 253 (R253X) in the predicted sequence of the
murine 2′,5′-oligoadenylate synthetase 1B (OAS1B) protein. We introduced by transgenesis the Oas1b cDNA
from MBT/Pas mice carrying the R253 codon (Oas1bMBT) into BALB/c mice homozygous for the X253 allele
(Oas1bBALB/c). Overexpression of Oas1bMBTmRNA in the brain of transgenic mice prior and in the time course
of infection provided protection against the neuroinvasive WNV strain IS-98-ST1. A 200-fold induction of
Oas1bMBTmRNA in the brain of congenic BALB/c mice homozygous for a MBT/Pas segment encompassing the
Oas1b gene was also efficient in reducing both viral growth and mortality, whereas a 200-fold induction of
Oas1bBALB/cmRNA was unable to prevent virally-induced encephalitis, confirming the critical role of the
R253X mutation on Oas1b activity in live mice.
© 2011 Elsevier Inc. All rights reserved.
West Nile virus (WNV) is a positive-sense, single-stranded RNA
flavivirus transmitted by mosquitoes that infects a wide range of
vertebrate hosts and causes severe illness in humans, including
encephalitis, meningitis, or flaccid paralysis. About 80% of WNV
infections are asymptomatic, 20% result in self-limited West Nile
fever, and b1% result in neurologic disease (Hayes and Gubler, 2006).
Host genetic factors might be important to control the susceptibility
and severity to WNV infection (Diamond et al., 2009). Our
laboratories reported that classical inbred strains such as BALB/c,
whose genome is 92% of Mus m. domesticus origin (Yang et al., 2007),
were highly susceptible to intraperitoneal infection with the
neuroinvasive and neurovirulent Israeli strain IS-98-ST1 of WNV
while mouse strains derived from wild progenitors of the M. m.
musculus (MBT/Pas) or Mus spretus (SEG/Pas) species were resistant.
We mapped the resistance locus to a critical interval of approximately
1 Mb that contains a cluster of 10 members of the Oas gene family
(Oas1a to Oas1h, Oas2 and Oas3) encoding 2′,5′-oligoadenylate
synthetases, and identified a premature stop codon within exon 4 of
the Oas1b gene, hence encoding a truncated version of OAS1B, lacking
~30% of its C-terminal domain (Mashimo et al., 2002). There was a
perfect correlation among the various inbred strains of mice between
the presence of this mutation and the susceptibility phenotype,
strongly supporting that the truncated Oas1b allele was responsible
for the susceptibility of laboratory mice to infection with the IS-98-
ST1 viral strain. Perelygin et al.(2002) simultaneously identified the
premature stop codon in C3H/He mice susceptible to infection with
WNV strain Eg101 and showed that viral growth was impaired in
C3H/He fibroblasts expressing a full-length OAS1B protein. In vitro
experiments further demonstrated that expression of full-length
OAS1B protein but not the C-terminally truncated form inhibits WNV
replication inside infected mouse cells (Kajaste-Rudnitski et al., 2006;
Lucas et al., 2003). Together, there is mounting evidence that the OAS
family may play a crucial role in antiviral host immunity to WNV
mediated by type-I interferons (Kristiansen et al., 2011). Consistent
with this finding, genetic variations in human and horse OAS1 are risk
factors for infectionwithneuropathogenic WNV(Lim et al.,2009; Rios
et al., 2010). To establish that the alteration in the coding sequence of
the Oas1b gene is causative of susceptibility to flaviviruses, Scherbik et
al. (2007a) replaced exons 4 and 5 in the genome of 129/Sv mice with
DNA that corresponds to the MBT/Pas allele and reported that the
Virology 417 (2011) 147–153
⁎ Corresponding author at: Institut Pasteur, Mouse functional Genetics, 25 rue du
Docteur Roux, 75015 Paris, France. Fax: +33 1 4568 8634.
E-mail address: firstname.lastname@example.org (J.-J. Panthier).
0042-6822/$ – see front matter © 2011 Elsevier Inc. All rights reserved.
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knock-in mice were resistant to lethal infection with neurotropic
flavivirus via the intracranial route.
In addition to the nonsense mutation, several nucleotide sequence
variantshave beenidentifiedin thepromoterregionof theOas1bgene
(Mashimo et al., 2003). Although none of them was consistent with
the pattern of resistance or susceptibility across the inbred strains
tested, we suspected that one or more of the SNPs could alter
activation of Oas1b transcription in response to viral infection. To gain
a better understanding of the molecular basis of Oas1b-mediated
WNV resistance in vivo, we took a complementary approach. A cloned
cDNA corresponding to the Oas1b mRNA expressed in resistant MBT/
Pas mice was introduced by transgenesis to induce constitutive
expression of the full-length coding sequence in an otherwise
susceptible genetic background (BALB/c). We report that up-regula-
tion of a wild-type Oas1b cDNA confers to BALB/c mice resistance
against lethal WNV-induced encephalitis.
Results and discussion
Full-length OAS1B is a key-factor for mouse resistance to WNV infection
Positional cloning identified Oas1b as a candidate gene accounting
for the resistance of wild-derived inbred strains of mice to infection
with WNV (Mashimo et al., 2002; Perelygin et al., 2002). To further
assess the role of the Oas1b gene during lethal infection, we produced
a C.MBT-Oas1b congenic line where the Oas gene cluster of the MBT/
Pas resistant strain was introgressed into the BALB/c susceptible
genetic background. C.MBT-Oas1b congenic mice survive an i.p.
inoculation of 1000 FFU of the virulent IS-98-ST1 WNV strain while
BALB/c mice die within 10 days post-infection (Fig. 1), indicating that
the MBT/Pas segment carries one or more determinant(s) of
resistance (Mashimo et al., 2002). Although this result demonstrates
the importance of the Oas gene cluster, it does not point unambig-
uously to the Oas1b nonsense mutation.
To address this issue, a transgene consisting of the CAG promoter
and Oas1b cDNA from MBT/Pas strain was constructed (Fig. 2) and
micro-injected into (C57BL/6 J×SJL/J)F2 fertilized eggs. Eggs were
implanted in pseudo pregnant females. Three resulting founder
animals harboured the entire transgene (Tg), as shown by PCR
analysis, and were crossed with BALB/c mice to generate transgenic
lines in a BALB/c genetic background. After twelve generations of
backcrossing, Tg/+ transgenic mice from each of the three lines were
examined for their susceptibility to lethal infection with WNV. C.MBT-
Oas1b congenic and BALB/c mice served as controls. Mice were
inoculated i.p. with 1000 FFU of the virulent WNV IS-98-ST1 strain. All
BALB/c mice (N=20) died within 10 days post-infection, whereas all
C.MBT-Oas1b mice (N=20) survived the infection, as expected. In
CAG::Oas1bMBTtransgenic line 1, 75.7% (25/33) of the Tg/+ mice
survived the infection (Fig. 1). There was no significant sex bias on
survival. Tg/+ mice from transgenic lines 2 and 3 also survived the
infection, though the percentage of survivors was lower (50 and 37%
on average, respectively) (Fig. 1). These data indicate that the CAG::
Oas1bMBTtransgene partially rescued the susceptible phenotype of
The CAG composite promoter has been shown to drive transgene
expression in the whole body of adults and in embryos (Kawamoto et
al., 2000; Kubo et al., 2002; Okabe et al., 1997). To assess CAG::
Oas1bMBTtransgene expression in vivo, RT-PCR experiments were
performed on various tissues of adult mice from each line. Organs
were harvested and total RNAs were extracted from the heart, lung,
liver, kidney, spleen and brain, RT-PCR assays were performed using
primer pairs specific for the transgene (primers c–d in Fig. 2C) and the
housekeeping aldolase gene, respectively. Fig. 3 shows that the
transgene was expressed at detectable levels in the heart, lung, and
brain in each line. However, transgene expression level was hardly
detected in the liver in all lines. The CAG::Oas1bMBTexpression was
not identical among the three lines suggesting that expression of the
transgene was dependent on the site of integration, as commonly
observed in transgenic mice (De Sepulveda et al., 1995). The more
resistant transgenic line was used in subsequent investigations. To
evaluate the expression of transgenic and endogenous Oas1b genes,
we measured Oas1b mRNA levels in peripheral organ (spleen) and the
central nervous system (brain) of BALB/c, C.MBT-Oas1b and CAG::
Oas1bMBTmice using primers that amplify the transcripts from the
endogenous Oas1bMBTand Oas1bBALB/cgenes, and from the Oas1bMBT
transgene (primers a-b in Fig. 2). mRNAs containing a premature
translation termination codon, such as Oas1bBALB/cmRNA, are
generally degraded by nonsense-mediated decay (NMD) (Matsuda
et al., 2008). Table 1 shows that, in the spleen, Oas1b mRNA level was
approximately two fold higher in BALB/c than in C.MBT-Oas1b mice
(pb0.05). This suggests that despite the premature translation
termination codon in exon 4 of Oas1b in BALB/c mice, the transcript
Fig. 1. Survival analysis of mice after West Nile virus infection. CAG::Oas1bMBT
transgenic mice from line 1 (N=33), line 2 (N=8) and line 3 (N=22), C.MBT-Oas1b
congenic mice (N=20) and BALB/c control mice (N=20) were inoculated with
1000 FFU of WNV IS-98-ST1 strain via the intraperitoneal route and monitored daily for
mortality during 14 days. CAG::Oas1bMBTtransgenic mice from lines 1, 2 and 3 were
significantly more resistant than BALB/c mice (pb10−7, pb10−8and pb0.005,
Fig. 2. Schematic structure of the endogenous Oas1b gene and CAG::Oas1bMBT
transgene. Structure of the endogenous Oas1b gene of BALB/c (A) and MBT/Pas
(B) mice. Exons 1 to 6 of Oas1b are shown in boxes. Introns are shown by broken lines.
The position of the CGA arginine codon in MBT/Pas mice that is mutated into TGA stop
codon in BALB/c mice is also indicated. The positions of primers used for detecting (RT-
PCR) and measuring (qRT-PCR) Oas1b mRNA are indicated. (C) The transgene contains
the CMV/β-actin (CAG) promoter with β-actin intron and the complete MBT/Pas Oas1b
cDNA and the SV40 polyadenylation sequence (SV40 p(A)).
D. Simon-Chazottes et al. / Virology 417 (2011) 147–153
was not degraded by NMD. This conclusion agrees with previous
detection of Oas1b mRNA in different tissues of BALB/c mice
(Mashimo et al., 2003; Perelygin et al., 2002), and the truncated
form of OAS1B in fibroblasts overexpressing Oas1bBALB/ccDNA
(Kajaste-Rudnitski et al., 2006). Oas1b is likely an additionalexception
to the rule for which nonsense codons trigger NMD (Matsuda et al.,
2008). In the brain of transgenic CAG::Oas1bMBTmice, Oas1b mRNA
levels were approximately 1,300 (i.e. 5,814/4.3) and 5,800 fold higher
than in the brain of C.MBT-Oas1b (pb0.001) and BALB/c mice
(pb0.001), respectively. Small significant difference in Oas1b mRNA
levels was also observed in the spleen of CAG::Oas1bMBTmice
compared to C.MBT-Oas1b (pb0.01). To determine whether the
expression of other Oas genes was affected by the increase in Oas1b
expression, we measured Oas1a mRNA levels in the spleen and brain
of BALB/c, C.MBT-Oas1b and CAG::Oas1bMBTmice. Table 2 shows that
Oas1a gene was expressed at similar levels in BALB/c, C.MBT-Oas1b
and CAG::Oas1bMBTmice, indicating that increase Oas1b expression
was not associated with variation in Oas1a expression.
Up-regulation of endogenous Oas1b in response to West Nile virus
Oas1b gene is induced in response to WNV infection in mouse
embryonic fibroblasts (Fredericksen et al., 2008; Scherbik et al.,
2007b). To determine the time course and level of endogenous Oas1b
gene expression in susceptible and resistant mice, we measured the
level of Oas1b mRNA in spleen and brain after lethal infection with
WNV. Susceptible BALB/c, resistant C.MBT-Oas1b, and partially
resistant CAG::Oas1bMBTmice were infected intraperitoneally with
1000 FFU of the virulent WNV IS-98-ST1 strain and Oas1b mRNA was
measured in samples harvested on days 1, 2, 4, 6, 8 and 10 post-
infection by qRT-PCR using primers specific for the endogenous Oas1b
transcripts (c–d′ in Fig. 2B). In susceptible BALB/c mice that carry a
nonsense mutation in the Oas1b coding sequence, we detected no
significant induction of Oas1bBALB/cmRNA in the spleen (Fig. 4A).
However, in the brain, the expression of Oas1bBALB/cmRNA was
increased12 fold onday2. Oas1bBALB/cmRNAlevels peakedonday8in
the brain (200-fold increase, pb0.001) (Fig. 4B), one day before the
death of most infected BALB/c mice (Fig. 1). In resistant C.MBT-Oas1b
mice, with an arginine codon replacing the stop codon in the Oas1b
coding sequence, Oas1bMBTmRNA levels were increased 260 to 600
fold in the spleen on days 1 and 2. At later stages, Oas1bMBTmRNA
levels in the spleen decreased gradually to reach the level seen in
uninfected controls (Fig. 4C). In the brain, the peak induction of
Oas1bMBTmRNA (200-fold increase; pb0.001) was observed on day 4.
Thereafter, Oas1bMBTmRNA levels decreased progressively even
though it still remained high on day 10 post-infection, 15 fold higher
than the level measured in uninfected controls (Fig. 4D). Partially
resistant CAG::Oas1MBTtransgenic mice are homozygous for the
Oas1bBALB/callele. In the spleen of CAG::Oas1MBTmice, Oas1bBALB/c
mRNA levels were moderately increased (7-fold) on day 1 and
decreased progressively thereafter (Fig. 4E). In the brain of CAG::
Oas1MBTmice, the level of Oas1bBALB/cmRNA increased until day 8 (16
fold compared to uninfected controls, Fig. 4F). Altogether, resistant
C.MBT-Oas1b mice were characterized by a significant induction of
Oas1bMBTexpression in the spleen and brain at an early stage post-
infection. By contrast, the expression of Oas1bBALB/cmRNA was not
significantly increased in the spleen of susceptible BALB/c mice. High
levels of Oas1bBALB/cmRNA in the brain at day 8 likely reflected very
high viral burden at this late stage of infection preceding death from
encephalitis. These data suggest that susceptibility in BALB/c mice
could result from the combined effects of the nonsense mutation in
the coding sequence and of one or more regulatory mutation(s) in the
promoter region of the BALB/c Oas1b gene (Mashimo et al., 2003).
Expression of mRNA encoding full-length OAS1B in transgenic mice
infected with WNV
we analyzed the kinetics of expression of Oas1bMBTmRNA in the spleen
Fig. 3. Expression of the Oas1bMBTmRNA in CAG::Oas1bMBTtransgenic mice. Agarose gel
showing RT-PCR products generated from total RNA collected from the heart, lung,
liver, kidney, spleen and brain of a mouse from transgenic line 1, line 2 and line 3, and
BALB/c (BALB) inbred line (C). RT-PCR primers were designed to amplify fragments of
the transgene Oas1b cDNA (343 bp) and the endogenous internal control aldolase (Ald)
cDNA (278 bp). M (marker) denotes the low range DNA ladder, 25–700 bp (Fermentas
GmbH, Courtaboeuf, France).
Relative levels of expression of the endogenous and transgenic Oas1b mRNA in the spleen
and brain of uninfected wild-type BALB/c, C.MBT-Oas1b congenic and CAG::Oas1bMBT
Mice Expressed Oas1b
Relative level of Oas1b
mRNA expression in
Relative level of
expression in the
1 [0.61–1.64]1 [0.59–1.71]
1.82 [1.02–3.25] 5814 [4965–6807]c
aTotal RNA from the same mice and organs was analyzed for the expression of
endogenous Oas1b mRNA (wild-type BALB/c, and C.MBT-Oas1b mice) and of both
endogenous and transgenic Oas1b mRNA (CAG::Oas1bMBTmice) by qRT-PCR. Data are
expressed as the relative increase (n-fold) compared with uninfected BALB/c mice.
Average values and 95% confidence intervals for the relative quantization values are
from ten mice.
Relative levels of expression of Oas1a mRNA in the spleen and brain of uninfected wild-
type BALB/c, C.MBT-Oas1b congenic and CAG::Oas1bMBTtransgenic mice.
MiceRelative level of Oas1a mRNA
expression in the spleena
Relative level of Oas1a mRNA
expression in the braina
1.14 [1.52–1.14] 1.27 [0.42–3.79]
aTotal RNA from the same mice and organs was analyzed for the expression of Oas1a
bData are expressed as the relative increase (n-fold) compared with uninfected
BALB/c mice. Average values and 95% confidence intervals for the relative quantization
values are from ten mice.
D. Simon-Chazottes et al. / Virology 417 (2011) 147–153
and brain of infected CAG::Oas1bMBTmice. CAG::Oas1bMBTmice were
infected i.p. with 1000 FFU of WNV IS-98-ST1 strain and Oas1bMBT
post-infection by qRT-PCR using primers specific for the transgenic
Oas1bMBTmRNA (c–d in Fig. 2C). In the spleen, transgenic Oas1bMBT
mRNAlevels were decreased atanearly stage, ondays 1 (20 fold) and 2
(approximately 500,000 fold). On later days, Oas1bMBTmRNA levels
were progressively restored to that of uninfected controls (Fig. 5A). In
the brain, Oas1bMBTmRNA levels also dropped off (160 fold) on days 1
and 2, but increased significantly thereafter to peak on day 6 post-
infection at 150 fold the level measured in the uninfected controls.
Thereafter, Oas1bMBTmRNA levels decreased progressively to the level
seen in the controls (Fig. 5B). Therefore, induction of the transgenic
Oas1bMBTmRNA in the spleen may not account for the survival of
transgenic BALB/c mice. These data suggest that the high expression of
Oas1bMBTmRNA in the brain prior and after the infection is critical for
the survival of CAG::Oas1bMBTmice.
To evaluate the effect of Oas1b mRNA expression on WNV
infection, we analyzed viral growth kinetics in the brain of infected
BALB/c, C.MBT-Oas1b and CAG::Oas1bMBTmice. Fig. 6 shows that the
viral burden increased significantly in BALB/c mice between day 6 and
day 8. The peak of viral burden in the brain of BALB/c mice correlated
with the 200 fold increase of Oas1bBALB/cmRNA expression (Fig. 4B).
By contrast, the viral burden did not vary between day 6 and day 8 in
the brain of C.MBT-Oas1b and CAG::Oas1bMBTmice. These data
suggest that the antiviral effect of Oas1bMBTgene expression
In the present study, we show that inducing constitutive
expression of an Oas1bMBTtransgene in an Oas1bBALB/cbackground is
able to confer resistance to an otherwise lethal infection with WNV.
Our results establish the functional role played by the premature stop
codon in exon 4 of Oas1b gene in the susceptibility of BALB/c mice but
also suggest impaired activation of the Oas1b gene in response to viral
infection in this strain. These findings are in agreement with in vitro
studies that showed reduce viral production in WNV-infected cells
overexpressing mRNA encoding the full-length OAS1B (Lucas et al.,
2003; Perelygin et al., 2002). In recent studies, Scherbik et al.(2007a)
Fig. 4. Levels of endogenous Oas1b mRNA expression in the spleen and brain of West Nile virus-infected mice. BALB/c (A, B), C.MBT-Oas1b (C, D) and CAG::Oas1bMBT(E, F) mice were
infected with 1000 FFU of the virulent IS-98-ST1 strain and euthanized on days 1, 2, 4, 6, 8 and 10. Total RNA from the spleen (A, C and E) and brain (B, D and F) was analyzed for the
expression of endogenous Oas1b mRNA by qRT-PCR. Data are expressed as the relative increase (n-fold) compared with uninfected controls. Data from ten mice are shown as
whisker-box plots. Open circles indicate outliers.
D. Simon-Chazottes et al. / Virology 417 (2011) 147–153
used a complementary experimental approach and showed that the
replacement of exons 4 and 5 of Oas1b gene in susceptible 129/SvJ
mice by the corresponding sequences from resistant mice results in
the acquisition of resistance to intracerebral infection by the
neurovirulent 17D strain of yellow fever virus. Together, the results
of knock-in and transgenesis experiments establish that expression of
a full-length OAS1B protein is a cornerstone in the resistance to
infection with flaviviruses.
Important for the outcome of the disease in transgenic mice were
the levels of Oas1bMBTmRNA in the brain prior to and after infection.
The 1300 fold increase in Oas1bMBTmRNA level in the brain of
and the further 150 fold increase at day 6 post-infection (Fig. 5B) were
able to control viral burden in the brain (see Fig. 6). This suggests that
towards later WNV infection. These data agree with recent studies
6 post-infection compared to the viral burden in the brain of BALB/c
mice. This suggests that the expression of full-length OAS1B protein in
peripheral organs of congenic and transgenic mice had no significant
the brain. At day 8, a significant increase in viral burden was observed
in the brains of BALB/c mice. By contrast, there was no increase in viral
burden in the brain of congenic and transgenic mice. These data
suggest that following infection with a neuroinvasive and neuroviru-
limit the burden of viral genome. Despite this protection by the CAG::
Oas1bMBTtransgene, approximately one-third of transgenic mice did
chromosomal segment carried by congenic mice, the Oas1bMBT
transgene conferred only partial resistance to WNV-infected BALB/c
mice. Although the reasons remain unclear, several differences
between congenic and transgenic mice could account for the disparity
in results. First, by contrast with endogenous Oas1bMBTgene, the
transgene expression driven by the hybrid cytomegalovirus (CMV)
enhancer/chicken β-actin (CAG) promoter was down-regulated in the
spleen and brain at an early stage post-infection. This decrease is most
likely due to inhibition of the CMV immediate early enhancer by the
cytokine response that follows the viral infection. Indeed, cytokines
induced by WNV infection, such as IFN-α and TNF-α (Fredericksen et
al., 2008) have been shown to reduce transgene expression from the
CMV promoter (Gribaudo et al., 1995; Qin et al., 1997). Second, the
activity of the transgene in the brain prior to infection varied from
mouse to mouse. Lower transgene expression could be responsible for
on average, susceptible transgenic mice died approximately two days
later than BALB/c mice (10.1±0.5 days for CAG::Oas1bMBTmice and
8.2±0.2 days for BALB/c mice), suggesting that transgenic Oas1bMBT
mRNA could provide some level of protection against the viral
infection to all individuals. Finally, the possibility that, in congenic
mice, there is a contribution to resistance by additional genetic
factors within the Oas cluster is a critical issue that remains to be
Materials and methods
The WNV IS-98-ST1 strain was isolated from the cerebellum of a
white stork during the 1998 outbreak in Israel and passaged three
times in a mosquito cell line (Malkinson et al., 2002) before its
characterization in mice (Lucas et al., 2003).
All studies on animals followed the guideline on the ethical use of
animals from the European Community Council Directive of 24
November 1986 (86/609/EEC). Animal experiments were approved
and conducted in accordance with the Institut Pasteur Biosafety
Committee. BALB/cAnNCrl (BALB/c hereafter) mice were purchased
from Charles River France Laboratories (L'Arbresle, France). Congenic
and transgenic mice were produced in the animal facilities at the
Fig. 5. Level of CAG::Oas1bMBTmRNA expression in West Nile virus-infected transgenic
mice. Mice were infected with 1000 FFU of the virulent IS-98-ST1 strain and euthanized
on days 1, 2, 4, 6, 8 and 10. Total RNA from the spleen (A) and brain (B) wasanalyzed for
the expression of transgenic Oas1b mRNA by qRT-PCR. Data are expressed as the
relative increase (n-fold) compared with uninfected controls. Data from ten mice are
shown as whisker-box plots. Open circles indicate outliers.
Fig. 6. Viral burden analysis in the brain of wild-type BALB/c, congenic C.MBT-Oas1b
and transgenic CAG::Oas1bMBTmice after West Nile virus infection. West Nile virus RNA
in the brain was determined from samples harvested on days 6 and 8 using qRT-PCR.
Data are shown as viral RNA equivalent of PFU per gram of tissue at the indicated time
point. Data from ten mice are shown as whisker-box plots. *: pb0.05.
D. Simon-Chazottes et al. / Virology 417 (2011) 147–153
Eight- to ten-week-old mice were used in all experiments.
Infection was performed by intraperitoneal inoculation of 1000 FFU
Generation of congenic mice
The 1.45 Mb chromosome 5 segment from the MBT/Pas genome
delimited by the D5Mit68 and D5Mit242 markers, that encompasses
27 genes, including the eight Oas1 paralogs, and the Oas2 and Oas3
genes, was introgressed into the BALB/c background following ten
successive backcrosses. Thereafter, mice heterozygous for the MBT/
Pas chromosomal segment were intercrossed to produce homozy-
gotes for the MBT/Pas segment that were maintained by sib mating.
The resulting congenic line is designated C.MBT-Oas1b.
Production of transgenic mice
The Oas1b cDNA from MBT/Pas strain (GenBank: AF466823) was
inserted into a pCAL3 expression vector (Niwa et al., 1991; Xu et al.,
2001) containing the CMV immediate early enhancer, chicken β-actin
promoter with β-actin intron and SV40 (late gene) polyadenylation
sequence. The entire insert with the promoter and coding region,
pCAG::Oas1bMBT, was linearized with PvuI restriction enzymes and
gel-purified. The purified fragment was injected into (C57BL/6 J×SJL/
J) F2 fertilized eggs. Incorporation of the transgene was assessed by
PCR analyses of ear DNA. PCR primers for the transgene detection
were as follows: forward primer, 5′AACCATGTTCATGCCTTCTTCT-3′;
reverse primer, 5′-AAGGAACACCACCAGGTCAG-3′. Three transgenic
founders were used to derive three independent congenic strains by
twelve generations of backcrossing onto the BALB/c background. All
transgenic mice used in this study were heterozygous for the
Quantification of Oas1a and Oas1b mRNA levels by qRT-PCR
Ten mice were used per time point. After extensive cardiac perfusion
with phosphate-buffered saline (PBS), organs were harvested in 1 ml
Trizol®(Invitrogen, Carlsbad, CA, USA) containing 0.4 g of glass beads
(212–300 μm ø, SIGMA) and homogenized for 40 s at 6.0 in FastPrep
Instrument (MP Biochemicals, Illkirch, France). Isolated RNA was
repurified using an RNeasy®Plus Mini Kit (QIAGEN, Valencia, CA, USA).
cDNA synthesis was performed with 5 μg RNA using RevertAidTMH
Minus First Strand cDNA Synthesis Kit (Fermentas Inc., Glen Burnie, MD,
USA). Five microliters of a 1:10 dilution of cDNA were used per qRT-PCR
reaction. The cDNA obtained from organs was quantified by real-time
qRT-PCR using MaximaTMSYBR Green/ROX qPCR Master Mix (Fermen-
To analyze the relative fold induction of endogenous Oas1b gene and
CAG::Oas1b transgene mRNA, aldolase (Ald) mRNA expression levels
were also determined for normalization by using the CTmethod (Livak
and Schmittgen, 2001). For Ald mRNA, the forward primer was 5′-
AGCAGAATGGCATTGTACCC-3′ and reverse primer was 5′-ACAG-
GAAAGTGACCCCAGTG-3′. For Oas1b mRNAs, five primers were
designed: a: 5′-GAGGTGCCGACGGAGGT-3′; b: 5′-TCCAGAT-
GAAGTCTTCCCAAAG-3′; c: 5′-CAGGATGCTCCAGAGTCAGACG-3′; d: 5′-
TGACACCAGACCAACTGGTAA-3′; and d′: 5′-GACAAGAGAAAGCCCA-
CACC-3′. Primers a and b amplify endogenous Oas1b gene and CAG::
Oas1bMBTtransgene mRNAs.Primers c and d′ amplifyendogenous Oas1b
mRNA. Primers c and d amplify CAG::Oas1bMBTtransgene mRNA.
Therefore, primers c–d and c–d′ allow distinguishing the mRNAs from
mRNA expression levels, the following primers were used: forward: 5′-
GGAGGCGGTTGGCTGAAGAGG-3′; reverse: 5′-GAACCACCGTCGGCA-
CATCC-3′. Reactions were quantified in triplicate and data was analyzed
with StepOnePlus software version 2.1 (Applied Biosystems).
Quantification of tissue viral burden
Tomonitor viral spread in thebrain,positive-strandviral RNA levels
were measured using Taqman qRT-PCR with the Hot Pol®Probe qPCR
Mix (Euromedex, Souffelweyersheim, France). The 5′-end of WNV
capsid mRNA was amplified from total RNA and its expression level
5′-CCTGTGTGAGCTGACAAACTTAGT-3′; reverse primer, 5′-
GCGTTTTAGCATATTGACAGCC-3′; probe, 5′-FAM-CCTGGTTTCTTAGA-
CATCGAGATCT-3′-TAMRA, as previously described (Linke et al., 2007).
Data analysis was performed using the MS Excel statistical package
(Microsoft Corporation). Kaplan–Meier survival data was analyzed by
the log rank test. Differences in relative expression were analyzed
using one-way analysis of variance (ANOVA) or non-parametric
We thank Sylvie Paulous for preparing the stock of WNV IS-98-ST1
strain. This work was funded by the Transverse Research Program No.
202, the genetics of host predisposition to infectious diseases, from
the Institut Pasteur. The Mouse Functional Genetics Unit is supported
by Merck Serono.
De Sepulveda, P., Salaun, P., Maas, N., Andre, C., Panthier, J.J., 1995. SARs do not impair
position-dependent expression of a kit/lacZ transgene. Biochem. Biophys. Res.
Commun. 211, 735–741.
Diamond, M.S., Mehlhop, E., Oliphant, T., Samuel, M.A., 2009. The host immunologic
response to West Nile encephalitis virus. Front. Biosci. 14, 3024–3034.
Fredericksen, B.L., Keller, B.C., Fornek, J., Katze, M.G., Gale Jr., M., 2008. Establishment
and maintenance of the innate antiviral response to West Nile Virus involves both
RIG-I and MDA5 signaling through IPS-1. J. Virol. 82, 609–616.
Gribaudo, G., Ravaglia, S., Gaboli, M., Gariglio, M., Cavallo, R., Landolfo, S., 1995.
Interferon-alpha inhibits the murine cytomegalovirus immediate-early gene
expression by down-regulating NF-kappa B activity. Virology 211, 251–260.
Hayes, E.B., Gubler, D.J., 2006. West Nile virus: epidemiology and clinical features of an
emerging epidemic in the United States. Annu. Rev. Med. 57, 181–194.
Kajaste-Rudnitski, A., Mashimo, T., Frenkiel, M.P., Guenet, J.L., Lucas, M., Despres, P.,
2006. The 2′,5′-oligoadenylate synthetase 1b is a potent inhibitor of West Nile virus
replication inside infected cells. J. Biol. Chem. 281, 4624–4637.
Kawamoto, S., Niwa, H., Tashiro, F., Sano, S., Kondoh, G., Takeda, J., Tabayashi, K.,
Miyazaki, J., 2000. A novel reporter mouse strain that expresses enhanced green
fluorescent protein upon Cre-mediated recombination. FEBS Lett. 470, 263–268.
Kristiansen, H., Scherer, C.A., McVean, M., Iadonato, S.P., Vends, S., Thavachelvam, K.,
Steffensen, T.B., Horan, K.A., Kuri, T., Weber, F., Paludan, S.R., Hartmann, R., 2010.
Extracellular 2′–5′ oligoadenylate synthetase stimulates RNase L-independent antiviral
Kristiansen, H., Gad, H.H., Eskildsen-Larsen, S., Despres, P., Hartmann, R., 2011. The
oligoadenylate synthetase family: an ancient protein family with multiple antiviral
activities. J. Interferon Cytokine Res. 31, 41–47.
Kubo, J., Yamanouchi, K., Naito, K., Tojo, H., 2002. Expression of the gene of interest
fused to the EGFP-expressing gene in transgenic mice derived from selected
transgenic embryos. J. Exp. Zool. 293, 712–718.
Lim, J.K., Lisco, A., McDermott, D.H., Huynh, L., Ward, J.M., Johnson, B., Johnson, H., Pape,
J., Foster, G.A., Krysztof, D., Follmann, D., Stramer, S.L., Margolis, L.B., Murphy, P.M.,
2009. Genetic variation in OAS1 is a risk factor for initial infection with West Nile
virus in man. PLoS Pathog. 5, e1000321.
Linke, S., Ellerbrok, H., Niedrig, M., Nitsche, A., Pauli, G., 2007. Detection of West Nile
virus lineages 1 and 2 by real-time PCR. J. Virol. Methods 146, 355–358.
quantitative PCR and the 2(−Delta Delta C(T)) Method. Methods 25, 402–408.
Lucas, M., Mashimo, T., Frenkiel, M.P., Simon-Chazottes, D., Montagutelli, X., Ceccaldi, P.E.,
Guenet, J.L., Despres, P., 2003. Infection of mouse neurones by West Nile virus is
modulated by the interferon-inducible 2′–5′ oligoadenylate synthetase 1b protein.
Immunol. Cell Biol. 81, 230–236.
Malkinson, M., Banet, C., Weisman, Y., Pokamunski, S., King, R., Drouet, M.T., Deubel, V.,
2002. Introduction of West Nile virus in the Middle East by migrating white storks.
Emerg. Infect. Dis. 8, 392–397.
D. Simon-Chazottes et al. / Virology 417 (2011) 147–153
Mashimo, T., Lucas, M., Simon-Chazottes, D., Frenkiel, M.P., Montagutelli, X., Ceccaldi, P.E., Download full-text
Deubel, V., Guenet, J.L., Despres, P., 2002. A nonsense mutation in the gene encoding
2′–5′-oligoadenylate synthetase/L1 isoform is associated with West Nile virus
susceptibility in laboratory mice. Proc. Natl. Acad. Sci. U. S. A. 99, 11311–11316.
Mashimo, T., Glaser, P., Lucas, M., Simon-Chazottes, D., Ceccaldi, P.E., Montagutelli, X.,
Despres, P., Guenet, J.L., 2003. Structural and functional genomics and evolutionary
relationships in the cluster of genes encoding murine 2′,5′-oligoadenylate
synthetases. Genomics 82, 537–552.
Matsuda, D., Sato, H., Maquat, L.E., 2008. Chapter 9. Studying nonsense-mediated mRNA
decay in mammalian cells. Methods Enzymol. 449, 177–201.
Niwa, H., Yamamura, K., Miyazaki, J., 1991. Efficient selection for high-expression
transfectants with a novel eukaryotic vector. Gene 108, 193–199.
Okabe, M., Ikawa, M., Kominami, K., Nakanishi, T., Nishimune, Y., 1997. 'Green mice' as a
source of ubiquitous green cells. FEBS Lett. 407, 313–319.
Perelygin, A.A., Scherbik, S.V., Zhulin, I.B., Stockman, B.M., Li, Y., Brinton, M.A., 2002.
Qin, L., Ding, Y., Pahud, D.R., Chang, E., Imperiale, M.J., Bromberg, J.S., 1997. Promoter
attenuation in gene therapy: interferon-gamma and tumor necrosis factor-alpha
inhibit transgene expression. Hum. Gene Ther. 8, 2019–2029.
Rios, J.J., Fleming, J.G., Bryant, U.K., Carter, C.N., Huber, J.C., Long, M.T., Spencer, T.E.,
Adelson, D.L., 2010. OAS1 polymorphisms are associated with susceptibility to
West Nile encephalitis in horses. PLoS One 5, e10537.
Scherbik, S.V., Kluetzman, K., Perelygin, A.A., Brinton, M.A., 2007a. Knock-in of the
Oas1b(r) allele into a flavivirus-induced disease susceptible mouse generates the
resistant phenotype. Virology 368, 232–237.
Scherbik, S.V., Stockman, B.M., Brinton, M.A., 2007b. Differential expression of
interferon (IFN) regulatory factors and IFN-stimulated genes at early times after
West Nile virus infection of mouse embryo fibroblasts. J. Virol. 81, 12005–12018.
Xu, Z.L., Mizuguchi, H., Ishii-Watabe, A., Uchida, E., Mayumi, T., Hayakawa, T., 2001.
Optimization of transcriptional regulatory elements for constructing plasmid vectors.
Gene 272, 149–156.
Yang, H., Bell, T.A., Churchill, G.A., Pardo-Manuel de Villena, F., 2007. On the subspecific
origin of the laboratory mouse. Nat. Genet. 39, 1100–1107.
D. Simon-Chazottes et al. / Virology 417 (2011) 147–153