Journal of General Virology (2007), 88, 476–480
Short
Communication
DOI 10.1099/vir.0.82439-0
Pregnancy increases the risk of mortality in
West Nile virus-infected mice
Laura Córdoba,1 Estela Escribano-Romero,1,2 Antonio Garmendia3
and Juan-Carlos Saiz1
Correspondence
Juan-Carlos Saiz
[email protected]
1
Departamento de Biotecnologı́a, Instituto Nacional de Investigaciones Agrarias (INIA),
Ctra Coruña km 7.5, 28040 Madrid, Spain
2
Centro de Investigación en Sanidad Animal (CISA), INIA, Valdeolmos, 28130 Madrid, Spain
3
Department of Pathobiology and Veterinary Sciences, University of Connecticut,
61 N. Eagleville Road, Storrs, CT, USA
Received 1 August 2006
Accepted 29 October 2006
West Nile fever outbreaks in the USA have caused over 700 human deaths, primarily due to
neurological disease. The usual transmission route of West Nile virus (WNV) involves mosquito
bites; however, alternative routes, including intrauterine infection, have also been reported. Here,
the pathogenicity of WNV in mice during gestation has been investigated. An extremely high
mortality rate was observed in pregnant mice (98 %, 60/61) compared with non-pregnant mice
(52 %, 28/53; P<0.001), independent of the infecting dose or the week of pregnancy. Antibody
titres were similar between pregnant and non-pregnant mice and between surviving and nonsurviving animals. WNV RNA titres in brains were also similar between pregnant and non-pregnant
mice. WNV RNA could be detected in placentas and fetuses. These observations suggest strongly
that, in the mouse model, pregnancy increases the risk of severe WNV infection and may help to
understand the pathogenic mechanisms involved in WNV infection during pregnancy.
Since it was first detected in New York in 1999 (Anderson
et al., 1999; Lanciotti et al., 1999), West Nile virus (WNV), a
mosquito-transmitted flavivirus, has spread all over the
USA, some provinces of Canada, northern Mexico, parts of
the Caribbean and Latin America, causing thousands of
deaths among wild birds and horses. In humans, WNV
infection is frequently unapparent or causes a relatively mild
febrile condition, but it may also cause fatal encephalitis
(Granwehr et al., 2004; Hayes & O’Leary, 2004). Thus far,
around 20 000 human cases have been reported in the USA
(http://www.cdc.gov). More than 8000 were classified as
neuroinvasive, which resulted in over 700 deaths. Up to
10 000 cases were classified as West Nile fever and more than
400 had an unspecified clinical presentation. To date, no
licensed human vaccine or prophylactic therapy is available
(Granwehr et al., 2004; Hayes & O’Leary, 2004). Even
though exposure to infected mosquitoes is the most
important risk factor for acquiring WNV infection, virus
transmission to humans through blood transfusion (Pealer
et al., 2003), organ transplantation (Iwamoto et al., 2003)
and breastfeeding (CDC, 2002a), as well as transplacental
infection during pregnancy (CDC, 2002b; Hayes & O’Leary
2004), has also been reported. Data from a recent study in
pregnant women shows detection of WNV in infants within
a month of delivery from WNV-positive mothers and
suggests congenital transmission of the virus (O’Leary et al.,
2006). However, little is known about the mechanisms of
transmission and it is unclear whether and how the virus
476
causes some of the abnormalities observed in infants
(O’Leary et al., 2006).
Mice are a suitable animal model for the study of WNV
infection in humans as some signs in WNV-infected mice
parallel those exhibited by humans with severe neuroinvasive disease, such as confusion, tremor of extremities and
paralysis (Granwehr et al., 2004). Moreover, in mice,
pregnancy somewhat resembles that of humans and the first,
second and third weeks of pregnancy are, in many aspects,
equivalent to the first, second and third trimesters of the
human gestational period. In an attempt to gain insights
into the incidence of WNV infection during gestation, we
infected pregnant mice (at different weeks of gestation) with
various doses of WNV. As controls, groups of male and nonpregnant female mice were also infected.
WNV strain NY99 flamingo 382-99 (Lanciotti et al., 1999),
kindly provided by Dr H. von Briesen (Georg-Speyer-Haus,
Frankfurt, Germany), was propagated and titrated on Vero
cells (Tardei et al., 2000). Eight- to ten-week-old BALB/c
(H-2d) mice were infected by intraperitoneal (i.p.) injection
with different doses of virus (102–108 p.f.u. per mouse) in
200 ml Dulbecco’s modified Eagle’s medium (DMEM)
containing 5 % fetal bovine serum (FBS). Non-infected
contact-control cage-mate mice were inoculated with
DMEM containing 5 % FBS. Virus manipulation and
mouse experimentation were carried out in our Biosafety
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Mortality in WNV-infected pregnant mice
Level 3 (BSL-3) containment facilities and were approved
by and performed according to the guidelines for animal
experimentation of the Animal Safety Committee of CISA
(Madrid, Spain). In some instances, mice were bred in house
and pregnancy was checked by the presence of vaginal plugs.
Mice were monitored daily for signs of illness. Typical
clinical signs of WN disease were observed among infected
mice, and animals that died of the disease presented ruffling,
hunchback posture and hindlimb weakness and paralysis
24–48 h prior to death. In contrast, none of the non-infected
contact-control cage mates developed disease signs.
Mortality rates and mean survival time (MST) values were
recorded and statistical comparisons between groups were
made by using x2 or Fischer’s tests for categorical variables
and the Mann–Whitney test (unpaired samples) for
quantitative variables. Values of P<0.05 were considered
significant. At indicated time points, animals were anaesthetized with halothane before bleeding or euthanasia.
Collection of tissue and blood was performed under sterile
conditions as reported by Julander et al. (2005).
Mortality rates and MST values recorded among nonpregnant mice dying of WNV disease (Table 1) were not
statistically significantly different from those described by
Diamond et al. (2003). However, at the lower doses,
mortality rates were slightly higher than those reported in a
further study (Wang et al., 2003). The slightly higher
mortality rates reported here could be due to differences
either in virus strain (NY99 or Sarafend), mouse strain
(BALB/c or C57BL76J), administration route (i.p., intravenous or subcutaneous) or a combination thereof. In fact, the
NY99 strain, which belongs to WNV lineage I, is more
virulent than some strains isolated in other continents
(Beasley et al., 2002). Actually, the MST recorded in the
present study was similar between the different groups of
non-pregnant mice and, in general, lower than those
described for the WNV lineage II Sarafend strain (Wang
et al., 2003).
Humoral and cellular response against viral proteins
contributes to protection and recovery from WN disease
(Granwehr et al., 2004). Serological analysis by ELISA (Ebel
et al., 2002) using heat-inactivated WNV as antigen (Blitvich
et al., 2003) showed that specific IgM and IgG antibodies
were elicited in all infected mice but, at a given infecting
dose, no significant differences in antibody titres were
observed between mice that died of WN disease and those
that survived (Fig. 1). IgG antibodies were detectable in the
latter for up to 4 months post-infection (p.i.), and these
animals were protected against challenge with a lethal dose
of 108 p.f.u. per mouse inoculated within 21–95 days p.i.
On the other hand, none of the non-infected contactcontrol cage mates developed specific antibodies. Thus,
contrary to what has been described in birds and alligators in
laboratory settings (Komar et al., 2003; Klenk et al., 2004),
no horizontal transmission of WNV was observed in mice.
Mortality rates in pregnant mice were extremely high (98 %,
60/61) compared with those in non-pregnant animals
(52 %, 28/53; P<0.001; Table 1), independent of the
infecting dose administered (92 vs 55 % at a dose of
102 p.f.u. per mouse, P=0.038; 100 vs 58 % at 103 p.f.u. per
mouse, P=0.023; and 100 vs 48 % at 104 p.f.u. per mouse,
P<0.001) or the week of pregnancy (first, second or third)
at which they were infected (data not shown). In contrast,
MST values, whilst slightly higher in non-pregnant mice
dying of WN disease, were not statistically significantly
different (Table 1).
Our results also showed that, at any given infecting dose,
specific IgM and IgG titres elicited in pregnant and nonpregnant mice were similar (Fig. 1). Therefore, it seems
unlikely that the high mortality found here in pregnant mice
Table 1. Mortality rates and survival time recorded in WNV-infected mice
NT,
Not tested.
Dose
(p.f.u. per mouse)
102
103
104
105
106
107
108
Non-pregnant
Percentage of dead mice
(no. dead/total)
55.5
58.3
47.8
50.0
66.6
83.3
100.0
(10/18)
(7/12)
(11/23)
(7/14)
(8/12)
(5/6)
(23/23)
Pregnant
MST* (days)
Percentage of dead mice
(no. dead/total)
9.4±2.93
10.2±1.66
9.7±1.71
9.1±1.45
9.2±0.78
8.6±0.49
8.2±1.29
91.6 (11/12)D
100.0 (10/10)d
100.0 (39/39)§
MST* (days)
8.7±0.86
8.9±0.54
9.3±1.97
NT
NT
NT
NT
NT
NT
NT
NT
*Values represent mean survival time (MST)±SD of mice that died up to 15 days post-WNV infection.
DP=0.038.
dP=0.023.
§P<0.001.
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477
L. Córdoba and others
Fig. 1. Scatter plot of the levels of specific IgG (squares) and
IgM (circles) antibodies at day 7 after infection with 104 p.f.u.
WNV per mouse. Data correspond to pregnant (empty symbols)
and non-pregnant (shaded symbols) mice that died of WN disease, and to surviving mice (filled symbols). Titres were determined by ELISA and are expressed as positive/negative (P/N)
values of each sample, calculated by dividing the mean absorbance of positive antigen-containing wells by the absorbance of
the negative antigen-containing wells (Ebel et al., 2002). The
dotted line represents the positive cut-off P/N value below
which results are considered negative. Thin and thick solid lines
represent the mean titre corresponding to animals that survived
or died of WN disease, respectively. Thin and thick dashed
lines show mean titres of pregnant and non-pregnant mice,
respectively. No significant differences were observed between
the different groups analysed.
was due to a deficient antibody response. Likewise, titration
of WNV RNA by real-time RT-PCR (Lanciotti et al., 2000)
in brains of a representative number of animals at the time of
death, although quite variable, did not show significant
differences between pregnant and non-pregnant mice
(Fig. 2).
In other disease models, there are indications that resistance
to infectious pathogens varies during the menstrual cycle
and pregnancy, suggesting a role of sexual hormones in
disease outcome to virus infection (Klein, 2000). However,
we did not find significant differences in mortality rates
between non-pregnant females (11/23, 48 %) and a limited
number of males (4/11, 36 %) or non-pregnant females (4/8,
50 %) pre-treated with an i.p. injection of 2 mg Depo
(dihydroxyprogesterone acetate), a long-acting progestational formulation used widely to facilitate infection in
animal models (Kaushic et al., 2003), that was administered
5 days before being exposed to WNV.
Pregnancy might affect the clinical course of virus infections
negatively (Gilbert, 2002). In animal models, an increased
disease severity during pregnancy has been reported after
infection with several flaviviruses, such as Japanese
encephalitis virus (JEV) (Mathur et al., 1981), St. Louis
encephalitis virus (SLEV) (Andersen & Hanson, 1975) and
478
Fig. 2. Scatter plot of the levels of WNV RNA in the brains of
a representative number of mice infected with 102, 103 or
104 p.f.u. WNV per mouse. Data represent titres of pregnant
mice (filled symbols) infected during the first (triangles), second
(squares) or third (circles) week of gestation, and non-pregnant
mice (empty symbols). Brains were harvested at the time of
death (8–10 days p.i.), homogenized and subjected to real-time
RT-PCR (Lanciotti et al., 2000) using a positive-control sample
of known titre. Values are given as genomic equivalents
(g tissue)”1. The dotted line indicates the limit of sensitivity of
the assay.
Murray Valley encephalitis virus (MVEV) (Aaskov et al.,
1981), but the proportion of dead animals did not reach that
observed here. In WNV infection, a recent analysis of the
effect of reactive immunoglobulin in fetal virus infection has
shown high mortality in a limited number of untreated
dams (Julander et al., 2005). Congenital infection of mice
with SLEV 8 days post-coitus (p.c.) resulted in infection of
both the placenta and the fetus (Aaskov et al., 1981).
Likewise, JEV (Andersen & Hanson, 1975) and WNV
(Julander et al., 2005) infect mouse fetuses more efficiently
during the first week of pregnancy than thereafter,
suggesting that fetal infection may differ at different stages
of placental development. Furthermore, WNV titres in the
placenta were higher and were detectable earlier after
infection than in other maternal organs (Julander et al.,
2005). Consistent with these observations, no WNV RNA
was detected in the brains, placentas or fetuses of a few dams
infected during the second or third week of gestation that
were euthanized 2–4 days p.i. In contrast, WNV RNA was
detected in the placentas (between 1.16105 and 1.66105
genomic equivalents g21) and the fetuses (between 8.96104
and 2.76105 genomic equivalents g21) of two mice infected
during the first week of pregnancy (6 days p.c.), euthanized
4 and 5 days p.i., respectively. Lack of virus detection in the
brain at this early time point is not surprising as, at the dose
administered (104 p.f.u. per mouse), it is too early in the
infection process for invasion of the brain. Replication of
WNV in the placenta might increase viral load in pregnant
mice early after infection, even before it could be detected in
the brains of the dams, favouring a high mortality rate.
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Journal of General Virology 88
Mortality in WNV-infected pregnant mice
Virus infection during pregnancy could have serious
consequences for fetuses and newborns (Koi et al., 2001).
Intrauterine fetal infection with several flaviviruses is often
associated with fetal mortality, abortion, preterm delivery of
stillborns and death of newborns at or shortly after birth, but
most babies showed an apparently normal life (Aaskov et al.,
1981; Andersen & Hanson, 1975; Julander et al., 2005;
Mathur et al., 1981). In the present report, all animals
infected during the first week of pregnancy died before
delivery. On the other hand, five of the 14 mice infected
during the second week of gestation delivered before dying
of WN disease (10 days p.i.). All pregnant mice infected
during the third week of gestation survived to deliver pups,
but only one dam, infected with the lowest dose (102 p.f.u.),
survived to the end of the experimental period (90 days).
This animal was actively infected, because specific antibodies were present in its serum and it was protected against
challenge with a lethal dose of WNV administered 2 months
after the initial infection. No overt signs of WN disease were
observed during the follow-up of infants born 3 days p.i. to
this surviving dam, and they showed specific IgG 1 month
after birth. In fact, they were protected against challenge
with a lethal dose of WNV inoculated 60 days after infection
of their mother.
Maternal infection by WNV during pregnancy has been
reported in humans (CDC, 2002b; Hayes & O’Leary, 2004).
Infection of the placenta and intrauterine transmission of
WNV to the fetus were first documented in a woman with
signs of WN disease, who was later diagnosed with
meningoencephalitis (CDC, 2002b; Hayes & O’Leary,
2004). Her infant was born at term with chorioretinitis
and severe cerebral abnormalities, although such abnormalities could not be associated conclusively with the virus
infection (Alpert et al., 2003). A case of a premature delivery
has also been documented in a WNV-infected woman and,
although her infant presented with neonatal respiratory
distress, no tests for WNV were performed (Hayes &
O’Leary, 2004). In another three pregnancies complicated
by WNV infection, no apparent abnormalities have been
observed in the newborns (Hayes & O’Leary, 2004). Lately, it
has been reported that none of 71 WNV-infected pregnant
women included in a retrospective study died of WN disease
and that most of their children were born healthy (O’Leary
et al., 2006). In this study, three cases of infant malformation
were observed, suggesting the possibility of congenital
infection with WNV. In any case, and because the
mechanisms of non-mosquito-borne transmission and the
effects and abnormalities seen in the infants remain largely
unknown, assessment of the fetus or child is recommended
when mothers are infected by WNV (CDC, 2004). Even
more, the CDC and the state health departments of the USA
are currently collecting clinical and laboratory data on
outcomes of pregnancies of WNV-infected women, and
clinicians are encouraged to report known or suspected
cases (O’Leary et al., 2006). Nevertheless, all of these
observations indicate that, in contrast to the elevated
mortality found here in WNV-infected pregnant mice, no
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increased mortality is observed in pregnant women
(O’Leary et al., 2006). Susceptibility to WNV infection in
inbred mice has been linked to the presence of point
mutations in the 29-59-oligoadenylate synthetase gene
(Mashimo et al., 2002), of which regulation by interferon
can be affected during pregnancy and, thus, these mutations
may account, to some extent, for the differences observed
between humans and mice.
In summary, and although care should be taken before
extrapolating our data to WNV-infected women, the high
risk of severe WN disease observed in pregnant mice
deserves further investigations, which should help to
understand better the pathogenic mechanisms implicated
in WNV infection during pregnancy in mice.
Acknowledgements
We are indebted to A. Canals and E. Domingo for making possible for
us the use of the BSL-3 facilities and for their continuous support. The
work was supported in part by a grant (AGL2004-06071) from the
Spanish Ministerio de Educación y Ciencia (MEC) to J.-C. S. and by
the Research Foundation, University of Connecticut, for support
through a Faculty Small Research Grant to A. G. L. C. has been
supported by a scholarship from INIA and EER by the ‘Juan de la
Cierva’ programme (MEC).
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